Protoplasma (2007) 230: 203Ð215 DOI 10.1007/s00709-006-0234-7 PROTOPLASMA Printed in Austria

Annexins: putative linkers in dynamic membraneÐcytoskeleton interactions in plant cells

D. Konopka-Postupolska

Laboratory of Plant Pathogenesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw

Received January 10, 2006; accepted March 14, 2006; published online April 24, 2007 © Springer-Verlag 2007

Summary. The plasma membrane, the most external cellular structure, proteins with similar characteristics in plant cells (Boustead is at the forefront between the plant cell and its environment. Hence, it is et al. 1989, Blackbourn et al. 1991). In vertebrates the annex- naturally adapted to function in detection of external signals, their trans- duction throughout the cell, and finally, in cell reactions. Membrane ins were grouped into 13 families. In contrast, plant annexins lipids and the cytoskeleton, once regarded as simple and static structures, seem to represent a relatively simpler, smaller, and less have recently been recognized as significant players in signal transduc- diverse family of proteins. Nevertheless, in all analysed tion. Proteins involved in signal detection and transduction are organised in specific domains at the plasma membrane. Their aggregation allows to plant species, at least two distinct proteins with molecular bring together and orient the downstream and upstream members of sig- masses between 33 and 36 kDa have been discovered nalling pathways. The cortical cytoskeleton provides a structural frame- (Smallwood et al. 1990, Shin et al. 1995, Proust et al. 1996, work for rapid signal transduction from the cell periphery into the Thonat et al. 1997). However, a search through the nucleus. It leads to intracellular reorganisation and wide-scale modula- tion of cellular metabolism which results in accumulation of newly syn- Arabidopsis thaliana genome, in which seven annexin thesised proteins and/or secondary metabolites which, in turn, have to be genes were found (Clark et al. 2001), showed that this distributed to the appropriate cell compartments. And again, in plant number can be even larger. Summarising, it seems to be cells, the secretory vesicles that govern polar cellular transport are deliv- ered to their target membranes by interaction with actin microfilaments. true for both vertebrates and plants that more than one anne- In search for factors that could govern subsequent steps of the cell re- xin is usually expressed at a given moment and in a par- sponse delineated above we focused on an evolutionary conserved pro- ticular cell type (so-called annexin fingerprint). This tein family, the annexins, that bind in a calcium-dependent manner to membrane phospholipids. Annexins were proposed to regulate dynamic indicates that in spite of significant functional homology, changes in membrane architecture and to organise the interface between individual annexins support distinct and divergent func- secretory vesicles and the membrane. Certain proteins from this family tions. Some data suggest that individual annexins are asso- were also identified as actin binding, making them ideal mediators in ciated with different cellular compartments possibly cell membrane and cytoskeleton interactions. conferring specificity of cellular response to the given stim- Keywords: Plant annexin; Actin microfilament; Stress response; An- ulus. A special family of plant vacuolar annexins, VCaBP, nexinÐactin interaction; Exocytosis. with a slightly larger molecular mass (ca. 42 kDa) was discovered in various plant species of the families Annexins in plant cells Solanaceae and Brassicaceae (Seals et al. 1994, Seals and Randall 1997). In mustard plants (Sinapis alba), annexin Annexins constitute a family of ubiquitous, calcium- and p28 was shown to be a part of the chloroplast translation membrane-binding proteins. They have been intensively apparatus (Pfannschmidt et al. 2000). Finally, the presence studied since the identification of the first annexin in animal of nuclear annexins has also been documented (Clark et al. tissues (Creutz et al. 1978) and subsequent recognition of 1998, Kovacs et al. 1998). Annexins have an evolutionary conserved overall struc- ture, with an about 70-amino-acid motif repeated four * Correspondence and reprints: Laboratory of Plant Pathogenesis, Insti- tute of Biochemistry and Biophysics, Polish Academy of Sciences, times within the molecule, and contain a discrete (neither Pawinskiego 5A, 02-106 Warsaw, Poland. EF-hand nor C2) calcium binding site (Fig. 1). Calcium 204 D. Konopka-Postupolska: Annexins in regulation of membraneÐcytoskeleton dynamics D. Konopka-Postupolska: Annexins in regulation of membraneÐcytoskeleton dynamics 205 binding induces structural changes resulting in protein regulation of membrane organisation, membrane traffick- translocation from the cytoplasm to the cell periphery. ing, interactions with the cytoskeleton, and secretion. In Crystallographic data reveal that membrane binding oc- time it became clear that certain plant annexins can also curs via formation of a ternary complex between annexin, function in plant stress response. Expression of different calcium, and the membranes (Swairjo et al. 1995). Annex- annexins was induced by osmotic stress (AnnMs2 from ins have been shown to preferentially bind to negatively alfa alfa [Kovacs et al. 1998]; AnnAt1 from A. thaliana charged membrane phospholipids (e.g., phosphatidylse- [S. Lee et al. 2004 and our unpubl. data]), which suggests