Cell Signaling by Rho and Miro Gtpases

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 320

Cell signaling by Rho and Miro GTPases

Studies of Rho GTPases in Cytoskeletal Reorganizations and of Miro GTPases in Mitochondrial Dynamics

ÅSA FRANSSON

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Till mina nära och kära

The big wheel keeps on turning On a simple line day by day The earth spins on its axis One man struggles while another relaxes

(Massive Attack, Hymn of the Big Wheel)

LIST OF PUBLICATIONS

This thesis is based on the following publications, which will be referred to in the text by their Roman numerals:

I. Pontus Aspenström, Åsa Fransson and Jan Saras “Rho GTPases have diverse effects on the organization of the actin filament system” Biochem J. 377:327-37, 2004.

II. Åsa Fransson, Aino Ruusala and Pontus Aspenström “Atypical Rho GTPases have roles in mitochondrial homeostasis and apoptosis” J Biol Chem. 278: 6495-502, 2003.

III. Åsa Fransson, Aino Ruusala and Pontus Aspenström “The Atypical Rho GTPases Miro1 and Miro2 have essential roles in mitochondrial trafficking” Biochem Biophys Res Commun. 344:500-10, 2006.

Reprints were made with permission from the publishers.

RELATED PUBLICATIONS

i. Pontus Aspenström, Åsa Fransson and Ninna Richnau “Pombe Cdc15 homology proteins: regulators of membrane dynamics and the actin cytoskeleton” Trends Biochem Sci.31:670-9, 2006.

ii. Ninna Richnau, Åsa Fransson, Khashayar Farsad and Pontus Aspenström “RICH-1 has a BIN/Amphiphysin/Rvsp domain responsible for binding to membrane lipids and tubulation of liposomes” Biochem Biophys Res Commun. 320:1034-42, 2004.

CONTENTS

ABBREVIATIONS ...... 13 INTRODUCTION ...... 15 1 The Ras Superfamily of GTPases ...... 15 1.1 Regulation and Localization of GTPases...... 16 1.2 Applied Mutations in GTPases...... 19 2 The Cytoskeleton ...... 19 2.1 Actin Polymerization...... 21 3 The Rho GTPases...... 21 3.1 Rho GTPases in Actin Dynamics ...... 24 3.1.1 Cell Migration...... 24 3.1.1.1 Protrusive Force...... 24 3.1.1.2 Formation and Turnover of Cell-Substrate Adhesions ...... 25 3.1.1.3 Cell Contraction...... 25 3.1.1.4 Directional Sensing ...... 26 3.1.2 Membrane Trafficking ...... 27 3.2 Additional roles for Rho GTPases...... 27 3.3 Rho GTPases in Diseases ...... 28 4 The Miro GTPases ...... 29 5 Mitochondria 30 5.1 Mitochondrial Dynamics ...... 31 5.1.1 Mitochondrial Transport ...... 32 5.1.1.1 Microtubule-Based Transport...... 32 5.1.1.2 Actin in Transport and Docking ...... 35 5.1.1.3 Links between Intermediate Filaments and Mitochondria ...... 36 5.1.2 Mitochondrial Fusion...... 36 5.1.3 Mitochondrial Fission ...... 39 5.2 Mitochondria-Shaping Proteins in Cell Death...... 41 5.3 Mitochondrial Dynamics in Disease...... 41 PRESENT INVESTIGATION...... 44 Aims ...... 44 Paper I: Rho GTPases have Diverse Effects on the Organization of the Actin Filament System ...... 44

Paper II: Atypical Rho GTPases have Roles in Mitochondrial Homeostasis and Apoptosis...... 45 Paper III: The Atypical Rho GTPases Miro-1 and Miro-2 have Essential Roles in Mitochondrial Trafficking ...... 46 REFLECTIONS ...... 49 ACKNOWLEDGEMENTS ...... 52 REFERENCES...... 54

ABBREVIATIONS

Arp Actin-related protein ALS Amyotrophic lateral sclerosis ATP Adenosine triphosphate Bcl-2 B-cell lymphoma 2 BTB Broad Compex/Tramtrack/Brick à brac DRF Diaphanous-related formin Drp1 Dynamin-related protein-1 EGF Epidermal growth factor GAP GTPase-activating protein GDF GDI-displacement factor GDI Guanine nucleotide dissociation inhibitor GDP Guanosine-5’-diphosphate GEF Guanine nucleotide exchange factor GRIF1 GABAA receptor interacting factor 1 GTP Guanosine-5’-triphosphate IF Intermediate filament JIP JNK-interacting protein JNK c-Jun N-terminal kinase MAP Microtubule-associated protein Mfn Mitofusin Miro Mitochondrial Rho MLC Myosin light chain OIP106 O-linked N-acetylglucosamine transferase interacting protein 106 Opa1 Dominant optic atrophy associated protein PDGF Platelet-derived growth factor PLD Phospholipase D Ras Rat Sarcoma viral oncogene homolog Rho Ras homologous ROCK Rho-associated kinase ROS Reactive oxygen species VDAC Voltage-dependent anion-selective channel WASP Wiskott-Aldrich syndrome protein WAVE WASP-like verprolin-homologous protein

INTRODUCTION

Cells in multicellular organisms like humans exhibit a wide variety of distinct shapes and internal organizations. With delicate timing and precision, cells alter their structure and behavior in response to internal and extracellular signals. Cells adapt to their ever-changing environment by changing polarity, crawling, dividing, transporting organelles, extending and retracting processes and establishing and breaking contacts with neighboring cells. All these processes require extensive remodeling of the cytoskeleton, a dynamic network of different types of protein filaments extending throughout the cytosol. Cytoskeletal remodeling consumes energy. Mitochondria are the power plants of the cell converting the energy of food into the high-energy compound, ATP, used to fuel cell reactions. Mitochondria acquire specialized conformations and undergo changes in intracellular distribution, often in response to the metabolic needs of the cell. Defects in either cytoskeletal regulation or mitochondrial dynamics inevitably lead to diseases.

This thesis will focus primarily on the biological functions of two groups of Ras superfamily GTPases: Rho and Miro. The Rho GTPases respond to extracellular signals and transduce these signals to cytoskeletal rearrangements, whereas the Miro GTPases are regulators of mitochondrial movement and morphology.

1 The Ras Superfamily of GTPases The founding members of the Ras superfamily are K-Ras, H-Ras and N- Ras, identified in the early 1980 as oncogenes mutated in human cancers of different origins (Krontiris et al. 1981; Perucho et al. 1981; Shih et al. 1981). The oncogenic properties of these proteins triggered interest in exploring the biological roles of related proteins.

Members of the Ras superfamily function as molecular switches and alternate between an active, GTP-bound, and an inactive, GDP-bound state (Takai et al. 2001). In humans, the Ras superfamily of proteins comprises more than 150 members that can be divided into six subfamilies: Ras, Rab, Arf, Ran, Rho and Miro. Members of the Ras subfamily regulate gene

15 expression, Arf proteins control microtubule dynamics and, together with Rab, regulate intracellular vesicle trafficking, whereas Ran masters nucleocytoplasmic transport, mitotic spindle and nuclear envelope assembly (reviewed in Takai et al. 2001). The Rho GTPases govern a number of cellular functions involved in actin cytoskeleton remodeling. In addition, Rho GTPases influence cell cycle progression, cell survival and gene transcription. The Miro proteins act as regulators of mitochondrial dynamics. Thus, in virtually all aspects of cell behavior Ras superfamily proteins participate as control elements.

1.1 Regulation and Localization of GTPases With the exception of Ran, membrane-association is a prerequisite for the function of the members of all Ras superfamily branches (Wennerberg et al. 2005). Many Ras-related proteins act on the plasma membrane, but in addition or instead, some localize to specific intracellular membrane compartments. Ras superfamily proteins attach to membranes via their posttranslationally modified lipid moieties (reviewed in Seabra 1998; Pechlivanis et al. 2006; Wright et al. 2006). The primary translation product of most Ras and Rho GTPases terminates in a CAAX (C, cysteine; A, aliphatic acid; X, any amino acid) motif. After synthesis on free polysomes in the cytoplasm, a GTPase serves as substrate for a pair of transferases that attach a cholesterol-related isoprenoid group to the cysteine residue in the CAAX-motif (McTaggart 2006). Whereas the Ras proteins are prenylated with a 15-carbon farnesyl, the Rho GTPases are generally modified with 20- carbon geranylgeranyl. Prenylation targets the proteins to the endoplasmic reticulum (ER) where the -AAX portion is proteolytically removed and the exposed cysteine residue is carboxylmethylated.

The targeting of CAAX-proteins to their desired membrane compartment is emerging as a relatively complex matter, where secondary membrane- targeting motifs are involved (ten Klooster et al. 2007). Some Rho and Ras proteins have one or two cysteine residues positioned just upstream of the CAAX-motif that becomes palmitoylated and serve as a secondary membrane targeting motif, whereas in other proteins a polybasic lysine/arginine stretch helps to determine the correct targeting (Hancock et al. 1990; Adamson et al. 1992; Williams 2003). The Arf proteins lack a CAAX-box but instead have a glycine residue in their N-terminal that becomes modified with myristic acid (D'Souza-Schorey et al. 1995). Rab proteins are generally geranylgeranylated on two C-terminal cysteine residues (Farnsworth et al. 1991; Farnsworth et al. 1994).

The activity of the common GTPases is tightly controlled by a multitude of regulator proteins designated as guanine nucleotide exchange factors (GEFs)

16 and GTPase activating proteins (GAPs). Rho and Rab GTPases are further regulated by a third class of regulators called guanine nucleotide dissociation inhibitors (GDIs).

In response to extracellular stimulus-activation of various cell surface receptors, typical Ras-related GTPases are activated by their cognate GEFs at cellular membranes. GEFs promote activation of the GTPases by catalyzing the release of GDP. Since the intracellular GTP levels are much higher than the GDP levels, the majority of proteins will be reloaded with GTP and thereby enter their active state. Upon GTP-binding, GTPases undergo a conformational change that enables interaction with and activation of downstream effectors. The GTPase signaling is terminated by a GAP, which enhances the, normally very low, intrinsic ability of GTPases to hydrolyze GTP.

