Thesis

Characterization of α-actinin as a member of the spectrin superfamily of proteins in "Neurospora crassa"

COTADO-SAMPAYO, Marta

Abstract

Nous avons étudié le rôle de l' α-actinine dans le développement de "Neurospora crassa". A l'aide des outils bioinformatiques, nous avons trouvé un gêne codant pour l'α-actinine puis nous avons caractérisé ce dernier biochimiquement. Nous avons déterminé sa localisation "in situ" et "in vivo" pendant la germination et pendant la croissance hyphale. Bien que le rôle exact de l'α-actinine n'ait pas pu être totalement élucidé, l'étude phénotypique du transformant ("knock-out" partiel de l'α-actinine) nous amène à penser que cette protéine participe avec l'actine à la coordination de la germination, la formation des septa et l'établissement des ramifications hyphales lors de la croissance du champignon. En outre, nous avons pu déterminer que l'α-actinine est la seule protéine appartenant à la superfamille des spectrines chez les champignons filamenteux, les levures et les Oomycètes et représente un membre "primitif" de cette superfamille.

Reference

COTADO-SAMPAYO, Marta. Characterization of α-actinin as a member of the spectrin superfamily of proteins in "Neurospora crassa". Thèse de doctorat : Univ. Genève, 2008, no. Sc. 3976

URN : urn:nbn:ch:unige-18247 DOI : 10.13097/archive-ouverte/unige:1824

Available at: http://archive-ouverte.unige.ch/unige:1824

Disclaimer: layout of this document may differ from the published version.

1 / 1 UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Département de botanique et biologie végétale Professeur Reto J. Strasser Laboratoire de bioénergétique et microbiologie Dr François Barja

Characterization of α-actinin as a member of the spectrin

superfamily of proteins in Neurospora crassa

THÈSE présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Marta COTADO-SAMPAYO de Ourense (Espagne)

Thèse n° 3976

Genève Repromail, Université de Genève 2008

Les minutes passées à réfléchir au contenu d’une erreur marquent plus profondément les esprits que les heures passées à ingurgiter des théorèmes exacts. Car ces minutes sont accompagnées d’une émotion, d’une révolte intérieure…….. Albert Jacquard « L’Équation du nénuphar »

REMERCIEMENTS

Je désire d’abord remercier les membres du Jury: le Dr. Roland Beffa, le Professeur William Broughton, le Professeur Reto Strasser et le Dr. Francisco Barja qui ont accepté de lire et d’évaluer ce travail. Je tiens à exprimer ma gratitude au Dr. R. Beffa qui a soigneusement veillé à la bonne organisation et rédaction de ce manuscrit et au Professeur R. Strasser pour son soutien.

L’aboutissement de cette thèse a été rendu possible par le soutien porté par mon superviseur de thèse, Francisco Barja. Arrivée à Genève comme étudiante Erasmus, un peu désorientée et avec un projet incertain, Francisco m’a ouvert les portes de son laboratoire et m’a donné sa confiance pour entreprendre une thèse. J’apprécie que malgré toutes les difficultés rencontrées, il m’ait toujours soutenue dans tous mes efforts et ma motivation pour mener à bien à ce travail. Ce projet de recherche qui se termine maintenant est le résultat de tout le «groupe de Francisco». Grâce à la bonne entente de l’équipe propice à un travail fructueux, il est encore possible de nos jours de travailler dans une ambiance de convivialité et de confiance.

C’est donc avec une particulière gratitude que je remercie Malou Chappuis et Ariane Fehr pour leur aide efficace. Merci à Cristina Andrés pour tous les bons moments partagés et pour son si précieux soutien dans les moments difficiles.

Le mérite de cette thèse est à partager avec le Dr. Ruben Ortega qui m’a aidée par ses conseils judicieux et ses critiques constructives tant dans la réalisation pratique que dans la discussion. Et encore plus important il ne m’a pas laissé « achicopalarme ».

Je remercie aussi vivement le Dr. Mukti Ojha pour son aide savante et amicale, et qui m’a sans cesse encouragée en me guidant et me donnant des conseils avisés.

Un grand merci aussi aux membres du groupe de Bioimagerie, le Dr. Christophe Bauer et Jérôme Bosset pour sa disponibilité. À Mike Parkan pour m’avoir auxilié avec beaucoup de patience avec mes « lacunes » dans le domaine d’informatique et bien sur pour sa bonne humeur.

Je tiens aussi à remercier tous mes collègues du laboratoire de Bioénergétique (Abdallah Oukarroum, Georgina Ceppi, Madeleine Fontana, Marie-France Blanc, Gert Schansker et Dina Hanggraini) ainsi que ceux du département de Biologie végétale, spécialement Christophe Dunand et Sonia Guimil, pour tout le soutien qu’ils m’ont apporté. Enfin, ma reconnaissance va aussi aux nombreux membres du laboratoire, aussi bien anciens qu’actuels, qui ont également participé plus au moins directement à ce travail. Ainsi, je remercie Arlette Cattanéo, Pilar Okenve, Edurne Martinez, Idoia Alonso, Javier Remiro, Greta Rubio, Enrique Raposo, Loreto Naya, Lucia Soliño, Marta Alonso, Sibylle Baruchel (Schindhelm), Aurélia Weber, Catherine Wilson, Marco Dias, Géraldine Martinelli et Fabrizio Molino pour leur aide scientifique mais aussi pour tous les bons moments passés ensembles.

Bien sûr une pensée pour mes amis, spécialement Maria del Mar, Ana “pajaros”, Txema, Cristina “bailarina”, Urko, Ana “Barja”, Gorge Faustino et Sébastien qui ont suivi de très près l’aventure de ma thèse.

Un grand merci à Manu pour tout l’amour qu’il m’a donné malgré les heures que mon travail lui a volé.

Enfin, merci à ma famille pour leur soutien et leurs encouragements.

Con gran alegría y satisfacción, dedico este trabajo a mis padres.

CONTENTS

Résumé 3

Summary 5

List of original publications 6

Abbreviations 7

1. GENERAL INTRODUCTION 9

1.1. CYTOSKELETON 9

1.2. ACTIN CYTOSKELETON 11

1.2.1. Actin 11 1.2.2. Actin binding proteins 13 1.2.3. Spectrin superfamily 13 1.2.4. α-Actinin 17 1.2.4.1. Functions 1.2.4.2. Isoforms 1.2.4.3. “Atypical” α-actinins

1.3. WHY STUDY FUNGI AS A MODEL OF TIP GROWTH? 20

1.3.1. Filamentous fungi 21 1.3.2. Cytoskeleton in fungi 22 1.3.3. Actin cytoskeleton in fungi 23 1.3.3.1. Actin 1.3.3.2. Actin binding proteins

2. BACKGROUND AND AIMS OF THE STUDY 25

3. MATERIALS AND METHODS 27

4. RESULTS 37 4.1. Identity of anti-αβ-spectrin immunoreacting peptides 37

in fungi and Oomycetes (Publications I and II)

4.2. α-Actinin orthologs in fungi (Publication III) 51

4.3. Characterization of α-actinin from Neurospora crassa 53

(Publication IV)

5. DISCUSSION, CONCLUSIONS AND PERSPECTIVES 67

6. REFERENCES 75

ANNEXE 95

Résumé

Caractérisation de l’α-actinine, une protéine membre de la superfamille des spectrines chez Neurospora crassa

La superfamille des spectrines est composée par des protéines qui lient l’actine. Elles participent à l’organisation du cytosquelette et interagissent avec d’autres protéines ou structures comme la membrane plasmique. Les protéines de ce groupe possèdent trois domaines: N-terminal « CH- domain » (Calponin Homology domain), C-terminal « EF-hand motifs » et un domaine central composé d’un nombre variable de « spectrin repeats ». Avec ces caractéristiques, la spectrine, l’α- actinine et la dystrophine/utrophine ont toujours été considérées comme des membres de la superfamille des spectrines. Ces protéines ont été décrites dans la plupart des cellules eucaryotes. Néanmoins, chez les plantes et les champignons, la présence de spectrine a été démontrée sur la base des résultats obtenus par des techniques immunologiques, souvent en utilisant des anticorps commerciaux polyclonaux. Ainsi, la présence des protéines «spectrin-like » chez les champignons filamenteux, les levures et les Oomycetes est mise en doute car ces protéines ont seulement été identifiées à l’aide d’anticorps dont la spécificité n’était pas suffisamment établie. De plus, chez ces organismes, après l’analyse des génomes complètement séquencés, aucun gène codant pour la spectrine n’a été trouvé. Par analyse de spectrométrie de masse, nous avons identifié la protéine « spectrin-like » comme étant le facteur d’élongation 2 (EF 2) chez Neurospora crassa. Par ailleurs, nous avons aussi identifié une protéine correspondant à la protéine de choc thermique (Hsp70) chez l’oomycete Phytophthora infestans. La caractérisation du peptide reconnu chez N. crassa par l’anticorps anti-αβ-spectrine ainsi que la réactivité croisée de cet anticorps ont été amplement traités dans la partie 4.1 de cette thèse, ainsi que dans les publications I et II.

De plus, en utilisant les outils de bioinformatique (BLAST) nous avons trouvé dans la base de données génomique de N. crassa (http://www.broad.mit.edu/annotation/fgi/) un gène (ncu06429.4) qui code pour une protéine similaire à l’α-actinine. Nous avons utilisé la séquence de cette protéine pour chercher leurs orthologues chez les champignons. L’α-actinine semble être le seul membre de la superfamille des spectrines. Néanmoins, cette protéine n’est pas présente dans tous les champignons ; chez certaines levures du groupe des Saccharomycotina, il n’y a pas d’évidence prouvant l’existence d’α-actinine. Dans ces champignons cette protéine semblerait avoir été perdue lors de l’évolution du fait que d’autres protéines liant l’actine pourraient complémenter sa fonction. Ce sujet a été traité en détail dans la partie 4.2 de cette thèse et dans la publication III.

Suite à notre étude bioinformatique nous avons pu identifier un gène codant pour l’α-actinine, membre le plus « primitif » de la superfamille des spectrines. Cette protéine a été identifiée, localisée in situ et in vivo et caractérisée biochimiquement chez N. crassa.

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Bien que le rôle exact de l’α-actinine chez N. crassa n’aie pas pu être totalement élucidé, l’étude phénotypique du transformant (« knock-out » partiel de l’α-actinine) nous amène à penser que l’α- actinine participe avec l’actine à la coordination d’activités telles que la germination, la formation des septa et l’établissement des ramifications hyphales lors de la croissance. En outre, à la différence d’autres organismes, chez N. crassa l’α-actinine est une protéine essentielle, car son absence (« knock-out » complet de l’α-actinine) est létale pour le champignon.

4

Summary

Cell shape, cell division, polarization and tip growth are processes that have been widely studied using fungi as models. The organization of the cytoplasm and the coordination of cell activity during the life cycle of fungi are poorly understood but are believed to depend on a cytoskeletal system. The cytoskeleton in fungi is composed principally of microfilaments and microtubules. The dynamics and function of these structures are regulated by associated proteins. These proteins were first described using immunological techniques but their existence can now be verified by the availability of a number of complete fungal and plant genome sequences. The existence of an actin-binding “spectrin- like” protein in plants, fungi, Oomycetes and lower eukaryotic organisms has been reported. However, these reports were solely based on immunological studies and evidence for a gene coding for spectrin was not presented. In fungi and Oomycetes another member of the spectrin superfamily, α-actinin, was first proposed by us to be the immunoreactive peptide and we gave this the name “spectrin- related” protein. However, further studies demonstrated that α-actinin is not related to the protein that is recognized by anti-spectrin antibodies in fungi and Oomycetes. Instead, this immunoreactive peptide turned out to be a cross-reacting protein not related to the spectrin superfamily.

Assessing the characteristics of α-actinin in fungi may provide further insights into the biology of fungi and also help to establish new links between developmental complexity and genome evolution. In the work described here we studied the features of the α-actinin orthologous group in fungi. Using Neurospora as a model for filamentous fungi we studied the ability of this protein to bind actin and calcium. Our results on the localization of α-actinin and the phenotype of the α-actinin knock-out strain suggest that α-actinin is essential for conidial germination and septum formation during hyphal growth. The fact that α-actinin is also localized along the peripheral region suggests that this protein may have additional functions.

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List of original publications

The present thesis is based on the following original articles (see annexe), which are referred to in the text by their Roman numerals:

I. Cotado-Sampayo M., Ojha M., Ortega Perez R., Chappuis M-L., Barja F., 2006. Proteolytic cleavage of a spectrin-related protein by calcium-dependent protease in Neurospora crassa. Curr. Microbiol. 53: 311-316.

II. Cotado-Sampayo M., Okenve Ramos P., Ortega Perez R., Ojha M., Barja F., 2008. Specificity of commercial anti-spectrin antibody in the study of fungi and oomycetes: cross-reaction with proteins other than spectrin. Fungal Genet. Biol. 45 (6): 1008-1015.

III. Cotado-Sampayo M., 2008. Features of α-actinin in fungi and Oomycetes. Under revision.

IV. Cotado-Sampayo M., Ortega Perez R., Seum C., Ojha M., Barja F., 2008. Characterization of Neurospora crassa α-actinin. Under revision.

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Abbreviations aa Amino acid ABD Actin-binding domain ABP Actin-binding protein Aip1 Actin-interacting protein 1 Amp Ampicillin ARP Actin related protein ATP Adenosine-5'-triphosphate BLAST Basic local alignment search tool BCIP Bromo-chloro-indolyl phosphate bp Base pairs BSA Bovine serum albumin °C Degree Celsius CBD Calcium-binding domain cDNA Complementary DNA CDP Calcium-dependent protease GCY Glucose Casein hydrolase Yeast extract GFP Green fluorescent protein GST Glutathione S-transferase CH Calponin homology CIP Calf intestinal phosphatase cm Centimeter 2D Two dimensional Da Dalton DAB 3,3'-Diaminobenzidine tetrahydrochloride dH2O Distilled water dist. Distilled DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DNAse Deoxyribonuclease dNTP Deoxyribonucleotide triphosphate DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid EF Elongation factor e.g. “exempli gratia” (for example) EGTA Ethyleneglycol-bis(2-aminoethylether)-N,N'-tetraacetic acid EST Expressed sequence tag et al. “et alii” (and others) FGSC Fungal genetics stock center Fig. Figure FITC Fluorescein-isothiocyanate g Gram GFP Green fluorescence protein GST Glutathione S-transferase GTP Guanosin-5'-triphosphate h Hour HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid His Histidine HMW High molecular weight Hsp Heat shock protein i.e. “id est” (that is) IPTG Isopropyl-β-D-thiogalactopyranoside k Kilo kDa Kilodalton

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KIF Kinesin family protein KO Knock-out l Liter LB Luria Bertani LMW Low molecular weight M Mol/l MALDI Matrix-assisted laser desorption/ionization min Minute μF Microfaraday µm Micrometer MIPS Munich center for protein sequences MOPS Morpholinopropanesulfonic acid Mr Relative molecular mass MS Mass spectrometry MYA Million years ago nm Nanometer NMDA N-methyl-D-aspartate OD Optical density PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffer saline PCR Polymerase chain reaction PDA Potato Dextrose Agar pH Potential of hydrogen pI Isoelectric point PMSF Phenylmethylsulfonyl fluoride pNA para-Nitroaniline PVDF Polyvinylidene difluoride PVP Polyvinyl polypyrrolidone RNA Ribonucleic acid RNAse Ribonuclease rpm Revolutions per minute SDS Sodium dodecyl sulfate SEM Simple and efficient method for transformation sec Second SR Spectrin repeat siRNA Small interfering RNA TBS Tris buffer saline TCA Trichloroacetic acid TOF Time-of-Flight Tris Tris-hydroxymethyl-aminomethane TRITC Tetramethylrhodamine isothiocyanate Triton X-100 tert-Octylphenoxypolyethoxyethanol Tween 20 Polyoxyethylene(20) sorbitan monolaureate U Units V Volt v/v Volume per volume w/v Weight per volume WT Wild-type YpSs Yeast protein Soluble starch

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1. GENERAL INTRODUCTION

1.1. CYTOSKELETON

The cytoskeleton is fundamental to the intracellular organization, plays an important role in cell division and also in the communication of the cell with its environment. A large number of studies have been performed in recent years in order to identify proteins involved in the cytoskeleton and to understand its role in the physiology of eukaryotic cells. Processes such as the establishment of cellular shape, cell locomotion, endo- and exocytosis, signaling, intracellular transport and cell division depend on this complex network of protein filaments that extends throughout the cytoplasm (Schliwa, 1986; Lloyd, 1991; Qualmann and Kessels, 2002; Smythe and Ayscough, 2006; Lanzetti, 2007). A substantial amount of molecular, biochemical and physiological information has been obtained on the cellular organization of the cytoskeleton. Knowledge of cytoskeletal and associated proteins is also important to gain a better understanding of numerous human diseases that depend on cytoskeleton dysfunction. Abnormal phosphorylation of a microtubule associated protein, Tau, is associated with Alzheimer neurofibrillary pathology (Goedert et al., 1996; Strong et al., 2006; von Bernhardi, 2007) and point mutations in cardiac actin have been detected in familial hypertrophic cardiomyopathy (Mogensen et al., 1999; Vang et al., 2005; Monserrat et al., 2007).

The cytoskeleton is composed of three major types of protein filaments: actin filaments (microfilaments), microtubules and intermediate filaments (Figure 1). Each type of filament is formed as a chain of protein monomers and can be built into a variety of structures depending on its associated proteins.

Intermediate filaments are polymers of elongated fibrous protein monomers, such as vimentin, keratin and lamin, which belong to a family of structurally and genetically related proteins. Intermediate filaments are found in metazoans. Different families of intermediate filaments are expressed in different cell types. One essential role of these filaments is to distribute tensile forces across cells in a tissue.

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A B

Figure 1. Cytoskeleton in eukaryotic cell. A. Organization of actin, microtubules, and intermediate filaments within a cell. B. Confocal image of an endothelial cell where actin filaments are shown in red, microtubules in green, and the nuclei in blue (web images in the public domain).

Microtubules are hollow tubes formed by the assembly of heterodimers of α- and β-tubulin. The heterodimers are arranged in longitudinal rows called protofilaments. Thirteen protofilaments are assembled in a parallel fashion around a hollow core that is approximately 25 nm in diameter. The microtubules are polar structures with two distinct ends: a fast-growing plus end and a slow-growing minus end. This polarity is an important consideration in determining the direction of movement along microtubules. The “minus” end in the cell starts in the MTOC (microtubule organizing center). Oakley and Oakley (1989) identified a third type of tubulin, named γ-tubulin. Microtubules participate in chromosome segregation during cell division, transport of vesicles and organelles, and cilia and flagella movements.

Microfilaments are 4 to 7 nm wide filaments formed by actin monomers (G-actin), a globular protein of 43 kDa. Microfilaments (F-actin) are the principal components of the actin cytoskeleton. The actin cytoskeleton shows an organizational flexibility that allows cells to assume many shapes. In motile cells like fibroblasts actin participates in locomotion. In mammals and many other organisms, actin is required for muscle contraction, cytokinesis, cell-substrate interactions, endocytosis and secretion.

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1.2. ACTIN CYTOSKELETON

1.2.1. Actin

Microfilaments are polymers of actin monomers that, together with a large number of actin-binding proteins, form the actin cytoskeleton (dos Remedios et al., 2003; Dominguez, 2004).

Actin monomer structure

Monomeric G-actin has dimensions of ~67 × 40 × 37 Å and a molecular mass of about 43 kDa. About 40% of the structure consists of α-helices (Otterbein et al., 2001; dos Remedios et al., 2003). The actin monomer contains four subdomains (Figure 2), a central cleft contains a high-affinity binding site for a nucleotide (ATP or ADP) and a cation (usually Ca2+ or Mg2+). Many of the known actin-binding proteins bind to the same loci, in the hydrophobic cleft between subdomains 1 and 3, and therefore can be expected to compete for this binding site (Dominguez, 2004). Subdomain 2 contains a DNAse-I- binding loop that participates in the intra-strand interactions between F-actin subunits. The function of the DNAase I loop is unknown. However, DNAse I is a valuable tool to measure the G-actin content of actin solutions. When DNAase I is bound by actin it no longer has the capacity to cleave DNA (Lazarides and Lindberg, 1974; Hitchcock, 1980) and it can therefore be used to titrate actin.

Actin isoforms

The actins are classified into three groups according to their isoelectric point: α-, β- and γ-isoforms. In mammals, there are at least six different actin isoforms, each encoded by a separate gene, and these differ by <10% of the amino acid sequence (Vandekerckhove and Weber, 1978). In plants the number of genes is higher; e.g. Petunia contains >l00 actin sequences in its genome (Baird and Meagher, 1987) and other plant species, such as soybean, tobacco, potato, rice, and lodgepole pine, also appear to have dozens of actin genes (Meagher, 1991; Thangavelu et al., 1993; Meagher and Williamson, 1994).

The presence of an ancestral actin protein in bacteria was first suggested by Bork et al. (1992). This protein, called MreB, was proposed to be the prokaryotic origin of the actin cytoskeleton (van den Ent et al., 2001). Although the amino acid sequence homology of the MreB to eukaryotic actin is limited to 15%, their overall size and shape are markedly similar (van den Ent et al., 2001). Furthermore, MreB has similar actin-like cytoskeletal roles (Jones et al., 2001; Carballido-Lopez, 2006; Pradel et al., 2006; Graumann, 2007; Vats and Rothfield, 2007).

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Figure 2. Ribbon representation of the structure of uncomplexed actin in the ADP state (Otterbein et al., 2001).

Actin polymerization

Actin polymers assemble spontaneously through non-covalent interactions between monomeric subunits and thus form highly dynamic structures with turnover at both ends. Actin monomers polymerize under physiological conditions, i.e. high ionic strength (KCl concentrations >50 mM), neutral or slightly acidic pH, high Mg2+ levels and elevated temperature (Asakura et al., 1960; Grazi and Trombetta, 1985). The assembly of actin monomers (G-actin) into filaments (F-actin) occurs in three steps: (1) a slow initial association into a dimer, (2) the formation of a more stable trimer that represents the nucleus of polymerization, and (3) the elongation phase. Each actin monomer can bind ATP. When an actin monomer is incorporated into the polymer the ATP is hydrolyzed to ADP (Carlier, 1990, 1992; Carlier and Pantaloni, 1997; Romero et al., 2004; Zheng et al., 2007). The elongation rate is directly proportional to the concentration of free actin and only ATP-actin monomers are likely to participate in polymerization (Pollard, 1986). At the steady-state concentration, the rate of actin assembly is the same as the rate of actin disassembly and the actin filaments thus have a constant length. This phenomenon is known as treadmilling and it acts like a motor for cell motility and pathogen locomotion (Disanza et al., 2005).

Several actin-binding proteins exist in vivo and these regulate different aspects of actin dynamics and will be discussed in the next section.

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1.2.2. Actin-binding proteins

Monomer availability for polymerization is regulated by actin monomer binding and actin filament capping proteins (Weber et al., 1999; Pollard and Borisy, 2003). Capping proteins are F-actin-binding proteins that interfere with the growth of an actin filament by blocking one of its ends. The activities of these proteins are often regulated by signaling molecules and ions such as Ca2+ (Lehrer, 2002; Oertner and Matus, 2005; Lange and Gartzke, 2006).

Actin-binding proteins can be grouped into three groups according to their function: (1) those that participate in the formation of filaments from G-actin and the subsequent stability of these filaments, (2) motor proteins that use F-actin for traction and (3) those that connect actin filaments to the cell membrane or cross-link actin filaments to form different structures such as bundles, branching filaments and three-dimensional networks. Examples of the latter group are all the members of the spectrin superfamily.

1.2.3. Spectrin superfamily

The spectrin superfamily is composed of spectrin, α-actinin, dystrophin and utrophin. These proteins are involved in the organization of the actin cytoskeleton. Proteins belonging to the spectrin superfamily have three characteristic domains. They contain an N-terminal actin-binding domain (ABD) and a C-terminal calcium-binding domain (CBD) linked by a rod domain. In spectrin, where the basic unit is a heterodimer of α- and β-spectrin, the actin- and calcium-binding domains are both in the N-terminal region of the dimer (Figure 3).

The actin-binding domain contains two calponin homology domains (CH1 and CH2 domains), both with a low sequence similarity and functional diversity despite the predicted similarity in secondary structure. Actin-binding studies on the isolated binding domain of α-actinin have shown that the two domains have different roles in actin-binding (Way et al., 1992; McGough et al., 1994; Lorenzi and Gimona, 2008). The CH1 domain by itself has a reduced affinity for F-actin. The CH2 does not have any intrinsic actin-binding activity but contributes substantially to the interaction of the complete actin-binding domain, perhaps by acting as a locator of low affinity docking sites on the actin filament (Djinovic-Carugo et al., 1997; Bañuelos et al., 1998; Gimona et al., 2002). The fact that the single calponin domain lacks actin-binding activity suggests the possibility that CH domains have additional functions.

The calcium-binding domain is composed of EF-hand motifs located at the C-terminus (except for spectrin, where is absent on the β-subunit). The EF-hand motif found in the spectrin superfamily

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shares structural homology with calmodulin. For this reason, the calcium-binding domain has also been named the calmodulin-like domain.

EF-hand regions are usually paired helix-loop-helix structures involved in the coordination of up to two divalent cations, usually calcium but occasionally magnesium (Tufty and Kretsinger, 1975). The binding of calcium to the EF-hands induces a conformational change that is implicated in the regulation of the actin- binding activity of the protein (Lundberg et al., 1992; Trave et al., 1995). However, divergent evolution has led in some members of the spectrin superfamily to a set of EF- hands that no longer chelate calcium (Nakayama and Kretsinger, 1994).