rine). Plant annexins are fairly abundant cellular proteins that they might participate in drought resistance. Some (Clark and Roux 1995) and should thus be considered as a data indicate also that AnnAt1 can act at a crossroad be- very important element of calcium signalling pathways. tween auxin and abscisic acid signalling (Bianchi et al. On the basis of immunocytochemical experiments, it has 2002). Enhanced expression of annexin was also reported been concluded that in plant cells, annexins are localised after different treatments that led to accumulation of reac- mainly in the cytoplasm. When calcium levels increase, tive oxygen species during defence response in tomato they are moved towards the cytoplasmic surface of certain plants (Xiao et al. 2001), and after salicylic acid and hy- membrane structures, mainly the plasma membrane, but drogen peroxide treatment in Arabidopsis plants (Gidrol the presence of an annexin that binds specifically to the et al. 1996). It is worth mentioning that those two an- outer membrane of the chloroplast envelope was also re- nexins, namely, p34 from tomato cells and AnnAt1 from ported (Seigneurin-Barny et al. 2000). On the other hand, A. thaliana, represent close homologs (84% of homology experimental data show that particular proteins, although on protein level). AnnAt1 also has the ability to protect lacking defined targeting sequences, are present in non- heterologous cells from the consequences of oxidative stimulated cells in different cellular compartments. Proteo- stress. Molecular mechanisms of this protection have not mic analysis revealed that, e.g., AnnAt1 from A. thaliana been elucidated, although different hypotheses are consid- was present in the fraction of cell wall proteins (P. Woj- ered, beginning from an intrinsic peroxidase activity of taszek, Adam Mickiewicz University, Poznan, Poland, pers. AnnAt1 (Gidrol et al. 1996). An indirect effect via modu- commun.), integral membrane proteins (Santoni et al. 1998, lation of calcium signalling that results in the lowering of S. Lee et al. 2004), in central, vegetative vacuoles (Carter superoxide production and reduction of protein kinase C et al. 2004), as well as in the nuclear matrix (A. Jerz- activity was also proposed (Kush and Sabapathy 2001, manowski, Institute of Biochemistry and Biophysics, Polish Janicke et al. 1998). It is also possible that it can be a non- Academy of Sciences, Warsaw, Poland, pers. commun.). specific consequence of membrane lipid protection Mimosa annexin exhibits dayÐnight changes in distribution: against oxidative stress. Mammalian annexin A5 was during the daytime it is localised on the cell periphery, shown to bind, with affinity similar to that of phos- while at night it stays in the cytoplasm (D. Hoshino et al. phatidylserine, to malondialdehyde adducts, a major prod- 2004). Additional analyses are necessary to establish uct of lipid peroxidation generated by the nonenzymatic whether this microcompartmentalisation is also true for the reaction of polyunsaturated fatty acids with molecular other plant annexins. oxygen (Balasubramanian et al. 2001). In contrast to free Despite several years of investigation, the primary radicals, lipid peroxides are long-lived and can thus dif- physiological function for annexins has not yet been elu- fuse from the site of origin and exert deleterious effects cidated. It is generally assumed that annexins are impli- and/or activate stress-related pathways in surrounding tis- cated in several processes related to membranes, including sues. If AnnAt1, and possibly other annexins, shares this

Fig. 1. Alignment of the deduced amino acid sequences of human (A1, A2, A4, A5, A6, A7, A11, A13) and plant (Arabidopsis thaliana AnnAt1 to -7, Capsicum annuum Ca_p32 and p38, Lycopersicon esculentum Le_p34 and p35, Gossypium hirsutum Gh1, Nicotiana tabacum Nt_VCaBP, Solanum tuberosum St_p34, and Medicago sativa MsAnn annexin genes obtained using T-COFFEE (Notredame et al. 2000). Potential functional domains are indicated as follows: Ca2-binding sites of type II G-X-GTD-{ca. 38}-E/D are shown in black, potential actin binding motifs (IRI and/or LLYLCG GDD) are shown in italics, putative PIP2 binding domain (R/K)LXXX(K)X(K)(R/K) is underlined, generic motif for N-myristoylation site in AnxA13. GenPept accession numbers of human annexins are as follows: AnxA1, NP_000691; AnxA2, NP_001002858; AnxA4, NP_001144; AnxA5, NP_001145; AnxA6, NP_004024; AnxA7, NP_004025; AnxA11, NP_665876; and AnxA13, NP_004297. Locus numbers of Arabidopsis annexins are as follows: AnnAt, At1g357201; AnnAt2, At5g65020; AnnAt3, At2g38760; AnnAt4, At2g38750; AnnAt5, At1g68090; AnnAt6, At5g10220; and AnnAt7, At1g10230. GenPept accession numbers of plant annexins are as follows: Ca_p32, CAA10210; Ca_p38, CAA10261; Le_p34, AAC97494; Le_p35, AAC97493; Nt_VCaBP42, AAD24540; St_p34, ABB02651; and MsAnn, CAA72183. Due to lack of space only the first half of the doubled annexin AnxA6 molecule is depicted in the scheme 206 D. Konopka-Postupolska: Annexins in regulation of membraneÐcytoskeleton dynamics malondialdehyde-binding activity, it could protect cells Annexins and membrane dynamics from oxidative stress by hampering the propagation of signalling molecules derived from polyunsaturated fatty Plant cellular membranes contain striking amounts of acids. both structurally and functionally varied lipids that are