In a resting cell, the Rho GTPases (as well as the Rab GTPases) are maintained as cytosolic complexes with a GDI. GDIs block GDP release and mask the prenylated C-terminal tail of the GTPase required for membrane localization (Keep et al. 1997). GDIs have been proposed to mediate the specific delivery of Rho GTPases to restricted membrane compartments by interacting with yet unidentified receptors (Dransart et al. 2005). At the membrane interface, the Rho GTPase/GDI complex dissolves. This step is triggered by interactions with proteins termed GDI- displacement factors (GDFs), and/or through kinase-mediated phosphorylation of GDI (Takahashi et al. 1997; Mehta et al. 2001; DerMardirossian et al. 2004). After completion of its work and GTP hydrolysis, the Rho GTPase re-associates with GDI to be extracted from the membrane. It has been suggested that phosphorylation of the Rho GTPases might be a mechanism that induces the GDI-binding (Lang et al. 1996; Forget et al. 2002; Ellerbroek et al. 2003; Tu et al. 2003). GDIs can also interact with GTP-bound GTPases and inhibit both GTP hydrolysis and interaction with effectors (Hart et al. 1992; Chuang et al. 1993; Hancock et al. 1993; Sasaki et al. 1993; Faure et al. 1999). The cycling of Ras-related GTPases is illustrated in Figure 1.

17 bc

GTPase GEF GTPase Effector GTP GDP adGTP Cellular Outcome

GTPase Pi GTPase GDP GAP GTP

GDF Pi GDI GDI GTPase GTPase GDP GDP fe

Figure1. The cycling of Ras superfamily GTPases. (a) Ras superfamily proteins attach to membranes via their posttranslationally modified lipid moieties. (b) Upon cell activation, membrane-anchored GTPases are activated by their cognate GEFs, which catalyze the release of GDP, thereby allowing the GTPase to rebind the more abundant GTP. (c) In their active, GTP-bound, state the GTPases propagate downstream signaling through interactions with effectors. (d) The signaling is terminated by a GAP, which stimulates the hydrolysis of the GTPase-bound GTP to GDP. (e) GTPases of the Rho and Rab family are further regulated by GDIs, which extract inactivated GTPases from membranes and sequester them in the cytosol. (f) GTPases can be liberated from their GDIs by GDI displacement factors (GDFs). (a) After GDI dissociation, the GTPases insert into their appropriate target membrane and are ready for another round of activation. Pi = inorganic phosphate.

RhoGAPs and RhoGEFs are typical multifunctional proteins embracing multiple domains (for RhoGAP reviews, see Peck et al. 2002; Moon et al. 2003 and for RhoGEF reviews, see Erickson et al. 2004; Buchsbaum 2007). Genome analysis has predicted the presence of about 80 human genes encoding RhoGAPs (Bernards 2003). Also the RhoGEF family is large, currently represented by 69 members of the Dbl family and 11 members of the more recently discovered Dock family (Erickson et al. 2004; Rossman et al. 2005). Only for Rho, Rac and Cdc42 more than 50 different effectors have been identified (for extensive reviews of effector proteins, see Van Aelst et al. 1997; Aspenstrom 1999; Bishop et al. 2000; Schmitz et al. 2000). The RhoGDIs constitute a family with three mammalian members: the ubiquitously and abundantly expressed GDI, the haematopoietic cell- selective GDI and GDIparticularly expressed in lung, brain and testis

18 reviewed in Sasaki et al. 1998; Olofsson 1999; DerMardirossian et al. 2005; Dovas et al. 2005)In addition of being regulated by GAPs, GEFs and GDIs, several Rho GTPases are highly regulated at the level of transcription or translation (Wennerberg et al. 2004).

Most Ras superfamily members alter their conformation upon GDP binding, primarily in two regions called switch I and switch II. In Rho GTPases, the switch I region extends from amino acid residue 26 to 45 (Rac1 numbering), while the switch II region spans residue 59 to 76 (Rac1 numbering). The switch I region has a pivotal role in the interaction with many effector proteins, and is hence denoted the effector region (Wittinghofer et al. 1996). In some cases, even the switch II region seems to act in effector binding. The switch regions can also act as docking sites for GEFs (Gao et al. 2001) and GDIs (Hoffman et al. 2000).

1.2 Applied Mutations in GTPases A very powerful way to study the function of GTPases is to use two types of mutants: constitutively active mutants (i.e. G12V or Q61L in Ras and Rac), which are unable to hydrolyze GTP and therefore reside constantly in the GTP-bound state, and dominant-negative mutants (i.e. T17N in Ras and Rac), which have low affinity for nucleotides and therefore exist mainly in an inactive nucleotide free state, where they are suggested to compete with the normal GTPase for binding to GEFs. It has to be taken into consideration that dominant-negative mutants may target GEFs important also for other GTPases. Constitutively active mutants, defective in GTP hydrolysis, do not exactly reflect GEF-mediated activation of the protein. Another type of mutation (F28L) was generated in Cdc42 to more closely mimic natural activation of Rho GTPases (Lin et al. 1997). Such mutants undergo enhanced intrinsic GDP/GTP transition, whereas the normal GTP hydrolysis is maintained. In fact, these “fast-cycling” mutants have been postulated to better reflect GEF-mediated activation than the constitutively active GTPase-defective mutants, indicating that the ability to cycle is important for full function of the Rho GTPases (Paper I; Reinstein et al. 1991; Lin et al. 1999; Wu et al. 2000).

2 The Cytoskeleton The ability of eukaryotic cells to form into a variety of shapes and perform coordinated and directed movement depends on elaborate arrays of dynamic protein fibres, collectively called the cytoskeleton. The cytoskeleton also serves as tracks for intracellular trafficking. The cytoskeleton is composed of three distinct, yet interconnected, types of elements: actin microfilaments,

19 microtubules and intermediate filaments. Proteins belonging to the Rho GTPases are vital regulators of the actin filament system, and have also been implicated in the regulation of microtubules and intermediate filaments. Miro proteins have functions in the transport of mitochondria along the microtubule system.

Actin filaments are formed by the polymerization of actin monomers (G- actin) into filaments (F-actin) (reviewed in Schmidt et al. 1998). Actin filaments are polar with a fast-growing end (also known as barbed or plus end), and a slow-growing end (also known as pointed or minus end). Many cellular events, such as phagocytosis, cell adhesion and cell division, involves actin cytoskeleton rearrangements (Castellano et al. 2001; Jaffer et al. 2004; Li 2007). During cell movement, actin assembly generates cell protrusions at the cell front and association of actin with myosin motors generates contractile forces required for retraction of the cell rear (Ridley 2001). Additionally, some cargo transport occurs along actin, with myosin motors (Cheney et al. 1993; Wells et al. 1999).

Microtubules are cylindrical tubes built from / tubulin dimers (reviewed in Gundersen 2002). Like actin filaments, microtubules are polar, that is, they have a dynamic plus (fast growing) end and a more stable minus (slow growing) end. Usually, the minus end originates from the centrosome close to the cell nucleus and the fast growing (and fast shrinking) end radiates toward the cell periphery. Microtubules function as tracks, along which ATP-dependent motors transport organelles and vesicles around the cell. Transport toward the plus end of the microtubules (anterograde transport) is mainly achieved by kinesin, whereas transport toward the minus end (retrograde transport) is mainly achieved by dynein. Crosstalk, both direct and mediated via signaling molecules exists between microtubules and actin microfilaments, for instance actin and microtubules cooperate to polarize cells (Palazzo et al. 2002; Rodriguez et al. 2003; Etienne-Manneville 2004; Gundersen et al. 2004).

The third set of cytoskeletal elements is the intermediate filaments (IFs), a collection of fibers composed of a variety of related proteins. IFs provide the cell with tensile strength (reviewed in Coulombe et al. 2000). IFs differ from the microfilaments and microtubules in that they are non-polar (with identical ends), and have not been implicated in intracellular transport. Among the IFs are the keratins, which strengthen mammalian epithelial cells and form the resilient material of hair and nails; the vimentins that maintain cell integrity of mesoderm-derived cells; the neurofilaments that provide strength to the long axons of neurons and the lamins that stabilize the inner membrane of the nuclear envelope. Beyond their function in providing a supporting framework within cells, IFs can sequester, position

20 or act as scaffolds for signaling molecules, including stress activated kinases (Pallari et al. 2006).

2.1 Actin Polymerization The initial “nucleation phase” in the formation of a new actin filament is thermodynamically unfavorable. The main kinetic barrier to nucleation is the formation of an actin dimer. Two major mechanisms of de novo nucleation have been identified, employing either the nucleation factor Arp2/3 or Diaphanous-related formins (DRFs) (Pollard 2007). Both types of nucleation factors depend on Rho GTPase signaling for activity. Once the first few actin monomers have been tied together, actin monomers rapidly self assemble into filaments. During filament elongation, the barbed ends need to be protected against blockage by abundant capping proteins (Schafer et al. 1996; Kwiatkowski 1999).

Actin polymerization driven by the Arp2/3 complex creates a branched network. The Arp2/3 complex can bind to the side of a preexisting filament and initiate the formation of a new filament, which branches out from the parent filament (Mullins et al. 1997). In addition, it has been proposed that Arp2/3 can interact with the barbed end of an existing filament to initiate a new branched filament (Pantaloni et al. 2000). The Arp2/3 complex comprises seven subunits, including the actin-related proteins Arp2 and Arp3. Arp2/3 alone poorly nucleates actin. For efficient nucleation, Arp2/3 needs to be stimulated by nucleation-promoting factors, such as the WASP/WAVE family of Rho GTPase effectors. Addition of nucleation- promoting factors brings Arp2 and Arp3 closer together, so that they form a pseudoactin dimer that can act as an actin nucleus (Goley et al. 2004; Rodal et al. 2005).

The DRF family includes the Dia, DAAM and FRL formins in mammals, which are dimeric proteins that nucleate unbranched filaments. They remain bound to the fast-growing barbed end of the filament and thus prevent capping. Actually, some formins may have their primary role in filaments elongation and not in the nucleation process. Several formins also bundle, sever or depolymerize actin. (For formin reviews, see Higgs 2005; Faix et al. 2006; Kovar 2006).