α-actinin (100 kDa)

CH CH EF EF

CH-domain Plekstrin Homology EF-hand Src3 Homology Septrin repeat β-spectrin (220 kDa) WW domain ZZ domain

Cys-rich domain α-spectrin (240 kDa)

Dystrophin (420 kDa)

Utrophin (395 kDa)

Figure 3. Schematic representation of the different members of the spectrin superfamily (adapted from Broderick and Winder, 2002).

The rod domain is composed of several repeats called spectrin repeats because they were initially described in spectrin (Speicher and Marchesi, 1984). The folding of the spectrin repeats consists of three α-helices in a coiled-coil assembly, where the three helices wrap around each other (Pascual et al., 1996; Djinovic-Carugo et al., 1999, 2002; Broderick and Winder, 2005). The number of spectrin repeats varies within the spectrin superfamily and ranges from 2–4 in α-actinin to 17–24 repeats in spectrin, utrophin and dystrophin (Broderick and Winder, 2005). The repeat length has also been

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found to differ between α-actinin, spectrin, and dystrophin, with 114–125, 106, and 109 residues, respectively. This is mainly due to differences in the inter-helical loop length (Parry et al., 1992).

Spectrin superfamily evolution

Since gene codings for any member of the spectrin superfamily of proteins have not been found in the bacterial and plant genomes available to date, it has been suggested that this superfamily appeared in a primitive unicellular organism belonging to the animal kingdom (Virel and Backman, 2004). The amino acid sequences and the structure analysis of spectrins, α-actinin and utrophin/dystrophin proteins suggest that all three protein families arose from a single common ancestral protein that was α-actinin-like (Byers et al., 1989 and 1992; Dubreuil, 1991).

Phylogenetic analysis indicated that the α-actinin-1 from the protozoan Entamoeba histolytica is the earliest diverging α-actinin, followed by the α-actinin of Encephalitozoon cunculi (Virel and Backman, 2004). These two proteins have the shortest rod domain, with only one spectrin repeat. The presence of five repeats in the rod domain of Trichomonas vaginalis α-actinin has been suggested (Addis et al., 1998; Bricheux et al., 1998), but only the first of these repeats shows some similarity with those found in other α-actinins (Bricheux et al., 1998). An intragenic duplication gave rise to two spectrin repeats (SR1 and SR4). This group of α-actinins is present in fungi and E. histolytica (α- actinin-2). A subsequent, second, intragenic duplication added two more repeats (SR2 and SR3) (Virel and Backman, 2004, 2007).

The most likely scenario for the evolution of the spectrin superfamily of proteins is that it began with the introduction of seven repeats between repeat 2 and 3 of α-actinin, producing an elongated “α- actinin”. At this point the dystrophin/utrophin lineage presumably diverged from the αβ-spectrin lineage (Figure 4). The next step consisted of a duplication of the seven-repeat block in the elongated “α-actinin” and the insertion of a repeat between the two seven-repeat blocks. The protein was split into two parts at this inserted repeat (Figure 4A). Modern α- and β-spectrin evolved out of these two different fragments. One α-helix (a0) of the cleaved repeat became the N-terminal end of what is now known as α-spectrin and the two last α-helices (b17) became the end of what is now known as β- spectrin (Figure 4A). A triple helix bundle, which is the characteristic structure of an entire repeat, is completed when b17 binds to a0 to form the spectrin heterotetramer (Figure 4B) (Pascual et al., 1997; Thomas et al., 1997; Viel, 1999).

The evolution of the spectrin superfamily can be divided into two phases. A first active phase is characterized by intragenic duplication and concerted evolution. In this phase gene duplication produced the α-actinin, dystrophin/utrophin and spectrin lineages. Just before arthropod/vertebrate

15

divergence the evolution of the spectrin superfamily entered a new, more stable phase (~500 MYA) (Thomas et al., 1997).

A

B

Figure 4. A. Model for the evolution of the spectrin superfamily of proteins (adapted from Pascual et al., 1997; Thomas et al., 1997; Virel and Backman, 2004). B. Spectrin heterotetramer (the functional unit of spectrin). All the SR dark and light blue are similar.

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The three lineages of the spectrin superfamily of proteins are present in all metazoans. In sequenced bacterial and plant genomes, there is no evidence of genes coding for any member of the spectrin superfamily. In protozoans, fungi and Oomycetes the superfamily is represented by the α-actinin protein (Virel and Backman, 2004, 2007; Publications I and II). However, in plants, fungi and Oomycetes the presence of 240–220 kDa spectrin-like proteins has been reported based on studies using anti-spectrin antibodies (Michaud et al., 1991; Reuzeau et al., 1997; Kaminskyj and Heath, 1995; Holzinger et al., 1999; Degousée et al., 2000; Heath et al., 2003; Slaninová et al., 2003). This discrepancy between the bioinformatics data and the immunological studies will be discussed in more detail in Publications I and II.

The following section is focused on the basic principles of α-actinin structure and function. More information about this protein is given in Publications III and IV, which represent the main part of the thesis.

1.2.4. α-Actinin

α-Actinin is the smallest member of the spectrin superfamily. The functional unit is a homodimer (Blanchard et al., 1989; Viel, 1999; Ylänne et al., 2001). There is antiparallel binding of two α-actinin monomers, with the actin-binding domain (ABD) of one monomer facing the calcium-binding domains (CBD) of the other (Figure 3). This organization gives α-actinin the ability to cross-link actin filaments in a calcium-dependent manner (Tang et al., 2001). Another group of regulators, phosphoinositides, are also able to regulate the interaction of α-actinin with actin filaments but these can also regulate the association/dissociation of α-actinin with integrins, another class of α-actinin- binding proteins (Fukami et al., 1992; 1996; Fraley et al., 2005). α-Actinin can also be phosphorylated by kinases such as focal adhesion kinases. Focal adhesion plaques are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix. The phosphorylation of α-actinin reduces its affinity for actin and prevents its localization to focal adhesion plaques (Izaguirre et al., 2001; von Wichert et al., 2003). Moreover, an increase in the level of α-actinin phosphorylation on tyrosine 12 of the ABD weakens the linkages formed between integrins and the cytoskeleton and alters the focal adhesion dynamics (Rajfur et al., 2002; von Wichert et al., 2003). These observations support the possibility that the phosphorylation of α-actinin may serve to modulate the coupling/uncoupling of integrins to the cytoskeleton (Zhang and Gunst, 2006).

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1.2.4.1. Functions

α-Actinin has many biological functions. In striated muscle it is the major thin filament cross-linking protein in the muscle Z-disc (Suzuki et al., 1976; Fay et al., 1983; Luther, 2000), connecting actin filaments of adjacent sarcomeres. In non-muscle cells, α-actinin is a major component of stress fibers, a contractile structure analogous to the more organized units found in striated muscle cells (Otey and Carpen, 2004).

α-Actinin is also found in adhesion sites close to the plasma membrane, where it cross-links cortical actin to different adhesion and trans-membrane proteins, such as catenin (Knudsen et al., 1995) and integrins (Otey and Carpen, 2004), and serves as a linker between trans-membrane receptors and the cytoskeleton. In synapsis α-actinin-2 may play a role in the localization of the neurotransmitter receptor NMDA and its modulation by Ca2+ (Wyszynski et al., 1997; Rycroft and Gibb, 2004; Franzot et al., 2005).

It has been proposed that α-actinin participates in cytokinesis in fission yeast (Wu et al., 2001).

In addition, a number of important human diseases are caused by α-actinin dysfunction. Mutations in α-actinin-4 cause a form of familial focal segmental glomerulosclerosis (FSGS) (Kaplan et al., 2000), which is a common nonspecific renal lesion characterized by regions of sclerosis in some renal glomeruli and often results in loss of kidney function. Different studies have found a relationship between α-actinin dysfunction and different types of cancer, such as colorectal cancer (Honda et al., 2005; Craig et al., 2007), lung cancer (Honda et al., 2004), breast cancer (Guvakova et al., 2002) and neuroblastoma (Nikolopoulos et al., 2000).

1.2.4.2. Isoforms

Four isoforms of α-actinin exist in almost all vertebrate organisms: α-actinins-1, -2, -3 and -4 (MacArthur and North, 2004) classified on the basis of their Ca2+ affinity. α-Actinins-1 and -4 are generally located in non-muscle cells and they have conserved EF-hand motifs that bind Ca2+. In contrast, the muscle-isoforms α-actinins-2 and -3 have lost their Ca2+-binding ability. Furthermore, alternative splicing generates additional isoforms in some vertebrates (Parr et al., 1992; Kremerskothen et al., 2002; Honda et al., 2004). Only one gene coding for α-actinin has been found in the invertebrate organisms studied to date. Therefore, the three isoforms described for Drosophyla are probably due to the occurrence of alternative splicing (Roulier et al., 1992).

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1.2.4.3. “Atypical” α-actinins

The rod domain of “atypical” α-actinins reported for some protozoa (Trichomonas vaginales and Entamoeba histolytica), fungi and Oomycetes (Addis et al., 1998; Bricheux et al., 1998; Wu et al., 2001; Virel and Backman, 2004, 2006, 2007; Virel et al., 2007; Publication I) is composed of one or two spectrin repeats instead of the four observed in classical α-actinins (Virel and Backman, 2007) (Figure 5). In these organisms there is generally only one α-actinin gene; the presence of different isoforms due to alternative splicing has not been reported. However, in the protozoa Entamoeba histolytica two proteins coded by two different genes have been characterized (Virel and Backman, 2006, Virel et al., 2007). “Atypical” α-actinin seems to represent the only member of the spectrin superfamily in protozoa, fungi and Oomycetes (Virel and Backman, 2004; Publication I). It has been proposed that this is the ancestor from which dystrophin, utrophin and spectrin evolved in two phases (see section 1.2.3).

Figure 5. Diagrammatic representation of the “atypical” α-actinins. (CH: Calponin homology; SR: spectrin repeat; CC: coiled coil region; CBD: Calcium-binding domain). The CC region from Phytophthora infestans is shorter than that in T. vaginalis, corresponding to the accommodation of three and four putative SR, respectively.

The controversial reports of spectrin-like proteins in fungi and the relevance of the atypical fungal α- actinins for the understanding of the evolutionary history of this superfamily make these organisms an excellent model to obtain further insights into the evolution of this superfamily. The study of the function of α-actinin in tip growth could reveal new roles for this actin-binding protein.

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1.3. WHY STUDY FUNGI AS A MODEL OF TIP GROWTH?

Fungi represent the second largest group of organisms after insects, with about 1.5 million species – most of which are filamentous fungi (Hawksworth, 2001). Fungi play an important role in the decay of organic material and nutrient recycling.

The relationship between plants and fungi is sometimes positive, leading to the formation of symbiotic structures inside plant roots. The mycorhizae form a group of organisms that are capable of such a positive interaction. In other cases, fungi can be pathogenic agents and cause significant damage to agricultural crops. Plant diseases caused by fungi include rusts, smuts, and leaf, root, and stem rots. Fungi are also agents of animal diseases. From evolution point of view, fungi are more chemically and genetically similar to animals than other organisms, making fungal diseases difficult to treat.

Fungi are also important in the pharmaceutical and food industries. They are used to produce enzymes and secondary metabolites such as antibiotics and also participate in beer, champagne, cheese and bread production (Hesseltine, 1965; Hersbach et al., 1984; Mapari et al., 2005; Schuller and Casal, 2005; Menacho-Márquez and Murguía, 2007; Wang and Lin, 2007).

The experimental tractability of fungi makes these organisms among the most important models in fundamental research. Important knowledge in biochemistry, genetics and molecular biology has been acquired from studies on fungi. Fungal cellular physiology and genetics share key components with animal cells, including multicellularity, cytoskeletal structures, development and differentiation, sexual reproduction, cell cycle, intercellular signaling, circadian rhythms, DNA methylation, regulation of gene expression through modifications of the chromatin structure, and programmed cell death (Colot and Rossignol, 1999; Borkovich et al., 2004; Dunlap and Loros, 2004; Galagan et al., 2003; Galagan and Selker, 2004). The shared origins of the genes responsible for these fundamental biological functions in humans and fungi continue to make the study of the fungal genes of vital interest to human biology.

Hyphae of filamentous fungi belong to the most polarized cellular structures found in nature. The study of fungal tip growth has been widely used to increase our understanding of the physiology of other highly polarized cells, such as root hairs, pollen tubes and neurons. Common aspects of, as well as differences in, cytoskeleton organization and function between these types of cells have provided important insights into the relationship between the cytoskeleton and cell growth.

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1.3.1. Filamentous fungi

In filamentous fungi polar extension is needed for vegetative growth and the development of complex tissues. For several reasons, tip-growing cells represent an ideal system to study cell expansion. Fungi concentrate their growth machinery at one cell surface site and only at this site does robust growth occur. Localized growth implies that all material required for growth has to be present at, or delivered to, the surface area where cell expansion takes place. Furthermore, the growth rate of the expanding cell surface area in a tip growing cell is much higher than the growth rate of a cell that distributes the growth machinery more or less evenly over its surface. This allows changes in growth rate and direction to be observed more easily.

When the spores of filamentous fungi germinate, nuclear division is accompanied by a series of ordered morphological events, including the switch from isometric to polar growth. As growth continues, the hyphae become compartmentalized with the addition of more septa, and lateral branches emerge from basal compartments.

Most of the studied filamentous fungi are of industrial interest, e.g. Aspergillus nidulans and Ashbya gossypii (Steiner et al., 1995; Harris, 1997; Wendland et al., 1999; Momany and Taylor, 2000; Kaminskyj, 2001; Goldman and Kafer, 2004; Guest et al., 2004; Oakley, 2004; Gattiker et al., 2007). Other examples are plant pathogens such as Uromyces appendiculatus, Magnaporthe grisea, Botrytis cinerea and Ustilago maydis (Barja et al., 1998; Dijksterhuis, 2003; Hamer and Talbot, 1998; Banuett and Herskowitz, 2002; Silva et al., 2006; Ebbole, 2007; Klosterman et al., 2007). Examples of non- pathogenic fungi include the ascomycete N. crassa (Borkovich et al., 2004; Galagan and Selker, 2004; Dunlap, 2006; Dunlap et al., 2007) and the chytridiomycete Allomyces arbuscula (Ojha, 1996; Ojha and Barja, 2003).

The ascomycete N. crassa is the main experimental model used in this work, but other members of this “phylum” have also been studied (M. grisea and B. cinerea). The complete sequence of these three fungal genomes has been annotated by the Broad Institute (http://www.broad.mit.edu/annotation/fgi/).

N. crassa is an ascomycete that grows on semisolid media by forming colonies that spread. This fungus was first described as an infectious agent in French bakeries and today is used in diverse research programs.

The ascomycete Magnaporthe grisea is a plant pathogenic fungus that is responsible for an important disease in rice, rice blast, but can also infect other agriculturally important cereals. This fungus has been used as a model to understand plant-pathogen interactions.

Botrytis cinerea is another plant pathogen that affects many plant species, although its most notable hosts are wine grapes. The fungus is usually referred to by the name of its asexual (anamorph) form

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because the sexual phase is rarely observed. The sexual form (teleomorph) is called Botryotinia cinerea.

In the work described here we also studied the chytridiomycete Allomyces arbuscula, which is a water fungus that is distributed throughout the world and is particularly abundant in warm climates. The filamentous cells of Allomyces divide in a characteristic dichotomous pattern. The life cycle can alternate between two stages, gametophytic and sporophytic, reflecting the capacity for both sexual and asexual reproduction. The reproductive structures are located at the end of the hyphae and are separated by complete septa.

Phytophthora infestans, an oomycete in the kingdom of Stramenopila, has a filamentous phenotype but is no longer considered as a fungus. In contrast to the fungi, Oomycetes are more closely related to plants than to animals. Whereas fungal cell walls are made primarily of chitin, the cell walls of Oomycetes are built mainly of cellulose. Ploidy levels and biochemical pathways are different between these two kingdoms. P. infestans is an important potato pathogen that causes late blight and has considerable economic impact.

The mode of growth of filamentous fungi is supported by the extreme polarization of the cytoskeleton and endo-membrane network, which allows the long-range transport of vesicles containing precursors required for apical extension to the tip region (Bourett and Howard, 1991; Roberson and Vargas, 1994).

1.3.2. Cytoskeleton in fungi

The principal components of the fungal cytoskeleton are microtubules and microfilaments. The presence of intermediate filaments is more controversial and has only been reported in a few studies (May and Hyams, 1998; McConnel and Yaffe, 1993; Geitmann and Emons, 2000). These cytoskeletal elements are required for a variety of cellular processes including polarized growth, organelle movement and positioning, secretion, endocytosis, cell division and chromosome segregation. As in other eukaryotic cells, fungal microtubules are known to function in mitosis and chromosome segregation (Morris and Enos, 1992; Thaler and Haimo, 1996; Jung et al., 1998). The importance of microtubules for the establishment of polar growth and tip growth varies depending on the fungal species (Oakley and Morris, 1980; Caesar-Ton That et al., 1988; Barja et al., 1993; Sawin and Nurse, 1998; Heath et al., 2000; Horio, 2007). Microtubules cooperate with microfilaments to control cell shape, cell division and intracellular transport (Momany and Hamer., 1997; Schott et al., 2002; Bretscher, 2005).

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1.3.3. Actin cytoskeleton in fungi

1.3.3.1. Actin

Fungi contain either one or only a few actin genes (Tarkka et al., 2000; Helgason et al., 2003). In N. crassa the presence of three isoforms, α- β- and γ-actins, has been described by Barja et al. (1991). Nevertheless, as in other filamentous fungi and yeast there is only one bona fide actin gene, indicating that the three isoforms may be products of post-transcriptional modifications. However, actin-related proteins such as ARP 1, ARP 3 and RO 4 have recently been identified in Neurospora. Some of these proteins share the same molecular weight and sequence similarity as actin (Robb et al., 1995; Tinsley et al., 1998; Lee et al., 2001). Therefore, the reported three isoforms found in Neurospora could be the result of a cross-reaction of anti-actin antibodies with these actin-related proteins.

F-Actin appears in fungi in two principal forms: patches and cables (Heath et al., 2000; Walker and Garrill, 2006). Patches are usually localized in growing regions of yeast or filamentous fungi. Cables are described in yeast in a polarized manner from the bud along the mother cell. The organization observed in yeast is less evident in filamentous fungi such as N. crassa or Aspergillus (Xiang and Plamann, 2003), where the actin cytoskeleton appears principally as patches associated with the hyphal tip and cell cortex (Barja et al., 1991, 1993; Heath et al., 2000). The absence of actin bundles in some filamentous fungi has been explained in terms of the difficulty of preserving these structures during the preparation of samples used for immunofluorescence microscopy (Heath, 1987; Harold and Harold, 1992; Kaminskyj and Heath, 1994). However, in Aspergillus short actin cables were described in the tip region of the hyphae (Pearson et al., 2004; Virag and Harris, 2006). In the basidiomycete Ustilago maydis and the Oomycetes both structures, cables and patches, form the actin cytoskeleton (Bachewich and Heath, 1998; Banuett and Herskowitz, 2002; Walker et al., 2006).

Actin is also localized in the cytoplasm and forms a diffuse network of microfilaments in the septa of filamentous fungi (Harris et al., 1994; Capelli et al., 1997; Rasmussen and Glass, 2005, 2007) and in the contractile actin rings of yeast (Pruyne and Bretscher, 2000; Schott et al., 2002; Kamasaki et al., 2007).

The actin-binding proteins (ABP) form part of the actin cytoskeleton. These regulate its dynamics and mediate the interaction with other proteins. The actin cytoskeleton also contains actin related proteins (ARPs).

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1.3.3.2. Actin-binding proteins

ABPs have been described principally in yeast. There are, e.g., twinfilin and cofilin/ADF, which sever actin filaments (Moon et al., 1993; Okada et al., 2006; Moseley et al., 2006); profilin and formin, which regulate the assembly of G-actin into filaments (Haarer et al., 1990; Sagot et al., 2002; Evangelista et al., 2003; Kovar et al., 2005; Takaine and Mabuchi, 2007). In addition, there are capping proteins, which regulate microfilament length (Amatruda and Cooper, 1992; Sizonenko et al., 1996; Nakano and Mabuchi, 2006), fimbrin and α-actinin, which promote the bundling of microfilaments (Adams et al., 1989; Wu et al., 2001; Goodman et al., 2003), and myosin, which is involved in organelle movement (Watts et al., 1987; Matsui, 2003). Some ABPs were first described in Saccharomyces as Aip1 (Amberg et al., 1995) and were shown to collaborate with cofilin, capping the barbed ends of filaments severed by cofilin (Okada et al., 2006; Okreglak and Dubrin, 2007).

As far as filamentous fungi are concerned, there are some reports on proteins involved in the assembly and stability of actin filaments such as formin. A. nidulans and N. crassa encode a single formin (Xiang and Plamann, 2003). The A. nidulans formin, SepA, localizes to both septation sites and hyphal tips, suggesting that filamentous fungi use site-specific regulatory mechanisms to control formin- mediated actin polymerization (Harris et al., 1997; Sharpless and Harris, 2002). Genes encoding for myosin proteins have been found in the genomes of N. crassa and A. nidulans (Xiang and Plamann, 2003; Steinberg, 2007) and has been characterized in these two organisms (van Tuinen et al., 1986; McGoldrick et al., 1995; Osherov et al., 1998; Yamashita et al., 2000; Takeshita et al., 2002) and in other filamentous fungi (Woo et al., 2003; Schuchardt et al., 2005; Weber et al., 2006). In N. crassa, an actin-binding protein of 47 kDa with the same intracellular distribution as actin has been reported (Capelli et al., 1997). This protein, which is named p47 and identified as EF1α (elongation factor 1α) (Taillefert, 1988), also binds to calmodulin (CaM). The actin-p47-CaM complex may participate in the relationship between the actin cytoskeleton and protein synthesis machinery.

So far, only a relatively small number of ABPs has been identified in filamentous fungi. However, the completion of their genome sequence will allow the identification of a large number of the ABPs in these organisms. For example, most of the Saccharomyces genes coding for the actin cytoskeleton, including the actin-binding proteins, have orthologs in N. crassa (Borkovich et al., 2004).

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2. BACKGROUND AND AIMS OF THE STUDY

The cytoskeleton in fungi is not yet well understood. The functions of actin and tubulin have been established to some extent by observing the effects of anti-cytoskeleton drugs on cell phenotype or on the in situ localization of these proteins (Caesar-Ton That et al., 1988; Hoang-Van et al., 1989; Barja et al., 1993; Riquelme et al., 1998; Torralba et al., 1998; Czymmek et al., 2005). Tools, such as constructs of green fluorescence protein (GFP)-fusion proteins, improvements in mutant constructs and bioinformatics have revealed more details on the composition and dynamics of the cytoskeleton network. Nowadays, we try to understand the complexity of this structure through the interaction that occurs between cytoskeletal proteins and other cellular structures, a process that is mediated by a plethora of associated proteins.

We were interested in a group of actin-binding proteins, the spectrin superfamily, that participate in the organization of the actin cytoskeleton and its connection with the plasma membrane in metazoans. In the case of neurons, several studies report the essential role of spectrin in tip growth (Morris, 2001; Spira et al., 2003). Initial studies on “spectrin-like” proteins in other highly polarized cells like root hairs, pollen tubes and oomycetes and fungal hyphae assign “spectrin-like” roles in the process of apical growth (Michaud et al., 1991; Kaminskyj and Heath, 1995; Bisikirska and Sikorski, 1997; Reuzeau et al., 1997; De Ruijter et al., 1998, 2000; Holzinger et al., 1999; Degousée et al., 2000; Braun, 2001; Heath et al., 2003; Slaninová et al., 2003; Toquin et al., 2006). In this thesis I provide strong evidence for the absence of spectrin genes in any of the completed plant and fungal sequenced genomes. Furthermore, the antibody used to report the presence of spectrin in these organisms cross- reacts with proteins other than spectrin. α-Actinin is the only member of the spectrin superfamily in fungi and this represents an early step in the present model for the evolution of this superfamily.

The goal of this thesis was to characterize the α-actinin protein in Neurospora crassa and to establish the relationship between α-actinin and actin. This would allow a better understanding of the function of the actin cytoskeleton in processes such as spore germination or growth of the mycelium tip. In addition, elucidation of the structure and function of α-actinin in N. crassa will allow a comparison with other better characterized metazoan models and lead to the possible discovery of new functions for this protein in fungi. The first target of the study was to determine the veracity of the presence of a “spectrin-like” protein in fungi and Oomycetes and to identify the peptide detected by the commercial anti-spectrin antibody (commonly used so far in the scientific comunity) in these organisms.

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For this purpose we planned to:

• Look for the gene coding for a putative spectrin in the completed fungal genome databases.

• Perform mass spectrometric analysis on the peptide reacting with the anti-αβ-spectrin antibody in N. crassa and P. infestans.

• Use bioinformatics tools to study the features of the α-actinin protein in fungi and Oomycetes.

• Construct a GST Neurospora α-actinin fusion protein for biochemical characterization.

• Produce polyclonal anti-α-actinin using the GST-fusion protein as an immunogen for further biochemical characterization of the fungal α-actinin.

• Localize the protein in situ by immunodetection.

• Construct a GFP-α-actinin fusion protein for localization of the protein in vivo.

• Study of α-actinin knock-out Neurospora strain to provide an insight into protein function.

In this thesis I provide strong evidence for the absence of spectrin genes in any of the completed plant and fungal sequenced genomes. Furthermore, the antibody used to report the presence of spectrin in these organisms cross-reacts with proteins other than spectrin. α-Actinin is the only member of the spectrin superfamily in fungi and this represents an early step in the present model for the evolution of this superfamily. Studies on this fungal protein may help to provide more details of the evolutionary history of the spectrin superfamily as well as to give a greater insight into the function of the actin cytoskeleton in fungi.