Fig. 2 AÐC. Schematic representation of raft structures and mechanism of their interac- tions with annexins. Rafts represent laterally organised lipid microdomains enriched in sphingolipids and sterols (liquid order phase). A specific set of proteins interact with rafts through transmembrane domains or lipid an- chor (GDI-tail for extracellular and double acyl for intracellular proteins). Due to their ability to concentrate signaling molecules and to interact with the actin cytoskeleton, rafts were proposed to function as signaling centers. In animal cells, annexins were pro- posed to be important for raft function, gov- erning their aggregation and influencing cy- toskeleton interactions. There are some data suggesting that in plant cells, annexins may support similar functions. A Upon initial cal- cium elevation, different calcium-binding proteins, including annexins, are activated. This can result in triggering of specific intra- cellular transduction pathways, e.g., phos- phoinositide signaling. At the same time, a particular annexin (A1) with the highest cal- cium affinity is translocated to the plasma membrane and binds to negatively charged phospholipids in the inner leaflet. B Once bound, annexin A1 can oligomerise on mem- brane surfaces, thus initiating raft clustering and stabilisation. In turn, further calcium el- evation can lead to the activation of another protein from this family, with slightly differ- ent characteristics and binding preferences. After translocation, A2 can bind to another type of negatively charged phospholipid, PIP2, that is enriched within sterol-rich do- mains. C In this localisation, annexin A2 can serve as a platform for actin assembly, pro- mote cytoskeleton rearrangement, and regu- late its binding with membranes. Thus, new signaling pathways can be constituted. More- over, raft clusters (macrorafts) are further sta- bilised by hindering their lateral mobility by association with the cytoskeleton. Addition- ally, transporting vesicles that moved along microfilaments can be directed to the center of exocytosis D. Konopka-Postupolska: Annexins in regulation of membraneÐcytoskeleton dynamics 207 distributed asymmetrically among different membranes. rafts) to fulfill the signalling function. Clustering can be For example, cholesterol and phytosterols are localised accomplished from both sides of the plasma membrane, mainly in the plasma membrane, within the outer leaflet and some experimental evidence indicates that annexins, of the bilayer, while phospholipids are more or less uni- acting from the inner leaflet side, promote lipid raft for- formly distributed in the inner leaflet. Moreover, even mation and influence their dynamics. Indeed, certain ver- within a single membrane, lipids can be organised later- tebrate annexins (A2, A6, and A13b) were shown to be ally to form discrete domains that can support particular present in a Triton X-100-insoluble membrane fraction cellular functions. One of the most widely studied types that contains raft domains (Harder and Gerke 1994, Lafon of such lateral organisation results from the association of et al. 1998, Draeger et al. 2005). It was proposed that an- sphingolipids parallel to one another, probably through nexins oligomerise on the membrane surface resulting in weak interactions between their carbohydrate heads. the formation of a two-dimensional lattice (Lambert et al. Empty spaces between associated sphingolipids are filled 1997) that can serve as a platform for raft aggregation into with stiff sterol molecules arranged perpendicularly to the larger domains (Oliferenko et al. 1999). It is worth stress- bilayer forming a so-called liquid ordered structure, called ing that several plant annexins were shown to form rafts (Simons and Ikonen 1997, Harder and Simons 1997, homodimers and oligomers in solutions, which is a prereq- Munro 2003, Edidin 2003) (Fig. 2). In such an ordered uisite for raft aggregation (Hoshino et al. 1995, Hofmann arrangement, the lateral movement of lipids is not im- et al. 2002). Moreover, the presence of a protein sheet on paired so that rafts can easily move in the plane of the sur- membrane surface can induce segregation of certain pro- rounding, more fluid bilayer and aggregate into larger teins and modulate enzyme functions (Andree et al. 1992, domains. Rafts are especially abundant in the plasma Dubois et al. 1998). In turn, raft binding can also modify membrane but can also be found in secretory and endocytic the properties of annexins. For example, annexin A2 was pathways (Simons and Toomre 2000). Although rafts de- identified as a one of the main cellular substrates for velop exclusively in the outer leaflet of a bilayer, they are pp60c-Src kinases (Hubaishy et al. 1995), which is concen- able to organise, in a still unknown way, the lipids of the in- trated in cholesterol-rich domains. ner leaflet. Recently, Gri et al. (2004) found that in Jurkat One has to keep in mind that a single cell expresses T cells the domains at the outer and inner leaflets are physi- several annexins at the same time and they may act in syn- cally coupled and this coupling requires cholesterol. ergy to support cellular processes, although their mode of The results of several studies suggest that this lateral action could be slightly different. The most detailed and assembly of sphingolipids and sterols may be involved in compelling picture depicting the function of annexins in transport of newly synthesised material to the cell surface membrane dynamics emerged from smooth muscle cells in polarised and nonpolarised vertebrate cells (Musch (Babiychuk and Draeger 2000, Draeger et al. 2005). The et al. 1996, Simons and van Meer 1988, Yoshimori et al. initial calcium signal (up to 300 nM) induces annexin A2 1996, Zegers and Hoekstra 1998) and in organising the translocation to the rafts, which promotes their aggrega- cellular machinery for exo- and endocytosis (Schnitzer tion and formation of macrorafts. As the calcium concentra- et al. 1996). Due to this ability to selectively aggregate tion increases further (up to 600 nM), the second annexin, special proteins, rafts were also proposed to function in A6, translocates to the plasma membrane, where it binds cell signaling (Parton and Simons 1995, Simons and in the vicinity of the macrorafts and induces the formation Toomre 2000). Protein association with lipid rafts is medi- of a mechanical link coupling the actin-based cytoskele- ated by glycosylphosphatidylinositol (GPI) anchoring ton and the membrane (Babiychuk et al. 1999), thus sta- (Mayor et al. 1994), which results in coclustering of dou- bilising the macrorafts. Further elevation of the calcium ble acylated proteins on the cytosolic surface (Resh 1999). signal above 1000 nM results in annexin A5 binding to Association with the inner leaflet could also be governed glycerophospholipids, which may additionally stabilise by direct protein interaction with cholesterol (Parton et al. the macrorafts. It is not clear if a similar step-by-step mech- 1994). The newest measurements, using single-particle anism also operates in other less specialised cell types, but tracking, showed that at steady state, rafts are generally the fact that single cells usually express several annexins dif- small, with a diameter of about 50 nm (which corresponds fering in calcium affinity and other properties (e.g., the abil- to about 3500 lipids [Pralle et al. 2000]), exist for less ity to bind to the cytoskeleton) supports this notion (Fig. 2). than 1 min, and contain no more than 10Ð30 protein mole- The presence of lipid microdomains in plant cell mem- cules (Varma and Mayor 1998). Thus, single rafts are too branes has been recognised only recently. With respect to small and have to coalesce into bigger domains (macro- lipid and protein contents, they seem to have characteris- 208 D. Konopka-Postupolska: Annexins in regulation of membraneÐcytoskeleton dynamics tics similar to those of animal and yeast rafts, although distributed more or less uniformly within the inner leaflet, discrete differences have also been reported (Peskan et al. while rafts, enriched in sphingolipids and sterols, develop 2000, Mongrand et al. 2004, Borner et al. 2005). They con- in the outer leaflet. However, it was shown that under cer- tain significant amounts of GPI-anchored proteins (Borner tain conditions, individual annexins could display wider et al. 2005), a large number of signalling molecules (re- binding preferences than it had been concluded from ceptor kinases with leucine-rich repeats), several compo- in vitro experiments with artificial membranes. Annexins nents of downstream signalling pathways (small and A2 and A6 were shown to interact directly with choles- heterotrimeric G proteins), and finally proteins involved in terol in native membranes isolated from chromaffin gran- stress response (similar to hypersensitive-stress-response- ules and CHO endosomes, respectively (Ayala-Sanmartin related proteins from Zea mays) (Shahollari et al. 2004, et al. 2000, Diego et al. 2002). In turn, annexin A2 pro- Borner et al. 2005). Elicitation of tobacco cells with cryp- tects membrane cholesterol from extraction with cy- togein results in recruitment of NADPH oxidase to the clodextrin (Ayala-Sanmartin et al. 2001, Mayran et al. phytosterol-rich domains (Mongrand et al. 2004), indicat- 2003). Furthermore, even low concentrations of choles- ing that upon stimulation, plant rafts are able to mobilise a terol-sequestering agents like filipin or digitonin quantita- specific set of proteins. Recently, Bhat et al. (2005) tively released annexin 2 from the membranes (Jost et al. showed that infection with a fungal pathogen leads to re- 1996, Harder et al. 1997). These data suggest that at least distribution and polarisation of sterols in the plasma mem- certain annexins can bind to rafts through direct interac- brane of epidermal cells. This is accompanied by re- tion with cholesterol. On the basis of recent experiments, distribution of plasma membrane proteins and followed another possibility was also indicated. Annexin A2 was by focal accumulation of callose deposition at the site of shown to directly interact with another membrane compo- pathogen entry. In line with the observation that elicitors nent known to be located in rafts, phosphatidylinositol induce NADPH recruitment to membrane microdomains 4,5-bisphosphate (PIP2), despite the fact that the protein (Mongrand et al. 2004), these data indicate that generation lacks a typical PIP2-interacting domain (Hayes et al. of phytosterol-rich macrodomains may participate in the 2004, Rescher et al. 2004). Recently, Gokhale et al. plant defence response. (2005) proposed that PIP2 binding could be governed by a Far-reaching similarities between plant and animal rafts cluster of cationic residues localised on the convex sur- raise the possibility that regulatory processes that govern face of the AnxA2 molecule (Fig. 1). In addition to the raft dynamics may also be similar. Thus, plant annexins well established role of PIP2 as a precursor of lipid-de- could also be involved in the