3 The Rho GTPases The mammalian Rho GTPases are encoded by 20 distinct genes, and can be divided into subfamilies (Figure 2): the Rho subfamily (RhoA, RhoB and RhoC), the Rac subfamily (Rac1, Rac2, Rac3 and RhoG), the Cdc42

21 subfamily (Cdc42, TC10 and TCL), the Wrch-1/Chp subfamily (Wrch-1 and Chp), the Rnd subfamily (Rnd1, Rnd2, and RhoE/Rnd3), the RhoBTB subfamily (RhoBTB-1 and RhoBTB-2) and the RhoD/Rif subfamily (RhoD and Rif) (Wherlock et al. 2002; Fransson et al. 2003; Wennerberg et al. 2004; Aspenstrom et al. 2007; Boureux et al. 2007). RhoH/TTF is an outsider not belonging to any of the subfamilies. Most typical Rho GTPases consist of 190-250 amino-acid residues. In these proteins, only short extensions flank the GTPase domains. Within their GTPase domains, the Rho GTPases display 40-95 % sequence identity with each other and share about 30 % sequence identity with Ras (Wennerberg et al. 2004). Rho proteins have a unique surface-exposed alpha-helical region that distinguishes them from other Ras-related proteins. This region is called the insert domain, spans amino acids 124-135 (Rac1 numbering), and has been described roles in activation of effectors (Freeman et al. 1996; Zong et al. 2001; Walker et al. 2002). In general, each activation/inactivation cycle of Rho proteins comprises both GDP/GTP and cytosol/membrane exchange. However, in some cases these two cycles seem to be uncoupled. Interestingly, TCL appears to be regulated only by GTP/GDP exchange and to be constantly membrane-targeted (de Toledo et al. 2003). . Some Rho GTPases are regulated in very different ways from the classical ones, and therefore they are called atypical. This category of proteins includes RhoH, Wrch-1, Chp and members of the Rnd and Rho BTB subfamilies (Aspenstrom et al. 2007). Apart form their GTPase domain; the atypical members often contain additional motifs. These proteins do not follow the general scheme of GEF and GAP-stimulated GDP/GTP cycling. Members of the Rnd and RhoH families have amino acid substitutions in sites important for GTPase activity, that render them GTPase defective and subsequently constitutively activated (Li et al. 2002; Chardin 2006). Rnd and RhoH members are instead tightly regulated at the level of transcription. Possibly, the RhoBTBs reside constitutively in a GTP-bound state, since also this group of proteins carries activating amino acid substitutions at the sites important for GTPase activity (Aspenstrom et al. 2007). Wrch-1 has elevated rate of nucleotide exchange ability, and consequently exists predominantly in its GTP-loaded form (Saras et al. 2004; Shutes et al. 2004). In some cases, atypical Rho GTPases might be controlled by phosphorylations or protein-protein interactions, involving types of domains not represented in their classical counterparts (Riento et al. 2005; Aspenstrom et al. 2007). Chp and Wrch-1 depend on palmitoylation, not isoprenylation, to be targeted to membranes (Berzat et al. 2005). Since Chp and Wrch-1 are not posttranslationally prenylated, they are not likely to be regulated by GDIs. Concordantly, GDIs have been reported unable to extract Chp from membranes (Chenette et al. 2006).

22 TCL TC10 Cdc42 ”Classical” Rho GTPases Rif (RhoF) Rac1 Rac2 Rac3 RhoD RhoG

RhoB RhoC Chp RhoA Wrch1

RhoH Rnd3 /RhoE ”Atypical” Rho GTPases Rnd2 Rnd1 RhoBTB2 RhoBTB1

Figure 2. Dendritic tree representing the 20 members of the Rho GTPase family. Adapted from Aspenström et al. 2007.

Rho GTPases act as molecular switches in signal transduction pathways that link plasma membrane receptors to actin cytoskeleton rearrangements. They are involved in the control of a diverse array of cellular phenomena, including cell morphology, cell polarity, cell adhesion, cell migration, axon guidance, cytokinesis, angiogenesis, host-pathogen interactions and intracellular membrane trafficking events (Etienne-Manneville et al. 2002; Jaffe et al. 2005). Rho GTPases lead the way in almost every aspect of cell migration, and hence, play pivotal roles during embryonic development, immune responses and wound repair (Grose et al. 1999; Fenteany et al. 2004). Actin filaments are not the only cytoskeletal elements that can be regulated by Rho proteins; microtubules, and possibly also intermediate filaments, can be subjected to regulation by Rho-regulated pathways (Goto et al. 2000; Hollenbeck 2001; Wittmann et al. 2001; Chan et al. 2002; Kawauchi et al. 2008). Apart from their cytoskeletal functions, Rho GTPases also play roles in cell cycle progression, cell survival and gene transcription (Van Aelst et al. 1997; Villalonga et al. 2006).

The best-studied members of the Rho GTPases are RhoA, Rac1 and Cdc42, initially identified because of the profound and distinct effects they exert on the organization of the actin filament system (Hall 1998; Schmitz et al. 2000). However, Rho GTPases not belonging to the classical triad of

23 GTPases have started to attract attention. Some of them appear to have signaling properties and effects on the cytoskeleton that overlap with those of RhoA, Rac1 and Cdc42, whereas others give rise to novel responses (reviewed in Aspenström et al. 2004; Wennerberg et al. 2004; Aspenstrom et al. 2007). Rho GTPase signaling is complex and involves feedback loops, cooperative signaling pathways and intracellular compartmentalization. Crosstalk occurs between the different Rho GTPases as well as between Rho GTPases and other members of the Ras superfamily (Matozaki et al. 2000).

3.1 Rho GTPases in Actin Dynamics The initial studies on Rho GTPases showed that RhoA promotes assembly of contractile actin-myosin bundles (stress fibers) and formation of integrin based focal adhesions, Rac1 stimulates actin-rich sheet-like protrusions (lamellipodia) and Cdc42 gives rise to finger-like protrusions (filopodia) with parallel bundles of actin (Ridley et al. 1992; Ridley et al. 1992; Kozma et al. 1995; Nobes et al. 1995).

3.1.1 Cell Migration Cell migration is crucial for embryonic development, wound repair and inflammatory immune response (Ridley 2001). Cell migration also occurs in human diseases; in tumor metastasis, atherosclerosis and chronic inflammatory diseases, such as rheumatoid arthritis. The mechanism of cell crawling involves the formation of directed protrusions at the front, in combination with adhesion to the surrounding substrate, contraction of the cell body and detachment of the trailing edge. Each of these steps requires actin reorganizations mediated by Rho GTPases.

3.1.1.1 Protrusive Force Crucial for migration is protrusion at the front of a cell, driven by actin polymerization coupled to cell adhesion. Sheet-like extensions at the leading edge of a cell, known as lamellipodia, provide the driving force necessary for migration. Lamellipodia contain extensively branched filaments of actin. The force that pushes the plasma membrane forward is generated by treadmilling of the actin filament array. Lamellipodia formation is dependent on Arp2/3-based actin polymerization, mediated by Rac1 via its WASP-like verprolin-homologous protein (WAVE) effector (Hahne et al. 2001). Implicated in lamellipodia formation are also divergent Rho GTPase signaling pathways that act through LIM kinase to inhibit the actin depolymerizing factor cofilin, thereby leading to actin stabilization required for lamellipodia protrusion (Mouneimne et al. 2004; DesMarais et al. 2005; Huang et al. 2006). Inhibition of Cdc42 reduces lamellipodia (Kurokawa et al. 2004); however, a requirement of the Cdc42 effector neural Wiskott-

24 Aldrich syndrome protein (N-WASP) for lamellipodia formation is controversial (Kawamura et al. 2004; Sukumvanich et al. 2004; Innocenti et al. 2005). RhoG seems to contribute to the activation of Rac1 in migrating cells (Katoh et al. 2006).

3.1.1.2 Formation and Turnover of Cell-Substrate Adhesions A cell adheres to substratum at specific foci, where the cytoskeleton is linked via transmembrane receptors (integrins) to the extracellular matrix. Establishment of new adhesion sites at the leading edge as well as degradation of contacts at the rear of the cell is a prerequisite for cell migration.

Adhesion sites are initiated underneath lamellipodia and filopodia as focal complexes; structures that contain integrins and a few accessory proteins. Active Rac1 is required for the assembly of focal complexes but whether Rac1 is directly involved in this process, or focal complexes are unable to form in the absence of lamellipodia is not yet elucidated (Nobes et al. 1995; Allen et al. 1997; Rottner et al. 1999; Ridley 2001). Focal complexes either form and quickly dissolve or they persist and differentiate into larger and stable focal adhesions. RhoA controls the maturation of focal complexes into focal adhesions via its immediate effectors Rho-associated kinase (ROCK) and Dia1 (Ridley et al. 1992; Hotchin et al. 1995; Bershadsky et al. 2006). Dia1 plays a dual role in the regulation of focal adhesions, since it also coordinates focal adhesion turnover in the tail of a migrating cell (Bershadsky et al. 2006).

3.1.1.3 Cell Contraction Contraction of the cell body and retraction of the rear are required for movement. Stress fibers are the major contraction element in cells and they are formed of actin and myosin filaments. The sliding of myosin fibers along the actin filaments, which leads to a shortening of the stress fibers, generates the contractile force for cell movement. Stress fibers are actin- myosin bundles that attach to the plasma membrane at points of focal adhesions.

Rho activates ROCK that in turn phosphorylates myosin phosphatase. This results in reduced phosphorylation of myosin light chain (MLC) and thus an increased activation of the protein (Noda et al. 1995; Kimura et al. 1996; Hartshorne et al. 1998; Somlyo et al. 2000). In some cases, ROCK is capable of direct phosphorylation of MLC (Kureishi et al. 1997; Totsukawa et al. 2000). The net effect of MLC phosphorylation is cellular contractility and stress fiber formation, due to increased interaction between the MLC and actin filaments. The activity of Dia1 is also required for the formation of

25 stress fibers. This protein drives actin polymerization from the focal contacts (reviewed in Pellegrin et al. 2007).

3.1.1.4 Directional Sensing For directed movement, cells need to sense and respond to guidance cues in their surroundings. Filopodia, finger-like protrusions on the plasma membrane are considered to serve as sensory organelles, probing for the occurrence and concentration of chemoattractants. Filopodia have a core of parallel actin bundles. The mechanism by which filopodia detect the presence of effector molecules and transduce these signals to the cell body is not yet resolved. However, one interesting discovery is that epidermal growth factor receptors (EGFR) situated on filopodia undergo systematic retrograde transport after binding to EGF (Lidke et al. 2005). It seems likely that such retrograde transport in filopodia is a general mechanism by which cells can probe areas, far from the cell body, for the presence of effector molecules. For a long time, Cdc42 has been thought of as the main inducer of filopodia. However, Cdc42-depleted cells can still form filopodia (Czuchra et al. 2005). The Rho GTPases Rif, RhoD and Wrch1 are all capable to form filopodia (Paper I; Saras et al. 2004; Shutes et al. 2004; Pellegrin et al. 2005).

Cdc42 activates Arp2/3 actin polymerization, through its hematopoietic- specific effector WASP or the closely related ubiquitously expressed N- WASP (Stradal et al. 2006). How does activation of Cdc42 cause the unbranched, bundled filaments in filopodia? There are several mechanisms proposed. In one model, filopodia are formed by Arp2/3 complex-mediated nucleation, followed by rearrangements of the dendritic network into bundles (Svitkina et al. 2003; Korobova et al. 2008). However, experiments in Arp2/3-depleted cells have provided conflicting results concerning whether filopodia or only lamellipodia were affected (Di Nardo et al. 2005; Korobova et al. 2008; Steffen et al. 2006). An alternative model suggests that formins are solely responsible for filopodia induction (Faix et al. 2006).

