Role of plant myosins in motile organelles: Is a direct interaction required?

Role of plant myosins in motile organelles: Is adirect interaction required?Limor Buchnik, Mohamad Abu‐Abied and Einat Sadot*

The Institute of Plant Sciences, The Volcani Center, ARO, PO Box 6, Bet‐Dagan 50250, Israel.

Einat Sadot

*Correspondence:[email protected]

Abstract Plant organelles are highly motile, with speedvalues of 3–7mm/s in cells of land plants and about20–60mm/s in characean algal cells. This movement is believedto be important for rapid distribution of materials around thecell, for the plant’s ability to respond to environmental bioticand abiotic signals and for proper growth. The main machinerythat propels motility of organelles within plant cells is based on

the actin cytoskeleton and its motor proteins the myosins.Most plants express multiple members of two main classes:myosin VIII and myosin XI. While myosin VIII has beencharacterized as a slow motor protein, myosins from class XIwere found to be the fastest motor proteins known in allkingdoms. Paradoxically, while it was found that myosins fromclass XI regulate most organelle movement, it is not quite clearhow or even if these motor proteins attach to the organelleswhose movement they regulate.

Keywords: Motility; myosin; organelleCitation: Buchnik L, Abu‐Abied M, Sadot E (2014) Role of plant myosinsin motile organelles: Is a direct interaction required? (2014). J IntegrPlant Biol XX: XXX–XXX. doi: 10.1111/jipb.12282Edited by: Tobias Baskin, University of Massachusetts Amherst, USAReceived May 16, 2014; Accepted Aug. 31, 2014Available online on Sept. 5, 2014 at www.wileyonlinelibrary.com/journal/jipb© 2014 Institute of Botany, Chinese Academy of Sciences

INTRODUCTION

Plant myosins belong to two main groups of unconventionalmyosins: myosin XI and myosin VIII, both closely related tomyosin V (Berg et al. 2001; Reddy and Day 2001; Fothet al. 2006). The Arabidopsis thaliana myosin family comprises17 members: 13 myosin XI variants (myosin XI‐A–K, XI‐1, andXI‐2) and four myosin VIII variants (ATM1, ATM2, myosin VIII‐A,and VIII‐B) (Reddy and Day 2001; Peremyslov et al. 2011), nowrenamed following a comprehensive phylogenetic analysis(Muhlhausen and Kollmar 2013) but still preserve their oldnames in this review.

The A. thaliana myosins have a conserved motor domainwith ATPase and actin‐binding activities, a number of IQdomains to bind myosin light chains, a coiled‐coil domain fordimerization, and a specific tail that binds different cargoes(Tominaga and Nakano 2012). Using these functional domains,myosins convert chemical energy from ATP hydrolysis intophysical movement along actin filaments (Tominaga et al.2003; Ito et al. 2007, 2009), carrying membrane‐boundorganelles or RNA/protein complexes with their tails (Li andNebenführ 2008; Steffens et al. 2014).

Plant myosin XI were found to be involved in polar growth,which involves the preferential delivery and accumulation ofwall and membrane materials to the correct location via thesecretory and endocytic pathways (Samaj et al. 2005). Cells ofroot hairs, pollen tubes andmoss protonemata are typical longcells that elongate in a polar tip growth manner (Samajet al. 2004; Vidali and Bezanilla 2012; Guan et al. 2013; Hepler