regulation of raft dynamics rived signalling molecules, it is known to act as a lo- in plant cells. So far only a very limited amount of co- calised regulator of membrane events (vesicle fusion, localisation data supports this idea. In plant cells, annexins fission, and endocytosis) (Cullen et al. 2001, Martin are concentrated in the region of polarised growth, and in 2001). Hence, Rescher and Gerke (2004) proposed that vertebrate cells, raft domains are known to be especially recruitment of annexin A2 into rafts could be a two-step abundant in such region. Recently, Clark et al. (2005) process. After docking to the membrane through interac- showed colocalisation of AnnAt1 and AnnAt2 with ara- tions with negatively charged phospholipids, the annexin binogalactan proteins in Arabidopsis cells. Since classical could be subsequently recruited into rafts via direct bind- arabinogalactan proteins are predicted to have a GPI an- ing to PIP2. It remains to be established whether PIP2 chor, they may be a constituent of rafts. This may suggest binding is a generic feature of the whole family or just the that plant annexins could also be connected with spe- exclusive property of AnxA2. Since PIP2 is known to be cialised raft domains and are therefore involved in their an important regulator of cytoskeleton function, the ability functions. of annexins to interact with PIP2 suggests that annexins However, the very basic question concerning the mech- can connect rafts with the cortical cytoskeleton and organ- anism of annexin interaction with lipid rafts still remains ise the membraneÐcytoskeleton interface. In line with this open. Only in the case of annexin A13b, this interaction reasoning, certain members of the vertebrate annexin fam- can be, at least partially, attributed to single-protein ily were shown to copurify with cytoskeleton proteins myristoylation (Fig. 1) (Wice and Gordon 1992) (typically (Gerke and Weber 1984, Shadle et al. 1985, Mangeat double acylation is necessary to confer raft association), 1988), and perturbation of membrane cholesterol resulted whereas other proteins from this family do not undergo in the release of annexin A2 together with the actin cy- even such posttranslational modifications. In principle, an- toskeleton (Harder et al. 1997, Zeuschner et al. 2001). In nexins bind to negatively charged phospholipids that are summary, it seems that in vertebrate cells, annexins facili- D. Konopka-Postupolska: Annexins in regulation of membraneÐcytoskeleton dynamics 209 tate rapid clustering of rafts in response to calcium eleva- typic domain of this type and quite divergent peptides sup- tion and stabilise such structures by hindering lateral port such function in different proteins. In the case of ver- movements of lipids within the bilayer by attaching them tebrate annexin A2, the actin binding domain was mapped to the cytoskeleton. Although the significance of annexin experimentally to the very last C-terminal-sequence non- interaction with the actin cytoskeleton is not completely apeptide LLYLCGGDD (Fig. 1). A second peptide, VLIR- clear, the linkage of annexins both to membranes and to IMVSR, localised a little towards the centre of the protein the actin cytoskeleton could be important for cortical cy- molecule, was shown to be involved in actin bundling toskeleton rearrangement in response to different stimuli (Jones et al. 1992, Filipenko and Waisman 2001). The for- as well as for cytoskeleton interaction (see below). mer domain, only slightly modified, is also present in ani- mal annexins A5 and A6 that are known to interact with microfilaments, but no similar sequence was detected in Annexins and actin cytoskeleton dynamics plant annexins. Instead, a conserved amino acid motif IRI, Several vertebrate annexins, namely, A1, A2, A5, and previously identified as the smallest region of the putative A6, were shown to bind in a Ca2-dependent manner to F-actin binding site of myosin (Suzuki et al. 1987), was filamentous actin both in vitro and in vivo (Schlaepfer proposed to support this function. It was found in the 3rd et al. 1987, Khanna et al. 1990, Traverso et al. 1998, endonexin repeat in almost all plant annexins identified up Babiychuk et al. 1999, Tzima et al. 2000). Actin binding to now and in a subset of vertebrate annexins (A1, A2, A6, is regulated also by posttranslational modification of an- A10, and A13) (Fig. 1). Direct experimental evidence sup- nexin proteins, e.g., phosphorylation (Glenney and Tack porting IRI-mediated attachment is lacking. Interestingly, 1985, Jost and Gerke 1996), S-glutathionylation (Caplan maize annexins p33 and p35, which share the IRI domain, et al. 2004) and poly-/multiubiquitination (Lauvrak et al. failed to interact with F-actin (Blackbourn et al. 1991). 