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3. MATERIALS AND METHODS

Materials

Unless otherwise indicated, chemicals were obtained from Sigma-Aldrich/Fluka, Merck, Bio-Rad, Amersham, and Roche Diagnostics (Mannheim). Restriction enzymes and buffers as well as other DNA-modifying enzymes were purchased from New England Biolabs (Frankfurt am Main), Promega and Roche. For the PCR-reactions the Primus 25 advanced PCRSystem (PeqLab) was used. Antibodies were obtained from Sigma-Aldrich. Oligonucleotides were provided by Microsynth (http://www.microsynth.ch/).

Fungal strains and cell culture

Neurospora crassa

Wild type N. crassa (FGSC 262, strain St. Lawrence STA 4) was obtained from the Fungal Genetics Stock Center, School of Biological Sciences, Kansas City, MO. In order to produce large quantities of macroconidia, the fungus was first grown on solid “Davis and De Serres” medium (Davis and De Serres, 1970), for 3 days at 33°C in the dark and then at 25°C in artificial light for 4 days. Conidia were harvested and inoculated at an inoculum density of 5x106 conidia/ml of Vogel’s minimal medium (Vogel, 1956) enriched with 1.5% or 2% sucrose. The culture was incubated at 30°C for 0, 6, 12, 18 hours on a rotary shaker at 150 rpm. “Davis and De Serres” medium with slight modification (1 liter)

5 g Na and K Tartrate, 3 g NaNO3, 0.5 g MgSO4.7H2O, 0.1 g CaCl2.2H2O, 3 g KH2PO4, 0.1 g 1 NaCl, 10 g Sucrose, 10 ml Glycerol, 0.1 ml Oligoelements* , 0.1 ml FeCl3 (sol. 0.5%), 0.05 ml Biotine*2. The pH value was adjusted to 5.6 with 1N KOH. Medium was dispensed in 150 ml Erlenmeyer (20 ml/Erlemeyer) and solidified with 2% agar. Vogel’s miminal medium (1 liter)

2 g NH4NO3, 2.5 g Citrate de Na.2H2O, 5 g KH2PO4, 0.2 g MgSO4.7H2O, 0.1 g CaCl2.2H2O, 0.1 ml Oligoelements*1, 50 μl Biotin*2 .

*1Solution of oligoelements (100 ml)

5.0 g Citric Acid, 5.0 g ZnSO4, 1,0 g Fe(NH4) 2 (SO4) 2.6 H2O, 5 mg H3BO3, 0.25 g CuSO4.

H2O, 0.05 g MnSO4.2H2O, 0,05 g Na2MoO4.H2O. *2Solution of Biotin Biotin 1 mg, in 10 ml 80% Ethanol (stock 4°C, during 6 months)

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Magnaporthe grisea

M. grisea (wild type P1.2 strain) was kindly donated by Dr. M-H. Lebrun (Unité Mixte de Recherche, Centre National de la Recherche Scientifique/ BayerCropScience, Lyon). M. grisea was grown on solid rice medium with small pieces of filter for a few days until the filters were colonized and white mycelium appeared.

For storage the colonized filters in solid rice medium containing M. grisea were put into a sterile container and placed for at least 3 days in an oven at 37°C until they were dried, then stored at –20°C.

Mycelia for protein extraction were obtained from liquid culture in TNK medium containing 0.2% Yeast extract and 1% Glucose (Ou, 1985). The cultures were allowed to grow for 48 hours in the dark in a shaker (150 rpm). The average weight of mycelium collected per liter of liquid medium was 2.5 g.

Solid Rice Medium (200 ml) 4 g Rice powder, 0.4 g Yeast extract, 4 g Agar. TNK Medium (1 liter)

2.0 g NaNO3, 2.0 g KH2PO4, 0.5 g MgSO4.7H2O, 0.1 g CaCl2.2H2O, 4 mg FeSO4.7H2O, 1 ml Oligoelements*. *Solution of Oligoelements (100 ml)

0.79 g ZnSO4.7H2O, 60 mg CuSO4.5H2O, 10 mg H3BO3, 20 mg MnSO4.2H2O, 14 mg

NaMoO4.2H2O.

Botrytis cinerea

B. cinerea (BO47) was kindly donated by Dr. R. Beffa (BayerCropScience, Lyon). Mycelia were grown in solid potato dextrose agar medium (Difco) for one week on the bench with day periods of light. Spores were then harvested by flooding the culture with water and separated from the mycelial fragments by filtration. Spores obtained from a Petri dish (9 cm diameter) culture were used to inoculate 200 ml of liquid potato dextrose broth medium (24 g/l, Difco). Mycelia were harvested after 24 hours of growth with agitation (150 rpm) at 25°C.

Allomyces arbuscula

A. arbuscula strain Burma LD was grown on filter pads placed on solid YpSs medium (Emerson, 1941). Zoospores were induced by shifting the filters in sterile Petri-plates containing 30-40 ml sterile

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distilled water (water active liberation of zoospores) according to a procedure described by Ojha and Turian (1981). Zoospores were inoculated in CGY medium (Turian, 1963) and grown for periods of 6, 12 and 18 hours with forced aeration.

Solid YpSs (1 liter) (Emerson, 1941)

7.5 g Soluble starch, 2 g Yeast extract, 0.5 g K2HPO4, 0.25 g MgSO4.7H2O, 15 g Agar.

GCY Medium (1 liter)

1.0 g K2HPO4 * , 0.2 g MgSO4.7H2O, 0.1 g NaCl, 0.1 g CaCl2.2H2O, 0.02 g FeCl3.6H2O, 3.0 g Casein Hydrolysate, 5.0 g Sucrose, 0.1 g Yeast extract.

The pH was adjusted to 6.8 with 125 μl/l of 12 N HCl. *K2HPO4 was dissolved in water, sterilised separately and added to medium before use.

Phytophthora infestans

P. infestans (PT78) isolate was kindly donated by Dr. R. Beffa (BayerCropScience, Lyon). Mycelium was first grown on pea-agar (125 g of pea cooked and grinded in 1 liter of H2O, 2% agar). Sporangial inoculum was prepared from a 8 to 12 days culture. The sporangia were detached from the mycelia by flooding the culture with water and separated from the mycelial fragments by filtration. Sporangia were inoculated in V8 liquid medium (50 ml tomato juice/liter of distilled water, the pH was adjusted to 5 if necessary) at a final concentration of 105 sporangia/ml. Mycelia were harvested after 72 h of growth in the dark at 20°C without agitation.

Protein extraction

To optimise the conditions for spectrin extraction, we tested different experimental conditions and protocols for cell disruption including:

a) several detergents: Triton-X-100, Triton-X-114, Empigen 1%, Octyl β-D glucopyranoside,

b) Sodium Dodecyl Sulfate (SDS) extraction. Dry fungal powder was resuspended in buffer containing 4% SDS, 5% 2-β−mercaptoethanol, 5% sucrose, 10 mg of insoluble polyvinil polypirrolidone (PVP) and boiled for 3 min before centrifugation (13000 x g; 20 min),

c) Trichloroacetic Acid (TCA)-Acetone extraction (Granier, 1988). The dry powder was resuspended in buffer containing 10% TCA, 0.07% 2-β−mercaptoethanol in cold acetone and kept at -18°C for 1h. After 15 min centrifugation at 20000 x g the supernatant was removed and the pellet was rinsed for 1 h at -18°C with cold acetone

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containing 0.07% 2-β−mercaptoethanol. The rinsing solution was removed with caution and the pellet vacuum-dried for 1 h. The pellet was resuspended in extraction buffer [5 mM Hepes-KOH pH 7.5; 2 mM EGTA, 2 mM dithiothereitol (DTT)],

d) different concentrations of NaCl (0 to 100 mM) in 20 mM Tris-HCl pH 7.5,

e) cells fixed in 3% paraformaldehyde before extraction,

f) cell wall digestion with Lysing enzyme (Sigma 5 mg/ml) for 20 min before extraction,

g) buffer with Vanadate, a phosphatase inhibitor, in order to reduce the susceptibility of spectrins to cleavage by μ-calpain (Nedrelow et al., 2003),

h) protein extraction from the cell wall-less mutant Neurospora crassa (FGSC 1118 fz;sg;os-1).

The standard procedure adopted to study the evolution of spectrin in growing mycelia was the following: cells from different stages of development were harvested by filtration through Millipore filters (pore size 0.5-1 μM; Millipore Corporation, Bedford, MA /USA), washed twice with distilled water, frozen in liquid nitrogen and ground in a mortar kept at a low temperature using liquid nitrogen. The frozen powder was suspended in cytoskeleton stabilizing buffer (2-4 ml per mg powder) of Abe and Davies (1995) with slight modifications, containing 5 mM Hepes-KOH, pH 7.5, 250 mM sucrose,

15 mM Mg(OAc)2, 2 mM EGTA, 2 mM dithiothreitol (DTT), 25 mM K2O5S2, 10% glycerol and 0.5% polyvinylpyrrolidone and the following protease inhibitors: 1 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride (PMSF), 2 μg/ml each of leupeptin, chymostatin, trasylol; or Complete EDTA-free Protease Inhibitor Cocktail Tablets (Roche Applied Science). The homogenates were incubated at 4°C with gentle agitation for 20 min and then centrifuged at 6000xg for 15 min at 4°C. The supernatant was recovered for in vitro proteolysis, analysis by SDS-PAGE and immunoblotting. The protein concentration in supernatants was measured according to Bradford (1976) with Bovine Serum Albumine (BSA) as a standard.

One-dimensional gel electrophoresis

The proteins in the crude extract were separated on SDS-PAGE and native gels, according to Chrambach and Rodbard (1971) and Laemmli (1970), respectively. The gels were stained with Coomassie brilliant blue R-250. Samples of 50 μg or 10 μg of protein were loaded on each lane of a 15x10 cm gel or mini-gels respectively. Various molecular weight markers were utilized (for SDS- PAGE gels: Precision Plus Protein Standards from BioRad, LMW-SDS Marker Kit from Amersham Biosciences and Prestained Protein Marker IV from PeqLab. For non-denaturing gels: HMW Native Marker Kit from Amersham Biosciences).

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Two-dimensional electrophoresis

Proteins were first separated on isoelectrofocusing (IEF) gels according to O’Farrell (1975). The first dimension gel was prepared in glass tubes and loaded with 300 μg of protein. The run condition used was 400 V at room temperature for 19 hours and 1 hour at 500 V.

After equilibration, the gels were placed in a 6.5-10% SDS-PAGE gel according to Chrambach and Rodbard (1971) in order to separate the proteins by their molecular weight. Gels could be stored for at least one month at -20°C.

Gels where stained with Coomassie Blue or transferred to the appropriate membrane (nitrocellulose for immunoblot assays or PVDF, ImmobilonP for EDMAN-sequencing).

Immunoblotting

The proteins were electrophoretically transferred to nitrocellulose as described by Towbin et al. (1979). After blocking with TBS (10 mM Tris pH 7.5, 0.15 M NaCl) 0.5% Tween-20 and 5% BSA, the nitrocellulose sheet was incubated with the primary antibody and then with goat peroxidase- conjugated secondary antibodies (dilutions of antibodies were prepared in TBS + 0.5% BSA + 0.5% Tween-20). Washing solution was the same as blocking solution without BSA. The proteins were revealed with 0.5 mg/ml of 3,3’-diaminobenzidine tetrahydrochloride (DAB) in 100 mM Tris-HCl, pH

7.5, containing 0.03% H2O2.

Mass spectrometric identification and EDMAN sequencing analysis of proteins

Protein spots corresponding to the immunoreacting peptides were excised from the 2D-gels and used for further analysis. Proteins were subjected to trypsin digestion and subsequent identification by MALDI-TOF MS, performed as a service by Alphalyse A/S (Odense, Denmark) and by the Section of Pharmaceutical Sciences (University of Geneva). For N-terminal amino acid sequencing, also known as EDMAN sequencing, the spots were transferred to a PVDF membrane. Analyses were performed by the Analytical Research Services (University of Basel, Switzerland).

Immunofluorescence

Cells were fixed with 3% paraformaldehyde (v/v) in 50 mM phosphate buffer, pH 7.4, for 30 min to 1 hour at room temperature. Partial digestion of the cell wall was performed by incubation of the

31

mycelia for 5 to 10 minutes in a solution (5 mg/ml) of lysing enzyme (Sigma), in phosphate buffer, pH 6.5. The cells were rinsed three times with phosphate buffer and plasma membrane permeabilized with 0.1% Triton X-100 in the same buffer for 10 min. Triton was then removed by five washes in phosphate buffer. All operations were performed at room temperature.

Permeabilized cells were blocked in phosphate buffer containing 3% BSA and then incubated 2 hours at room temperature with the first antibody prepared in the same buffer. Samples were rinsed in phosphate buffer and re-incubated for 1 h at RT with fluorescein-isothiocyanate (FITC) or crystalline tetramethylrhodamine isothiocyanate (TRITC) goat anti-rabbit antibodies (Sigma F9887 and T5268, respectively). After a final rinse in phosphate buffer, the cells were mounted in p-phenylenediamine- glycerol (1 mg/ml p-phenylenediamine in 78% glycerol) containing 2.5 μg/ml DAPI for staining the nuclei and examined with an epi-illumination microscope (Zeiss axioplan) or Leica SP2 Confocal microscope. Both microscopes were equipped with selective filter combinations to visualize FITC and TRITC-fluorescence patterns.

Cells treated for 4 hours with sordarin (50 μg/ml), a specific inhibitor of EF2, were also used in immunofluroescence assays and compared with untreated hyphae.

Electron microscopy and immunogold

Cells were fixed at room temperature by the addition of a concentrated solution of fixative to obtain a final concentration of 0.5% glutaraldehyde (v/v), 4% paraformaldehyde (v/v). This was done to maintain the integrity of the culture and to minimize any alteration due to fixation. Samples were collected by centrifugation, pre-embedded in 1.5% agar at 46°C, dehydrated respectively in 70% and 100% ethanol (for 30 min each), in 2:1 (v/v) ethanol : LR White resin (Polysciences), 1:1 (v/v) ethanol : LR White, followed by 1:2 (v/v) ethanol : LR White for 1h and finally in 100% LR White for 24 h at 50°C for polymerization.

Ultrathin sections were made and taken on nickel grids, incubated for 2 h at room temperature in 50 mM phosphate buffer (K2HPO4/KH2PO4) pH 7.0 containing 2% BSA, 0.05% (v/v) Tween-20, and then for a further 2 h with specific antibodies. The sections were rinsed with 0.05% Tween-20 in 50 mM phosphate buffer pH 7.0 and incubated with the secondary antibody conjugated to 20 nm gold particles. After incubation, sections were successively washed with phosphate buffer, stained for 10 min in 2% uranyl acetate, 5 min in Reynold’s lead citrate and examined at 60 kV in a Philips M410 transmission electron microscope.

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Proteolysis of cell-free extracts by the calcium-dependent protease CDP II

The cell-free extract, obtained as described earlier, was digested with 0.11 μg/μl CDP II (Ojha and Wallace, 1988) (specific activity: 21 mM para-Nitroaniline (pNA) released μg-1 min-1) in 20 mM Tris- HCl, pH 7.4, containing 4 mM EGTA, 1% β-mercaptoethanol and 10 mM free Ca2+. The reaction was performed in a total volume of 75 μl, at enzyme: protein ratios of 1:100, 1:50 and 1:10. After incubation at 37°C for 30 min, the reaction was stopped by the addition of 25 ml of 4X loading buffer (Laemmli, 1970). The proteins were denatured in boiling water for 5 min, separated by SDS-PAGE, transferred to nitrocellulose membranes and immunorevealed with spectrin polyclonal antibodies.

Reaction mixtures either without calcium or with leupeptine (10 mM), an inhibitor of CDP II activity, were used as controls.

In situ proteolysis by CDP II

Hyphae from 12 h cultures were prepared as described earlier for immunofluorescence. Before blocking, the cells were incubated for 1 hour at 37°C with CDP II in the enzyme reaction buffer described above. The hyphae were then washed five times, for 5 min each, with phosphate buffer and immunorevealed as described earlier (Ojha and Barja, 2003).

Construction of Neurospora GST-fusion proteins

Total RNA was isolated from hyphae after 18 hours of growth using TRIZOL reagent (Sigma) according to the supplier’s protocol and treated with DNAse (Ambion, Cat. 1906). cDNA synthesis was done using the Promega reverse transcription kit (A3802). The primers used for amplification of the coding sequences of elongation factor 2 (ncu07700.4) and the full length and fragments of the α- actinin (ncu06429.4) are shown in Figure 6A, where the restriction site added in the PCR product to perform the cloning into the expression vector is also indicated. As the ncu06429.4 gene is 3084 nucleotides in length, we took advantage of an AatII site in the middle of the sequence to make the construct in two steps. The PCR fragments were first cloned in the plasmid pGEMTeasy (Promega) and then digested by BamHI and NotI to be introduced in the pGEX4T.1 plasmid (Amersham, Biosciences) opened with these two enzymes.

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A

B

Figure 6. Primers used for Neurospora GST-fusion protein constructs. A. Table with all the primers, indicating the name of the target Neurospora gene, the name and sequence of the primer, and the restriction enzyme name corresponding to the restriction site introduced with each primer. B. Chart of primer position in the gene coding for α-actinin protein.

Bacterial transformation pGEMTeasy constructs were transformed in Escherichia coli DH5α using the SEM-method (Inoue et al., 1990). SEM-competent E. coli cells were thawed on ice. 100 µl of cell suspension were mixed with 5 µl (10 ng) of the ligation (pGEMTeasy constructs). The bacteria were then incubated on ice for 30 min, at 42°C for 2 min and put immediately on ice for 5 min. The cells were then plated on LB 2% agar plates containing 100 μg/ml Ampicillin (LB-Amp). For electro-transformation of bacterial cells with the plasmid containing the expression constructs, electrocompetent E. coli cells (BL.21) were thawed on ice. 45 µl of cell suspension was mixed with 5 µl of DNA (10 ng approximately) and placed in a pre-chilled sterile electroporation cuvette (BioRad, distance between the electrodes: 2 mm). After a pulse of 2.5 kV and 25 µF, 950 µl of LB medium was

34

added immediately. The cells were gently shaken for 45 min at 37°C and then plated on LB-Amp agar plates.

Fusion protein expression After selection of clones expressing the fusion protein, growth conditions were evaluated for optimal expression. In most cases, we adopted the following protocol: the bacteria were grown in LB-Amp liquid medium at 37°C to an A600 of 0.4-0.5, and then protein induction was performed by adding 0.1 M Isopropyl-βD-thiogalactopyranoside (IPTG) at 20 °C for 10 hours to avoid inclusion body formation. Proteins from induced and uninduced recombinant E. coli BL21 were extracted by sonication in extraction buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, and 1 mM EDTA), centrifuged, and separated on SDS-PAGE gel. The GST-α-actinin was purified by chromatography on GSTrap FF affinity columns.

Immunization and preparation of anti-serum against Neurospora α-actinin Antibody production was performed as described by Barnes et al. (1998). Prior to immunization, sera of two rabbits were tested against N. crassa extracts for background exclusion or cross reactivity. The rabbits were immunized for a period of one month, with 450 μg of GST-α-actinin protein. The sera were tested weekly against the antigen preparation by Western blotting, starting from day 15 following the first immunization.

Construction of Neurospora α-actinin-GFP fusion proteins The α-actinin-egfp gene fusion was constructed by PCR amplification of the sequence encoding the α- actinin gene (ncu06429.4), including the 930 bp of the N-terminal extension before the region coding for the first CH-domain. The PCR was carried out using the oligonucleotides Actinin_forward (5’- CGCCGCGGATCCTGGAGATGCTGGGGGTGGAG-3’) and Actinin_reverse (5′-GT CACGTTAATTAAATGATACCCATTCGGCTT -3′). This led to the insertion of BamHI and PacI restriction sites (underlined) used to clone the gene in the egfp gene containing plasmid pMF272 constructed by Freitag et al (2004).

Transformation of Neurospora crassa Transformation of N. crassa his-3 mutant (FGSC 9014; ridRIP1 mat A his-3) and heterokaryon transformant selection were performed as described previously (Margolin et al., 1997; Freitag et al., 2004).

Acquisition of live cell images Cultures were prepared in liquid medium as describe above. For acquisition of images during long times (12 hours), germinating spores were deposited on 8 chambered coverglass (Lab-Tek®, ref

35

155411) and covered with a small block of Vogel 1.5% agar medium in order to maintain the cells in the same plane. GFP fluorescence was recorded with a Leica AF6000LX microscope. For the acquisition of images during shorter times, 12 hour-old hyphae were deposited in similar culture chambers as described above and observed with Leica SP2 microscope. Images were processed with Adobe Photoshop 6.0.

Neurospora crassa heterokaryon knock-out (KO) strain We asked the Neurospora Genome Project (Colot el al., 2006) to construct an α-actinin KO strain. It is now available at the Fungal Genetic Stock Center (FGSC11835) as a heterokaryon because the homokaryon KO strain was lethal.

Cell wall stain with calcofluor Slides coated with Vogel’s medium containing 1.5% sucrose were inoculated with conidia and grown at 25°C for 8 hours in a wet chamber. The mycelium was stained with 10 μM calcofluor (Fluorescent Brightener 28) for 3 minutes and covered with a coverslip. An Orthoplan epi-illumination microscope (Leica) equipped with fluotar optics and a selective filter combination was used to visualize calcofluor fluorescence patterns. Fluorescent micrographs were obtained with a Leica-DFC490 camera and processed with Adobe Photoshop 6.0.

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4. RESULTS

4.1. Identity of anti-αβ-spectrin immunoreacting peptides in fungi and Oomycetes

Immunoblot

The spectrin immunoanalog proteins in fungi showed varying molecular weights under denaturing conditions. In the ascomycetes N. crassa and M. grisea, a single band of 100 kDa was detected with the anti-chicken αβ-spectrin (Fig. 1B in Publication I; Fig. 1A in Publication II), three bands corresponding to 65, 105 and 230 kDa in the chitrydiomycete A. arbuscula (Figure 7), and a single lower band corresponding to a relative mass of 67 kDa was observed in the oomycete P. infestans (Fig. 1A in Publication II). Further studies in N. crassa suggested that this protein was present in the same amount in all states of development during the vegetative life cycle (Fig. 1B in Publication I). The specificity of the antibody used for the characterization of the spectrin immunoanalog protein in fungi was demonstrated using human erythrocyte spectrin in a competition assay, showing progressive diminution of the immuno-reacting band with increasing concentration of the competing human spectrin (Fig. 1D in Publication I). However, the reaction of the antibody with the purified human protein was weaker than with the fungal peptide (Fig. 1C in Publication I).

When crude extracts from Neurospora were immunoblotted in non-denaturing conditions two bands were found (Figure 8, lane b). In an SDS-PAGE gel the lower band was confirmed to correspond to the 100 kDa peptide (Figure 8, lane c) and the higher one corresponded to a 70 kDa peptide in denaturing conditions (Figure 8, lane d), similar to a minor band that sometimes appeared in immunoblotted crude extracts of Neurospora (Figure 8, lane e).

The Isoelectric point (pI) of this peptide was analyzed in a 2D gel; the predominant immuno-reacting band had a molecular weight of 100 kDa and a pI in the range 6.5– 7.0 (Fig. 1E in Publication I; Fig. 2A in Publication II). The spot obtained in the 2D gels was subsequently identified by two different techniques: (a) MALDI-TOF MS and (b) EDMAN sequencing. In both cases the results indicated that the 100 kDa protein detected by the polyclonal anti-chicken αβ-spectrin corresponded to the elongation factor 2 (EF2) in N. crassa (Fig. 2A and C in Publication II). The EF2 of Neurospora has a predicted molecular weight of 95 kDa and a pI of 6.3.

The GST-elongation factor 2 fusion protein was also recognized by the commercial anti-spectrin antibody used in this work (Fig. 1B in Publication II). An additional band of about 65 kDa, which reacted strongly with the antibody, was co-purified with the fusion protein. We analyzed this peptide to determine if it was a degradation product of the GST recombinant protein or a protein from the bacterium. Analysis by MALDI-TOF MS revealed it to be GroEL from Escherichia coli.

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The immunoreacting band in extracts from P. infestans differed considerably from the band obtained in the analyzed Ascomycetes and did not correspond to the theoretical Mr and pI of EF2 in these organisms. For this reason, we also attempted to identify this peptide and it was found to be heat shock protein Hsp70 (Fig. 2B-C in Publication II).

38 Figure 7. Immunoblot of the Allomyces arbuscula protein revealed with anti-chicken αβ- spectrin antibody. Both lanes correspond to different crude extracts prepared under the same conditions.

39

Figure 8. Relationship between the peptides from Neurospora revealed with the anti-chicken αβ-spectrin antibody in non-denaturing (lanes a and b) and denaturing (lanes c-e) conditions. (lane a) N. crassa crude extract on a native coomassie stained gel; (lane b) immunoblot; lanes c and d correspond to the bottom and top bands from the native gel, respectively; (lane e) N. crassa crude extract. Mr refers to the relative mass of the markers in the native PAGE gel.

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Immunofluorescence

In all of the studied fungi the spectrin immunoanalog protein was localized at the peripheral region of the cell and concentrated in the hyphal tip during the exponential phase of growth. A localization study on this protein in N. crassa (Fig. 2A–E in Publication I) and A. arbuscula (Figure 9) during development showed an iso-diametric signal in the germinating conidia. When the polarized germ tube appeared, the fluorescence was concentrated in the peripheral region of the hyphae. In some cells we observed that the fluorescence signal was concentrated as a cap in the hyphal tips and “branch initials”. This concentrated signal may correlate with high growth activity. The same localization was found in M. grisea (Figure 10) and P. infestans (Figure 11).