The filopodia formed by different Rho GTPases differ in morphology. Cdc42-induced filopodia are short and thick cell extensions, originating from the lamellipodia. Such filopodia contain focal complexes and adhere to substratum (Pellegrin et al. 2005). Rif (Rho in filopodia), on the other hand, induces many long apical filopodia that lack focal complexes. The Rif- induced structures are mediated through actin polymerization by the formin protein Dia2 (Pellegrin et al. 2005). It is not clear whether Cdc42-induced filopodia-formation can be mediated by Dia.

26 3.1.2 Membrane Trafficking Actin remodeling is required in the majority of membrane trafficking events, including endocytosis, exocytosis and intracellular transport (Lanzetti 2007). For instance, during endocytosis, local actin assembly participates in the pinching off of vesicles and the movement away from the donor membrane. Cdc42 stimulates endo- and exocytosis via N-WASP (Symons et al. 2003; Bader et al. 2004; Gasman et al. 2004). Further, Cdc42 acts in Golgi to ER transport in an N-WASP dependent manner (Luna et al. 2002). The Cdc42-related proteins, TCL and TC10 are also implicated in membrane trafficking events. TCL regulates the movement of early endosomes (de Toledo et al. 2003), whereas TC10 has important functions in exocytosis where its GTP hydrolysis is supposed to promote vesicle fusion (de Toledo et al. 2003). Constitutively active RhoA and Rac1 mutants have been shown to inhibit clathrin-mediated endocytosis (Lamaze et al. 1996). Both RhoD and RhoB localize to endosomes and through recruitment of Dia1 and a Dia2 splice variant, respectively, they are hypothesized to induce actin coat assembly on the endosomes (Murphy et al. 1996; Ellis et al. 2000; Gasman et al. 2003; Fernandez-Borja et al. 2005). The presence of an actin coat promotes association of these vesicles with the actin cytoskeleton, thereby slowing endosomal dynamics. Through its effect on vesicle trafficking, RhoB may contribute to the regulation of receptor tyrosine kinases, such as EGF and PDGF, and their downstream signaling cascades (Gampel et al. 1999; Huang et al. 2007). RhoBTB1 and RhoBTB2 localize to vesicular structures, and have been suggested roles in microtubule-mediated transport (Chang et al. 2006). Finally, RhoG appears to regulate microtubule-based transport of lysosomes (Vignal et al. 2001).

3.2 Additional roles for Rho GTPases Beyond their roles in cytoskeletal rearrangements, Rho GTPases are vital regulators of a wide range of fundamental cellular functions. One important assignment is to control the activity of a variety of transcription factors such as SRF, c-Jun and NF- B (Coso et al. 1995; Hill et al. 1995; Minden et al. 1995; Perona et al. 1997). Moreover, Rho GTPases control cell cycle progression and growth (reviewed in Welsh 2004; Villalonga et al. 2006). In addition, Rho GTPases serve functions in cell survival, by either promoting or antagonizing apoptosis (Embade et al. 2000; Zhu et al. 2007).

Atypical Rho GTPases have structural and functional features very distinct from the other Rho GTPase members. For instance, the haematopoetic- specific RhoH contributes to thymocyte selection and T-cell receptor signaling (Dorn et al. 2007). Additionally, RhoH antagonizes the classical Rho GTPases by inhibiting Rho, Rac and Cdc42-dependent activation of

27 NF- B and p38-mitogen-activated protein kinase (p38MAPK) (Li et al. 2002). The newly identified RhoBTBs have atypical traits. RhoBTB2 is the best- characterized family member and appears to play roles in protein degradation where it forms a functional ubiquitin ligase complex together with cullin-3 (Wilkins et al. 2004). In addition to a GTPase domain, RhoBTB proteins encompass proline-rich motives and two so called Broad Compex/Tramtrack/Brick à brac (BTB) domains, a domain type found to mediate homomeric or heteromeric protein-protein interactions (Collins et al. 2001).

3.3 Rho GTPases in Diseases Aberrant Rho GTPase signaling can contribute to a wide variety of disorders, including cancer and neurodegenerative diseases (Boettner et al. 2002; Linseman et al. 2008). Oncogenic Ras leads to activation of Rho GTPases, which in turn activate a spectrum of functions that contribute at different stages of cancer development (Ridley 2004). Indeed, a synergistic participation of Rho GTPases is required for Ras-induced malignant transformation (reviewed in Bar-Sagi et al. 2000; Lambert et al. 2002; Giehl 2005). Abnormal signaling through Rho GTPases can be involved in tumor initiation and growth by promoting cell cycle progression (Sahai et al. 2001; Liberto et al. 2002). Furthermore, Rho GTPases mediate cytoskeletal rearrangements crucial for cell migration, a prerequisite for invasiveness and subsequent metastasis (Price et al. 2001). Rho GTPases can also encourage cell migration by other means, by stimulating downregulation of cell-cell adhesions and degeneration of extracellular matrix (through activation of matrix metalloproteinases) (Fukata et al. 2001; Zhuge et al. 2001).

Alterations of Rho GTPase expression have been observed in various human cancers, including those as genetically diverse as those occurring in breast, colon, lung and pancreas (reviewed in Lin et al. 2004; Ellenbroek et al. 2007). RhoA, RhoB, RhoC, Rac1, Rac3, RhoG and Cdc42 are overexpressed in breast tumors and RhoA overexpression is associated with advanced stages of the disease (Fritz et al. 1999; Fritz et al. 2002). RhoC is overexpressed in 90 % of inflammatory breast cancer (van Golen et al. 1999) and has been implicated to control production of angiogenic factors (van Golen et al. 2000). A splice variant of Rac1, Rac1b, is upregulated in some breast and colon carcinomas (Jordan et al. 1999; Schnelzer et al. 2000). This splice variant has elevated GTPase exchange activity and is unable to bind GDIs (Matos et al. 2003; Fiegen et al. 2004). RhoH is the only Rho GTPase that has been found genetically altered in cancers. Rearrangements and mutations of RhoH result in development of multiple myeloma, non-Hodkin’s lymphoma and diffuse large B-cell lymphoma (Sahai et al. 2002). The exact role of RhoH rearrangements in pathology has

28 not yet been elucidated. Finally, RhoBTB-2 and RhoB have tumor suppressor properties (Prendergast 2001; Hamaguchi et al. 2002; Huang et al. 2006; Sato et al. 2007).

The Rho GTPases, their regulators and effector molecules provide a large number of promising targets for therapeutic intervention. Specific inhibitors of individual proteins in the Rho GTPase pathways are predicted to have great therapeutic effects on a diverse array of diseases (Olson 2008).

4 The Miro GTPases The Miro GTPases (Mitochondrial Rho) were identified in a screen for new Ras-like GTPases (Paper II). Members of this subfamily are conserved between yeast and human. Two genes encoding Miro proteins exist in human: Miro1 and Miro2. The Miro proteins contain two GTPase domains that flank a pair of calcium binding EF-hands. The N-terminal GTPase domain shows similarity to Rho GTPases, and therefore the Miro GTPases were initially classified as Rho GTPases (Paper II). However, because of their distinct structural and functional properties the Miro proteins are now considered to constitute a separate subfamily (Wennerberg et al. 2004; Boureux et al. 2007). Miro proteins lack the Rho specific insert region, a unique feature that distinguishes the Rho family of proteins from their Ras relatives. Moreover, they lack Switch 2 domains (G3 motif) and are devoid of a CAAX-box. Instead of a CAAX-box, these proteins harbor a transmembrane domain in their C-terminus. The Miro proteins are inserted in the outer mitochondrial membrane by their transmembrane domain, exposing their other domains to the cytosol. The specific structure of Miro implies that these proteins are regulated in a distinct way compared to other Ras-like GTPases.

Proteins of the Miro family act as potent regulators of mitochondrial dynamics in several ways. Yeast Miro, Gem1p, is essential for the maintenance of tubular mitochondrial morphology and contributes to mitochondrial inheritance, a process depending on actin cables (Frederick et al. 2004; Frederick et al. 2008). Flies with homozygous dMiro mutations die due to numerous defects as larva or early pupa. In fly larvae lacking Miro, mitochondria in neurons and muscles are limited to the cell body and are depleted from axons and presynaptic terminals (Guo et al. 2005). In fly and human, Miro orthologs regulate mitochondrial movement along the microtubule system (Guo et al. 2005; Paper III). Drosophila Miro shows binding to an adaptor protein called Milton, which in turn forms a complex with kinesin heavy chain (Rice et al. 2006). Human Miro proteins interact with the Milton-related adaptor proteins OIP106 and GRIF1 (Paper III).

29 Miro and Milton-related proteins modulate motor protein attachment to mitochondria.

Ectopic expression of Miro in COS7 cells caused dramatic effects on the mitochondrial morphology (Paper I and Paper II). Miro1 expression evoked the formation of interconnected tubular mitochondrial networks. Strikingly, Miro1 or Miro2 constructs carrying a constitutively active mutation (Miro1/P13V; Miro2/A13V) in their N-terminal GTPase domain, caused perinuclear aggregations of mitochondria in a high percentage of cells. Hence, the activity of the N-terminal GTPase domain seems important for the function of Miro. To some extent, mitochondrial clustering could also be seen in cells expressing the corresponding dominant negative (T18N) Miro variants. Perhaps, the clustering can result from a traffic jam in organelle transport, which can occur at both high and low Miro activities.

New data demonstrate that proteins of the Miro subfamily can affect mitochondrial movement and morphology in a calcium dependent manner (G. Hajnóczky, personal communication). Miro overexpression, in H9c2 cells, enhanced mitochondrial movement at resting cytoplasmic calcium levels, whereas it promoted mitochondrial arrest at elevated calcium levels. For the arrest to happen, Miro with functional EF-hands and N-terminal GTPase domain was required. Possibly, calcium binding by the EF-hands of Miro triggers the release of GRIF/OIP106 from Miro, thus causing the calcium dependent arrest. In addition, Miro overexpression promoted mitochondrial tubulation at basal calcium concentrations in primary neurons, whereas it at high calcium levels caused enhanced mitochondrial fission. Interestingly, it was also uncovered that Miro proteins affect the mitochondrial calcium uptake, an effect that was independent of the calcium-binding activity.

5 Mitochondria Mitochondria are organelles of elaborate structure that play key roles in many fundamental biological processes. One chief assignment of these remarkable organelles is to convert energy from nutrient molecules into the energy-yielding molecule ATP that can be used to drive cellular reactions. Importantly, mitochondria play critical roles in calcium homeostasis and in apoptosis. Mitochondria contain DNA that code for a subset of the mitochondrial proteins but the main part of the mitochondrial proteins is coded for in the nucleus. The mitochondrial DNA is circular and resides in the matrix. Impaired mitochondrial function is implicated in a diverse array of diseases, as well as in aging (Beal 2007; Haas et al. 2007; Leuner et al. 2007; Marin-Garcia et al. 2008).