2005). Annexins A1 and A2 not only bind but also induce However, under the experimental conditions of that study, bundling of actin microfilaments in vitro (Schlaepfer et al. substantial amounts of actin remained in the supernatant 1987, Glenney et al. 1987, Filipenko and Waisman 2001). and could retain annexins and prevent coprecipitation. Be- Besides actin, particular annexins can also bind accessory sides, in those experiments heterologous actin from rabbit proteins and hence influence the cytoskeleton dynamics muscle cells was used, potentially impairing the interac- indirectly. Annexin A1 binds profilin, modulating its in- tion with plant annexin. Summarising, further experiments hibitory effect on actin polymerisation. In turn, profilin are necessary to precisely characterise the mechanism of strongly inhibits annexin self-association and impairs its plant annexin interactions with actin. Moreover, it is neces- ability to aggregate liposomes (Alvarez-Martinez et al. sary to undertake colocalisation studies of the annexins 1996, 1997). Furthermore, annexins A2 and A6 have been and actin filament distribution to assess the significance of shown to interact with spectrin, which is a member of the in vitro binding data for cellular events. two-dimensional network that lines the inner surface of For several in vivo experimental systems employing ver- most metazoan cells (Bennet 1990). This raises the possi- tebrate cells, the interactions of annexins with the cy- bility that annexins could be engaged in remodelling of toskeleton were shown to take place exclusively on the this network, which is a prerequisite for the occurrence of membrane surface. Annexins are specifically recruited to exocytosis and endocytosis (Kamal et al. 1998). those areas of cellular membranes that are associated with In contrast, only a few plant annexins, namely, p34 and the actin cytoskeleton, like nascent cell-to-cell contacts p35 from tomato (Calvert et al. 1996), zucchini (Hu et al. (Hansen et al. 2002, Burkart et al. 2003, D. Lee et al. 2004). 2000), and mimosa plants (D. Hoshino et al. 2004), were Moreover, they localise to sites of dynamic actin assembly shown to bind filamentous actin (F-actin) but not globular platforms at cellular membranes, i.e., motile pinosomes actin in a Ca2-dependent manner. Attachment to the mi- (Merrifield et al. 2001) and pedestals induced by en- crofilaments did not impair enzymatic activity of p34 and teropathogenic Escherichia coli (Zobiack et al. 2002). At p35 (ATPase/GTPase) from tomato in contrast to mem- the same time, they are not found in cytosolic, actin-con- brane binding that abolished it completely. Mimosa an- taining structures, like stress fibres (Gerke and Moss 2002) nexin induced bundling of actin in vitro and was shown to or actin tails that propel intracellular movement of Listeria be involved in pulvinar nyctinastic movements (D. Hoshino species (Merrifield et al. 2001). In summary, it seems that et al. 2004). in vivo membranes are necessary to provide a platform for It is not clear which protein domain is responsible for annexin-induced actin assembly. The recent discovery that annexin interaction with actin. There is no single proto- annexins (or at least annexin A2) can directly bind PIP2, 210 D. Konopka-Postupolska: Annexins in regulation of membraneÐcytoskeleton dynamics which is a well-known regulator of cytoskeletonÐmem- other words, the question is whether the isoforms are equiv- brane adhesion, shed some light on this requirement. It is alent to each other and, if so, to what extent. A number of widely accepted that activity of several cytoskeleton modu- observations from yeast and animal cells strongly suggest lating and anchoring proteins can be regulated by PIP2 that actin isoforms, although displaying about 87% homol- (Janmey and Stossel 1987, Yonezawa et al. 1990). Hence, ogy, could have unique properties. For example, wild-type rapid and localised changes in PIP2 concentration resulting yeast actin polymerised in vitro at a faster rate than muscle from Ca2-induced activation of protein kinase C, PIP ki- actin because of differences in the nucleation step (Buzan nases, and phosphatases can regulate the membrane attach- and Frieden 1996, Kim et al. 1996). For plant actin, the ment of the cortical cytoskeleton and facilitate changes in question regarding the functional equivalency of different membrane curvatures. Annexins can mediate actin binding isoforms remains open. The available data come exclu- with membranes via interaction with PIP2, thus anchoring sively from in vivo experiments with loss-of-function mu- the actin cytoskeleton in dynamic regions engaged in exo- tants and are contradictory. Disruption of most of the actin and endocytosis and coupling together membrane and genes (ACT1, -2, and -4) had only a mild effect on plant actin-connected signalling cascades. morphology (McKinney et al. 1995, Gilliland et al. 2002), Another mechanism ensuring the specificity in actin–an- which suggests that certain isoforms can easily substitute nexin interactions is also worth mentioning. It was shown for each other. Moreover, ectopic expression of both vege- that in vertebrate cells, annexins can, in certain cases, dis- tative and generative isoforms complemented phenotype al- play selectivity toward particular actin isoforms and thus teration in the act2-1 mutant and restored normal root hair bind exclusively to a specific pool of cellular actin. Reloca- elongation impaired after mutation (Gilliland et al. 2002). tion of annexin A5 to the cytoskeleton in human platelets is On the other hand, ectopic expression of generative actin mediated by the specific interaction with actin isoform ACT1 had a great impact on plant morphology, caused (Tzima et al. 1995). In smooth muscle cells, annexin A2 dwarfing and the alteration of some organs (Kandasamy was shown to interact in a different way with a spatially et al. 2002), hence testifying in favour of functional non- segregated “contractile” and “cytoskeletal” actin pool equivalency. (Babiychuk et al. 2002). Preferential association of annex- Another question is whether there are cellular mecha- ins with a particular type of filaments would result in speci- nisms that