et al. 2013). Knocking down the two myosin XIs in mossresulted in severe loss of cell polarity (Vidali et al. 2010) and A.thaliana knocked out for myosin XI‐K and XI‐2 had shorter roothairs (Ojangu et al. 2007; Peremyslov et al. 2008). In addition,root hairs in xik3 plants grow more slowly and stop growthsooner than those of wild type plants (Park and Nebenführ2013) suggesting a role for myosins in polar growth. Also, whendouble, triple, and quadruple myosin‐knockout A. thalianaplants were generated, it was found that the more myosingenes were knocked out, the smaller and shorter themesophyll and epidermal cells, as well as the whole plants,became (Prokhnevsky et al. 2008; Peremyslov et al. 2010;Ojangu et al. 2012), indicating on a role of myosin XI in polardiffuse growth too. In an elegant study, the head domain ofmyosin XI‐2 was replaced with either that from the high‐speedmyosin XI from the alga Chara corallina or that from the low‐speed myosin Vb from Homo sapiens (Tominaga et al. 2013).Interestingly, cytoplasmic streaming, cell length and plant sizeincreased in plants expressing the high‐speed chimera anddecreased in those expressing the low‐speed chimera(Tominaga et al. 2013), indicating the importance of cyto-plasmic streaming and myosins to polar growth.

While accumulated data indicate thatmyosins XI propel themotile organelles (Sparkes 2011; Tominaga and Nakano 2012;Madison and Nebenführ 2013), and cytoplasmic streaming(Shimmen and Yokota 1994, 2004; Shimmen 2007; Esseling‐Ozdoba et al. 2008; Woodhouse and Goldstein 2013), themachinery by which this is manifested is not entirelyunderstood.

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In contrast to myosin V and other unconventional myosinsthat attach to organelles via adaptor proteins (Li andNebenführ 2008) and move them along actin tracks, thesituation that has been recently uncovered in plants seemsdifferent.

A family of myosin receptors was found (MyoB), themembers of which are membrane proteins with a conserveddomain DUF593 (Peremyslov et al. 2013). Colocalization studiesof MyoB1/2 with a full‐length functional myosin XIK identifiednew vesicles, not labeled with other known markers, whichwere determined as transport vesicles. It was found thatmyosin XIK predominantly attaches to these transport vesiclesand therefore it was concluded that their movement propelsthe cytoplasm and indirectly the other known vesicles andorganelles to which no myosin XIK binding was observed(Peremyslov et al. 2013, Figure 1). This is an unexpectedproposed mechanism of organelle movement, which isdifferent from that described in the text books.

Other studies using a full‐length functional myosin XIshowed diffused cytoplasmic distribution rather thanspecific colocalization with motile organelles (Vidali et al.2010; Park and Nebenführ 2013). This led to the suggestionthat the excess of myosin molecules in the cytoplasm,which are ready to act, or inhibited by a head to tail interaction(Avisar et al. 2012), might mask specific accumulation onthe surface of organelles. In contrast, studies using anti-bodies and truncated recombinant fragments of myosin tailsshowed myosin‐organelle colocalization. However, thesestudies have attracted criticism because of lack of antibodyspecificity and because truncated myosin tails are notnormally expressed in cells and might exhibit unspecificinteractions.

Here we review the accumulated evidence for both directand no direct interaction between myosins and plantorganelles and outline the open questions in the field.

ROLE OF MYOSINS IN ENDOPLASMICRETICULUM MOTILITYThe endoplasmic reticulum (ER) is the first organelle in thesecretory pathway (Sparkes et al. 2009a). It resides in twocompartments: the submembrane compartment (cortical ER),which is a less mobile fraction, and the cytoplasmic strands—the highly mobile fraction (Quader and Schnepf 1986;Stromgren Allen and Brown 1988). The cortical ER is madeup of a polygonal network of tubules that expands throughoutthe cell with cisternae (flat membrane sheets) positionedinside the polygons. It was hypothesized that the less motilecortical ER is anchored to the plasma membrane (Quaderet al. 1987) and later, a candidate protein myosin VIII, ATM1,which was found to colocalize with ER junctions (Golombet al. 2008), was suggested to bind the ER to the plasmamembrane (Sparkes et al. 2009c). Recently NET3c (a familymember of the actin binding NET proteins that link actin tomembrane compartment), and VAP27 (vesicle associatedprotein 27) were found to mediate the link between the ERand the plasmamembrane (Wang et al. 2014) and it is intriguingto checkwhethermyosin VIII is part of this complex. ER bindingby myosin at the plasmodesmata was also suggested (Radfordand White 1998, 2011; Baluška et al. 2000, 2001), and sincemyosin VIII was localized to the plasmodesmata (Baluška et al.2000, 2001; Golomb et al. 2008; Haraguchi et al. 2014), itbecame a putative candidate for this binding. Analysis of theknockout plant of all five myosin VIII transcripts of Physcomi-trella patens revealed that this class has a role in developmentand hormone homeostasis (Wu et al. 2011). The analysis of thequadruple mutant of all myosins VIII of Arabidopsis is still to bedone.