As they were growing, Neurospora hyphal cells were treated with sordarin – a drug that blocks the interaction of EF2 with ribosomes – and the immunoreacting peptide was delocalized to the cytoplasm (Fig. 3 in Publication II).

Immunogold

Immunogold labeling was performed in ultra-thin sections of N. crassa (Fig. 3 in Publication II) and A. arbuscula (Figure 12). This study confirmed the association of the protein detected by the anti- chicken αβ-spectrin antibodies with the peripheral region of the cell. Control experiments with anti- spectrin antibody pre-absorbed with human spectrin or the use of only secondary antibodies showed the absence of labeled grains, thus confirming the specificity of the primary antibody (data not shown).

In vitro and in situ digestion with CDP II

Incubation of a crude extract from N. crassa with the protease CDP II under optimal conditions for enzyme activity resulted in a diminution of the relative intensity of the band at low enzyme to protein ratios (1:100, 1:50) and the complete absence of a band at a ratio of 1:10. The protein remains unaffected in the absence of Ca2+ in the reaction mixture or in the presence of a specific protease inhibitor (Leupeptin). Incubation of the extract without CDP II did not show any appreciable proteolysis of the immuno-reacting protein, indicating that there was no other endogenous protease targeting this protein under the test conditions (Fig. 4A in Publication I).

The digested protein in vitro appears as dots localized within the peripheral region of the cell instead of localization along the plasma membrane and capped at the tip (Fig. 4B-C in Publication I).

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The immunorevealed protein with anti-chicken αβ-spectrin antibody and the protease CDP II were co- localized in growing tips, as shown in Fig. 2F–I in Publication I. Outside of this region the localization differs: the immunoreacting protein was predominantly associated with the plasma membrane whereas CDPII showed diffuse cytoplasmic distribution.

44 Figure 9. Immunolocalization of the protein revealed with anti-chicken αβ- spectrin antibody in Allomyces arbuscula. A) Isodiametric distribution in germinating zoospore with high fluorescence signal concentration along the plasma membrane. B) Predominant antigen localization in growing rhizoid and tip of early stages of germ tube formation. C) Distribution along the plasma membrane after 12 hours of growth. D) Predominant localization in hyphal tips after 18 hours of culture growth (window: detail of rhizoid). Bar 10 μm

45

Figure 10. Immunolocalization of the protein revealed with anti-chicken αβ-spectrin antibody in Magnaporthe grisea. Fluorescence signal distributed along the plasma membrane (A-C) and in a branching tip (D). Bar 10 μm.

Figure 11. Immunolocalization of the protein revealed with anti-chicken αβ-spectrin antibody in Phytophthora infestans. Fluorescence signal distributed along the plasma membrane in the apical (A) and sub-apical region (B). Bar 10 μm.

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Figure 12. Immunogold labeling of the protein revealed with anti-chicken αβ-spectrin antibody in Allomyces arbuscula. A) General view of the apex showing predominance of the antigen along the peripheral region of the cell, B) higher magnification of a portion of the tip. C) Thin section of a rhizoid showing distribution of the signal along the plasma membrane. (cw: cell wall; pm: plasma membrane; m: mitochondria; n: nucleus)

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4.2. α-Actinin orthologs in fungi

Spectrin-like proteins have so far only been described in plants and fungi (Publication II, Table 1) using the criteria of cross-reactivity with commercial anti-spectrin antibodies. We have demonstrated here that the antibody predominantly used for these studies (anti-chicken αβ-spectrin) cross-reacts with other proteins. A search in the fungi and oomycetes databases was initiated to verify the presence of spectrin-related proteins in these organisms.

The first (unsuccessful) approach to search for spectrin-like proteins in fungi (M. grisea and N. crassa genome sequences) and Oomycetes (Phytophthora sojae and Phytophthora ramorum genome sequences, partial EST sequences of P. infestans) involved a homology search (BLAST tool) using the entire spectrin amino acid sequences from human and chicken spectrins. The screening of the N. crassa genomic database using the sequence of the domains defining the spectrin family (CH-domain, spectrin repeats and EF-hand domain) gave a gene, ncu06420.4, coding for a hypothetical protein with the three domains. This protein was closer in structure and sequence to an α-actinin, another member of the spectrin superfamily. We used this protein to find its orthologs in other fungi and Oomycetes (Publication III, Table 1).

This orthologous group is currently collected in the MIPS database and has been the object of two recent publications concerning the bioinformatics approach (Virel and Backman, 2007; Publication III).

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4.3. Characterization of α-actinin from Neurospora crassa

Over-expression of α-actinin and α-actinin domains in a GST system and antibody production

The GST fusion proteins constructed were found to vary in their level of expression and solubility. In general, both expression and solubility increased when the culture time was prolonged at low temperature (25°C). The full length α-actinin-GST protein, containing the N-terminal extension and characteristic domains (Figure 6B, Material and Methods), was used as an immunogen to produce specific antibodies against N. crassa α-actinin. The resulting polyclonal antibody was named anti- Neurospora α-actinin and has been used for several biochemical analyses described in Publications II and IV and in this part of this work.

Immunochemical detection of proteins reacting with anti-Neurospora α-actinin antibody

The anti-Neurospora α-actinin antibody reacted against α-actinin from different ascomicetous fungi. In N. crassa the antibody detected an 80 kDa peptide corresponding in molecular weight to the protein containing the two N-terminal CH-domains, the rod domain and C-terminal EF-hand motifs (Figure 13; Fig. 1B, lane d in Publication II). In Magnapothe grisea the antibody detected a 72 kDa peptide also corresponding to a protein with the specific α-actinin domains (Figure 13; Fig. 1B, lane e in Publication II). The antibody was highly reactive against the 80 kDa α-actinin protein from a Botrytis cinerea crude extract (Figure 13). In Phytophthora infestans an immunoreacting band with anti- Neurospora α-actinin antibody was not obtained under the test conditions used (Fig. 1B, lane f in Publication II).

We also tested the antibody against the three over-expressed fragments of the N. crassa α-actinin, corresponding to the Actin-Binding Domain (ABD), the rod domain and the Ca2+-binding domain. The antibody showed poor affinity for ABD (approaching the background signal). The principal epitopes for the anti-Neurospora α-actinin antibody seem to be in the rod and Ca2+-binding domains (Figure 14).

Characterization of Neurospora α-actinin properties

The actin- and calcium-binding properties of Neurospora α-actinin have been described in detail in Publication IV. The results show that calcium binds to α-actinin from N. crassa with the same affinity as the α-actinin from chicken gizzard. Since we used recombinant GST-α-actinin for this binding assay, the GST protein was used as a negative control and shows that this tag does not have affinity for calcium (Fig. 4 in Publication IV). A co-sedimentation assay and electron microscopy were used to analyze the actin-binding properties of Neurospora α-actinin. This protein binds actin in a calcium-

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dependent manner (Fig. 2 in Publication IV) and cross-links the actin microfilaments to organize them in parallel structures as bundles (Fig. 3B in Publication IV).

Localization of α-actinin in N. crassa

Neurospora α-actinin clearly localized within the septum (Fig. 5C-D and Fig. 7A-B in Publication IV; Figure 15). Results obtained from the in vivo localization revealed that the α-actinin is only present at this location during septum formation (Figure 15; Fig. 7 in Publication IV). Immunofluorescence and GFP signal were found at the germination site in the conidia and in the tip of the emerging tube (Fig. 5A and Fig. 6A–C in Publication IV). α-Actinin immunolocalizes at the peripheral region of the cell in growing hyphae (Fig. 5B in Publication IV). This localization was not confirmed by the α-actinin GFP fluorescence.

Phenotype of the heterokaryon α-actinin knock-out strain of Neurospora

We approached the Neurospora Genome project (Colot et al., 2006) to create an α-actinin knock-out strain. The heterokaryon knock-out strain was deposited in the FGSC. The homokaryon strain was defined as lethal. The heterokaryon mutant in Davis and De Serres medium (1970) showed a shortening of aerial hyphae (Fig. 8A in Publication IV) and a delay in colony expansion in Vogel 2% saccharose plates in comparison to the wild type (Figure 16). The morphology of the hyphae was different with respect to their branching pattern, showing a predominantly dichotomous phenotype compared to sympodial branching in the wild type (Fig. 8B in Publication IV).

Immunochemical characterization of “α-actinin-GFP-expressing” and “heterokaryon α-actinin knock-out” strains of N. crassa

We examined the crude extract of the α-actinin-GFP-expressing and wild type (wt) Neurospora strains with the anti-Neurospora α-actinin antibody. This antibody reacted with the 80 kDa peptide both in the wild type and the transformed strains (Figure 17, lanes a', b'), but additional bands appear at 110 and 140 kDa in the transformed strain (Figure 17, lanes b'). In an attempt to clarify the identity of these two bands we used a commercial anti-GFP antibody, which reacted in this same crude extract with the 110 and 140 kDa bands (Figure 17, lane d’). In order to confirm the identity of these two bands as the recombinant GFP-fusion proteins proposed in Figure 17, it will be necessary to perform sequence analysis.

In a crude extract from the heterokaryon α-actinin knock-out strain of N. crassa, the anti-Neurospora α-actinin antibody gives a faint signal that could reflect its reduced gene copy number when compared with wild type (Figure 17, lane c').

54 Figure 13. Immunoblot of α-actinin from different ascomycetous fungi. (Lane a) N. crassa; (lane b) M. grisea; (lane c) B. cinerea.

Figure 14. Immunoreactivity of anti-Neurospora α-actinin antibody with the different domains of α-actinin. (Lane a) full-length GST-α-actinin; (lane b) GST-Actin-binding domain; (lane c) GST-rod domain; (lane d) GST-Calcium-binding domain. (Arrowhead shows the position of the GST-Actin- binding domain).

55

Figure 15. Confocal images of germinating conidia expressing α-actininGFP showing its localization during the septa formation. The GFP signal appears at the site of septum formation and disappear following completion of the septum after 10 min. (Arrow: septum). Bar 5 μm.

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Figure 16. Growth on solid medium of the Neurospora heterokaryon α-actinin knock-out strain compared to the wild type.

59

kDa 170 140 kDa GFP

116 110 kDa GFP

76 80 kDa

a b c a’ b’ c’ d d'

Figure 17. Anti-Neurospora α-actinin immunoreacting peptides in the heterokaryon α- actinin knock-out and α-actinin-GFP containing strains compared to the wild type. Immunoblot of the protein revealed with anti-Neurospora α-actinin (left panel) and anti-GFP ( right panel, lane d’) antibodies. Lanes a-a’ corresponds to the wild type strain; lanes b-b’, to the α- actinin-GFP containing strain and c-c’ to the heterokaryon α-actinin knock-out strain; lane d-d’ is the same as b-b’.

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Immunolocalization of α-actinin and actin in Botrytis cinerea

N. crassa was the principal model in this work for the characterization of α-actinin. The study of theis protein in other fungi could give us more insights into the role of this actin-binding protein in fungi. As the Western analysis of the whole crude extract proteins from B. cinerea resulted in a strong reacting band with the anti-Neurospora α-actinin antibody, we have performed the in situ localization of the α-actinin in this ascomycetous fungus. After 12 h of growth the cultures were in the exponential growth phase. In this phase α-actinin was localized throughout the cytoplasm and concentrated in the septum and tip region (Figure 18A–D). Actin principally localized as cortical patches and occasionally an immuno-fluorescence signal was detected in the septum (Figure 18E–F).

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Figure 18. Immunolocalization of Botrytis cinerea α-actinin (A-B) and actin (C-D). α-Actinin localization in the tip region (A) and in the septum of the hyphae (B). Actin was mostly concentrated in the septum and as dots in the apical region (C-D). Bar 5 μm.

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5. DISCUSSION, CONCLUSIONS AND PERSPECTIVES

Discussion

Identity of the spectrin-like protein in fungi and an Oomycete

Our experimental models are filamentous fungi, mainly Neurospora crassa and Magnaporthe grisea. In these organisms, the existence of a fibrous network of peripheral proteins known as membrane skeleton has been proposed by Degousée et al. (2000) and Torralba and Heath (2001). According to these authors, this membrane cytoskeleton is likely to be composed mainly of actin, spectrin and integrin. In support of this hypothesis studies have been published describing the presence of high molecular weight spectrin in plants, fungi and Oomycetes that reacts with anti-spectrin antibodies (Table 1 in Publication II). Our results using the same primary antibody that was used to report the presence of spectrin-like proteins in these organisms, indicated that the major immunoreacting peptide is a 100 kDa protein in N. crassa and M. grisea and a 60 kDa protein in the oomycete P. infestans (Publications I and II). Despite a great deal of effort, we were unable to confirm the presence of a high molecular weight spectrin-like protein as described by the authors cited above. Furthermore, in the available sequenced genome database for fungi and Oomycetes, a putative gene coding for high molecular weight spectrins was not found. This contradicts previous reports of a spectrin-like gene product of 240–220 kDa in fungi (Kaminskyj and Heath, 1995; Degousée et al., 2000; Heath et al., 2003; Slaninová et al., 2003; Toquin et al., 2006). The peptides reacting with the anti-αβ-spectrin antibody in N. crassa and the oomycete P. infestans are not related to the spectrin superfamily of proteins but to two different proteins: EF2 in N. crassa and Hsp70 in P. infestans (Publication II). The three proteins; spectrin, EF2 and Hsp70, were found to share one characteristic: a predicted chaperone activity due to a hydrophobic region. Further, another anti-β-spectrin antibody has been reported to cross-react with the E. coli chaperone, GroEL (Czogalla et al., 2003).

The localization of N. crassa EF2 and P. infestans Hsp70 in the peripheral region of the cell is difficult to interpret, especially for EF2. Heat shock proteins have previously been found to localize in the cell walls of fungi (López-Ribot and Chaffin, 1996; López-Ribot et al., 1996; Purin and Rillig, 2008) where they participate in processes associated with fungal cell wall biosynthesis. However, the localization of EF2 to the peripheral region is not consistent with its function in protein elongation, associated with ribosomes. Localization of elongation factors to the plasma membrane has been found in bacteria (March and Inouye, 1985). All living cells utilize conserved systems responsible for membrane protein biogenesis. In eukaryotic cells, integral membrane proteins, as well as many secreted proteins, are targeted to the endoplasmic reticulum. Bacteria utilize a similar pathway, however in these organisms ribosome target to the cytoplasmic membrane (Herskovits and Bibi, 2000, 2002 and reference cited therein). In fungi, as in the other eukaryotic cells, the relationship between protein synthesis and the plasma membrane is less probable and has not been probed.

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The existence of other unknown functions for EF2 that may explain this unexpected localization cannot be ruled out. EF2, as other elongation factors (the EF1α and EF1β) is an actin-binding protein (Yang et al., 1990; Bektaş et al., 1994; Condeelis, 1995; Gross and Kinzy, 2005). The EF1α has also other functions besides its role in the elongation phase of protein synthesis and its ability to bind actin (Yang et al., 1990). It has also been described as an activator of the phosphatidylinositol-4-kinase (Yang et al., 1993).

The explanation for the results of the anti-spectrin antibody cross-reactivity is discussed in Publication II.

Features of α-actinin in fungi and Oomycetes

The absence of spectrins in fungi does not exclude the possibility that other members of the spectrin superfamily may be involved in actin cytoskeleton organization and function. In 2001 Wu et al. reported the presence of a gene coding for an α-actinin-related protein (Ainp1) in Schizosaccharomyces pombe but observed that its ortholog is absent in the budding yeast Saccharomyces cerevisiae. Complete genome sequences of filamentous fungi were not available at the time until the publication of the N. crassa genome in 2003 (Galagan et al., 2003). In recent years other fungal genomes have been completely sequenced and this enabled us to compare orthologs in these fungi and Oomycetes. In a recent study on the evolution of α-actinins, Virel and Backman (2004) provided a direct evidence for the presence of α-actinin orthologs in two fungi: N. crassa and S. pombe. While our work on the evolution of α-actinin in fungi using CH-domain sequences was in progress, a paper by the same authors appeared documenting actinin orthologs in fungi using the rod domain criteria (Virel and Backman, 2007). We extended these observations by including a representative from the Chytridiomycota and carried out a detailed analysis of α-actinin in Saccharomycotina. In this sub-class, which includes very diverse organisms (Dujon, 2006), we found only one species, Yarrowia lipolitica, that contains a gene coding for α-actinin. There is no gene coding for a bona fide α-actinin in S. cerevisiae (Wu et al., 2001; Virel and Backman, 2004) or in the more recently sequenced genomes of Kluyveromyces lactis, Candida glabrata and Ashbia gossypii. In other yeasts, we found hypothetical proteins with one conserved N-terminal CH-domain in C. albicans and two in Pichia stipitis, Debaryomyces hansenii and Candida sp. (Candida guilleimondi and Candida tropicalis). Spectrin repeats or C-terminal EF-hand motifs were not found. The estimated molecular weights (Mw) of these proteins are between 70–80 kDa, similar to the estimated Mw of fungal α-actinins. However, this does not confirm their identity as actinin-related proteins. It is clear that there are other proteins having CH-domains besides spectrin superfamily members, e.g., calmin, enaptin and nuance. These proteins have been described as having two N-terminal CH-domains and a C-terminal trans-membrane domain(s) (Gimona et al., 2002 and references cited therein). However, these proteins generally have higher molecular weight than expected for fungal α-actinin. Other

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proteins with two N-terminal CH-domains, not mentioned in the review of Gimona et al. (2002), are the cortexillins I and II, present in the cellular slime mold Dictyostelium discoideum (Faix et al., 1996). We used the blast tools of NCBI (National Center of Biotechnology Information, http://www.ncbi.nlm.nih.gov/) and also found these proteins in another cellular slime mold Polysphondylium pallidum as well as in an amoeba Entamoeba histolytica. The sequences found, however, code for a protein of lower molecular wight, about 40 kDa and are implicated in cytokinesis (Faix et al., 1996) and mechanical properties of the cell cortex (Simson et al., 1998). We did not find any orthologs of these proteins, containing N-terminal CH-domains, in the fungal species examined.

The existence of “atypical” α-actinins with only one or two conserved N-terminal CH-domain(s) can be explained by evolutionary pressure where genes become highly diverged, modified beyond recognition or completely lost. Gene loss during evolution in several lineages is not a rare event and has been shown to occur for many genes (Aravind et al., 2000; Roelofs and Van Haastert, 2001; Krylov et al., 2003).

Despite the fact that the relationship between these proteins in the Saccharomycotina and other fungi is not clear, we can hypothesize that they have evolved from a common ancestor through two different pathways (as shown in Fig. 1 in Publication III). One pathway led to the loss of the α-actinin gene, as in S. cerevisiae, or severely modified, as in Candida albicans and the yeasts mentioned above. However, other yeasts such as Y. lipolytica followed another pathway in which the gene was preserved. This hypothesis is supported by the fact that even though we cannot recognize any functional domain in the middle and C-terminal regions of these hypothetical proteins, the gene is still present with a relatively large ORF. The absence of a “classical” fungal α-actinin in nearly all Saccharomycotina may be due to a complementation in function by other proteins, possibly fimbrin, as happens in S. pombe (Wu et al., 2001) or other actin-binding proteins (Rivero et al., 1999). Fimbrin is interesting from this point of view since it contains two conserved domains that are also found in α- actinin but with a reverse orientation, i.e., N-terminal Ca2+-binding EF-hand motifs and C-terminal actin-binding CH-domains. α-Actinin is present in all fungi examined, except most members of the Saccharomycotina, where α-actinin was found only in Y. lipolytica.

In other fungi, α-actinin seems to be highly conserved and display a significant sequence homology to chicken and human α-actinins. The estimated molecular weight for fungal α-actinin varies in the range 70–85 kDa. Some ascomycetes such as N. crassa, M. grisea, Sclerotinia sclerotia and Fusarium graminearum have N-terminal extensions of varying length and do not have sequence homology to any known protein. Considering the size of these N-terminal extensions, the expected protein should vary between 80 to 110 kDa. The α-actinins of these four species contain a second putative ATG-start codon located immediately upstream of the first CH-domain. The molecular weight of the protein resulting from transcription starting at the second start codon fits perfectly with the molecular weight

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of other fungal α-actinins. The anti-Neurospora α-actinin antibody reveals bands of 80 and 72 kDa, respectively, in N. crassa and M. grisea, suggesting that the expressed protein should have initiated at the second ATG-start codon under the conditions used in this work. The N-terminal extension could be a regulatory element in the mRNA, an alternate use of the two ATG start codons or a pro-domain in the protein, cleaved immediately after protein synthesis. The latter hypothesis is supported by the analysis of crude extracts N. crassa recombinant α-actinin-GFP expressed in E.coli. In these extracts three bands with relative masses of 80, 110 and 140 kDa were revealed with the anti-Neurospora α- actinin antibody. These peptides could correspond to the native short α-actinin, the short α-actinin- GFP and full-length α-actinin-GFP, respectively (the presence of the GFP tag in these polypeptides was confirmed using anti-GFP antibody). The presence of full-length and short α-actinin-GFP suggests that the cell possesses the enzymatic machinery necessary to cleave the pro-domain of the protein. These results do not rule out the possibility of an alternate use of the two in-frame methionine start codons. Indeed, such a phenomenon has been described for another protein, frequency (FREQ), implicated in the regulation of the circadian clock in Neurospora (Diernfellner et al., 2005). In this protein, the alternate use of the start codons of FREQ is regulated by one intron and the UTR-region.

Biochemical characterization of N. crassa α-actinin

The interaction of α-actinin with actin was calcium dependent, as demonstrated by co-sedimentation assays. It was shown that α-actinin has the ability to cross-link F-actins and to organize microfilaments in a parallel way. Actin filaments cross-linked by α-actinin were 15–25 nm apart. The distance between the filaments was shorter than those observed in other actin-α-actinin complexes (30–40 nm) (Taylor and Taylor, 1993; Tang et al., 2001; Hampton et al., 2007), but agrees with the difference in size between α-actinin of higher eukaryotic cells and the shorter fungal α-actinins, which contain only half the number of spectrin repeats (Virel and Backman, 2007). The calcium dependence of Neurospora α-actinin in actin-binding assays and the insensitivity of vertebrate α-actinin support the idea that the calcium-dependent regulation of actin-binding was lost during the invertebrate– vertebrate divergence (Dixson et al., 2003; Virel and Backman, 2004).

Localization of α-actinin in N. crassa

The localization of proteins can provide information on their function. Two approaches were used to localize α-actinin in N. crassa: in situ detection, using specific antibodies, and in vivo GFP fluorescence localization. α-Actinin was found to immunolocalize to some septa. In a manner consistent with the immunofluorescence results, the GFP signal was also transiently found in the septa during their formation. Other proteins required for septation in filamentous fungi, such as formin SepA (Sharpless and Harris, 2002) and actin (Momany and Hamer, 1997; Rasmussen and Glass, 2005), show the same pattern. α-Actinin also localizes in emerging tubes and the apical region of the hyphae,

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suggesting a role for the protein during germination and tip growth. The peripheral localization in the hyphae suggests additional roles in cellular physiology required for optimal growth. One can speculate that these functions are related to the lethal phenotype of the homokaryon knock-out mutant.

However, this localization was not observed in vivo, probably because the GFP signal was too weak to be detected or that the GFP tag partially modified the localization of the fusion protein (Ikonen et al., 1995; Schneider et al., 2000).

Function of N. crassa α-actinin

The knock-out phenotype was lethal. It is difficult to assume that the lethal phenotype is due only to a supposed role in septum formation. The aseptate mutants in Neurospora (Rasmussen and Glass, 2005; 2007) and other filamentous fungi (Ayad-Durieux et al., 2000; Wendland and Philippsen, 2002; Kim et al., 2006) are viable.

Although the exact function of α-actinin in Neurospora is still not clear, we can predict that a collaboration with actin plays a major role. The results from protein localization suggest a role in cytokinesis, which would agree with the function of its orthologs in S. pombe (Wu et al., 2001). However, differences can be expected because cytokinesis in filamentous fungi and yeast are two principally different mechanisms (Walther and Wendland, 2003). Moreover, α-actinin function in yeast and also in D. discoideum can be complemented by other actin-binding proteins, notably fimbrin (Wu et al., 2001; Rivero et al., 1999), resulting in non-lethal phenotypes for α-actinin knock-out mutants in these organisms.

The dichotomous phenotype of the α-actinin heterokaryon knock-out strain may be a consequence of splitting at the tip, also called “dichotomous branching”. This process has been observed in several other filamentous fungal mutants in which polarity maintenance proteins were targeted (Sharpless and Harris, 2002; Geissenhöner et al., 2001; Han and Prade, 2002). A similar phenotype has been observed in Neurospora treated with Cytochalasin A (Riquelme et al., 1998) and Neurospora actin mutants (Virag and Griffiths, 2004), suggesting that α-actinin together with actin may play a role in branching. However, other putative roles can be expected.

The list of newly identified proteins and molecules interacting with α-actinin has increased over the last several years, confirming that α-actinin is not merely an actin-binding protein but a scaffold for other protein-protein interactions connecting the cytoskeleton to diverse signaling pathways (Critchley and Flood, 1999; Otey and Carpen, 2004).

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Conclusions

The conclusions drawn from this work are as follows:

• There are no genes coding for spectrin in fungi and Oomycetes.

• The anti-spectrin antibody that has been used to demonstrate the presence of spectrin-like proteins in fungi and Oomycetes cross-reacts with proteins other than spectrin.

• In fungi, there is evidence for a gene coding for a spectrin superfamily protein. This protein has been identified as α-actinin.

• α-Actinin gene is not present in all fungi. In some saccharomycotina yeasts it has been lost.

• N. crassa α-actinin has a molecular weight of 80 kDa and pI of 5.9.

• N. crassa α-actinin has the ability to bind actin in a calcium-regulated manner.