The mitochondria embrace two specialized membranes, one smooth outer membrane and one very convoluted inner membrane. The two membranes delimit the functionally distinct intermembrane space (the area between the outer and the inner membrane) and the matrix (the area encircled by the inner membrane). The inner membrane has numerous invaginations, or cristae. The cristae are connected to the intermembrane space by narrow tubular junctions. The cristae expand the surface area on which the ATP production takes place. Mitochondria in cells that consume high levels of energy, such as muscle cells, have more cristae than typical mitochondria (Vogell et al. 1963).

5.1 Mitochondrial Dynamics Mitochondria are remarkably dynamic organelles that constantly change their shape, number and distribution to fulfill various cellular needs. The shape of the mitochondria is cell-type dependent. Even in the same cell, mitochondria can display a range of morphologies, from small round entities to highly elongated tubules. While most fibroblasts exhibit tubular and interconnected mitochondrial networks (Amchenkova et al. 1988), mitochondria are densely arranged along the myofibrils in heart and skeletal muscle cells (Segretain et al. 1981) and wrapped around the base of the flagella in sperm cells (Cardullo et al. 1991).

Mitochondria are actively transported around the cell along the cytoskeleton to subcellular locations where energy or calcium buffering is required. The mitochondria morphology is controlled by continual cycles of precisely regulated fusion and fission. Exchange of genomes between fusing mitochondria may be an efficient way to compensate for defects caused by mitochondrial DNA (mtDNA) mutations (Nakada et al. 2001). Highly elongated mitochondrial network may represent a transport system that, for instance, enables rapid transmission of membrane potential significant distances across the cell (Skulachev 2001). Fission allows mitochondria to perform discrete functions. Extensive mitochondrial fragmentation is an event in many apoptotic responses as well as in hyperglycemic conditions (Frank et al. 2001; Yu et al. 2006). During cell division, tubular mitochondria have been described to fragment in early mitotic phase in several cell lines (Barni et al. 1996; Margineantu et al. 2002). In the daughter cells, mitochondria fuse to form tubes again.

Mitochondria cannot be synthesized de novo but are generated by division. Old mitochondria are autophagocytosed by lysosomes in a process for long assumed to be random. However, increasing evidence indicates that

31 autophagocytosis of mitochondria can occur through a selective process called mitophagy (Kim et al. 2007; Kissova et al. 2007).

Efficient mitochondrial trafficking is particularly important in neurons, where mitochondria are obligated to travel considerable distances along axons to supply synaptic endings with energy, needed for neurotransmitter release and recycling (Chen et al. 2006). Mitochondria further play an important role in the regulation of neuronal calcium homeostasis (Wang et al. 2003). Mitochondria undergo fast anterograde transport along parallel microtubules whose plus-ends are directed toward the axon terminal. Derailed mitochondrial dynamics is correlated with several neurodegenerative diseases (reviewed in Chang et al. 2006). Mitochondria with high membrane potential are transported in the direction of the growth cone, whereas old or damaged mitochondria with low membrane potential move in the direction of the cell body (Miller et al. 2004).

5.1.1 Mitochondrial Transport Mitochondria are anchored and transported along cytoskeletal elements. Long and rapid transmission of mitochondria in animal cells is microtubule- based (Couchman et al. 1982; Morris et al. 1995; Rube et al. 2004), whereas actin serves as tracks for short range transport of mitochondria to areas where the microtubules do not reach. During stationary periods, the mitochondria have been found docked to actin. The mitochondrial transport system differs between species. In plants and in certain fungi, including the budding yeast Saccharomyces cerevisiae, the transport of mitochondria is generally mediated by actin filaments. In the fission yeast Schizosaccharomyces pombe, on the contrary, mitochondrial mobility appears to be motor independent and driven by microtubule polymerization (Yaffe et al. 2003). Merging evidence implicates that also intermediate filaments can associate with mitochondria.

5.1.1.1 Microtubule-Based Transport Different cargos are transported along the microtubule system with different motors. Members of two kinesin families: kinesin-1 (KIF5B) and kinesin-3 (KIF1B) have been implicated in anterograde transport of mitochondria in mammals (Nangaku et al. 1994; Tanaka et al. 1998). Dynein is the driving force of retrograde mitochondrial transport. Interestingly, kinesin-1 is critical for the retrograde dynein-driven transport of mitochondria. Further, inhibition of the dynein motor impairs minus end-directed movement as well as plus end-directed movement of mitochondria (Varadi et al. 2004). This cooperative regulation of the kinesin and dynein activities proposes that the two motors may bind together in a large motor complex (Varadi et al. 2004). Several adaptor proteins, linking mitochondria to microtubule motors, have been revealed during the last couple of years.

One big step toward a revelation of the mechanism underlying microtubule- based mitochondrial movement was the demonstration of a direct interaction in fly between Milton and the mitochondrial-anchored dMiro (Giot et al. 2003; Glater et al. 2006). Milton is a Drosophila protein required for mitochondrial transport to nerve terminals, which does not link directly to mitochondria. Milton interacts with the kinesin heavy chain of a kinesin-3 member (KIF1B) (Stowers et al. 2002). This represents a novel type of cargo binding, as kinesins typically bind to cargo by their light chains. The transport with Milton seems to be kinesin light chain-independent (Glater et al. 2006; Rice et al. 2006). Milton has several isoforms and it is suggested that Milton might exert isoform-specific effects on mitochondrial transport (Cox et al. 2006). Whereas one major isoform promotes plus end directed kinesin-mediated transport, another isoform inhibits kinesin-linkage to mitochondria. The latter Milton variant may instead mediate minus-directed transport. The Miro and Milton adaptor complex may determine which motor is active. The closest Milton-related proteins in human are GRIF1 and OIP106. GRIF1 and OIP106 have been reported to interact with kinesin-1 family members, and to indirectly couple to mitochondria (Brickley et al. 2005). Both human Miro1 and Miro2 bind to GRIF1 and OIP106 (Paper III). The mitochondrial transport along microtubules is depicted in Figure 3.

The axonal transport of mitochondria has been shown in several cell types to be regulated by calcium, where maximal movement is obtained at resting calcium concentration and where physiological rises of calcium concentration arrest mitochondria (Rintoul et al. 2003; Yi et al. 2004; Brough et al. 2005; Quintana et al. 2006). Calcium is a universal intracellular messenger and, hence, an essential regulator of many cellular processes that demand energy. Mitochondria recruitment to such areas is necessary to enhance local energy supply and calcium buffering. Intriguingly, Miro mediates the calcium dependent arrest of mitochondria (G. Hajnóczky, personal communication). To mediate the mobility inhibition, Miro is depending on functional EF-hands.

Mitochondrion

Miro GRIF-1 / OIP106

Kinesin heavy chain Anterograde transport to nerve terminals Microtubule -+

Figure 3. Long-range transport of mitochondria occurs along the microtubule system. Miro and GRIF1/OIP106 proteins mediate microtubule-motor attachment to mitochondria. Here is anterograde transport with kinesin heavy chain depicted; however, Miro and GRIF/OIP106 may in addition act in retrograde transport. Miro proteins are integral outer mitochondrial proteins, with two GTPase domains and two EF-hand motifs. At elevated calcium levels, Miro GTPases govern mitochondrial arrest and in this process, utilize their EF-hands.

Although Miro and the Milton-related proteins seem to form a key-universal adaptor complex linking motors to mitochondria, other adaptor complexes might exist. A mitochondria-specific isoform of Kinectin, a protein known to link kinesin to ER, has been identified (Santama et al. 2004). Furthermore, recent evidence indicates that the mitochondria-targeted protein Syntabulin has affinity for the heavy chain of the kinesin-1 family member KIF5B (Cai et al. 2005). Lastly, a fly protein called APLIP (Drosophila JNK-interacting protein-1 (JIP1)) has been proposed to be involved in retrograde transport of mitochondria (Horiuchi et al. 2005). JIPs have earlier been shown to attach certain cargoes, such as vesicles, to kinesin-1 light chain (Verhey et al. 2001). JIPs are scaffolding proteins for JNK pathway kinases, which are not simple hitchhikers but actively regulate the cargo/JIP1/kinesin-1 interaction. JIPs alone can not activate motors but when interacting with the brain specific fasciculation and elongation protein zeta-1 (FEZ1) they efficiently activate kinesin motor activity (Blasius et al. 2007).

Microtubule-associated proteins (MAPs) can influence mitochondrial transport. MAPs compete with the binding of the motors to the microtubules and depending on which MAP that is involved, anterograde or retrograde transport can be affected (Jimenez-Mateos et al. 2006). The level of the membrane lipid phosphatidyl inositol 4,5-biphosphate (PtdIns(4,5)P2) has been shown to regulate the balance between plus end and minus end directed transport, where sequestration of (PtdIns(4,5)P2) by pleckstrin homology domains enhanced anterograde transport (De Vos et al. 2003). Mitochondria moving to the axon terminal have higher membrane potential than those in retrograde transport. Interestingly, a Drosophila dynein light chain, Tctex, appears to associate with mitochondria through the Voltage- dependent anion-selective channel (VDAC) (Schwarzer et al. 2002). VDAC is a pore forming protein in the outer mitochondrial membrane, which adopts an open state when the membrane potential is low or zero and a closed conformation when the membrane potential is high (for recent VDAC reviews, see Shoshan-Barmatz et al. 2006; Lemasters 2007). Dynein may prefer the closed conformation of VDAC and thereby enable the elevated retrograde transport of mitochondria with low respiration.

Stationary mitochondria have previously been reported anchored to actin filaments (Chada et al. 2004). However, a recent finding indicates that also the microtubule system may serve functions in mitochondrial docking (Kang et al. 2008). Within axons, a neuron specific protein called Syntaphilin that resides in the outer mitochondrial membrane docks mitochondria through an interaction with microtubules.

5.1.1.2 Actin in Transport and Docking In humans, short range movement of mitochondria depends on actin, which allows the organelles to reach areas in the cell that lack microtubules (Morris et al. 1995; Ligon et al. 2000; Langford 2002; Hollenbeck et al. 2005). Translocation along actin is probably achieved by myosin V motors in higher eukaryotes (Langford 2002). In addition, actin is proposed to serve a function in tethering mitochondria at their sites of action (Chada et al. 2004).