Close examination of tobacco leaf epidermal cells enabledcharacterizing the dynamics of the cortical ER (Sparkes et al.2009c). Consistent with the membrane‐anchored model,cortical ER dynamics seemed not to consist of a shift of thewhole network relative to the plasmamembrane but rather, tobe based on remodeling of the polygon network due to thedynamic rearrangement of tubules, nodes and cisternae(Sparkes et al. 2009c). When these dynamic changes werecharacterized by persistency maps, which show the persis-tence of tubule and cisternae, it was found that in the presence

Figure 1. The possible models for organelle movement(A) Each organelle is attached to amyosin XI motor protein andis translocated along actin fibers. Organelles move in variabledirections due to the variable directions of actin fibers. Theviscous cytoplasm is entrained by themotion of the organelles,resulting in cytoplasmic streaming. Unbound myosin mole-cules, which might be head‐to‐tail folded in an inactive form(Avisar et al. 2012), are present in the cytoplasm. (B) Onlyspecific transport vesicles are bound to myosin XI andtranslocated along actin fibers. The other organelles movepassively in different orientations depending on the cyto-plasmic flows and turbulences created by the transportvesicles (Peremyslov et al. 2013). Alternatively, organellesacquire their motility by transiently attaching to the transportvesicles (Peremyslov et al. 2013).

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of myosin XI‐K tail dominant negative fragment or afterdisruption of actin with latrunculin B, persistency of ER tubulesincreased (Sparkes et al. 2009c). Recently, a more detailedanalysis was done in which regions of persistent tubules,cistrnae, and puncta were measured, the overall morphologyof tubule and cisternae were determined and total ERmovement was followed. It was found that myosins XI differin their ability to influence the ER network dynamics; while atail fragment of XIA had no effect, tail fragments of myosins XI‐K, XI‐C, XI‐1, and XI‐E increased persistency, that of myosin XI‐2reduced movement and in the presence of myosin XI‐I tailmajor increase in ER cisternae area occurred compared to wildtype plants (Griffing et al. 2014).

In addition, movement of the highly mobile ER incytoplasmic strands (which look like, and are termed ERstrands) was carefully measured in A. thaliana epidermal cellsof cotyledonary petioles (Ueda et al. 2010). It was shown thatthe ER in these strands moves with distinct orientations andvelocities. When the movements of such ER strands werefollowed in several single, double, and triple mutants ofmyosin XI‐K, XI‐1, and XI‐2, suppression of ER movement wasdetected in that targeting myosin XI‐K and was strongest inthe triple mutant. Myosin XI‐K also affected the pattern of theER and cofractionated with the ER in a microsomal fraction-ation assay (Ueda et al. 2010). This indicated a closerelationship between the ER and myosin XI‐K. Further supportfor this putative interaction was obtained when an antibodywas generated against a 175‐kDa myosin heavy chain,which was biochemically isolated from BY‐2 cells. Coimmu-nostaining using this specific anti‐myosin XI antibody revealedcolocalization with the ER (Yokota et al. 2009). Furthermore,full‐length myosin XI‐K labeled with YFP (yellow fluorescentprotein) partially colocalized to the ER (Peremyslovet al. 2012). The myosin XI‐I homolog of maize, Opaque1(O1), was also shown to cofractionate with the ER, upon itsmutation cisternal ER became dilated and a dominantnegative tail domain of O1 inhibited ER movement (Wanget al. 2012).