• In N. crassa α-actinin localizes in situ to: (1) the site of the initiation of germ tubes in germinating spores, (2) within the peripheral region of growing hyphae, and (3) in septa during their formation.

• Localization in situ was confirmed in vivo, with the exception of the plasma membrane localization.

• Deletion of the α-actinin gene in N. crassa is lethal suggesting that at least in this organism its function cannot be complemented by other actin-binding proteins.

• Although the role of α-actinin in N. crassa has not been precisely determined, localization and characterization of the knock-out heterokaryon suggests that the protein participates together with actin in the coordination of cellular activities such as germination, septum formation and branching.

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Perspectives

Our results represent an important step towards understanding the role of actin cytoskeleton in the spatial organization of hyphae during cell life cycle. Future experiments will be aimed at determining the function of the α-actinin N-terminal extension. First, it will be necessary to determine if this extension present in the mRNA is a UTR-region, a coding sequence with an alternative start-codon usage or a pro-domain of the protein. To achieve this, 5' RACE (Rapid amplification of 5' complementary DNA ends) experiments will allow us to determine the start-codon of α-actinin mRNA. Further, point mutations in the first putative start-codon will confirm the location of the translation initiation and answer whether the N-terminal extension is a pro-domain of the protein.

Studies into the effects of actin inhibitors on in situ and in vivo α-actinin localization will provide a greater insight into the cooperation between these two proteins, α-actinin and actin. Other GFP- constructs, such as N-terminal GFP tagging of the “full-” and “short-length” α-actinins, will help us to understand why α-actinin localization to the peripheral region has not been found in vivo. Comparison of the three transformed strains can provide information about the role of the N-terminal α-actinin extension.

Considering that α-actinin deletion is lethal in N. crassa, future experiments using siRNA may help to elucidate the function of this protein.

The evolutionarily related α-actinins characterized in N. crassa and S. pombe seem to differ in their cellular localization, function and indispensability. Studies of these proteins in other fungi may help to better understand their role as components of the actin cytoskeleton in these organisms.

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ANNEXE

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CURRENT MICROBIOLOGY Vol. 53 (2006), pp. 311–316 DOI: 10.1007/s00284-006-0057-y Current Microbiology An International Journal ª Springer Science+Business Media, Inc. 2006

Proteolytic Cleavage of a Spectrin-Related Protein by Calcium-Dependent Protease in Neurospora crassa

M. Cotado-Sampayo,1 M. Ojha,2 R. Ortega-Prez,1 M.-L. Chappuis,1 F. Barja1 1Laboratory of Bioenergetics and Microbiology, University of Geneva, 10 Chemin des Embrouchis, CH-1254 Jussy-Geneva, Switzerland 2Department of Biochemistry, University of Geneva, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland

Received: 3 February 2006 / Accepted: 18 May 2006

Abstract. To investigate the functional significance of a cytoskeletal spectrin-like protein, we studied its localization pattern in Neurospora crassa and sought the answer to whether it is a substrate for another apically localized protein, the calcium-dependent protease (CDP II). Immunoblots of crude extracts from exponentially growing mycelia, separated by one- and two-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis using antichicken a/b-spectrin antibodies, revealed a single band of approximately relative mass (Mr) 100 kDa with an isoeletric point (pI) in the range of 6.5 to 7.0. Despite rigorous efforts, we could not confirm the presence of an Mr 240- to 220-kDa spectrin-like protein in N. crassa. The immunofluorescence- and immunogold-labeling Mr 100-kDa protein showed its predominance along the plasma membrane of the conidia during the swelling phase of germination. In contrast, in the germ tubes and the growing hyphae, the localization was polarized and concentrated mainly in the apical region. The in vitro proteolysis experiments showed that indeed this protein is a preferred substrate of CDP II which is, as mentioned previously, also localized in the apical regions of the hyphae. These results indicate a putative functional relationship between these two proteins (spectrin-like protein and CDP II) in the dynamics of tip growth.

The filamentous fungi represent a large group of apical region of Allomyces, an aquatic fungus, and shown organisms that grow and colonize substrates by that their presence or absence is related to growth [4, 20– branching and apical elongation. Cytoskeletal elements 24]. An immunoanalogue of CDP II was also discovered are considered to be involved in this process and have and shown to be exclusively localized in the apical been the subject of intensive investigation. Ultrastruc- regions of the hyphae of an ascomycete fungus, Neu- tural and immunologic studies of the apical zone, for rospora crassa, and a phytopathogenic basidiomycete, example, have shown that this region is rich in cyto- Uromyces appendiculatus [25]. Therefore, this colocal- skeletal elements, such as actin [1–4], tubulins [5–11], ization of cytoskeletal proteins and CDP might have integrins, and spectrins [12–15]. These proteins are some functional significance. Indeed, we have shown considered to provide stability to the growing hyphal selective proteolysis of a-tubulin by CDP II in the apical tubes by the linkage among themselves and to the region of the growing hyphae and cell-free extracts [24]. plasma membrane. For the hyphae to grow, this linkage We sought to determine whether other apically must be reorganized. localized cytoskeletal proteins are also targets of this Another important factor implicated in regulation of protease. Because a spectrin-like protein has been polarized growth and branch formation is calcium sig- shown to be localized in the apex of the growing hyphae naling [16–19]. We have reported the presence of two of N. crassa using antichicken a/b spectrin antibodies calcium-dependent proteases (CDPs I and II) in the [14], we re-examined the spatial localization of this protein and CDP II during early development of N. crassa and studied the proteolysis of the former Correspondence to: F. Barja; email: [email protected] in situ as well as in cell-free extracts by this protease. 312 CURRENT MICROBIOLOGY Vol. 53 (2006)

Materials and Methods initiate the proteolytic activity. The reaction was performed in a total volume of 75 ll at enzyme-to-protein ratios of 1:100, 1:50, and 1:10. Organism and culture conditions. A wild-type strain of N. crassa After incubation at 37C for 30 minutes, the reaction was stopped by (FGSC 262, strain St. Lawrence STA4) was used in this study. the addition of 25 ll4· loading buffer [29]. The proteins were Production of conidial inocula and culture growth conditions were as denatured in boiling water for 5 minutes and separated by SDS-PAGE, described previously [26]. transferred to nitrocellulose membranes, and immunorevealed with spectrin polyclonal antibodies. Reaction mixtures either without Protein extraction and analysis. Crude extract was prepared calcium or with leupeptin (10 lM), an inhibitor of CDP II activity, according to Abe and Davies [27] with slight modifications. Briefly, were used as controls. the buffer contained 5 mM HEPES-KOH at pH 7.5, 250 mM sucrose, 15 mM Mg(OAc)2, 2 mM EGTA, 2 mM dithiothreitol, 25 mM K2O5S2, 10% In situ proteolysis by CDP II. Hyphae from 12-hour cultures were glycerol, 0.5% polyvinylpyrrolidone, and protease inhibitors (1 mM prepared as described previously for immunofluorescence. Before benzamidine, 2 mM phenylmethylsulfonyl fluoride, and 2 lg/ml each blocking, the cells were incubated for 1 hour at 37C with CDP II in for leupeptin, chymostatin, and trasylol). Protein concentration was the enzyme reaction buffer described previously, and washed with determined according to Bradford [28]. Sodium dodecyl sulfate– phosphate buffer five times for 5 minutes each, and then polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional immunorevealed as described earlier [24]. analysis were done according to Laemmli [29] and OFarrell [30], respectively.

Immunoblotting. Proteins from unstained monodimensional and bidimensional gels were electrophoretically transferred to Results nitrocellulose membranes (BA85, pore size 0.45 lm), using the Proteins from the cell-free extract, separated in nonde- transfer buffer described by Burnette [31], and immunoblotted as naturing PAGE and immunoblotted with chicken described earlier [24]. Antichicken a/b spectrin (Sigma S1390) and antirabbit IgG antibodies coupled to horseradish peroxidase were used antispectrin polyclonal antibodies, showed two immu- at dilutions of 1:800 and 1:2000 in Tris-buffered saline, respectively. noreacting bands (Fig. 1A). The lower band (arrow) from nondenaturing gel was cut and migrated in SDS-PAGE; a Immunofluorescence microscopy. Conidia were suspended in Vogels growth medium, and a sample was taken immediately, which single strong band of Mr 100 kDa appeared (data not represented the start point (0 hours). Further samples were taken at 6, shown). In direct SDS-PAGE of the cell-free extract, a 12, and 18 hours of culture growth and fixed instantaneously by the single heavily stained band of Mr 100 kDa was also ob- addition of paraformaldehyde in the culture medium to a final served. The intensity of this band did not change signifi- concentration of 3%. cantly in preparations obtained from cells either during Primary antichicken spectrin antibodies and secondary goat an- tirabbit fluorescein isothiocyanate (green, Sigma F9887) or crystalline germination or exponential growth (Fig. 1B). Human tetramethylrhodamine isothiocyanate (red, Sigma T5268) antibodies erythrocyte spectrin, used as a positive control, revealed were used at dilutions of 1:50 and 1:100, respectively, in phosphate two bands of Mr 240 and 220 kDa each (Fig. 1C). buffer [24]. These two secondary antibodies were used to differentiate The specificity of immunoreaction was demon- the distribution of spectrin-like protein and CDP II in the colocaliza- strated using human erythrocyte spectrin in a competi- tion experiments. The immunolabeled cells were examined with a microscope (Zeiss axioplan) equipped for epi-illumination with fluotar tion assay. The results showed a progressive diminution optics and selective filter combinations. Spectrin-like protein was re- of the immunoreacting band with increasing concentra- vealed using Sigma antichicken a/b spectrin and anti-CDP II using tion of the competing human spectrin (Fig. 1D). In two- laboratory stock of anti-CDP II prepared from Allomyces arbuscula. dimensional gels, the predominant immunoreacting Colocalization was done using respective primary antibodies. The band was Mr 100 kDa, with a pI in the range of 6.5 to images were taken with a Hamamatsu color, chilled 3 CCD camera, developed by Raster Optics video captor and treated by the program 7.0, in addition to a faint 80-kDa band with similar pI, Adobe PhotoShop 7. representing probably a degradation product of the ma- jor immunoreacting protein (Fig. 1E). Electron microscopy and immunogold labeling. Spores (0 hours) and 6-hour germinated conidia were used for immunogold labeling of the Immunofluorescence of the protein revealed with spectrin-like protein using antichicken spectrin antibodies diluted to 1:50 polyclonal antispectrin antibodies was intense all around in phosphate buffer and secondary goat antirabbit antibodies conjugated the peripheral regions of the isodiametric germinating to 20 nm gold particles and diluted to 1:30 in the same buffer. The conidia, representing the swelling phase of germination experimental procedure used has been described in detail elsewhere [24]. (earliest visible phase) (Fig. 2A). With the appearance Sections were examined at 60 kV using a Philips M400 transmission of germ tube outgrowth representing the polarized electron microscope. growth phase, much of the fluorescence was concen- Proteolysis of cell-free extract by CDP II. The cell-free extract, trated in the plasma membrane of the germ tubes obtained as described in the section on protein extraction and analysis, (Fig. 2B). In the cells from the 12- and 18-hour cultures, was digested with CDP II (laboratory stock purified from A. arbuscula as described in Ojha and Wallace [20], specific activity 21 lM representing filamentous hyphal growth and corre- paranitroaniline released lg–1min–1) in a reaction mixture containing sponding to exponential growth phase, the fluorescence 20 mM Tris-HCl at pH 7.4, 4 mM EGTA, 3 mM MgCl2, and 1% b- was also polarized and concentrated as a cap in the 2+ mercaptoethanol. 6 mM CaCl2 was added to obtain 5 mM free Ca to hyphal tips and branch initials (Figs. 2C through 2E). M. Cotado-Sampayo et al.: Spectrin-Related Protein As Target of Calcium-Dependent Protease 313

Fig. 2. (A through E) Immunolocalization of a N. crassa spectrin-like protein revealed with polyclonal antichicken a/b-spectrin in cells of different stages of growth. (A) Conidia. (B) Germinating conidia with germ tube. (C and D) Growing hyphae. (E) Tip of branch initial. Fig. 1. Immunoblot of a N. crassa spectrin-like protein revealed with Bar = 10 lm. (F through I) Colocalization of CDP II and spectrin-like anti–a/b-spectrin antibodies. (A) Native PAGE of crude extract pro- protein in N. crassa hyphae. (F) Localization of spectrin-like protein. teins; arrow indicates the band that gives the 100-kDa peptide in the (G) Localization of CDP II. (H) Merge of two proteins in the tip of the SDS-PAGE gel. (B) SDS-PAGE of crude extract proteins from 6, 12, germ tube. (I) Merge of two proteins in the branch initials. and 18 hours of culture development. (C) Human erythrocyte spectrin Bar = 10 lm. as control (1 lg). (D) Specificity assay of spectrin polyclonal anti- bodies in N. crassa with antibodies preadsorbed to human erythrocyte spectrin: (a) human erythrocyte spectrin revealed with unadsorbed antibodies; (b) crude extract with unadsorbed antibodies; (c and d) of germination showed uniform distribution in the cor- antibodies preincubated with human erythrocyte spectrin at ratios of tical region and along the plasma membrane (Fig. 3). 1:0.8 and 1:2, respectively. Preincubation of antibodies was performed Control experiments with antichicken antibody pread- for 30 minutes at room temperature before using them in immunoblot sorbed with human spectrin, or the use of only second- reactions. (E) Two-dimensional analysis of a N. crassa spectrin-like protein revealed with anti a/b-spectrin antibodies. The pI range of the ary antibodies, showed an absence of labeled grains protein is 6.5 to 7.0. indicating the specificity of the primary antibodies (data not shown). Proteolysis of 100-kDa protein by CDP II in the In the colocalization experiments shown in Figs. 2F presence of Ca2+ was evident by the diminution of the through 2I, both spectrin-like protein (green) and CDP II relative intensity of the band at low enzyme-to-protein (red) in 6-hour germinating conidia and branch initials ratios (1:100 and 1:50) and complete absence at the ratio of the hyphae, were abundantly present in the same re- of 1:10 (Fig. 4A, c through e). Under similar experi- gion of the cell, i.e., the growing tip. There was, how- mental conditions, but in the absence of Ca2+ or in the ever, clearly one difference: the predominance of presence of leupeptin, this protein remained unaffected spectrin-like protein along the plasma membrane and a (Fig. 4A, f through g). Incubation of the extract without more diffuse distribution of CDP II in the cytoplasm as CDP II did not show any appreciable proteolysis of the evident in the merge of the localization of the two immunoreacting protein, indicating there was no other proteins (Figs. 2H through 2I). endogenous protease targeting this protein under the test Immunogold labeling the protein in ultrathin sec- condition used. An examination of the relative intensity tions of conidia in the isodiametric and polarized phases of Ponceau-stained bands after transfer from SDS-PAGE 314 CURRENT MICROBIOLOGY Vol. 53 (2006)

Fig. 3. Immunogold labeling of a N. crassa spectrin-like protein revealed with anti–a/b- spectrin antibodies during development. (A) Ultrathin section of conidia showing isodiametric distribution along the plasma membrane. (B) Distribution along the plasma membrane shown at a higher magnification of a portion (A). (C) Labeling along the plasma membrane in a section of conidia after 6 hours of germination. (D) Higher magnification of a portion of (C). m = mitochondria; n = nuclei; pm = plasma membrane; v = vacuole; cw = cell wall. to nitrocellulose membrane did not show any massive C-terminal prodomain, two calponin homology domains, general proteolysis. a rod domain composed of two spectrin repeats, and a Fixed and permeabilized cells incubated with CDP N-terminal EF band domain. The gene NCU03992.2 II in the enzyme reaction mixture and processed for codes for a protein (i.e., theoretical Mr 72 kDa) corre- immunofluorescence using antispectrin antibodies sponding to fimbrin and comprised of four calponin showed partial digestion of this protein where it ap- homology domains and no spectrin repeat units. Western peared as faint fluorescent dots (Fig. 4C) instead of blot analysis of the cell-free extracts of N. crassa—using continuous fluorescence along the plasma membrane the same primary antibody that was previously used to and capped localization at the tip observed in untreated recognize spectrin epitopes in plants [32–36], i.e., the cells (Fig. 4B). This pattern was similar to the one noted oomycetes Saprolegnia ferax [12] and green algae in our previous work with tubulins, in which b-tubulin, [37]—yielded evidence for the occurrence of only one which is not affected by CDP II, had no modification in related protein of Mr 100 kDa. This protein could cor- its hyphal distribution, whereas a-tubulin appeared as respond to the gene product NCU06429.2. Contrary to faintly dispersed fluorescent dots in the cytoplasm. published results [14] using the same antibodies and the same organism (N. crassa), we did not find the presence Discussion of an Mr 240- to 220-kDa protein. The spatial localization of the Mr 100-kDa protein A search in the genomic database of N. crassa for the by immunofluorescence showed that it is localized in the spectrin superfamily proteins yielded two genes, the apical zone of the fungal hyphae (especially along the NCU06429.2 and NCU03992.2. The gene NCU06429.2 plasma membrane) confirming the published results codes for a hypothetical protein corresponding to [14]. The plasma membrane localization of the spectrin- a–actinin, i.e., theoretical Mr 110 kDa. This protein has a like protein was more evident in the images obtained M. Cotado-Sampayo et al.: Spectrin-Related Protein As Target of Calcium-Dependent Protease 315

from the immunogold-labeling experiments. This localization may confer stability to plasma membrane by anchoring it to other cytoskeletal proteins. It is to be expected that proteolysis is necessary to weaken the attachment of cytoskeletal proteins to plasma membrane or other underlying hyphal tube structures and permit the expansion of the growing hyphal apex. Proteolysis of this plasma membrane–associated protein and possible weakening of the membrane skeleton may also facilitate the access of intracellular vesicles to the plasma mem- brane and allow its expansion. This is of importance because the tip of the hyphae has been shown to be a site of membrane insertion by exocytosis processes [38, 39]. The question arises, what is the identity of the protease responsible for this proteolysis? The apical localization of CDP II, as shown in Fig. 2H, essentially confirmed our previous finding reported earlier [25]. The colocal- ization of the spectrin-like protein and CDP II in the N. crassa hyphal apices (Fig. 2I) suggests that this spectrin- like protein at some point in development is proteolyzed by CDP II and regulated in its function. Considering the in vitro and in situ proteolysis of this spectrin-like protein and its colocalization with CDP II, we suggest that it is a likely candidate for one of the specific substrates in vivo.

ACKNOWLEDGMENTS

We gratefully acknowledge financial support from the Academic Society of Geneva (F. B.) and National Science Foundation Grant No. 3100-056786.99 (M. O.). Thanks are due to R. Strasser for interest in the project and encouragement, A. Cattaneo for technical assistance, and A. Fehr for secretarial assistance.

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Fungal Genetics and Biology 45 (2008) 1008–1015 www.elsevier.com/locate/yfgbi

Specificity of commercial anti-spectrin antibody in the study of fungal and Oomycete spectrin: Cross-reaction with proteins other than spectrin

Marta Cotado-Sampayo a, Pilar Okenve Ramos a, Rube´n Ortega Perez a, Mukti Ojha b, Francßois Barja a,*

a Laboratory of Bioenergetics and Microbiology, University of Geneva, ch. des Embrouchis 10, CH 1254 Jussy-Geneva, Switzerland b Department of Biochemistry, University of Geneva, Quai Ernest Ansermet 30, CH 1211 Geneva4, Switzerland

Received 18 December 2007; accepted 13 February 2008 Available online 21 February 2008

Abstract

Spectrin was first described in erythrocytes where it forms a filamentous network in the cytoplasmic face of the plasma membrane and participates in the membrane’s structural integrity in addition to controlling the lateral mobility of integral membrane proteins. In fungi, spectrin-like proteins have been described in the plasma membrane, concentrated mainly in the region of maximum apical expansion. This localization led to the idea of a spectrin based membrane skeleton in fungi participating in mechanical integrity of the plasma mem- brane, generating and maintaining cell polarity. The occurrence of spectrin-like proteins in filamentous fungi, yeasts and Oomycetes, however, is questionable since the presence of such proteins has only been demonstrated with immunochemical methods using antibodies whose specificity is unclear. There is no evidence of a gene coding for the high molecular weight ab-spectrin in the genome of these organ- isms. Mass spectrometric analysis of the anti ab-spectrin immunoreacting peptides from Neurospora crassa and Phytophthora infestans identified them as elongation factor 2 (NCU07700.4) and Hsp70 (PITG_13237.1), respectively. An attempt was made to correlate the reactivity of anti-spectrin antibody to a common feature of these three proteins i.e., spectrin, elongation factor 2 and heat shock protein 70, in that they all have a hydrophobic region implicated in chaperon activity. Ó 2008 Elsevier Inc. All rights reserved.

Keywords: Spectrin; Neurospora crassa; Elongation factor 2; Heat shock protein 70; Polyclonal antibody

1. Introduction therefore the cell shape. (Steck, 1989; Lee et al., 1993; Viel and Branton, 1996; Bennett and Baines, 2001). Spectrin is a major constituent protein of the metazoans The presence of spectrin in non-animal cells has been cell membrane, first purified to homogeneity from red demonstrated mainly through studies employing immuno- blood cells. It is an elongated heterodimer composed of chemical techniques (Western blot, immunofluorescence non-identical a and b subunits (about 30% identity) with and/or immunogold) using commercial anti-spectrin anti- molecular weight of 240 and 220 kDa estimated, respec- bodies (Table 1). The proteins recognized have been tively, from electrophoretic mobility and shown to localize reported as spectrin-like proteins in protists (Hemphill at the cytoplasmic face of the plasma membrane. It also et al., 1991; Ghazali et al., 1995; Holzinger et al., 1999), interacts with a wide variety of proteins creating a cellular plants (Michaud et al., 1991; Faraday and Spanswick, network (Bennett and Gilligan, 1993; Winkelmann and 1993; Bisikirska and Sikorski, 1997; De Ruijter et al., Forget, 1993). These interactions with cytosolic and mem- 1998, 2000), Oomycetes (Kaminskyj and Heath, 1995) brane proteins control the elasticity of the lipid bilayer and and fungi (Degouse´e et al., 2000; Slaninova´ et al., 2003; Cotado-Sampayo et al., 2006). However, our search for * Corresponding author. Fax: +41 22 37 93756. the bona fide spectrin in fungal-specific and general dat- E-mail address: [email protected] (F. Barja). abases (Broad Institute Database, MIPS and NCBI) using

1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2008.02.003 M. Cotado-Sampayo et al. / Fungal Genetics and Biology 45 (2008) 1008–1015 1009