allow sorting of different isoforms to separate ficity of cell response to a given stimulus. Although there is microfilaments. Double mutants act2-1 act7-1 devel- no direct experimental evidence testifying to the presence oped a more severe phenotype than single mutants, which of different types of actin microfilaments in plant cells, the authors interpreted as evidence of synergism in the ac- there are some data supporting such reasoning. Plants ex- tion of those two actins and argued against sorting of iso- press multiple actin isoforms encoded by an ancient gene variants into specific filaments (Gilliland et al. 2002). Even family (Meagher and McLean 1990). In the model organ- if so, induction of ACT7 gene expression in response to ism A. thaliana, there are 8 genes (Meagher and Fechheimer hormonal treatment may result in formation of different fil- 2003) expressed in a tissue-specific manner which thus can aments as a simple consequence of its over-representation be grouped in two general classes: generative and vegeta- of one monomer in the total pool. Hence, it is tempting to tive (ACT2, -7, and -8) (An et al. 1996). Of the vegetative speculate that isoforms that are produced or modified in re- isoforms, ACT2 and ACT8 are the most closely related, sponse to external stimulation (Dantan-Gonzalez et al. with only a single amino acid substitution (McDowell et al. 2001, Wan et al. 2005) or hormonal induction (McDowell 1996), but the expression of ACT8 is more restricted and et al. 1996, Kandasamy et al. 2001) can give rise to new takes place only in a subset of ACT2-expressing tissues (An types of actin microfilament that are functionally different et al. 1996). In contrast, ACT7 is expressed predominantly or may interact with separate sets of actin-binding pro- in developing tissues, in dividing and expanding cells and teins. Alternatively, even a small contribution of a certain in response to most phytohormones (McDowell et al. 1996, isoform to the general pool of monomers can modify fila- Kandasamy et al. 2001). This unequal expression of actin ment properties due to its selective recruitment into a given isoforms in individual Arabidopsis leaf cells was confirmed region of microfilament. It was shown that there are some with single-cell RT-PCR (Laval et al. 2002). There is an on- mechanisms that govern the formation of microfilaments going debate concerning the purpose of expressing several with distinct compositions despite the availability of differ- almost identical isoforms in a single cell at the same time. ent monomers. Actins from rabbit muscle cytoskeleton and One of the possibilities is that despite significant similari- plant pollen cells do not form heteropolymers in vitro, and ties, particular isoforms can differ in at least one activity. In the introduction of rabbit actin into plant cells had a detri- D. Konopka-Postupolska: Annexins in regulation of membraneÐcytoskeleton dynamics 211 mental effect on the cells’ cytoarchitecture (Jing et al. were detected by autoradiography of de novo synthesised 2003). Thus, if plant annexins share the ability to preferen- cell wall polysaccharides. Comparison of those two pat- tially bind with different actin isoforms with animal coun- terns revealed significant overlapping, thus indicating that terparts, it is possible that they may also differentially the localisation of annexins is temporally and spatially regulate actin interaction with membranes in stress condi- coupled with secretion of cell wall material. Discrete dif- tions. Further analysis of plant annexin preferences toward ferences that were observed in localisation of particular actin isovariants are necessary to elucidate if such a mech- annexins, e.g., only AnnAt1 was expressed in developing anism could really be valid. sieve elements, additionally confirmed that despite func- tional equivalency, some annexins also fulfil specific func- tions in different cell types during plant development. Annexins in membrane trafficking All those data combined testify that annexins can par- Annexins can also regulate membrane dynamics by influ- ticipate in regulated secretion in plant cells. The question encing membrane turnover during exo- and endocytosis. regarding the exact role of annexins in vesicular traffick- The story began with the isolation of the first annexin, ing still remains open. To secrete their cargo, transporting synexin (annexin A7), as a protein participating in Ca2- vesicles have to arrive at the target membrane first. In regulated exocytosis in chromaffin cells (Creutz et al. 1992). plant cells this transport is supported by microfilaments Since then new data has demonstrated that this is actually (Taylor and Hepler 1997, Battey and Blackbourn 1993). the case and that annexins function in the process of Cytoskeleton proteins provide a mechanism for vesicle vesicular transport in all eukaryotic cells. For plants, im- movement, but they are not able to support fusion. Mem- munolocalisation studies showed that annexins are highly brane fusion is an energetically unfavourable process and concentrated at the extreme tip of polarised growing cells is accomplished by a special set of proteins called like pollen tubes of Lilium longiflorum (Blackbourn et al. SNAREs (Rothman 1994). Special sets of complementary 1992), maize roots, and fern rhizoids (Clark et al. 1994, SNAREs from different membranes interact, which re- 1995). Their localisation can be changed upon external sults in docking and permits membrane fusion (Hughson stimulation, e.g., in touch response of Bryonia dioica 1999, Mellman and Warren 2000, Chen and Scheller (Thonat et al. 1993, 1997) and during gravity sensing in 2001). This is accomplished first by the juxtaposition of pea plumules (Clark et al. 2000). Annexins were found to interacting organelles and then merging of the external be abundant in secretory cells of different types (outer leaflets of the two apposing bilayers (hemifusion). In the cells of root caps in maize and pea plants, developing vas- next step, merging of the two internal lipid leaflets occurs, cular tissues in Arabidopis seedlings, and maize egg cells) thereby completing the full fusion and establishing direct (Clark et al. 1992, 2000, 2001, 2005; Okamoto et al. 2004). continuity between the two previously separate compart- And finally, annexin expression was elevated during fruit ments. Because of their ability to bind calcium and mem- ripening when massive structural remodelling of the cell brane lipids, annexins have been proposed to function as wall took place (Wilkinson et al. 1995, Proust et al. 1996). CAPS (Ca2-dependent activator of protein secretion) in Considering all those data, it has been proposed that an- Ca2-dependent exocytosis (Zorec and Tester 1992, Thiel nexins may play a key role in cell expansion and organ et al. 1994, Carroll et al. 1998). They can mediate hemifu- growth due to their function in Golgi-mediated secretion sion, thus enhancing the possibility of further full-fusion of polysaccharide precursors for cell wall synthesis. Such events governed by SNARE proteins. In tight junction of activity has been confirmed both in vitro and in vivo. epithelial cells, annexin A2 was shown to promote stable Maize and green pepper annexins were shown to induce hemifusion, anchoring the cytoplasmic leaflets of secre- aggregation of artificial liposomes or plant secretory vesi- tory vesicles to each other or to the plasma membrane cles (Blackbourn and Battey 1993, T. Hoshino et al. 1995). (D. Lee et al. 2004). Strong support for this hypothesis Additionally, maize annexin p35 potentiated the Ca2-dri- has come from experiments with heterologous expression ven exocytosis in maize root cap protoplasts (Carroll et al. of mammalian annexins in yeast cells that do not express 1998). More functional analysis performed recently by endogenous proteins from this family. Annexins interacted Clark et al. (2005) revealed that such a scenario seemed to in a specific manner with the cellular secretory machinery be really operating. Localisation of particular annexins (Creutz et al. 1992) despite the lack of an endogenous (AnnAt1 and AnnAt2) in young Arabidopsis seedlings proteinaceous target, which strongly suggested that this was monitored by immunocytochemistry with specific an- effect was due to interactions of annexins with membrane tibodies and sites of active secretion of cell wall material elements of the yeast exocytosis machinery. 212 D. Konopka-Postupolska: Annexins in regulation of membraneÐcytoskeleton dynamics

Further perspectives Balasubramanian K, Bevers EM, Willems GM, Scroit AJ (2001) Binding of annexin V to membrane products of lipid peroxidation. Biochem- Despite nearly 30 years of investigation, we are still far istry 40: 8672Ð8676 from a comprehensive description of annexin function in Battey NH, Blackbourn HD (1993) The control of exocytosis in plant cells. New Phytol 125: 307Ð338 cell processes. Still much more is suggested than has been Bennett V (1990) Spectrin-based membrane skeleton: a multipotential established and much more remains to be done. Like adaptor between plasma membrane and cytoplasm. Physiol Rev 70: actin, annexins are so similar that they can easily substi- 1029Ð1065 Bhat RA, Miklis M, Schmelzer E, Schulze-Lefert P, Panstruga R (2005) tute for each other, but this does not exclude the possibil- Recruitment and interaction dynamics of plant penetration resistance ity that particular proteins are also specialised in specific components in a plasma membrane micro-domain. Proc Natl Acad Sci functions. Recently recognised active interplay between USA 102: 3135Ð3140 membranes and the cytoskeleton makes annexins particu- Bianchi MW, Damerval C, Vartanian N (2002) Identification of proteins regulated by cross-talk between drought and hormone pathways in larly interesting as they may function as a linker and mod- Arabidopsis wild-type and auxin-insensitive mutants, axr1 and axr2. ulate communication and cooperation between those two Funct Plant Biol 29: 51Ð61 cellular compartments. Their fusogenic activity also al- Blackbourn HD, Battey NH (1993) Annexin-mediated secretory vesicle aggregation in plants. Physiol Plant 89: 27Ð32 lows them to participate in secretion, which is particularly Blackbourn HD, Walker HJ, Battey NH (1991) Calcium-dependent important for plant cell growth. Understanding of an- phospholipid binding proteins in plants. Planta 184: 67Ð73 nexin-mediated signal transduction could allow manipula- Blackbourn HD, Barker PJ, Huskisson NS, Battey NH (1992) Properties and partial protein sequence of plant annexins. Plant Physiol 99: tion of plant stress response and in the future help in 864Ð871 obtaining more resistant crops. Borner GHH, Sherrier DJ, Weimar T, Michaelson LV, Hawkins ND, MacAskill A, Napier JA, Beale MH, Lilley KS, Dupree P (2005) Analysis of detergent-resistant membranes in Arabidopsis: Acknowledgments evidence for plasma membrane lipid rafts. 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