In addition, it was shown that ER tubules can bereconstructed in vitro by applying shear force generated bythe acto‐myosin system (Yokota et al. 2011). ER‐tubule‐likestructures were also formed behind Golgi bodies, which weretranslocated through the cytoplasm by laser tweezers(Sparkes et al. 2009b). However, the ER tubules were stilldynamic when the Golgi bodies were deconstructed byBrefeldin A (Sparkes et al. 2009c), suggesting that ER dynamicsis independent of that of Golgi bodies. Conversely, it wasshown that the movement of Golgi, mitochondria andperoxisomes is influenced by that of the ER. More strikingly,it was found that an ER membrane‐associated dynamin likeRHD3 regulates the ER remodeling in an actin independentmanner and that this dynamics affect the cytoplasm streaming(Stefano et al. 2014).

Taken together, it is strongly suggested that ER dynamicsdepends on class XI myosins, albeit not exclusively. In addition,there are no explicit indications for direct attachment, or viaadaptor proteins, of myosins XI or VIII to the ER. How canmyosins power ER movement without a direct interaction?One scenario might be via regulation of ER‐associated actinfilaments, an activity that has been recently described formammalian myosin 1c (Joensuu et al. 2014).

ROLE OF MYOSINS IN THE MOTILITY OFGOLGI, MITOCHONDRIA, ANDPEROXISOMESThe association of plant myosins with different organelles wasfirst predicted by immunohistochemical studies using heterol-ogous anti‐myosin antibodies prepared against non‐plantmyosins. These studies showed the labeling of unidentifiedorganelles in the pollen tubes of Lilium longiflorum andNicotiana alata (Tang et al. 1989; Miller et al. 1995), as well as ofamyloplasts andmitochondria in the pollen tubes ofHyacinthusorientalis and Helleborus foetidus (Heslop‐Harrison and Heslop‐Harrison 1989). Specific anti‐myosin XI antibodies labeledunidentified organelles (Liu et al. 2001) and mitochondria(Wang and Pesacreta 2004) in maize root tip cells, as well assome organelles in BY‐2 cells (Yokota et al. 2009). Specific anti‐myosin XI‐2 (Mya2) labeled peroxisomes in A. thaliana cells(Hashimoto et al. 2005).

These data are supported by findings obtained fromstudies in which fluorescent proteins were fused to the taildomains of various myosins. Fluorescent subdomains of XI‐1and XI‐2 colocalized with peroxisomes and occasionally withGolgi bodies (Li and Nebenführ 2007). In another study, amyosin XI‐2 tail domain, labeled peroxisomes, and that ofmyosin XI‐J seemed to sometimes colocalize with Golgi andmitochondria, while YFP‐XI‐K occasionally colocalized withGolgi bodies (Reisen and Hanson 2007). In agreement with theobservations of XI‐2 colocalization with peroxisomes, RabC2a,which was found to interact with myosin XI‐2 tail in a yeasttwo‐hybrid assay, was also localized to the peroxisomes(Hashimoto et al. 2008). Another study used fusions offluorescent proteins to a sub‐domain from different membersof myosin XI family that is homologous to the organelle‐inheritance domain of the myosin V, myo2p, from yeast. Theseshort fragments, termed PAL domain, from myosins XI‐1, XI‐B,XI‐D, XI‐F, XI‐G XI‐H, XI‐I, and XI‐K were colocalized to Golgibodies, those from XI‐C, XI‐E, XI‐G, XI‐H, XI‐I, and XI‐Kcolocalized to the mitochondria, and only the fragment fromXI‐F colocalized to peroxisomes (Sattarzadeh et al. 2013).