Table 1 Reports of spectrin-like proteins in fungi, Oomycetes, plants and low eukaryotes organisms Organism Antibodies Molecular weight (kDa) Localization Reference Neurospora crassa Anti ab-Spectrin Sigma 220, 240, 100 Plasma membrane Degouse´e et al. (2000) S1390 Apex Anti ab-Spectrin Sigma 100 Plasma membrane Cotado-Sampayo S1390 Apex et al. (2006) Geotrichum candidum Anti ab-Spectrin Sigma Plasma membrane Heath et al. (2003) S1390 Patches in the apex Saccharomyces Anti ab-Spectrin Sigma 220, 240, 60, several low Plasma membrane Slaninova´ et al. (2003) cerevisiae S1390 molecular weight bands Vacuolar membrane Cytoplasmic Schizosaccharomyces Anti ab-Spectrin Sigma 220, 240, 60, several low Plasma membrane Slaninova´ et al. (2003) japonicus S1390 molecular weight bands Cytoplasmic Septum Saprolegnia ferax Anti ab-Spectrin Sigma 246, several low molecular Plasma membrane Kaminskyj and Heath S1390 weight bands (1995) Anti-Spectrin (ICN, St. Laurent) Phytophtora infestans Anti ab-Spectrin Sigma 240, 220, 100, 70, 50, 30 Plasma membrane Toquin et al. (2006) S1390 Tomato plants Anti b-Spectrin 240, 220 Plasma membrane Michaud et al. (1991) Vivia sativa Anti ab-Spectrin Sigma Root hair tips De Ruijter et al. S1390 (1998) Anti-Spectrin Sigma S1515 Pisum sativum Anti ab-Spectrin (Lorenz Native: 800, 280, 170, 110, Bisikirska and et al., 1995) 70 Sikorski (1997) Onion cells Anti ab-Spectrin S3396 Native: 210/230 zone, 580, Associated to plasma membrane. Reuzeau et al. (1997) Anti ab-Spectrin S1515380 and smaller bands 100, Endoplasmic reticulum and 130, 170 endomembrane system Green algae, Anti ab-Spectrin Sigma 220, 120, 70 Plasma membrane Holzinger et al. (1999) Desmidiaceae S1390 Anti-Spectrin Sigma S1515 Endoplasmatic reticulum Anti-Spectrin Sigma S3396 Vesicles Chara globularis Anti ab-Spectrin Sigma 195, 170 Endoplasmatic reticulum Braun (2001) S1390 (ER) agregates Tip of rhizoids Anti-Spectrin Sigma S1515 195, 170, 110 conserved sequence of CH-homology domain did not show 2. Materials and methods any gene coding for spectrin. A protein with features of the spectrin superfamily has been proposed to exist in the 2.1. Strains and culture conditions Ascomycete Neurospora crassa (NCU06429.4) and the Oomycete Phytophtora infestans (PITG_13237.1), since an Wild-type strain N. crassa (FGSC 262, strain St. Law- immunoreacting peptide was revealed with anti-spectrin rence STA4) was used in this study. Production of conidial antibodies (Degouse´e et al., 2000; Cotado-Sampayo inocula and culture conditions were as described previously et al., 2006; Toquin et al., 2006). This protein, however, (Ortega Perez et al., 1994). M. grisea was kindly given by is smaller in molecular weight than the fungal spectrins Dr. M.-H. Lebrun (Unite´ Mixte de Recherche, Centre reported earlier (Kaminskyj and Heath, 1995; Degouse´e National de la Recherche Scientifique/BayerCropScience, et al., 2000). In more recent studies this protein has been Lyon). The fungus was inoculated on solid rice medium considered to be an a-actinin related protein (Cotado-Sam- and allowed to grow in the dark at 27 °C for a few days payo et al., 2006; Virel and Backman, 2004 and in MIPS until white mycelia appeared. In sterile conditions, small and Broad Institute data bases). pieces were cut and inoculated in 200 ml of Tanaka mini- In this report, using the filamentous fungi N. crassa and mal medium (Ou, 1985) with 0.2% yeast extract and 1% Magnaporthe grisea and the Oomycete, P. infestans,we sucrose. The culture was grown for 48 h with agitation demonstrate that the polyclonal anti-chicken ab-spectrin (150 rpm) in the dark. Mycelia were collected by filtration antibody does not react with a-actinin (NCU06429.4), and weighed. the spectrin superfamily protein present in these organisms. The P. infestans strain was kindly provided by Dr. R. Further, we clarify the identity of the protein that reacts Beffa (Unite´ Mixte de Recherche, Centre National de la with anti- chicken spectrin antibody and demonstrate that Recherche Scientifique/BayerCropScience, Lyon) and the reacting protein is EF2 in Neurospora and Hsp70 in grown on pea-agar medium. Sporangial inoculum was pre- Phytophthora. pared from a 8–12 days culture, detached from the mycelia 1010 M. Cotado-Sampayo et al. / Fungal Genetics and Biology 45 (2008) 1008–1015 by flooding the culture with water and separated from the the supplier’s protocol and treated with DNAse (Ambion, mycelial fragments by filtration. Sporangia were inoculated Cat. 1906). cDNA was synthesized using Promega reverse in V8 liquid medium (50 ml tomato juice/liter of distilled transcription kit (A3802). Amplifying primers for the cod- water, pH adjusted to 5 if necessary) at a final concentra- ing sequence of the elongation factor 2 were: forward 50- tion of 105 sporangia/ml. Mycelia were harvested after GCGCGCGGATCCATGGTCAACTTCACGATTGAC 72 h of growth in the dark at 20 °C without agitation. G-30 and reverse 50-ATAAGAATGCGGCCGCTTAGA GCTTGTCGTAGT AG-30. Amplification of a-actinin 2.2. Protein extraction and analysis was performed in two steps, taking advantage of an inter- nal AatII site. Primers used for the amplification were: for- To optimize the preservation of proteins against prote- ward, 50-CGCCGCGGATCCATGGAGATGCTGGGG olysis during the harvest of mycelia, grinding and suspen- GTGGAG-30 and reverse, 50-TCACGGACGTCCACTTC sion of the mycelial powder in the extraction buffers, GTCATAGCACG-30 and, forward 50-AAGTGGACGTC different protocols were followed. These included rinsing CGTGATTTCAGCGGCAG-30 and reverse 50-ATAAGA of mycelia with protease inhibitors before freezing in liquid ATGCGGCCGCCTAATGATACCCATTCGGCTTG-30. nitrogen as described by Kaminskyj and Heath (1995), The BamHI, NotI and AatII digestion sites are respectively quick freezing, use of different detergents and TCA–ace- underlined, in bold and in italics. Amplification program tone extraction (Granier, 1988); in all representing some was: denaturation at 94 °C for 3 min, 30 cycles at 94 °C 10 experimental conditions. Mycelia were always ground for 30 s, 55 °C for 50 s and 72 °C for 2 min, and a final in liquid nitrogen. The standard procedure finally adopted elongation step of 72 °C for 4 min. PCR fragments were was essentially as described earlier (Cotado-Sampayo et al., digested by BamHI and NotI, and cloned into pGEX4T- 2006). 1 (Amersham Biosciences). This recombinant plasmid Protein concentration was determined according to was used to transform competent Escherichia coli BL21 Bradford (1976). SDS–PAGE and 2-D analysis were done strain. To study the expression of the cloned gene, the according to Laemmli (1970) and O’Farrell (1975), transformed cells were grown in LB medium containing respectively. 100 lg/ml ampicillin and induced with 100 lM Isopropyl- b-D-thiogalactopyranoside (IPTG). Proteins from induced 2.3. MALDI-TOF MS analysis of anti-spectrin antibody and uninduced E. coli BL21 strain were extracted by soni- reacting proteins cation in extraction buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3, and 0.4 mg/ Protein identification was performed on several occa- ml of lysozyme), centrifuged and analyzed on SDS–PAGE sions in different laboratory services (collaboration with gel. Partial digestion was performed with thrombin (5 l/mg Lyon-Bayer Crop. Sciences, Lyon, France; Section of of the GST fusion protein) at 25 °C for 20 min in a final Pharmaceutical Sciences, University of Geneva and Alpha- volume of 20 ll. lyse A/S, Odense, Denmark). In brief, cross-reacting pro- tein bands were cut and subjected to matrix-assisted laser 2.5. Anti-N. crassa a-actinin antibody production desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting and iden- Purified GST-NCU06429.4 recombinant protein, veri- tified by matches across the peptide sequences in the fied by MALDI-TOF MS (Alphalyse A/S) was used to National Center for Biotechnology Information database immunize two rabbits according to Barnes et al. (1998). (Shevchenko et al., 1996). Corresponding entries were Sera from immunized rabbits were tested 20 days after searched in the Broad Institute Database (http:// immunization and pre-immune serum was used as control www.broad.mit.edu/annotation/fgi/). As control, known for background or cross-reactivity. EF2-GST fusion protein and GST were also sequenced to confirm the accuracy of the analysis. Alphalyse A/S 2.6. Immunoblotting analysis facility uses internal controls in each identification experiments. Proteins from unstained mono- and bi-dimensional gels The identity of the immunoreacting protein with anti- were electrophoretically transferred to nitrocellulose mem- spectrin antibody (S1390) from N. crassa was also con- branes (BA85, pore size 0.45 lm) using the transfer buffer firmed by EDMAN sequence analysis at Analytical described by Burnette (1981) and immunoblotted as Research and Services, University of Bern. described earlier (Ojha and Barja, 2003). The membrane was blocked overnight at 4 °C with 5% BSA in TBS-Tween, 2.4. Expression of N. crassa elongation factor 2 and a-actinin incubated for 2 h at room temperature with polyclonal anti-chicken ab-spectrin (Sigma S1390) or anti-N. crassa Elongation factor 2 and a-actinin in N. crassa are coded a-actinin antibody at 1:800 and 1:500 dilution in blocking by the genes ncu07700.4 and ncu06429.4, respectively. To buffer. Subsequently, the primary antibody was replaced prepare cDNA total RNA was isolated from hyphae after with anti-rabbit IgG antibodies coupled to horseradish per- 18 h growth using TRIZOL reagent (Sigma) according to oxidase (Sigma A8275) diluted at 1:2000 and reincubated M. Cotado-Sampayo et al. / Fungal Genetics and Biology 45 (2008) 1008–1015 1011 for 1 h. Following four washes in TBS, the membranes were treated with DAB-H2O2 to develop peroxidase activ- ity, as described earlier (Huber and Ojha, 1994). Although the anti-chicken ab-spectrin antibody (S1390), which has been extensively used by others and in our hands gave better resolution, was used in most experiments described here, we also tested monoclonal anti-spectrin antibody (Sigma S3396). The reactivity of this antibody was very poor (also reported by Slaninova´ et al., 2003) and therefore not continued.

2.7. Immunofluorescence microscopy

Mycelia from 10 h culture were treated for 2 h with sor- darin (Sigma S1442) at 50 lg/ml concentration; the control was a 12 h culture without treatment. Samples were fixed Fig. 1. Western blot analysis of crude extract proteins. (Lanes a and d) by the addition of paraformaldehyde to the culture med- Neurospora crassa, (lanes b and e) Magnaporthe grisea, (lanes c and f) ium to a final concentration of 3% (v/v). Phytophtora infestans, (lane g) over-expressed cloned GST-tagged a- Primary anti-chicken spectrin-antibody and secondary actinin in E. coli BL21. Proteins were immunoblotted with anti-chicken goat anti-rabbit fluorescein-isothiocyanate (FITC, green, ab-spectrin (A) anti-N. crassa a-actinin (B) antibodies. Sigma F9887) antibodies were used at dilutions of 1:50 and 1:100 in phosphate buffer (Ojha and Barja, 2003). (Fig. 2A) corresponding in the gel to three spots of 6.5, The fluorescent signal was analysed with a Leica TCS 6.6 and 6.8 (described earlier by Cotado-Sampayo et al., SP2 AOBS confocal microscope. Images were treated with 2006) and 64 kDa with a pI of 5.0 in P. infestans the program Adobe PhotoShop 7. (Fig. 2B). The different spots of 100 kDa protein, from N. crassa in 2-D gels, were analyzed by MALDI-TOF MS, first 3. Results as one sample, to discard the possibility that the results did not include a contaminant protein of 100 kDa, due to its rel- 3.1. Immunochemical detection of proteins reacting with atively ‘‘broad range” of pI (6.4–6.8), each spot were sepa- anti-chicken ab-spectrin and anti-N. crassa a-actinin rately analyzed and all showed to be EF2. MALDI-TOF antibodies MS analysis of the 64 kDa peptide from P. infestans resulted to be the heat shock protein 70 (Hsp70) (Fig. 2C). Neurospora crassa, M. grisea and P. infestans protein extracts, separated by SDS–PAGE, transferred to nitrocel- lulose membrane and immunoblotted with anti-chicken ab- 3.3. Neurospora crassa elongation factor 2(EF2) over- spectrin antibody showed a single band of about Mr expression and its reactivity to anti-chicken ab-spectrin 100 kDa both in N. crassa and M. grisea and a band of antibody 68 kDa in P. infestans (Fig. 1A, lanes a–c). The same pro- tein extracts blotted with anti-N. crassa a-actinin antibody The elongation factor 2 gene of N. crassa was cloned gave a reaction with a protein of 80 kDa in N. crassa, and expressed in E. coli BL21 as described in Section 2. 72 kDa in M. grisea and a band of 140 kDa in the extract Fig. 3A (lanes a–c) shows over-expression of the fusion of recombinant over-expressed GST-a-actinins which agree protein demonstrating the induction of a protein of with the relative mass deduced from the protein sequences 126 kDa corresponding to 26 kDa glutathione S-transfer- (Fig. 1B, lanes d, e and g). In P. infestans no immunoreact- ase (GST) and 100 kDa N. crassa elongation factor 2. ing band with the anti-N. crassa a-actinin antibody was Immunoblotting with anti-chicken spectrin antibody recog- obtained (Fig. 1B, lane f). nized the recombinant protein GST-EF2 (Fig. 3A lanes a0– The monoclonal anti-spectrin antibody did not reveal in d0 and 3B lane b), with the same intensity as the immuno- crude extract from N. crassa any protein of 240, 220 or reacting band from Neurospora protein extracts (Fig. 3A 100 kDa (results not shown). lanes d0–e0). However, the antibody recognized an addi- tional protein of about 63 kDa of the host cell (even better 3.2. Anti-chicken ab-spectrin cross-reacting proteins of N. than the 126 kDa polypeptide, Fig. 3A lanes a0–d0). crassa and P. infestans MALDI-TOF MS analysis revealed this protein to be the GroEL chaperone. Neurospora crassa and P. infestans protein extracts sepa- Neurospora crassa elongation factor 2 was cleaved from rated in 2D-gels and immunoblotted with anti-chicken ab- GST-fusion protein by partial digestion with the site-spe- spectrin antibody revealed a predominant polypeptide of cific protease, thrombin, and analyzed by Western blot 100 kDa with a pI between 6.5 and 6.8 in N. crassa with anti-chicken ab-spectrin antibody. As shown in 1012 M. Cotado-Sampayo et al. / Fungal Genetics and Biology 45 (2008) 1008–1015

Fig. 3. Western blot analysis using anti-chicken ab-spectrin antibodies. (A) Coomasie blue staining of total proteins in a 10% SDS–PAGE gel (lanes a–e), corresponding immunoblot (lanes a0–e0). Lanes a, b and c corresponds to different amount of partially purified GST EF2 protein (0.2 lg, 0.5 lg and 1 lg, respectively). Lane d corresponds to a mixture of crude extract of Neurospora crassa (10 lg) and 0.2 lg partially purified GST EF2 protein and lane e corresponds to a crude extract from N. crassa (10 lg). (B) Immonoblot of crude extract from N. crassa (lane a), purified GST EF2 (lane b) and purified GST EF2 partially digested with thrombin (digestion was performed to remove the GST component from the recombinant protein).

a cap (Fig. 4B). Incubation of the hyphae with sordarin (an antifungal compound that interacts specifically with the elongation factor 2 blocking its binding to the ribosome; Domı´nguez et al., 1999) caused a clear dislocation of pro- tein from the peripheral region and hyphal tips towards the cytoplasm (Fig. 4C and D).

4. Discussion

Fig. 2. Sequence analysis of anti-chicken ab-spectrin immunoreacting The presence of a spectrin related protein in fungi using peptides. Two-dimensionalgel analyses of Neurospora crassa (A) and Phytophtora infestans (B) protein extracts with anti-chicken ab-spectrin polyclonal anti-chicken ab-spectrin antibody has been antibodies. Arrows indicate the analyzed peptides. Table with results of reported by many authors (Degouse´e et al., 2000; Heath sequence analysis (C), each peptide is designated with the organism (Nc, et al., 2003; Cotado-Sampayo et al., 2006). However, in Neurospora crassa; Phi, Phytophthora infestans), and their pI, ‘‘Broad spite of the efforts to identify a spectrin sequence in the number” refers to Broad Institute Accession number of the protein. sequenced genomes of N. crassa, M. grisea or P. infestans no gene coding for the Mr 240–220 spectrin, as described Fig. 3B lane c the protein band of 100 kDa corresponding for higher eukaryotic cells, was found. An a-actinin related to EF2 was recognized by the antibody and had the same protein seems to be the only member of the spectrin super- molecular weight as the immunoreaction peptide from N. family protein present in fungi and Oomycetes, but it is not crassa protein extracts (Fig. 3B lane a). recognized by the anti-spectrin antibody. In N. crassa and M. grisea a protein with relative mass of 100 kDa shows strong reactivity to anti-chicken 3.4. Indirect immunofluorescence ab-spectrin antibody. The MALDI-TOF MS analysis of the Neurospora peptide reacting with the antibody corre- The localization of EF2 in the hyphae of N. crassa was sponds to elongation factor 2, a protein with apparently examined by indirect immunofluorescence microscopy with no similarity in function, structure or sequence with any anti-chicken spectrin antibody as primary antibody and protein belonging to the spectrin superfamily of pro- goat anti-rabbit conjugated to FITC as secondary anti- teins. These results were reproducible. Immunoblotting body. The antibody revealed continuous fluorescence in of cloned, expressed and purified elongation factor 2 the peripheral region (Fig. 4A) in the sub-apical region confirmed the identity of the immunoreacting peptide. but concentrated in the entire region of the hyphal tips as In P. infestans, however, the results of mass spectra M. Cotado-Sampayo et al. / Fungal Genetics and Biology 45 (2008) 1008–1015 1013

Considering that elongation factors, spectrin, heat shock proteins and GroEL all have a chaperone activity, we sus- pect that the anti-chicken ab-spectrin reacts with an epi- tope in a domain implicated in this activity, maybe located in a hydrophobic region. The localization of the immunoreacting protein with anti-spectrin antibody, identified now as EF2, was found along the peripheral part of the sub-apical region and as a cap in the tips during exponential phase of growth in Neurospora (Cotado-Sampayo et al., 2006). This pattern was also found in many fungi and other organisms (Table 1). The elongation factors have been described to be asso- ciated with the plasma membrane in bacteria. The LepA protein, proposed recently as a new elongation factor, EF4, (Qin et al., 2006), is localized in the plasma membrane of E. coli (March and Inouye, 1985). The association of DnaK from E. coli (Hsp70) to the plasma membrane has also been described (El Yaagoubi et al., 1994). About 47 kDa fragment of Hsp70 in Candida albicans (Mathews et al., 1998) and the 80 kDa homologue of Hsp70 in Histoplasma capsulatum (Gomez et al., 1992) have been shown to be localized in the cell wall. The pres- Fig. 4. Effect of sordarin treatment in the localization of the protein ence of the heat shock proteins in the cell wall of fungi has revealed with anti-chicken ab-spectrin antibodies in N. crassa. Non- treated (A and B) and treated (C and D) cells. Bar = 10 lm. been also described (Lo´pez-Ribot and Chaffin, 1996; Lo´pez-Ribot et al., 1996). The commercial antibody that we have employed is analyses of the immunoreacting band matched the heat polyclonal and produced using as antigen sequences from shock protein 70 (Hsp70). The relationship between two proteins, the a- and b-spectrin, that are similar in spectrins, the Neurospora EF2 and Hsp70 is difficult structure but not in sequence (30% identity). It has been to interpret. shown that polyclonal antibodies can cross-react with sev- It has been reported that GroEl from E. coli, a chapero- eral proteins sharing simple peptide sequences (Michaud nin belonging to the Hsp60 class of proteins, reacts with et al., 2003). Therefore, the results obtained with this poly- anti-b-spectrin antibodies (Czogalla et al., 2003). The com- clonal antibody must be interpreted with caution. mercial anti-chicken ab-spectin antibody used in the pres- ent study and the work of others reporting the presence Acknowledgments of spectrin in plants and fungi, clearly shows a strong reac- tivity to GroEl of E. coli (Czogalla et al., 2003). The evi- We gratefully acknowledge financial support from the dence that this commercial antibody reacts with GroEl Academic Society of Geneva (F.B.). Thanks are due to and Hsp70 and the fact that both proteins have a chaper- R. Strasser for interest in the project and encouragement, one function (Ellis, 1987; Craig, 2003) suggest that some M.-L. Chappuis for technical assistance, A. Fehr for secre- anti-spectrin antibodies could react with an eptitope from tarial assistance and B. Peck for reading this manuscript. chaperones. Indeed, EF-G, EF-Tu (the prokaryotic coun- terpart of EF2 and EF1a, respectively) and initiation factor References 2 (IF2), have a chaperone-like activity (Kudlicki et al., 1997; Caldas et al., 1998, 2000; Hotokezaka et al., 2002; Barnes, D.A., Tse, J., Kaufhold, M., Owen, M., Hesselgesser, J., Strieter, Malki et al., 2002). R., Horuk, R., Perez, H.D., 1998. Polyclonal antibody directed against Spectrin has also been reported to have a chaperone-like human RANTES ameliorates disease in the Lewis rat adjuvant- activity (Chakrabarti et al., 2001; Bhattacharyya et al., induced arthritis model. J. Clin. Invest. 101, 2910–2919. 2004). It has the hydrophobic binding sites for lipophilic Bennett, V., Gilligan, D.M., 1993. The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane. Ann. Rev. molecules through which it can interact with the denatured Cell Biol. 9, 27–66. proteins. These regions in a-andb-spectrins have been Bennett, V., Baines, A.J., 2001. Spectrin and ankyrin-based pathways: found to have sequence homology with known chaperone metazoans inventions for integrating cells into tissues. Physiol. Rev. 81 proteins (about >50% similarity and 30% identity). This (3), 1353–1392. indicates that there could be a putative chaperone-like Bhattacharyya, M., Ray, S., Bhattacharya, S., Chakrabarti, A., 2004. Chaperone activity and prodan binding at the self-associating domain domain in spectrin, located near the N-terminus of a-spec- of erythroid spectrin. J. Biol. Chem. 279 (53), 55080–55088. trin and the C-terminus of b-spectrin (Bhattacharyya et al., Bisikirska, B., Sikorski, A.F., 1997. Some properties of spectrin-like 2004). proteins from Pisum sativum. Z. Naturforsch. 52, 180–186. 1014 M. Cotado-Sampayo et al. / Fungal Genetics and Biology 45 (2008) 1008–1015

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Features of α-Actinin in Fungi and Oomycetes

Marta Cotado-Sampayo

Laboratory of Bioenergetics and Microbiology, University of Geneva, 10, chemin des

Embrouchis, CH-1254 Jussy-Geneva, Switzerland

Tel. +41 22 379 37 50, FAX: +41 22 379 37 50,

E-mail: [email protected]

Spectrin superfamily proteins are characterized by their domain structure: N-terminal calponin homology domains (CH) and C-terminal EF-hand motifs both linked together by a rod domain. Corresponding to these features, α-actinin, spectrin, utrophin and dystrophins are considered as closely related members (Blanchard et al. 1989; Pascual et al. 1997). These proteins are well known in metazoans where they frequently interact with actin and plasma membrane, and considered to play a role in cell-cell interactions and intercellular signaling (Otey and Carpen 2004). α-Actinin is a smaller member of the superfamily and its functional unity is a rod shaped homodimer with monomers assembling in opposite polarity to form the dimer

(Wallraff et al. 1986; Amos and Amos 1991).

In a recent study on the evolution of α-actinins, Virel and Backman (2004) gave a direct evidence for the presence of its orthologs in two fungi, Neurospora crassa and

Schizosaccharomyces pombe. Whilst our work was in progress on the orthologs and evolution of the α-actinin in fungi using CH-domain structure a paper by the same authors has appeared documenting actinin orthologs in fungi using the criterium of rod domain (Virel and Backman

2007). We have extended these observations by including a representative from Chytridiomycota

(Table 1) and a detailed analysis of α-actinin in Saccharomycotina. In this sub-class (Table 1 and 2 fig. 1), which includes very diverse organisms (Dujon 2006), we have found only one species,

Yarrowia lipolitica containing a gene coding for α-actinin. There is no gene coding for a bona fide α−actinin in Saccharomyces cerevisiae (Wu et al. 2001; Virel and Backman 2004) or in more recently sequenced genomes of Kluyveromyces lactis, Candida glabrata and Ashbya gossypii. In other yeasts, we have found hypothetical proteins with one conserved N-terminal α- actinin-like actin binding domain (CH-domain) in C. albicans and two in Pichia stipitis,

Debaryomyces hansenii and Candida sp (Candida guilleimondi and Candida tropicalis) but no spectrin repeats or C-terminal EF-hand motifs (Table 1). The estimated molecular weight (Mw) of these proteins is between 70-80 kDa, similar to the estimated Mw of the fungal α-actinins.

However, this can not be taken for sure as a criterion for actinin related protein. Obviously, there are other proteins having CH-domains besides spectrin superfamily members, like Calmin,

Enaptin and Nuance, described as having two N-terminal CH-domains and C-terminal trans- membrane domain(s) (Gimona et al. 2002 and references cited therein), but these proteins are generally of higher molecular weight than expected for fungal α-actinin. Other proteins with two

N-terminal CH-domains, not mentioned in the revision of Gimona et al. (2002), are two cortexillins, I and II, present in the cellular slime mold Dictyostelium discoideum (Faix et al.

1996). Using blast tools of NCBI, we have also found these proteins in another cellular slime mold Polysphondylium pallidum and in the ameba Entamoeba histolytica and, these are low molecular weight proteins of about 40 kDa and considered to be implicated in cytokinesis (Faix et al. 1996) and mechanical properties of the cell cortex (Simson et al. 1998). We have not found orthologs of any of these proteins, either high or low molecular weight, containing N-terminal

CH-domain, in the fungal species examined. 3

The “atypical” α-actinins with only conserved N-terminal CH-domain(s) can be explained as a process in evolution where genes are highly diverged or lost (or have modified beyond recognition). Gene losses during evolution in several lineages is not a rare event and has been shown to occur for many genes (Aravind et al. 2000; Roelofs and Van Hasteert 2001; Krylov et al. 2003).

Even though we are not certain of the relationship between these proteins in the

Saccharomycotina and other fungal α-actinins, we can hypothesize that they have evolved from a common ancestor through two different pathways. In one, the α-actinin seems to have been lost like in S. cerevisiae, or severely modified during evolution as in Candida albicans and the yeasts mentioned above but preserved in other, for example Y. lipolytica (fig. 1).

This hypothesis is supported by the fact that, even though we can not recognize any functional domain in the middle and C-terminal region of these hypothetical proteins, the gene is still present with a relative large ORF. Anyway, the absence of a “classical” fungal α-actinin in nearly all of Saccharomycotina might be due to a complementation in function with other proteins, maybe fimbrin as in S. pombe (Wu et al. 2001) or other actin binding proteins (Rivero et al. 1999). Fimbrin is interesting from this point of view since it has two conserved domains of α- actinin but with reverse polarity, i.e., N-terminal Ca2+ binding EF-hand motifs and C-terminal actin binding CH-domains.

We have also analyzed the α-actinin gene of other fungi and an Oomycete from the available genome database. As shown in Table 1 and figure 2, the sequence of α-actinin is organized into the three distinct domains: two N-terminal calponin homology domains (blue boxes), a rod domain (yellow boxes) and the C-terminal Ca2+-binding domain. The CH-domain is the best conserved region in the protein and contains three actin-binding sites (red boxes) able to bind one 4 actin monomer (Keep et al. 1999; Gimona et al. 2002). In most fungi rod domain, composed of two spectrin repeats, is the least conserved region. In Oomycetes we can expect the presence of four, given the size of the rod domain but only the first is relatively conserved and the other three are difficult to define. The spectrin repeats in fungi and Oomycetes loose some features such as tryptophan in position 17. However, spectrin repeats are defined more by their length and their

3D-structure than the sequence. In fungi they are of 101-115 amino acids in length and the structural prediction indicates that each domain contains three segments of α helix in the form of a three stranded coiled-coil.

The C-terminal calcium binding domain is composed of three EF-hand motifs (green boxes).