In S. cerevisiae, mitochondrial locomotion is highly actin-based. In dividing yeast cells, mitochondria are actively transferred to the emerging daughter cell along actin cables. Two mechanisms for movement have been proposed. In the first model, mitochondrial movement is brought about by a similar, but not identical, mechanism to that used by bacterial pathogens for propulsion in the infected host cell. Like pathogens, yeast mitochondria are proposed to be reliant on interactions with the actin nucleating enzyme Arp2/3, whose actin nucleation creates the force for locomotion

35 (Fehrenbacher et al. 2005). Whereas the movement of pathogens is randomly directed, mitochondria associate with actin cables for goal- directed movement. Three proteins situated on the outer mitochondrial membrane, Mdm10p, Mdm12p and Mmm1p, form a protein-complex referred to as the mitochore, that mediates ATP-dependent binding to actin filaments (Boldogh et al. 2003). According to the working hypothesis, the two protein complexes act together: the Arp2/3 generates the force for locomotion and the mitochore ensures directed movement through cyclic interaction with an actin filament. Higher eukaryotes lack homologues of the mitochore components. The second model for mitochondrial transport in yeast involves association with a motor (Hollenbeck 1996; Hollenbeck et al. 2005). Two proteins, a Rab GTPase, Ypt11p, and a peripheral outer mitochondrial protein, Mmr1p, can affect mitochondrial inheritance in a myosin V (Myo2p)-dependent manner (Itoh et al. 2004). Interestingly, recent results show that yeast Miro contributes to the Myo2p-dependent mitochondrial inheritance, independently of Ypt11p and Mmr1p (Frederick et al. 2008).

5.1.1.3 Links between Intermediate Filaments and Mitochondria A growing body of evidence indicates that intermediate filaments (IF) contribute in mitochondria support. Neurofilaments, neuronal IFs with stabilizing functions in axons, have been found intimately associated with mitochondria (Berezovskaya et al. 1995). Notably, mitochondria with high membrane potential showed elevated interaction to the neurofilaments (Wagner et al. 2003). Moreover, the ubiquitous IF member vimentin appears to play a role in mitochondria-microtubule interaction (Tang et al. 2007). Interestingly, deletion of the heart and muscle relevant IF protein, desmin, inhibits kinesin-1 recognition of mitochondria in heart muscle (Linden et al. 2001). One possibility is that IFs influence mitochondrial distribution through affecting microtubule motors (Boldogh et al. 2007).

5.1.2 Mitochondrial Fusion Mitochondrial fusion is an intricate matter that requires joining of both outer and inner membrane in a coordinated manner. During this process, the membrane potential has to be retained.

The first identified mediator of mitochondrial fusion is the Drosophila Fuzzy onion protein (Fzo) (Hales et al. 1997). This protein was found to be vital for Drosophila spermatogenesis where the mitochondria normally fuse into two giant mitochondria that coil around each other in a structure that resembles an onion slice when viewed in cross section. In Fzo mutants, fusion into the two giant mitochondria fails and instead many small mitochondria form a shape of a fuzzy onion. Fzo mutants are male sterile. Fzo is the founding member of the evolutionary conserved mitofusin family.

36 The mitofusin members are dynamin-related proteins that span the outer membrane twice, exposing a GTPase domain and two coiled-coil domains to the cytosol, and leaving a short loop in the intermembrane space. The mammalian counterparts of Fzo, Mitofusin1 and 2 (Mfn1 and Mfn2) appear to have both redundant and distinct functions in promoting mitochondrial fusion (Chen et al. 2003). Prior to fusion, Mfns on adjacent mitochondria dimerize by assembling an anti-parallel coiled-coil, thereby allowing mitochondrial tethering. Mfn1 and Mfn2 can assemble into hetero- or homooligomers. The GTPase domain plays a vital but unclear role in fusion. The Mfn1 is more efficient in tethering and has a higher GTPase hydrolysis activity than Mfn2 (Ishihara et al. 2004). Mfn2 appears to possess signaling functions that extends beyond fusion, such as in mitochondrial metabolism (Yu et al. 2006), cell-cycle progression (Linseman et al. 2008) and apoptosis (Neuspiel et al. 2005).

An intra-mitochondrial dynamin-like GTPase, Optic Atrophy 1 (Opa1) cooperates with the Mfn proteins in mitochondrial fusion (Cipolat et al. 2004). Opa1 is a bifunctional protein that independently from its function in fusion regulates inner membrane remodeling. Opa1 has at least eight splice variants, and each variant is found as several proteolytically processed isoforms. A combination of long and short Opa1 isoforms seems important for the fusion activity (Song et al. 2007). The metabolic status of mitochondria influences the Opa1 processing. Dissipation of membrane potential triggers several proteases to cut long Opa1 isoforms into short, thereby reducing the ability of Opa1 to act in mitochondrial fusion. Opa1 appears to shape the cristae and control the cristae junction openings by forming oligomeric complexes between inner membrane-anchored Opa1 and shorter forms of the same protein found in the intermembrane space (Frezza et al. 2006). Keeping tight the cristae, Opa1 sequesters most of the cell’s arsenal of the electron carrier cytochrome c in the cristae folds. In addition to Opa1, another inner membrane protein, Mitofilin, has been implicated in the control of inner membrane morphology (Odgren et al. 1996; Gieffers et al. 1997; John et al. 2005). Loss of Mitofilin results in changed cristae architecture and altered metabolism.

In yeast, the Opa1 ortholog, Mgm1p, is physically linked to the mitofusin Fzo1p, via a protein called Ugo1p (Sesaki et al. 2001; Wong et al. 2003; Sesaki et al. 2004). Through this association, the behavior of the inner and outer membrane during fusion might be coordinated. No human equivalent to Ugo1p has yet been identified. A cartoon of central components of mitochondrial fusion is presented in Figure 4. Illustrated in the picture is also Opa1, controlling the cristae junction openings.

Mitofusin GTPase domain

Coiled-coil

Membrane-bound Mitofusin Opa1 Opa1

Soluble Opa1

Cross-linker Cristae

Cytochrome c

IM Inner mitochondrial membrane

OM Outer mitochondrial OM IM Membrane

Figure 4. Mitochondrial fusion consists of outer membrane fusion followed by inner membrane fusion. Mfns and Opa1 act together in mitochondrial fusion. Mfns span the outer mitochondrial membrane twice, exposing their GTPase domain and two coiled-coil regions to the cytosol. The C-terminal coiled-coil mediates oligomerization between Mfns on adjacent mitochondria. The GTPase activity of Mfns has important but unclear function in fusion. Opa1 resides in the intermembrane space, where long isoforms are anchored to the inner membrane. A combination of long and short Opa1 isoforms is required for membrane fusion. In yeast, a protein called Ugo1p physically link the Mfn and Opa1 orthologues and most likely coordinates their function. No mammalian Ugo1p ortholog has yet been identified. In addition to inner membrane fusion, Opa1 controls the cristae junction openings by forming oligomeric complexes between inner membrane-anchored Opa1 and soluble forms of the same protein located in the intermembrane space. During healthy conditions, Opa1 sequesters cytochrome c in the cristae folds (Frezza et al. 2006).

The local lipid environment appears to play a role in mitochondrial fusion. A new and exciting discovery is that the generation of phosphatidic acid by a novel member of the phospholipase D (PLD) superfamily, is required for mitochondrial fusion (Choi et al. 2006). In many types of exocytosis, fusion of vesicles into target membranes requires PLD-generated phosphatidic acid (Vitale et al. 2001). Thus, mitochondrial fusion might have more in common with other membrane fusion events than was earlier believed to be the case. Mitochondrial PLD (MitoPLD) resides in the outer mitochondrial membrane where it hydrolyzes cardiolipin to produce phosphatidic acid.

38 This acid seems to act downstream of Mitofusin-mediated tethering, but the precise action remains to be determined. Based on its function in other types of membrane fusion, phosphatidic acid may activate or recruit fusion proteins or locally altering the curvature of opposing membranes in a way that facilitates fusion. Cardiolipin is mainly present in the inner mitochondrial membrane (Daum 1985). However, at contact sites between the inner and outer membrane, cardiolipin has been seen transduced from the inner membrane to the outer membrane (Ardail et al. 1990; Simbeni et al. 1991). Perhaps PLD restricts fusion to contact sites and thereby allows membrane fusion of the two membranes to occur in a coordinated way (Choi et al. 2006).

5.1.3 Mitochondrial Fission Similar to mitochondrial fusion, mitochondrial division requires a dynamin- related protein, designated Dynamin-related protein-1 (Drp1) in mammals and Dnm1p in yeast. Drp1/Dnm1p is recruited from the cytoplasm to fission foci, by the outer mitochondrial membrane integral Fis1 receptor. In yeast, Fis1p interacts with Dnm1p via either of the molecular adaptors Mdv1p or Caf4p (Cerveny et al. 2003; Yoon et al. 2003; Zhang et al. 2007). No human proteins similar to Caf4p or Mdv1p have been identified so far. Drp1 assembles into oligomeric spirals, wrapping around mitochondria. In a manner analogous to dynamin during endocytosis, the Drp1 complex is suggested to constrict and eventually divide mitochondria. Drp1 participates in the fission of ER and peroxisomes as well. How is fission of the inner membrane regulated? This is an unresolved question. A yeast protein called Mdm33p has been proposed to be involved, but the mechanism underlying the action of this protein remains enigmatic (Messerschmitt et al. 2003). Core components of mitochondrial division are illustrated in Figure 5.

39 a b

Drp1

Fis 1

Figure 5. (a) In fission, a dynamin-related protein, Drp1, is recruited from the cytosol to a mitochondrion, where it is proposed to oligomerize into ring- or spiral- like structures that constrict and eventually cleave the mitochondrion. In this model, Drp1 acts in a similar manner to the way dynamin functions during endocytosis. (b) Drp1 is linked to mitochondria by the integral outer membrane protein Fis1. The Drp1 ortholog in yeast, Dnm1p, is linked to Fis1p via either of the Mdv1p/Caf4p adaptor proteins. No functional mammalian homologue of Mdv1p or Caf4p has been revealed so far. OM = outer mitochondrial membrane, IM = inner mitochondrial membrane.

In addition to dynamin, two other components of the endocytic machinery, endophilin and synaptojanin, have variants that seem to take part in mitochondria fission. Recruitment of endophilin B1 has been suggested to be involved in outer mitochondrial membrane remodeling (Karbowski et al. 2004). During endocytosis, endophilins use their N-BAR domain to induce a curvature on membranes (Gallop et al. 2006). An isoform of synaptojanin, synaptojanin 2A, causes distinct effects on the mitochondrial morphology (Nemoto et al. 1999). In the formation of endocytic vesicles, synaptojanin act together with dynamin and endophilin I (Schuske et al. 2003).

Apart from their function in mitochondrial movement, Miro proteins can influence fission-fusion dynamics (Paper III; G. Hajnóczky, personal communication). At resting calcium concentrations, co-transfected Miro1 2 promoted mitochondrial fusion in neuronal processes, whereas Miro1 2 at elevated calcium levels promoted mitochondrial fission (G. Hajnóczky, personal communication). This bidirectional calcium-dependent control by Miro was proposed to be mediated through Drp1 suppression and activation, respectively.