Curiously, no colocalization with any of these organelleswas shown when the whole tail or the tail including the IQdomain of all 17 A. thaliana myosins was fused to greenfluorescent protein (GFP) or red fluorescent protein (RFP)(Sparkes et al. 2008; Avisar et al. 2009). Furthermore, when thefull‐length of myosin XI‐K was fused to GFP at its C terminusand expressed in the triple mutant xik/xi1/xi2, the dwarfphenotype of this mutant was rescued, indicating thefunctionality of the chimera (Peremyslov et al. 2012). However,the GFP signal of this functional full‐lengthmyosin only partiallycolocalized with the ER and very seldom with Golgi bodies(Peremyslov et al. 2012, 2013). Also, N‐terminal YFP fusion offunctional full‐length myosin XI‐K (Park and Nebenführ 2013)and GFP fusion of functional full‐length P. patens myosin XIa(Vidali et al. 2010), exhibited cytoplasmic localization in roothairs or protonemata, respectively.

Taken together, the truemyosin cytoplasmic distribution inthe different cells and the direct binding of myosins to Golgi,mitochondria, and peroxisomes remains to be verified.

The role of myosins in organelle movement was shownboth by the use of dominant negative truncated tail con-structs and in loss of function mutants. In the presence of

Role of plant myosins in organelle motility 3


overexpressed dominant‐negative tail constructs of myosinsXI‐K, XI‐F, and XI‐2 from Nicotiana benthamiana, the movementof Golgi, mitochondria, and peroxisomes was inhibited(Avisar et al. 2008). Similarly, using dominant‐negativeconstructs of all 17 A. thaliana myosins revealed that myosinsXI‐C, XI‐E, XI‐K, XI‐I, XI‐1, and XI‐2 were important for themotility of Golgi bodies and mitochondria in Nicotiana tabacumand N. benthamiana (Avisar et al. 2008, 2009; Sparkeset al. 2008. A reduction in organelle motility was also foundin the A. thaliana knockout plants xik and xi2 (Peremyslovet al. 2008) and in the double mutants xik/xi1, xik/xi2, and xi2/xib, while the xik/xi1 mutant showed the largest reduction inorganelle velocity (Prokhnevsky et al. 2008). Inactivation offourmyosins in the quadruplemutant (xi1/xi2/xii/xik) resulted indramatic arrest of Golgi and peroxisome motility, suggestingthat their trafficking is largely myosin‐dependent (Peremyslovet al. 2010). Curiously, Golgi, mitochondria, and peroxisomeshardly move in wild type P. patens protonemal cells (Furtet al. 2012).

Recently, using a yeast two hybrid screen, a family ofmyosin receptors was found, named MyoB (Peremyslovet al. 2013). It was found that myosin XI‐K, via the binding toMyoB1/2, localized to as‐yet unidentified transport vesicles thatwere not labeled with any of the known organelle or vesiclemarkers examined (Peremyslov et al. 2013). The MyoB1/2decorated transport vesicles were found to move in directtrajectories along actin fibers in myosin XIK dependentmanner. This observation, in combination with the lack offirm evidence of direct interaction of myosins XI with otherorganelles, led to the hypothesis that the transport vesiclesprovide the power for the hydrodynamic flowof the cytoplasmand that the movement of other known organelles is to alarge extent passive (Peremyslov et al. 2013). Alternatively,it was hypothesized that organelles might transiently attachto transport vesicle and are carried for a short distance(Peremyslov et al. 2013) (Figure 1). This suggested model isnovel and specific for plants.