The EF-hand motif consists of a twelve residue loop flanked on both sides by a twelve residue α- helical domain. The bioinformatic tools predicted only one putative calcium binding EF-hand domain. So far, occasionally a second putative EF-hand calcium insensitive domain has been detected with a very low score but is considered as a match primarily because EF-hand domain is known to be repeated and not likely to occur as a single copy in a protein. Using the pattern of

EF-hand motifs, PS00018 (http://us.expasy.org), a putative third C-terminal EF-hand domain was found, manually, about 70 nucleotides downstream from the first one. This domain, present only in Ascomycota, is probably calcium insensitive, given that position three, five and seven in the loop do not exactly correspond to the pattern of the canonical EF-hand domain.

Among the fungi examined here what is remarkable about Ascomycetes (N. crassa, M. grisea, S. sclerotia and F. graminearum) is the presence of an N-terminal extension upstream of the first CH-domain that has a low sequence similarity. It is variable in length and does not have any conserved domain motif (InterPro). This N-terminal region has not been considered in the annotation for Neurospora α-actinin in MIPS database, nevertheless we have demonstrated that it 5 is present in the mRNA of N. crassa (Cotado-Sampayo et al. 2006 and article in preparation).

The presence of N-terminal extension in some fungi could be attributed to a: UTR-region in the mRNA, a pro-domain of the protein or an alternate open reading frames (ORF). As an UTR- region, it will be too long (with 930 nucleotides length) and if one considers it to be translated as a pro-domain and cleaved afterwards, we do not know any putative enzyme cutting just upstream of the first calponin homology domain. In case of an alternative use of ATG-start, one can conclude that the full-length protein will be translated in special conditions since we have found only the short form of the protein in the crude extract from standard cultures of N. crassa (80 kDa) or M. grisea (72 kDa) (Cotado-Sampayo et al. 2008).

Our detailed analyses of the features of fungal α-actinin highlight the target for future experimental studies i.e. their biochemical and functional characterization in order to understand why they are absent or highly modified in most Hemiascomycetous yeast.

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LEGENDS

Table 1.

Accession Number, Domain Structure and Taxonomic position of organisms used in this study.

The Accession number for: Batrachochytrium dendrobatidis, Rhizopus oryzae, Neurospora 8 crassa, Magnaporthe grisea, Fusarium graminearum, Sclerotinia sclerotiorum, Botrytis cinerea,

Aspergillus nidulans, Candida sp., Cryptococcus neoformans, Coprinus cinereus, Chaetomium globosum and Phytophthora sp., sequences were obtained from the Broad Institute database

(http://www.broad.mit.edu/annotation/fgi/). Sequences for Yarrowia lipolitica and

Debaryomyces hansenii were obtained from the yeast specific database Genolevures

(http://cbi.labri.fr/Genolevures/). The sequences for Entamaeba cuniculi, Uromyces maydis,

Aspergillus fumigatus, Aspergillus oryzae, Schizosaccharomyces pombe and Pichia stipitis were derived from Swiss-prot protein database (http://ca.expasy.org/sprot/). No putative α-actinin gene was found in completed genomes of Saccharomyces cerevisiae, Candida glabatra,

Kluyveromyces lactis and Ashbya gossypii.

Structure domain is represented with rectangles for the CH-domain, ovals for spectrin repeats and squares for EF-hands.

FIG. 1.- The α-actinin protein evolution in hemiascomycetous yeast. The arrows indicate the major events in evolution.

FIG. 2.- Alignment of amino acid sequence of α-actinins from Ascomycetes. Domains are indicated in color boxes, CH-domain (blue), spectrin repeat (yellow) and EF-hand motif (green).

Acting Binding Sites (ABS) are indicated with black underlined red boxes.

Table 1

Taxon Organism Accesion no Domain structure

MICROSPORIDIA Entamoeba cuniculi Q8STW7 CH CH SR EF

Batrachochytrium dendrobatidis BDEG_05746 CHYTRIDIOMYCOTA CH CH SR SR EF

Rhizopus. oryzae RO3G_05027.1 ZYGOMYCOTA CH CH SR SR Phycomyces blakesleeanus Phybl1_37239 EF

Saccharomyces cerevisiae --

Candida glabatra --

Kluyveromyces lactis --

Ashbya gossypii --

Candida albicans CAWG_04250.1 CH HEMIASCOMYCOTA Candida tropicalis CTRG_01638.3

Candida guilleimondi PGUG_05348.1

CH CH Debaryomyces hanseni DEHA0C07491g

Pichia stipitis A3GG62

Yarrowia lipolitica YALI0F07601g ARCHIASCOMYCOTA Schizosaccharomyces pombe O13729 Chaetomium globosum CHGG_05684.1

Neurospora crassa NCU06429.4

Magnaporthe grisea MG06475.4

Fusarium graminarum FG07284.1

Sclerotinia sclerotiorum SS1G_00266.1

Botrytis. cinerea BC1G00894.1 CH CH SR SR EF EF EF

Aspergillus nidulans AN7707.2

Aspergillus fumigatus Q4WUF8

Aspergillus. oryzae Q2U7P8

Uromyces maydis Q4P6F1 CH CH SR SR EF BASYDIOMYCOTA Cryptococcus neoformans CNAG_02992.1

Coprinus cinereus CC1G_03395.1

Phytophthora sojae PSOJAE 137006

OOMYCOTA Phytophthora ramorum PRAMORUM 85609 CH CH SR EF EF

Phytophthora infestans PITG13237 CH: Calponin homology domain; SR: Spectrin repeat; EF: EF-hand motif.

Fig. 1

S. cerevisiae

C. glabatra Loss of α-actinin gene Loss of rod and K. lactis calcium binding domains in α-actinin A. gossypii

D. hansenii P. stipitis C. tropicalis C. guilleimondi C. albicans Y. lipolytica

Fig. 2

10 20 30 40 50 60 70 80 90 100 110 120 ----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:- Afumigatus/1-645 ------Aoryzae/1-645 ------Bcienrea/1-660 ------Ssclerotiorum/1-931 MFSALKKSLRIEPKVEAPKDVTSNTVAKEENVENVGIEDTIVVKDLDATRKEAEKEKVEDVREEDVL-VIKDLDAKWEERPDMRAEEEARDMEKGEDVK-GEEDTLVVKDLDVRVESEGVKEEDVE Cglobosum/1-643 ------Fgraminearum/1-889 ------MDAVRNSVQAFQGML------PL------QPGNLDGN-EKFPDFCLHSEALCSNDSHEDLEREQEQQHDKG------LHNKQSKQ Mgrisea/1-788 ------MDHQY------PH------DAGSVPSNREDWPLF------TNENSYHPG------GHTDNSLN Ncrassa/1-1027 -----MEMLGVEIGEPGPSVQSGQPAA------ALPDPATAPARAAAQSPTQSTNSTTST-RSGPASLARSRSSTAGASTTCSSRSWGETHSGGPAQVEKFPDLPTHTTSPEHQQQPLH Ylipolytica/1-616 ------Spombe/1-621 ------

130 140 150 160 170 180 190 200 210 220 230 240 250 ---|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|-- Afumigatus/1-645 ------Aoryzae/1-645 ------Bcienrea/1-660 ------Ssclerotiorum/1-931 MEVEYVN-EEVGEEEEEEEEGKREREPTITSNSTNYIGIPSPPSSEIGDESDDVI------GDEESEGEQINSTPGNDRCLVGSSSVTAANPDSEVNKNEETSISTTPRHSRAISPTPIENR Cglobosum/1-643 ------Fgraminearum/1-889 RHPHFHYPRPHSPSASISESSRL--SCISTA------SVSASSSTSSRVSSASFD--SFSTPI------SPTLDSSLHSPRSSLASNT--CSSPVSEFIAR-- Mgrisea/1-788 RPP------SL--SLSSTT------SSYD------FSDKDSVFDSQRAS------FIST-- Ncrassa/1-1027 HHPHPHH-RHHHHHQPASEAVRRDGTCAHTAE-----GTPTPPAAPAAPATPTPVPRLNTDHTELASPGPPPAASCPAIGRPPSLSIHSTASSSDHSSSLSDGDLVFDSRR--SS-----VVTS-- Ylipolytica/1-616 ------Spombe/1-621 ------

260 270 280 290 300 310 320 330 340 350 360 370 --:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:--- Afumigatus/1-645 ------MLTVEKSWVN Aoryzae/1-645 ------MLTVEKSWVN Bcienrea/1-660 ------MAFAEQQQWIT Ssclerotiorum/1-931 SHTQNQEKRISSSTADFEINAPSTPPATPPRLVRRERSASQREREILRSRQFSE---TARLQRSAEKPEKTDENLGLGKRRKRYQHRHKSK-GSNAGSGEALKAVEPLHEHQLGKMAFAEQQQWIT Cglobosum/1-643 ------MAFAEQQRWIT Fgraminearum/1-889 ------NRSLTGASLSS------VS------SSPPSSPTPRNRWDKPQPYAGRAQRSGSTTTNVSSPVGVHKQEQSSSPR--SF-RSNPIPADALAAVES-HDTKLIKMAFAEQQRWVT Mgrisea/1-788 ------SSSIDSCSIAC------GSPLPRSRSNAKQLALARLQRSGSNPVGLSSKKADSPSR-----K--RF-YSSGSAQEALAATEP-AGGGLTKM----EEDWVS Ncrassa/1-1027 ------ASTASAYNYPLS--PSCHQP------DSSPEHPLTRTRRDAKQLHAARLERSGSISLALASTKREAPSPALRNPT--RFQRASSEPTRAPKLVTE-QPAGFTKMAFAEQQQWIT Ylipolytica/1-616 ------MSLPQWIA Spombe/1-621 ------MQANQWQS

380 390 400 410 420 430 440 450 460 470 480 490 500 -|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|---- Afumigatus/1-645 VQQKTFTKWLNDKIKVRGI-LIDDLVTDLSDGVILIHLLEILGGESLGRYASKPKLRVQKFENVNKSLDFIRGRRIQMTNIGAEDIVDGNRKIILGLIWTLILRFTISDINAEGMTAKEGLLLWCQ Aoryzae/1-645 VQQKTFTKWLNDKLKVRRL-FIEDLVSDLSNGIILIHLLEILGGEPLGKYASNPRLRVQKFENVNKSLDTIKGRGIQMTNIGAEDVVDGNRKIILGLIWTLILRFTISDINEEGMTAKEGLLLWCQ Bcienrea/1-660 VQQKTFTKWLNTKIAHRKL-EVIDLVKDLSDGVILIHLLECLSNESLGRYAAKPKLRVQRFENANLSLDFIKSRGIQMTNIGAEDVVDGNRKIILGLIWTLILRFTISDINLEGMTAKEGLLLWCQ Ssclerotiorum/1-931 VQQKTFTKWLNTKIAHRKL-EVVDLVKDLSDGVILIHLLECLSNESLGRYAAKPKLRVQRFENANLSLDFIKSRGIQMTNIGAEDVVDGNRKIILGLIWTLILRFTISDINLEGMTAKEGLLLWCQ Cglobosum/1-643 VQQKTFTKWLNTKVEARGL-EVKDLVQDLSDGVMLIHLLECLSSESLGRYAAKPKLRVQRFENANRALDFIKSRGIQMTNIGAEDVVDGNRKIILGLIWTLILRFTINDINEEGMTAKEGLLLWCQ Fgraminearum/1-889 VQQKTFTKWLNTKIEARNL-EVKDLVKDLSDGVMLIHLLECLSHESLGRYASKPKLRVQKFENANTALDFVKSRGIQMTNIGAEDVVDGNQKIVLGLIWTLILRFTISDINEEGMSAKEGLLLWCQ Mgrisea/1-788 TQQKTFQKWANSKLAERSL-ETKNLVEDLKDGVLLIHLLECLASESLGRFASKPKLPVQQYENANTALGFIQSRGIRLTNCGAEDIVKGNRKIVLGLIWTLILRFTISDINEEGLTAKEGLLLWCQ Ncrassa/1-1027 VQQKTFTKWLNTKIEVRGL-EVKDLVKDLSDAVMLIHLLECLSGDSLGRYAAKPKLRVQRFENANLALNFIKSRGIQMTNIGAEDIVDGNRKIILGLIWTLILRFTINDINEEGMTAKEGLLLWCQ Ylipolytica/1-616 TQHKAFLRWANTYLEANQIGTMVSLETDFCDGVRLCQLIEIIGKESLGRYSGQPRMRFQMIENVNTALAFIRHRGVQLHNIGAEDICDGNLKLILGLLWILILRFTIEDISEEGLSAKEGLLLWCQ Spombe/1-621 VQNRTFTKWFNTKLSSRDLPSVFDLRKDLSDGILLIQLLEIIGDENLGRYNRNPRMRVHRLENVNKALEYIKSKGMPLTNIGPADIVDGNLKLILGLIWTLILRFTIADINEEGLTAKEGLLLWCQ

510 520 530 540 550 560 570 580 590 600 610 620 630

:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----| Afumigatus/1-645 RKTACYE-GVEVRDFSTSWNDGLAFCALLDIHRPDLIDFDALDKKDHRGNMKLAFDIATNEIGIPDLLDVDDVCDVAKPDERSLMTYIAYWFHAFSQLERVENAGRRVEKFINNMHGAWEMQNSYE Aoryzae/1-645 RKTACYE-EVEVRDFSTSWNDGLAFCALLDIHRPDLIDFDALDKKDHRGNMKLAFEIAANEIGIPDLLDVDDVCDVPRPDERSLMTYIAYWFHAFSQLERVENAGRRVEKFINNMHGAWEMQNSYE Bcienrea/1-660 RKTACYE-EVDVRNFTDSWNDGLAFCALLDIHRPDLIDYDTLDKDDHRGNMQLAFDIATKEIGIPALLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFVNNMQGAWEMQSAYE Ssclerotiorum/1-931 RKTACYE-EVDVRNFTDSWNDGLAFCALLDIHRPDLIDYDTLDKDDHRGNMQLAFDIATKEIGIPALLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFVNNMQGAWEMQSAYE Cglobosum/1-643 RKTACYD-EVDVRDFSASWNDGLAFCALLDIHRPDLIDYDALDKSDHRGNMQMAFDIAHKEIGIPKLLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFVNNMRGAWDMQSAYE Fgraminearum/1-889 RKTACYE-EVEVRDFSGSWNDGLAFCALLDIHRPDLIDYDALDKADHRGNMQLAFDIAHKEIGIPKLLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFVNNMQGAWEMQSAYE Mgrisea/1-788 RKTACYE-ECDVRDFSASWNDGLAFCALLDIHRPDLIDYDALDKTDHKGNMQMAFDIAHKEIGIPKLLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFVNNMQGAWEMQSAYE Ncrassa/1-1027 RKTACYD-EVDVRDFSGSWNDGLAFCALLDIHRPDLIDYDALDKSDHRGNMQLAFDIAHAEIGIPKLLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFFNNMQGAWEMQSAYE Ylipolytica/1-616 RKTAGYK-GVAVKDFSGSWSDGLAFCALLDKHRPDLIDFAQLDPTKPRENMELAISIATEQIGIPQILDVEDICGVAKPDERSVMTYVAYWFHAFSALDMIENAGRRLEKFVEMTSSAYAMQSGYE Spombe/1-621 RKTANYHPEVDVQDFTRSWTNGLAFCALIHQHRPDLLDYNKLDKKNHRANMQLAFDIAQKSIGIPRLIEVEDVCDVDRPDERSIMTYVAEYFHAFSTLDKVETAARRVERFTEVLMSTHDMKIDYE

640 650 660 670 680 690 700 710 720 730 740 750 ----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:- Afumigatus/1-645 RRMKELLRLIRAQREAWKNASFE--GTYKDAKEQARQFSLYKRNEKRRWVAEKSDLAALLGNIKTKLSTYRLRPYDPPEELRLEVCDNEWELLTRDEHERSQLINETIRDIKNALRRSFADKANDF Aoryzae/1-645 RRMKELLRLIRAQREEWKNASFE--GTYKDAKDQAFQFSLYKKKQKRQWVAEKSDLAALLGNIKTKLSTYRLRPYDPPAELSLEVCDQEWECLTRDEHERSQLINETIRDIKNALRRSFADKANDF Bcienrea/1-660 KRMRALLKSIKEQVITWQDATFE--GTYADAKKQATHFSSYKRGQKREWVAEKSDLAALLGNIKTKLSTYRLRAYDPPPELRLSVLDEEWAKLMKGEMARGQLINETIRDIKNALRRSFADKANDF Ssclerotiorum/1-931 KRMRALLKNIKEQVITWQDATFE--GTYVDAKKQASHFSSYKRGQKREWVAEKSDLAALLGNIKTKLSTYRLRAYDPPPELRLSVLDEEWALLMKGEMARGQLINETIRDIKNALRRSFADKANDF Cglobosum/1-643 RRMRELLKVIREQMESWQLAKFE--GTYTDAKAQAADFAAYKRGLKREWVAEKSELATLLGNIKTKLGTYRLRPYDPPAELRLEVLDQEWANLTKAEMARGQLINETIRDIKNALRKSFADKANDF Fgraminearum/1-889 RRMRALLKAIQERIEVWKEATFE--GTYTDAKAQLNQFFDYKRGKKREWVAEKSDLATLLGNIKTKLGTYRLRPYDPPAELSLDALEQRWAELASNEMRRAQLINETIRDIKNALRKSFADKANDF Mgrisea/1-788 RRMAALLQAIRAQVESWQQAKFE--GSYTDAKAQATDFASYKRGKKREWVAEKSELATLLGNIKTKLGTYRLRPYEPPAELRLDVLDGEWANLAAAEMKRGQLINETIRDIKNALRKSFADKANDF Ncrassa/1-1027 RRMAALLKAIREQVVSWKGSTFD--GTYADAKAQAFQFASYKKGKKREWVAEKSDLATLLGNIKTKLATYRLRPYDPPAHLRMEVLDDEWGNLSKAEMSRGQLINETIRDIKDALRKSFADKANDF Ylipolytica/1-616 ERMKALLKAIATQKEKWEQAADPEHLAYVEVKQQTAEHATFKIKTKREWTREKASLASLLGNIRTKLATYNLKEYSPPVGLRSADVEAAWKELHMGEINRSKLLNQSMRRLKESLRKRFADAANEF Spombe/1-621 SRMKRLLGSIARMQEYWHTVQFE--NNYTDVKSHSNNFAKFKATEKREWVKEKIDLESLLGTIQTNLKTYQLRKYEPPAGLKIVDLERQWKDFLSEEANQSKLINTHMREIKESMRIAFADRANSF

760 770 780 790 800 810 820 830 840 850 860 870 880 ---|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|-- Afumigatus/1-645 ALTLKTLSLAISGLDGDVEDQLEHVKRLNDNLPPLDAFLETIAELDEQCVEANIEENDFTTYTLDELSYELSLVKSSISKKLAFLENQLVARNMTNLTPIQLEEFESVFRHFDRDSSNTLHELEFS Aoryzae/1-645 ALTLKTLSLAISGLDGDVEDQLAHVKRLNDNLPPLDAFLDTIAEIDEQCEEANIEENDYTTYTLDELSYELSLVKSSISKKLAFLDNQLVARNMTNLTPIQLEEFESVFRHFDRDSSNTLHELEFS Bcienrea/1-660 ATTLNTMQLAISGLEGDVEDQLVHVRRLHDNLPPLNQFLDQIEAIDKKCEEANIEENDFTTYTYDELCYEMSLVKNSVAKKLAFLDNQVVARNMTNLTPIQLEEFESVFRHFDRDATNSLQELEFS Ssclerotiorum/1-931 ATTLNTMQLAISGLEGDVEDQLVHVRRLHDNLPPLNQFLDQIEAIDKKCEEANIEENDFTTYTYDELCYEMSLVKNSVAKKLAFLDNQVVARNMTNLTPIQLEEFESVFRHFDRDASNSLQELEFS Cglobosum/1-643 ALALNTIQLAISGLEGDVEDQLHHVRKLSENLPPLDAFLKTIAAVDAKCQEANIEENDFTTYTYDELCYELSLVKSSVSKKLAFLENQMVARNMTNLTPIQLEEFESVFRHFDRDDTNCLSELEFS Fgraminearum/1-889 AMALNTMQLALSGLDGDVEDQLHHVRKLSESLSPLDQYLDKISELDQKCQEANIEENDFTTYTYDELSYELGLVKTSVQKKLAFLENQMVARSMTNLTPIQLEEFESVFRHFDRDDTNSLQELEFS Mgrisea/1-788 ALALNTIQLAISGLEGDVEDQLHHVRKLSENLPPLDEYLKTIGAVDAKCQEANIEENDFTTYTYDELCYELGLVKSSVAKKLAFLDNQMVARNMTNLTPIQLEEFESVFRHFDRDDSNSLAELEFG Ncrassa/1-1027 ALALNTMQLAISGLEGDVEDQLHHVRKLSENLPPLDAYLKTIEAVDLKCQEANIEENDFTTYSYDELCYELSLVKSSVSKKLAFLENQMVARNMTNLTPIQLEEFESVFRHFDRDDTNSLSELEFS Ylipolytica/1-616 SDRLSVLSTAISQMDGPLEDQLEEIADISEKLRPLTEKIRLLKELDTSCVEANVEENDYTVYSYDELEYDLGLAKESVKKKLAFIENQIVARNMTNLTPIQLEEFESVFRHFDKSQHNALLESEFS Spombe/1-621 SKMLSTISNEITNLQGDWRDQLDHVEFLQEHLGPLEVELASVKVLYDNCFQAGIEENDYTMFSYEDLEHEFGITANIIANKIKYLENELLEREKRTLSKQELDGITKVFRHFEKKKSNMLNEVEFY

890 900 910 920 930 940 950 960 970 980 990 1000 --:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:--- Afumigatus/1-645 AALASLGLVYDEEEMHQVYVETCGPARLAQNAGVSFEQFIHFMVSVTEDQHTAEQVFQSFREVADGKPYVTELDLRHSLIPDEVIEHLVQTMPLHQGPDLLEDRDLPKYDYISFMEKMM Aoryzae/1-645 AALASLGLVYDEDEMHEVYVETCGPARLAQNAGVSFEQFIRFMVSVTEDQNTAEQVLQSFREVADGKPYVTELDLRHSLIPDEVIDHLVQTMPRHEVFDRGEDQNEPKYDYYSFMQKMM Bcienrea/1-660 AALASLGLVFSEDEMHDYFLDTSNGKDY-----VTFEEFIRFMVDVTEDQNTAEQVFQSFREVADGKPYVTEMDLRHSLVPDEVIEKLTQFIPIHKGPDLQEDRGMPQYDYISFMDKLL Ssclerotiorum/1-931 AALASLGLVFSEEEMHHYFLDTSNGKDY-----VTFEEFIRFMVDVTEDQNTAEQVFQSFREVADGKPYALDRWI------Cglobosum/1-643 AALASLGLIFSEDEMHDYFLDTSGGLDR-----VTFEQFIRFMVDVTEDQNTAEQVFQSFREVADGKPYVTEMDLRHSLVPDDVIEKLVEIMPGHSGPDMQSDRGQPQFDYIAFMDKMI Fgraminearum/1-889 AALASLGLVFSEDEMHDYFHATSGGRDY-----VTFEQFIRFMVDVTEDQNTAEQVYQSFREVADGKPYVTEMDLRHSLVPDEVIDQLVEIMPAHSGPDMSEDRGMPQYDYISFMEKLI Mgrisea/1-788 AALASLGVVFSEGEMHEYFVETAKGRDR-----ITFEQFIRFMVEVTEDQNTAEQVYQSFREVADGKPYVTEMDLRHSLVPDEVIDKLIEIIPAHNGPDTAQDRGMPQYDYIAFMDKFI Ncrassa/1-1027 AALASLGLVFSEDEMHEYFLSTSNGRDR-----VTFEQFIRFMVDVTEDQNTAEQVFQSFREVADGKPYVTEMDLRHSLVPDEVIEKLIEIIPKHTGPDMQSDRGMEQYDYIAFMEKLI Ylipolytica/1-616 GALASLGLVYSETEMHEVFQAASEGQVS-----VSFEQFITFMVEVTEDQLSAEQVLQSFAEVADGKMYVTELDLQNSLIPEPMIDQLKDTMP------KTADGFDYIAYMERLT Spombe/1-621 AALASLGLVYDTEEGTALFHRAANSEEG-----VTYERFTEIVMEELEDRDSARQVLYAFCDVADGKSYVTSDDLLRSQVRPNIVKFLECNMNKHS------EGLDYLTWIKQLL

1

Characterization of Neurospora crassa α-actinin

Marta Cotado-Sampayoa, Ruben Ortega Pereza, Mukti Ojhab, Carol Seumc,

François Barjaa*

aDepartment of Botany and Plant Biology, University of Geneva, ch. des Embrouchis

10, CH 1254 Jussy-Geneva, Switzerland bDepartment of Biochemistry, University of Geneva, Quai Ernest-Ansermet 30, CH

1211 Geneva 4, Switzerland cDepartment of Zoology and Animal Biology, University of Geneva, Quai Ernest-

Ansermet 30, CH 1211 Geneva 4, Switzerland

* Corresponding author. Fax: +41 22 379 3756.

E-Mail address: [email protected]

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Abstract

α-Actinin, an actin binding protein belonging to the spectrin superfamily is present in most eukaryotes except plants. It is composed of three domains: N- terminal CH-domains, a C-terminal calcium binding domain (with EF-hand motifs) and a central rod domain. α-Actinin from Neurospora crassa has been cloned and expressed as GST fusion protein for biochemical characterization and as GFP fusion protein for in vivo localization. In this report we show that the α-actinin from N. crassa has the ability to cross-link actin filaments in a calcium regulated manner just as some α-actinins from higher eukaryotes. The localization in situ and in vivo of α- actinin led us to propose an interaction of this protein with the actin cytoskeleton in the process of germination, hyphal elongation and septum formation. The deletion of the α-actinin gene in N. crassa has a lethal effect suggesting that its function is not complemented by other actin binding proteins as observed for other organisms such as Schizosaccharomyces pombe or Dictyostelium discoideum. These organisms can survive the knock-out of the α-actinin gene.