40 5.2 Mitochondria-Shaping Proteins in Cell Death Mitochondria-shaping proteins, including Opa1, Drp1 and Mfn2, interplay with the regulators of cell death. A hallmark of classical apoptosis is mitochondrial release of cytochrome c, which occurs through a two step process (Scorrano et al. 2002). The first step involves remodeling of the inner mitochondrial membrane, unravelling the cristae and thereby freeing cytochrome c to enter the intermembrane space. The second step is the release of cytochrome c into the cytosol via pores in the outer mitochondrial membrane. During healthy conditions, Opa1 sequesters cytochrome c in the cristae folds (Frezza et al. 2006). After apoptogenic signals (such as cBid treatment) the Opa1 oligomers dissociate, freeing cytochrome c to the intermembrane space. The mechanism underlying the Opa1 release remains an open question. Upon commitment to apoptosis, the pro-apoptotic Bcl-2 (B-cell lymphoma 2) members Bax and Bak translocate to mitochondria to form pores in the outer mitochondrial membrane. Close in time to the coalescence of Bax and Bak on the mitochondrial surface, excessive Drp1- dependent fragmentation of mitochondria occurs in many cases of cells undergoing apoptosis (for review, see Heath-Engel et al. 2006). In response to some pro-apoptotic stimuli, functional Drp1 is a prerequisite for propagation of the cell death signal (Frank et al. 2001; Lee et al. 2004; Estaquier et al. 2007). Interestingly, Drp1 has been implicated in inner membrane remodeling during apoptosis (Germain et al. 2005). Consequently, the pro-apoptotic role of Drp1 may be unrelated to its function in mitochondrial fission, and instead reside in its ability to remodel both the inner and outer mitochondrial membrane to promote cytochrome c release (Wasiak et al. 2007). Recent data suggest that Bax and Bak cause sumoylation of Drp1 and irreversible lock it on mitochondrial membranes. Bak can also interact with Mfn2 during apoptosis, probably to inhibit fusion (Yonashiro et al. 2006). In healthy cells, Bax is suggested to activate Mfn2, whereas after induction of apoptosis Bax is likely to adopt a conformation that instead is able to interact with Drp1 (Karbowski et al. 2006).

In contrast to the apoptosis-promoting function of Drp1 in response to specific lethal stimuli, Drp1-mediated mitochondrial fragmentation can inhibit apoptosis in response to a particular range of lethal stimuli that rely on intramitochondrial propagation of calcium waves (Szabadkai et al. 2004; Perfettini et al. 2005).

5.3 Mitochondrial Dynamics in Disease The dynamic properties of mitochondria are essential for mammalian development, normal neurological functions and apoptosis (reviewed in Detmer et al. 2007).

Disturbed mitochondrial dynamics during development can be fatal. Flies that lack either dMiro or Milton die during larval stages and mice that lack Mfn1, Mfn2 or Opa1 do not survive midgestation (Stowers et al. 2002; Chen et al. 2003; Guo et al. 2005; Alavi et al. 2007; Davies et al. 2007). Disturbed mitochondrial fission can also cause severe effects. Drp1 deprivation in Drosophila is semilethal, where the survivors have coordination problems and suffer from neurodegeneration (Verstreken et al. 2005). An infant with a dominant negative allele in Drp1 was recently reported (Waterham et al. 2007). This child suffered from a metabolic syndrome, reduced head growth and optic atrophy and died at 1 month of age.

Drosophila neurons that lack Miro or Milton fail to populate their synapses with mitochondria and show reduced synaptic transmission due to the distribution defects (Stowers et al. 2002; Guo et al. 2005). Moreover, Drosophila with homozygous loss-of-function mutations in Drp1 exhibit reduced mitochondrial transport to synapses (Verstreken et al. 2005). Inefficient transport of hyperfused mitochondria can possibly explain why a lack of the fission protein Drp1 results in disturbed mitochondrial distribution (Verstreken et al. 2005 and others).

Over time, even subtle perturbations of mitochondrial dynamics can give rise to severe effects in neurons. Several neurodegenerative diseases can be caused by inherited mutations in genes coding for mitochondrial dynamics proteins. Haploinsufficiency of either Mfn2 or the mitochondria-specific kinesin KIF1B cause Charcot-Marie-Tooth disease, the most commonly inherited neurological disorder. Charcot-Marie-Tooth patients gradually loose normal function of their hands/arms and feet/legs as nerves to the extremities degenerate (Zhao et al. 2001; Zuchner et al. 2004). Mutations in Ganglioside-induced differentiation-associated protein 1 (GDAP1), a protein that is suggested to play a role in mitochondrial fission, has also been linked to Charcot-Marie-Tooth disease (Cuesta et al. 2002; Niemann et al. 2005). Moreover, haploinsufficiency of Opa1 is the prevailing cause of autosomal dominant optic atrophy (ADOA), a heritable disease in which progressive degeneration of retinal ganglion cells ultimately results in blindness (Alexander et al. 2000).

Many lines of evidence suggest a central role of mitochondrial dysfunction in late-onset neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS), Huntington’s, Parkinson’s and Alzheimer’s (Lin et al. 2006). Impaired mitochondrial function can result in reduced energy supply, defective calcium-buffering, deregulated apoptosis and increased production of reactive oxygen species (ROS), effects that all theoretically could contribute to the progressive decline of neurons. ROS is a toxic by-product

42 of mitochondrial oxidative phosphorylation that can induce oxidative damage on cellular components (reviewed in Ott et al. 2007).

Accumulating data indicate that also aberrant mitochondrial dynamics can contribute to the pathogenesis of late-onset neurodegenerative conditions. For instance, derailed mitochondrial dynamics might be an initiating cause of Parkinson’s disease. It is well-known that both familial and sporadic types of Parkinsonism can be cased by loss-of-function mutations in the PTEN-induced kinase 1 (Pink1) or Parkin genes, which encode a mitochondrially localized serine/threonine kinase and a ubiquitin ligase, respectively (Kitada et al. 1998; Valente et al. 2004). According to a recent finding, Pink1 acts upstream of Parkin to stimulate mitochondrial fission, possibly though the activation of Drp1 (Poole et al. 2008). Furthermore, defects in mitochondrial trafficking appear to be an early event in Alzheimer’s disease. Small proteins called beta-amyloid aggregate in the brain of a person with Alzheimer’s disease. Interestingly, beta-amyloid was recently found to cause mitochondrial transport inhibition through signaling via GSK3 (Rui et al. 2006), a protein that earlier has been proposed to promote mitochondrial docking by phosphorylation of motor proteins (Morfini et al. 2002; Chen et al. 2007). Finally, in Huntington’s disease aggregated abnormal proteins in neuronal processes have been shown to physically block axonal transport of mitochondria and other cellular cargoes (Chang et al. 2006).

Understanding the regulation of mitochondrial movement may provide novel insights into treatment of several neurodegenerative diseases. Moreover, some mitochondrial dynamics proteins play important roles during apoptosis, and hence, are attractive targets to modulate cell death in cancer cells (Cereghetti et al. 2006).

43 PRESENT INVESTIGATION

Aims I. To compare all Rho GTPases effects on the actin cytoskeleton.

II. To explore functions of the novel Miro GTPases.

III. To examine the Miro proteins roles in mitochondrial dynamics.

Paper I: Rho GTPases have Diverse Effects on the Organization of the Actin Filament System In an effort to compare the effects of all Rho GTPase members in the same cell system, we transfected constitutively active Rho GTPases in porcine aortic endothelial (PAE) cells and examined their effects on the organization of the actin cytoskeleton.

In agreement with earlier findings, we found that active Rac members induced lamellipodia. In addition, Cdc42 and its close relative TCL triggered lamellipodia formation. The lamellae differed in shape, depending on which Rho GTPase that was expressed. Cdc42 is famous for its role in filopodia formation. In the present study, active Cdc42 induced lamellipodia rather than filopodia. However, cells transfected with Cdc42L28, a mutant that spontaneously undergoes GDP/GTP exchange, exhibited filopodia. Apparently, the GTPase cycling ability of Cdc42 is important for filopodia induction to occur.

Rho GTPases produce a variety of cell protrusions. Cells expressing either Rif or RhoD had long filopodia. Expression of Rnd proteins resulted in loss of the majority of cytoplasmic actin filaments and led to the formation of microvilli-like extensions on the dorsal side of the cell. Moreover, Wrch1 expressing cells had an extremely spiky phenotype. The several different categories of protrusions are likely to have specific functions, and each type requires the activities of distinct Rho GTPases.

44 RhoA-C are well known to trigger stress fiber formation. Active RhoA, RhoB and RhoC evoked a similar response in the assembly of filament bundles. Interestingly, other Rho GTPases could also give rise to actin bundles. Cells expressing active forms of Cdc42, TCL and Rac1-3 contained thick actin bundles. The actin bundles assembled in response to Cdc42, Rac1 and Rac2 were much thicker than classical stress fibers.

We next examined the Rho GTPases effects on the formation of cell-matrix adhesion structures. Cdc42 and Rac are known to regulate focal complexes, whereas Rho mediates maturation of focal complexes into focal adhesions. In line with this, active Cdc42, Rac1, Rac2, Rac3 and RhoG caused focal complexes at the leading edge of the cell, whereas cells expressing active RhoA-C had prominent focal adhesions and were small and up-rounded. In addition to Rho, also TCL, TC10 and Chp could induce the assembly of focal adhesions.

In the second part of the paper, we turned our focus to the Rho GTPase effectors. The activities of RhoA, Rac1 and Cdc42 have commonly been examined in GST pull-down assays with the Rho GTPase interacting regions of the effectors PAK1, WASP and Rhotekin, respectively. Here, we demonstrate that these effectors have a broader specificity than previously thought. Finally, we tested the binding of known Cdc42 effectors to four additional members of the Cdc42 subfamily in a yeast two-hybrid system experiment. In this assay, N-WASP was found to interact with TC10, TCL and Chp, whereas WASP bound only to TCL.

This is the first time the effects of all Rho GTPases have been compared side-by-side in the same cell type. We found overlapping and distinct functions of the different members.

Paper II: Atypical Rho GTPases have Roles in Mitochondrial Homeostasis and Apoptosis In the present study, we began a characterization of two novel human GTPases that we named Miro1 and Miro2 (for mitochondrial Rho). The Miro proteins are 60 % identical to each other and differ from the classical Rho GTPases in being much larger and in enclosing additional domains. In their N-terminal part, these proteins possess a GTPase domain related to the Rho GTPases. Intriguingly, the Miro proteins contain an additional GTPase domain, without homology to the Rho GTPases, in their C-terminal part. Intermingled between the two GTPase domains are two EF-hands, motifs

45 that confer calcium binding. Furthermore, the Miro proteins are devoid of the CAAX-box, a motif usually present in GTPases, which after posttranslational modification confers membrane targeting of the protein. Miro- like proteins are present in many eukaryotic organisms, indicating that Miro evolved early in evolution. Northern blot analysis revealed that mRNA of both Miro1 and Miro2 were present in most human tissues.