Keywords: α-actinin; Neurospora crassa; actin binding protein

1. Introduction

In Neurospora a wide variety of cell types is found during its cell cycle. For its vegetative and sexual phases 28 morphologically distinct cell types have been described (Britis et al., 2003). This complexity must be supported by a proper coordination between skeletal structures (cell wall and cytoskeleton) and signaling pathways. The actin cytoskeleton which participates in this coordination has a major

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role in the establishment of hyphal tips and the maintenance of polarized growth

(Barja et al., 1993; Heath, 1999; Heath et al., 2000; Virag and Griffiths, 2004). The dynamics of the actin cytoskeleton is regulated by a large number of actin-binding proteins (ABPs).

Taking advantage of the existence of complete genome sequences of several fungal genomes, many of these actin binding proteins were annotated by the Munich

Information Center for Protein Sequences (MIPS) database but their biochemical and functional characterization is not so well advanced. One of these proteins is α-actinin which has recently been presented in a study on its evolution, by Virel and Backman

(2004, 2007), and in our own work on the analysis of a putative spectrin-superfamily protein in fungi (Cotado-Sampayo et al., 2006, 2008).

α-Actinin belongs to the spectrin superfamily of which other members are spectrin, dystrophin and utrophin. All these proteins contain three functional and structural domains: two calponin homology (CH) domains at the N-terminal, a central rod domain containing spectrin-repeats and a C-terminal domain with EF- hand motifs. The functional unit of α-actinin is a homodimer of two anti-parallel monomers (Critchley and Flood, 1999; Djinovic-Carugo et al., 1999). As a consequence, α-actinin cross-links F-actin by binding with its each N-terminal end to an actin filament (Podlubnaya et al., 1975). The actin-binding ability is regulated by calcium but in skeletal muscle isoforms (α-actinin-1 and α-actinin-4) this regulation is calcium independent since their EF-hand motifs are non-functional (Blanchard et al., 1989).

α-Actinins has been described in most eukaryotic cells (Fryberg et al. 1990;

Barstead et al., 1991; Beggs et al., 1992; Mills et al., 2001; Virel and Backman,

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2006; Virel et al., 2007), in the protozoa Trichomonas vaginalis and Dictyostelium discoideum (Brier et al., 1983; Addis et al., 1998; Bricheux et al., 1998; Rivero et al.,

1999) and in the fission yeast, Schizosaccharomyces pombe (Wu et al., 2001).

Although its function is generally based on its ability to bind actin filaments, α- actinin is not merely a cross-linking protein. Recent studies have revealed an expanded number of α-actinin binding proteins, such as paladin, syndecan 4,

CaMKII, fesselin, and others that suggest variety of roles in the cell (Otey and

Carpen, 2004 and references cited therein; Pham and Chalovich, 2006; Bozulic et al.,

2007). Basically, α-actinin links the cytoskeleton with plasma membrane proteins, thus providing structural stability (Belkin and Koteliansky, 1987; Otey et al., 1990;

Knudsen et al., 1995; Rajfur et al., 2002; Dandapani et al., 2007). Some of these membrane proteins are receptors and α-actinin constitute a piece in the scaffold to integrate signaling pathways (Wyszynski et al., 1997; Dunah et al., 2000; Otey and

Carpen, 2004). In S. pombe α-actinin (Ainp1) seems to have a different function, playing a role in cytokinesis, but the interaction between this protein and the plasma membrane has not been established (Wu et al., 2001).

There is evidence for the presence in filamentous fungi of genes coding for α- actinins sharing high sequence similarity with Ainp1 of fission yeast (Virel and

Backman, 2004, 2007; Cotado-Sampayo et al, 2006, 2008). However, the biochemical and functional characteristics of these proteins are still unknown.

The N. crassa genome contains one open reading frame, potentially coding for an

α-actinin protein with an atypical N-terminal extension preceding the conserved CH- domain. In this report, we present a study on the α-actinin from N. crassa and

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provide insight in its domain structure, biochemical properties, localization and expression during development.

2. Materials and methods

2.1. Strains and culture conditions

Wild type N. crassa (FGSC 262, strain St. Lawrence STA4) and a heterokaryon

α-actinin knockout strain (FGSC11835, Colot et al., 2006) were obtained from the

Fungal Genetics Stock Center, School of Biological Sciences, Kansas City, MO. The procedure for the preparation of the conidial inoculum has been described earlier

(Cotado-Sampayo et al., 2006). Conidia were inoculated at a density of 5x106 conidia/ml in Vogel’s liquid medium (Vogel, 1956) containing 2% sucrose. The cultures were grown on a rotary shaker (150 rpm) at 30 °C for 6, 12 and 18 h.

2.2. Immunochemical characterization

Cells from different stages of development were harvested by filtration, washed twice with distilled water, frozen and ground in a mortar kept cool with liquid nitrogen. The frozen mycelial powder was suspended in cytoskeleton stabilizing buffer (Abe and Davies, 1995) with slight modifications as described earlier

(Cotado-Sampayo et al., 2006). The homogenate was incubated for 20 min at 4 °C while gently agitated and centrifuged at 6.000xg for 15 min at the same temperature.

Protein concentration in the supernatant was measured according to Bradford (1976) using bovine serum albumin (BSA) as standard. 2-Dimensional gel electrophoresis was carried out according to O’Farrel (1975); proteins were transferred to nitrocellulose membrane in a semidry system and stained with 0.05% (w/v) Ponceau

S in 3% (w/v) trichloroacetic acid (TCA). The membrane was blocked overnight at 4

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°C with 5% BSA in TBS-Tween, incubated for 2 hours at room temperature with polyclonal anti-Neurospora α-actinin antibody (Cotado-Sampayo et al., 2008) at

1:500 dilution, washed and re-incubated for 1 hour with anti-rabbit IgG antibodies coupled to horseradish peroxidase (Sigma A8275) diluted at 1:2000. Following 4 washes in TBS, peroxidase activity was assayed with 3,3’-diaminobenzidin tetrahydrochloride (DAB) and H2O2 as described earlier (Cotado-Sampayo et al.,

2006).

2.3. Construction of recombinant plasmids

Recombinant plasmids were constructed, A) to express and purify the fusion protein for biochemical analysis and B) to study in vivo expression:

A. Construction and expression of GST-α-actinin fusion protein for biochemical

analysis

The α-actinin entry in the Neurospora data base (NCU06429.4, Broad Institute) is a protein with two putative methionine start codons, giving two products with different predicted molecular weights (100 kDa and 80 kDa). The coding sequence of

GST-fusion proteins of both full- (100 kDa) and short-length (80 kDa) were amplified using the forward primers 5’-

CGCCGCGGATCCATGGAGATGCTGGGGGTGGAG-3’ for full- and 5’-

CGCCGCGGATCCATGGCTTTCGCAGAGCAACAAC-3’ for short- length α- actinin. The reverse primer 5’-

ATAAGAATGCGGCCGCCTAATGATACCCATTCGGCTTG-3’ was common for both constructions. The PCR product was cloned in pGEX4T.1 at BamHI and NotI restriction sites (underlined) and this construct was used to transform competent

Escherichia coli BL21 cells. For the expression of fusion proteins, transformed cells

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were inoculated in 0.5 to 1 liter LB medium containing 100 μg/ml ampicillin and incubated at 37 °C till an OD600 of 0.4–0.6 was reached. Expression of the protein was induced by the addition of IPTG (final conc. 100 mM) and the cultures were grown for a further period of 16 h at 23 °C, harvested by centrifugation, suspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 100 mM NaCl) and lysed by sonication. The soluble proteins were recovered by centrifugation (16.000xg, 15 min) and purified on GST-sepharose column. The construction of “short” GST-α-actinin, without the N-terminal extension, was used to characterize the actin- and calcium binding properties of the protein.

B. Construction of α-actinin-egfp gene containing plasmid and transformation

The α-actinin-egfp gene fusion was constructed by PCR amplification of the sequence encoding the α-actinin gene (ncu06429.4), including the 930 bp of the N- terminal extension, the region upstream of the sequence coding for the first CH- domain. PCR was carried out using forward primer 5’-

CGCCGCGGATCCATGGAGATGCTGGGGGTGGAG-3’ and reverse primer 5′-

GT CACGTTAATTAAATGATACCCATTCGGCTT-3′. This led to the insertion of

BamHI and PacI restriction sites (underlined) and used to clone the gene in the egfp gene containing plasmid pMF272 constructed by Freitag et al. (2004).

Transformation of the N. crassa his-3 mutant (FGSC 9014; ridRIP1 mat A his-3) and heterokaryon transformant selection were performed as described previously by

Margolin et al. (1997) and Freitag et al. (2004).

2.4. Biochemical analysis of α-actinin

Actin binding assays

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To monitor the co-sedimentation of Neurospora α-actinin with actin, the concentration of actin was maintained constant at 3.5 μM but the concentration of α- actinin was varied. G-Actin from rabbit muscle (Molecular Probes, Eugene, OR,

USA) and GST-α-actinin were mixed in actin binding buffer (5 mM Tris-HCl, pH

8.0, 0.2 mM ATP, 0.2 mM CaCl2, 0.5 mM β-mercaptoethanol) and polymerization of actin was initiated by the addition of KCl and MgCl2 to a final concentration of 100 mM and 1 mM, respectively. Samples were incubated at room temperature for 20 min and centrifuged for 30 min at 13,000 rpm in an Eppendorf centrifuge. The effect of calcium on the regulation of actin-binding capacity of α-actinin was tested by addition of 0.5 mM EGTA in all samples except for the one containing 3 mM CaCl2.

After centrifugation the supernatants and pellets were collected and electrophoresed in 10% SDS-PAGE and immunobloted with anti-actin antibody (Sigma A2668).

Cross-linking assay and electron microscopy visualization

For cross-linking assays, the “GST” tag from the recombinant protein, was removed with thrombin (5 units/mg protein for 30 min at 25 °C). The completion of the digestion was confirmed by gel SDS-PAGE (10%). The resulting α-actinin (2 mM) was mixed with 2 mM G-Actin in the actin binding buffer supplemented with actin polymerization solution (KCl and MgCl2, final concentrations of 100 mM and 3 mM, respectively). After incubation for 1 hour at room temperature, the mixture was stained with 0.5% sodium phosphotungstate (PTA) at pH 7.0 and deposited on a carbon-coated grid, and visualized at 60 kV using a Philips EM410 transmission electron microscope. A control without α-actinin was prepared under the same conditions.

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Calcium binding assays

The extent of calcium binding was determined using a 45Ca2+ overlay assay

(Maruyama et al., 1984). Briefly, 10 μg of GST-α-actinin was slot-blotted onto a nitrocellulose membrane. The membrane was equilibrated in imidazol buffer (10 mM

45 2+ imidazol, 5 mM MgCl2 and 30 mM KCl, pH 8.0) and then incubated with Ca

45 2+ (13 μCi/ml Ca ) as CaCl2. 10 μg of calmodulin and α-actinin from chicken gizzard

(Sigma A9776) were used as positive controls and GST (10 µg) as negative control.

2.5. Localization of α-actinin in situ

Samples for immuno-localization assays were prepared as described earlier

(Cotado-Sampayo et al., 2006). Anti-Neurospora α-actinin was used at a 1:20 dilution and FITC conjugated anti-rabbit secondary antibody at a dilution of 1:80.

2.6. In vivo expression of α-actinin-GFPand acquisition of live cell images

Cultures were prepared in liquid medium as described above. For long time image acquisition (12 hours), germinating spores were deposited in 8 chambered coverglass (Lab-Tek®,ref 155411) and covered with a small block of Vogel 1.5% agar medium in order to maintain the cells in the same plane. The GFP expression was recorded with a Leica AF6000LX microscope. For shorter time image acquisition, 12 hours hyphae were deposited in culture chambers as described above and followed with a Leica SP2 microscope. Images were processed with Adobe

Photoshop 6.0.

2.7. Cell wall staining with calcofluor

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Slides coated with Vogel’s medium containing 1.5% sucrose were inoculated with conidia and grown at 25 °C for 8 hours in a wet chamber. The mycelia were stained with 10 μM calcofluor (Fluorescent Brightener 28) for 3 minutes and covered with a coverslip. An Orthoplan epiillumination microscope (Leica) equipped with fluotar optics and a selective filter combination was used to visualize calcofluor fluorescence patterns. Fluorescent micrographs were obtained with a Leica-DFC490 camera and processed with Adobe Photoshop 6.0.

3. Results

3.1. Immunoblot

A Western blot of N. crassa crude extract and partial thrombin digested full length GST-α-actinin recombinant protein separated in 2D-gels showed three bands with different relative molecular weights and Isoelectric points (pI): the undigested

(140 kDa, pI 6.0), the thrombin cleaved recombinant protein (110 kDa, pI 5.9) and the “native” (80 kDa, pI 5.6) Neurospora α-actinin (Fig. 1A). These features correspond to the theoretical estimated values based on the sequences. The 140 kDa band agrees with the molecular weight of the fusion protein (110 kDa full length α- actinin plus 30 kDa of the GST), 110 kDa represents the full length α-actinin and the

80 kDa protein corresponds to the conserved region containing the characteristic domains of the α-actinins (Fig. 1B).

3.2. Actin-binding property

Our results show that actin sedimentation is modified by the presence of α- actinin and Ca2+. Low speed centrifugation sedimented neither G-Actin, nor F-Actin,

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nor fusion GST-α-actinin (Fig. 2a, b and c, respectively). Under the sedimentation conditions used the amount of the sedimented actin depended on the amount of the

GST-α-actinin when calcium was chelated with EDTA (Fig. 2d and e). In the presence of excess calcium, α-actinin failed to co-sediment with actin (Fig. 2f).

3.3. Cross-linking assay and electron microscopic visualization

Electron micrographs of negatively stained cross-linked samples also showed the affinity of α-actinin for actin filaments. α-Actinin cross-linked actin filaments and organized them in parallel structures as bundles (Fig. 3B). Bundle formation was observed when the ratio between actin and α-actinin was 1:1. High concentrations of actin relative to α-actinin resulted in a complete disorganization of the actin filaments (Fig. 3C). The inter cross-linker space between actin filaments was difficult to determine with precision but it was approximately between 15 and 25 nm.

3.4. Calcium-binding assay

45Ca overlay experiments showed that Ca2+ binds to α-actinin. For the same amount of protein the signal was higher for GST-actinin (Fig. 4b) than for commercial chicken gizzard α-actinin (Fig. 4a). Calmodulin was used as positive

(Fig. 4c) and purified GST as negative control (Fig. 4d).

3.5. α-Actinin localization

Immunofluorescence microscopy was used to detect the spatial localization of α- actinin in the germ tubes and growing hyphae of wild-type strain. The results showed that the protein was localized in the tip region (Fig. 5A), along the plasma membrane

(Fig. 5B) and the septa (Fig. 5C, D). This localization was confirmed by in vivo

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expressed α-actinin-GFP. GFP fluorescence was observed at the site of germ tube tips and the emerging tubes, (Fig. 6A, B, and C). In the growing hyphae α-actinin-

GFP was also located in the zone of hyphal fusions (Fig. 6F and Fig. 7C) and the septa (Fig. 7A, B). The localization in septa and the hyphal fusion zone seemed to disappear after completion of these structures. The plasma membrane localization was not observed in vivo in the α-actinin-GFP transformed strain (Fig. 6 and Fig.7).

3.6. Heterokaryon α-actinin knockout strain phenotype

The α-actinin knockout strain was deposited in the FGSC as heterokaryon (Colot et al., 2006), considering that the the homokaryon has a lethal phenotype because of poor or inexistent germination of the first generation of ascospores. Compared to the wild type, the heterokaryon showed shortened aerial hyphae (Fig. 8A) in Davis and

De Serres (1970) medium. The morphology of the hyphae was different with respect to their branching pattern. The mutant showed a predominantly dichotomous phenotype compared to sympodial branching in the wild type (Fig. 8B).

4. Discussion

The antibody prepared against α-actinin from Neurospora crassa reacted specifically with a 80 kDa protein in the Western blot of the crude extract. This relative molecular weight (Mr) agrees with the estimated molecular weight of the

Neurospora α-actinin annotated in the MIPS database (as an α-actinin related protein) based on the initiator methionine immediately upstream of the coding sequence for the first CH-domain. All close homologs in other fungi confirm that this methionine is the initiator and the resulting protein contains all the domains defining

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α-actinins. However, there are two start codons present in the ncu06429.4 gene annotation of the Broad Institute database and we have cloned the gene from the cDNA which included the first start codon indicating that this N-terminal extension is present in α-actinin mRNA and could be an UTR region or pro-domain of the protein. Alternatively, another interpretation could be that the translation of mRNA is initiated at both methionine codons located in the ORF. The UTR region hypothesis is not very likely for several reasons. The first reason is its length of 930 nucleotides.

5’UTR- are generally shorter than 3’UTR- regions and the average length is roughly

200 nucleotides (Pesole et al., 2000), although there are reports of unusually long

UTR regions in some genes (e.g. the cyclin CLN3 from S. cerevisiae (864 nucleotides) (Polymenis and Schmidt, 1997; Vilela and McCarthy, 2003) or frq

(frequency) from N. crassa with ~1.5 Kb (Diernfellner et al., 2005). The second reason is that the first 5’AUG could be a good initiator codon according to the predicted Kozak sequence of filamentous fungi (Bruchez, 1993). The putative full- length peptide would be of 310 amino acids longer than the short one. Alternative initiation of translation has been reported in N. crassa (Garceau et al., 1997;

Diernfellner et al., 2005) and could explain the presence of the two in frame ATG in a situation where the full-length α-actinin was translated under conditions other than those used in our analysis. The hypothesis of a pro-domain in the protein is more plausible even if we were not able to detect the product in the crude extracts. It might be that it was very unstable and quickly degraded.

The interaction of α-actinin with actin was negatively regulated by calcium, as demonstrated by the sediment assays. It was shown that the α-actinin has the ability to cross-link F-actin and organize the microfilaments in a parallel way. Cross-linked

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actin filaments with α-actinin are 15-25 nm apart. The distance between the filaments was shorter than observed in other actin-α-actinin complex (30-40 nm)

(Taylor and Taylor, 1993; Tang et al., 2001; Hampton et al., 2007), but agrees with the difference in size between α-actinin from higher eukaryotic cells and the shorter fungal α-actinins which contains only half the number of spectrin repeats (Virel and

Backman, 2007).

The calcium dependence of the Neurospora α-actinin and insensitivity of vertebrate α-actinin in actin binding assays, supports the idea that calcium dependent regulation of actin binding was lost in the vertebrate-invertebrate divergence (Dixson et al., 2003; Virel and Backman, 2004).

Higher levels of α-actinin were observed in germinating conidia at the site of hyphal tube emergence and were maintained in the tip of the emerging tubes suggesting that α-actinin plays a role in germination and branching. A similar localization has been observed for actin which was shown to be essential for germ tube initiation (Barja et al., 1993). α-Actinin was also localized along the plasma membrane in the tip region of the hyphae suggesting additional roles in cellular physiology. α-Actinin facilitates the stabilization of focal adhesion plaques in animal cells (Xu et al., 2000). The homological structures in fungi, are the actin plaques or dots (Torralba and Heath, 2001) where F-actin binding to the plasma membrane occurs (Hoch and Staples, 1983; Adams and Pringle, 1984; Kaminskyj and Heath,

1996). The connection of the α-actinin with the plasma membrane is mediated by other proteins such as β1,β3-integrin, vinculin, α-catenin (Otey and Carpen, 2004).

None of these proteins have homologs in the Neurospora genome but we can not exclude the presence of other unknown α-actinin partners (Otey and Carpen, 2004).

15

α-Actinin-GFP was not observed in the plasma membrane region. Is possible that the signals were too weak to detect or alternatively the GFP tag may have partially modified the localization of the fusion protein, a phenomenon that has been described before (Ikonen et al., 1995; Schneider et al., 2000). The localization of α- actinin in the septa was observed both in situ and in vivo. The disappearance of α- actinin from the septa once their formation is complete, has also been described for other proteins required for septation in filamentous fungi, such as formin SEPA

(Sharpless and Harris, 2002) and actin (Momany and Hamer, 1997; Rasmussen and

Glass, 2005, 2007).

The knock-out phenotype was lethal. It is unlikely that the lethal phenotype is based only on a role in septum formation. The aseptate mutants in Neurospora

(Rasmussen and Glass, 2005, 2007) and other filamentous fungi (Ayad-Durieux et al., 2000; Wendland and Philippsen, 2000; Kim et al., 2006) are viable.

The exact function of α-actinin in Neurospora is still not clear. We can assume that a major role is played by a collaboration between actin and α-actinin. The results on protein localization suggest a role in cytokinesis. This would agree with the function of its orthologs in S. pombe (Wu et al., 2001). However, differences can be expected since cytokinesis in filamentous fungi and yeast are two principally different mechanisms (Walther and Wendland, 2003). Moreover, the function of α-actinin in yeast and also in D. discoideum can be complemented by other actin-binding proteins, notably fimbrin (Rivero et al., 1999; Wu et al., 2001). This result in non- lethal phenotypes for α-actinin mutants in these organisms.

The dichotomous phenotype of the α-actinin heterokaryon knock-out strain is a consequence of a splitting at the tips. This is also called “dichotomous branching”,

16

and has been observed in several other filamentous fungi in which polarity maintenance proteins were mutated (Geissenhöner et al., 2001; Han and Prade, 2002;

Sharpes and Harris, 2002). A similar phenotype has been observed for Cytochalasin

A treated Neurospora (Riquelme et al., 1998) and Neurospora actin mutants (Virag and Griffiths, 2004) suggesting that α-actinin may also collaborate with actin in the formation of branching patterns.

However, other roles may be expected. The list of newly identified proteins and molecules interacting with α-actinin has increased over the last years, demonstrating that α-actinin is not merely an actin-binding protein but a scaffold for other protein- protein interactions and to connect the cytoskeleton to diverse signaling pathways (Critchley and Flood, 1999; Otey and Carpen, 2004).

Acknowledgment

We gratefully acknowledge financial support from the Academic Society of

Geneva (F. B.). Thanks are due to R. Strasser for interest in the project and encouragement, M-L. Chappuis for technical assistance, A. Fehr for secretarial assistance.

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Fig. 1. 2D-PAGE (10%) analysis of the N. crassa α-actinin and the GST-α-actinin

constructs. A. A mixture of N. crassa crude extract and partially digested GST-α-actinin

fusion protein immunoblotted with anti-Neurospora α-actinin antibody. B. Diagrammatic

representation of GST-α-actinin construction, bars underline the region of the protein

reacting with the anti-Neurospora α-actinin antibody shown in A. (GST, means

gluthathione S-transferase; CH, Calponin Homology domain; SR, spectrin repeat and EF,

refers to EF-hand motif).

Fig. 2. Analysis of α-actinin-actin interactions. SDS-PAGE (10 %) coomassie blue

stained gel (upper panel) of supernatants (sn) and pellets (p) of α-actinin-actin mixtures

and the corresponding immunoblot with the anti-actin antibody (lower panel). Lanes a, b

and c are 3.5 μM of G-actin, F-actin and GST-α-actinin respectively. Lanes d and e

represent reaction mixtures containing 3.5 μM actin and 2 μM (lane d) and 3.5 μM (lane

e) α-actinin. Lane f is the same as lane e but with excess calcium.

Fig. 3. Electron micrographs of α-actinin-F-actin cross-reaction products. A. 2 μM

actin; B. α-actinin to actin molar ration 1:1 (2 μM); C. α-actinin to actin molar ratio 1:2

(1 μM:2 μM). Bar 100 nm.

Fig. 4. Calcium-binding of N. crassa α-actinin probed by calcium overlay using

45Ca. a, 10 μg of GST-α-actinin; b, α-actinin from chicken gizzard; c, calmodulin and d,

GST protein were slot-blotted onto a nitrocellulose membrane.

Fig. 5. Immunolocalization of N. crassa α-actinin during different stages of growth.

Germinating conidia, emerging germ tubes and growing hyphae corresponding to 1 hour

(A) and 12 h (B-D) growth. Tip region of the hyphae with a branch initial (B).

Localization of α-actinin in the septum (C-D). Bar 5 μm.

Fig. 6. Time lapsed images of live cells for a duration of 5 hours. Laser scanning

confocal microscopy images of Neurospora hyphae expressing α-actinin-GFP. Images

correspond to intervals of approximately 1 hour. After one hour of growth the

fluorescence concentrated at the site of germination (A), this concentration was maintained until germ tube formation during the first 3 hours of growth (B-C), during hyphal growth no specific localization was found (D-E). The GFP signal accumulated at the fusion site of two hyphae (F). Bar 10 μm.

Fig. 7. In vivo localization of α-actinin in N. crassa. A-B. Transmission and GFP

signal is shown for each acquisition image; complete septum formation took less than 40

min. A. Time series corresponding to the first step of septum formation, the septum is not evident in the transmission images made during the first 20 min. B. Stack serie of the hyphae at 30 minutes, the fusion pore was visualized in the medial acquisition image. C.

confocal image showing a hyphal fusion using the transmission image (a), GFP signal (b)

and calcofluor stain (c).

Fig. 8. Phenotype of the α-actinin heterokaryon knock-out strain. A. Wild type (wt)

hyphae (left) and α-actinin heterokaryon knock-out strain (right). B. Calcofluor stained hyphae of N. crassa wt (a-b) and α-actinin heterokaryon knock-out strain (c-d). b and d are images at higher magnification of a and b. Bar 10 μm.