Many Rho GTPases play key roles in the organization of the cytoskeleton. We found that neither wild-type Miro1 nor Miro2 visibly affected the actin filament system or the micotubule system. Endogenous as well as ectopically expressed Miro localized to the mitochondria in both NIH3T3 cells and COS7 cells. In analogy with mutations employed for studies of other Rho GTPases, we mutated the Miro proteins in their N-terminal GTPase domain to attain constitutively active (Miro1/P13V; Miro2/A13V) and dominant-negative (Miro/T18N) forms. Transient transfections with Miro1/P13V caused collapse of the mitochondrial network in many transfected cells, and instead mitochondria were organized in large perinuclear assemblies. Most cells transiently transfected with Miro1/T18N exhibited a normal mitochondrial network but in some cases collapsed mitochondria were seen also under this condition. Miro1/P13V was present in the mitochondrial assemblies, but some protein was dispersed into the cytoplasm. We further demonstrated that an increased number of cells with cytoplasmic cytochrome c staining was seen 48 h after transfection. Since mitochondrial leakage of cytochrome c is a hallmark of apoptosis we stained Miro-transfected cells with the apoptotic marker M30. The Miro1/P13V population exhibited an increased apoptotic rate in comparison to cells expressing Miro1 wild-type or Miro1/T18N. By using caspase inhibitors we showed that this apoptosis was dependent on the caspase cascade.

To summarize, we identified an evolutionarily conserved family of Rho GTPases, Miro, which is a constituent of mitochondria and has a role in mitochondrial homeostasis and apoptosis. The GTP/GDP loaded status of the N-terminal GTPase domain of Miro1 is important for the heterogeneity of the mitochondrial network, and hence, mitochondrial homeostasis.

Paper III: The Atypical Rho GTPases Miro-1 and Miro- 2 have Essential Roles in Mitochondrial Trafficking In our previous study of Miro, we demonstrated that the Miro proteins affect the mitochondrial morphology and distribution (Paper II). The ambition in

46 the present work is to further explore the function of Miro in mitochondrial dynamics.

We set out to examine the importance of the separate domains of Miro for its effect on the mitochondrial morphology. For this, we used a variety of Miro variants, with mutations in the different domains, and analysed their effects on mitochondrial morphology in COS7 cells. Ectopic expression of Miro1/P13V frequently caused mitochondrial clustering close to the nucleus. In addition, this mutant could give rise to extremely elongated mitochondria. Often the two responses were present in the same cell. The formation of elongated mitochondria required Miro1 with functional N- terminal GTPase domain and EF-hand motifs. Miro2 overexpression could cause mitochondrial aggregation but never the formation of thread-like mitochondria. For neither Miro1 nor Miro2 the GTP-binding status of the C- terminal GTPase domain influenced the mitochondrial morphology.

Next we asked how the Miro proteins target to the mitochondria. We deleted the predicted transmembrane regions in wild-type Miro1 and Miro2, and ectopically expressed these mutated forms of Miro in COS7 cells. We found that the mitochondrial localization was lost and instead the mutated Miro proteins were dispersed all over the cytoplasm. Furthermore, ectopically expressed Miro1/P13V, mutated to lack the predicted transmembrane region, localized evenly throughout the cytoplasm and did not cause any aggregation of mitochondria. These observations demonstrate that Miro proteins are tethered by their transmembrane domain to the outer mitochondrial membrane and that mitochondrial-targeting is essential for the function of Miro.

When we initiated this work, a fly protein called Milton had been identified as a Miro-binding partner in a large-scale two-hybrid screen for proteins expressed in Drosophila (Giot et al. 2003). Milton was known to serve as a link between microtubule motors and mitochondria, but to be unable to bind mitochondria directly. We found that both Miro1 and Miro2 interact with the mammalian Milton-related proteins, GRIF1 and OIP106, suggesting that the human Miro GTPases constitute a connection between the mitochondria and the trafficking apparatus of the microtubules. Milton also shares significant homology with the Huntingtin-binding domain of the human Huntingtin associated protein-1 (HAP1). However, we could not detect any interaction between Miro and HAP1.

This paper provides evidence that human Miro members serve functions in the transport of mitochondria along the microtubule system. Moreover, Miro1 enhances the fusion-state of mitochondria and in this process, utilizes both its N-terminal GTPase domain and its EF-hands. The fact that Miro1

47 requires functional EF-hands to mediate the formation of highly elongated mitochondria indicates that Miro can be under control of calcium signaling pathways.

48 REFLECTIONS

Rho GTPases are encoded by 20 distinct genes in the mammalian genome. Despite the large number of Rho GTPases, most of the functional information has come from studies on the famous triad: RhoA, Rac1 and Cdc42. However, during the last couple of years, other members of the Rho GTPases have attained a slightly increased attention. New findings have resulted in that the concept of Rho GTPase regulated signaling pathways has broadened considerably. Further, there is an increased awareness that deregulated Rho GTPases occur in disease.

Different Rho GTPase members give rise to similar, but not entirely overlapping, responses in the organization of the actin filament system. This suggests that there exist more types of cytoskeletal organizations than what we commonly refer to as “lamellipodia”, “filopodia” and “stress fibers”. This brings up the necessity to study the Rho protein-induced actin reorganization with improved resolution in space and time. The atypical Rho GTPases undergo uncharacteristic ways of regulation and often display irregular functions. It is of importance to examine how these Rho GTPases are activated and inactivated. Moreover, it is essential to identify their binding partners in order to get a better picture of their function.

In a screen for novel Ras-related GTPases, we discovered the Miro GTPases. In our initial characterization, we established that the Miro proteins are essential for proper mitochondrial morphology and distribution. Overexpression of a constitutively active Miro1 mutant (Miro1/P13V) induced perinuclear clustering of highly fused mitochondria and resulted in an increased apoptotic rate. Several models of apoptosis are associated with extensive mitochondrial fission (Parone et al. 2006). Our finding support the idea that not all apoptotic pathways involve mitochondrial fission (Szabadkai et al. 2004). In line with our results, mitochondrial aggregation has been suggested to trigger apoptosis (Haga et al. 2003).

We showed that both human Miro1 and Miro2 interact with the milton- related proteins GRIF1 and OIP106, suggesting that human Miro GTPases form a link between mitochondria and the trafficking apparatus of the microtubules. Interestingly, Miro proteins have been demonstrated to regulate mitochondrial motility, as well as fusion-fission dynamics, in a

49 calcium-dependent manner (G. Hajnóczky, personal communication). Another exciting finding is that yeast Miro, Gem1p, contributes to proper mitochondrial inheritance in S. cerevisiae (Frederick et al. 2008). In S. cerevisiae, mitochondrial transport and inheritance is solely actin-based. This raises the possibility that Miro proteins, in addition to their interaction with microtubule motors, may modulate also the attachment of actin motors to mitochondria.

In order to achieve a deeper understanding of the functions of Miro, identifying binding partners is of great importance. In an attempt to isolate proteins interacting with Miro, we performed a two-hybrid screen with a constitutively active Miro1 mutant lacking its transmembrane domain (Miro1/P13V/ TM) as bait. Several potential binding partners were detected. Among the positive clones were the already identified Miro interactors GRIF1 and OIP106, indicating reliability of the screen. To validate our two-hybrid findings, we performed co-immunoprecipitations (Co-IP) with Miro. These results supported interactions between Miro1 and the two proteins, Centromere protein F (CENP-F) and LIM domain only 7 (Lmo7).

CENP-F is a nuclear matrix protein that just prior to mitosis localizes to the kinetochore regions of chromosomes and links these to dynein motors at spindle microtubules (Rattner et al. 1993; Feng et al. 2006; Vergnolle et al. 2007). This linkage is essential for proper chromosome segregation, since dynein contributes to the poleward movement of chromosomes along the spindle microtubules (Banks et al. 2001). Subsequently, after mitosis, CENP-F is thought to be degraded. What is the functional relevance of the interaction between CENP-F and Miro? We are planning to examine whether these proteins influence mitochondrial distribution during cell division. Mitochondrial inheritance has been studied quite extensively in yeast (Frederick et al. 2008), but how mitochondrial distribution is coordinated during cell division in higher eukaryotes remains to be clarified.

Lmo7 is a LIM domain containing protein that interacts with actin and the actin cross-linking protein -actinin (Ooshio et al. 2004). Lmo7 has been described roles in transcription regulation of muscle relevant genes (Holaska et al. 2006). In epithelial cells, Lmo7 appears to play a stabilizing function in the formation of cell-cell adherens junctions (Ooshio et al. 2004). Since Lmo7 is an actin binding protein, it might serve a function together with Miro in docking mitochondria to the actin filament system. We have detected overexpressed Lmo7 partially localized to mitochondria in COS7 cells.

50 Without a doubt, future studies of Miro will lead to a better understanding of the molecular mechanisms underlying the expanding field of diseases caused by defects in mitochondrial dynamics.

51 ACKNOWLEDGEMENTS

The work of this thesis has been carried out at the Ludwig Institute for Cancer Research in Uppsala. I would like to T H A N K the following people, without whom the work of this thesis would have been much less enjoyable or, in some cases not even possible:

My supervisor Pontus Aspenström for your never-ending support and excellent guidance. It has been great working with you!

Carl-Henrik Heldin, the director of the Ludwig Institute, for your scientific feedback throughout this work and for being a true inspiration.

My collaborators and co-authors: Aino, Jan, Ninna, and Pacho for fruitful teamwork.

All wonderful members of the MB lab: Aino, Annica, Latifa, Katarina, Marcia and Pontus for creating a familiar and stimulating working environment. I wish you the best of luck in the future at KI in Stockholm! Special thanks to Aino for introducing me to the lab when I first came and for being a true friend. Special thanks also to Katarina for reading and making important remarks on this thesis and for continuing the work on Miro. Aive, I still miss you in the MB-lab!

Christer, Emeli, Eva, Gullbritt, Ingegärd, Inger, Lasse, dator-Uffe, cykel-Uffe och Ulla och för superduperhjälp med allt som tänkas kan.

My fellow PhD students, past and present, for brightening my days.

Rosita for being a fantastic roommate.

Everyone at the Ludwig institute for making everyday life fun and special.

Gamla & nya Goda Vänner samt stora & små Kära Släktingar.

Den livsbejakande och ytterst värdefulla familjen Ljadas: Alide, Henrik, Maria & Ismael, Karin & Rein.

52 Mina bröder Daniel och Mattias för att ni är toppen. Min Pappa och Inger för ovärderlig support. Min underbara Mamma för allt och Lasse för att du är helt oslagbar! Mina vilda och fantastiska små flickor Alva och Svea och min finurliga och rakt igenom fina Magnus för att ni gör mig glad varenda dag. Tänk att ni finns - det är ju sån TUR!

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