ROLE OF MYOSINS IN THE MOTILITY OFSECRETORY AND ENDOCYTIC VESICLESArabidopsis thaliana full‐length and functional myosin XI‐K‐GFP(GFP fused to the tail) was found at the apex of elongating roothairs, overlapping with two exocytic markers, RabA4b andSCAMP2 (Peremyslov et al. 2012). In addition, a YFP‐XI‐K fusion(YFP fused to the head) partially overlapped with RabA4b andother tip markers in root hairs (Park and Nebenführ 2013).Moss GFP‐myosin XIa was also found at the apex ofprotonemal cells (Vidali et al. 2010; Furt et al. 2013) whereexocytosis and polar growth take place. Accordingly, myosinXI‐K cofractionated with RabA4b in A. thaliana leaf extract andwith Sec6, a component of the exocyst (Žárský et al. 2013), inthe root extract (Peremyslov et al. 2012). Moreover, in leavesofN. benthamiana, a GFP fused dominant‐negative construct ofmyosin XI‐K inhibited the movement of vesicles labeled withARA6, ARA7, and FYVE marking endosomes, SYP21 and SYP22marking the prevacuolar compartment, and SYP41 and RabA4bmarking the trans‐Golgi network (Avisar et al. 2012). However,no colocalization was observed between the GFP‐labeledmyosin XI‐K tail and these vesicles (Avisar et al. 2012).

Furthermore, no significant colocalization was found betweenthe full‐length myosin XI‐K fused to GFP and exocytic vesicleslabeled with VAMP721, clathrin‐coated endocytic vesicleslabeled with clathrin light chain 2 or vesicles labeled withflotillin, a marker of the clathrin‐independent endocyticpathway (Peremyslov et al. 2013). The accumulation of YFP‐XI‐K and (cyan fluorescent protein) CFP‐RabA4b fluctuatedindependently at the tip of root hairs and the distribution ofCFP‐RabA4b did not change in xik compared towild type plants(Park and Nebenführ 2013). Overall, it is suggested that myosinXI‐K is involved in propelling the movement of endocytic andexocytic vesicles, but it is still not clear whether it binds directlyto these vesicle and via which adaptor proteins.

ARE MYOSINS INVOLVED INCHLOROPLAST MOTILITY?To optimize light usage, chloroplasts move toward low light(accumulation response) and to minimize photo damage, theymove away from strong light (avoidance response) (Kong andWada 2013). Chloroplasts are surrounded by baskets of actinfilaments (Kandasamy and Meagher 1999; Krzeszowiecet al. 2007; Anielska‐Mazur et al. 2009) and disruption of actinby specific drugs inhibits chloroplast movement (Kandasamyand Meagher 1999). Further studies specified that chloroplastmovement is regulated by short chloroplast‐specific actinfilaments (cp‐actin) that accumulate on the chloroplastsurface in a light‐dependent manner (Wada and Suetsugu2004; Kong and Wada 2013). To move, chloroplasts need to betethered to the plasma membrane via cp‐actin and specificactin‐binding proteins such as chloroplast unusual positioning 1(CHUP1) (Wada and Suetsugu 2004; Kong and Wada 2013),which is an outer‐envelope chloroplast protein with actin‐binding activity, kinesin‐like protein for actin‐based chloroplastmovement 1 (KAC1) and KAC2 (Suetsugu et al. 2012), whichare essential for chloroplast association with the plasmamembrane, and THRUMIN1, which is associated with theplasma membrane and regulates actin bundling (Whippoet al. 2011).

Contradictory data have been accumulated both regardingmyosin’s co‐localization with chloroplasts and regarding theirdirect role in chloroplastmovement. Immunostainingwith anti‐animalmyosin antibodies (Przemyslaw et al. 1996; Krzeszowiecand Gabryś 2007) or specific anti‐myosin XI (Wang andPesacreta 2004) or anti‐myosin VIII (Wojtaszek et al. 2005)antibodies suggested an association of myosin with plastidsand chloroplasts. Furthermore, when YFP was fused to adomain of the myosin XI‐F tail (of both A. thaliana and N.benthamiana), fluorescence was associated with N. benthami-ana leaf chloroplasts (Sattarzadeh et al. 2009). Similarly, GFPfused to a tail domain homologous to myosin XI‐2 from N.benthamiana decorated N. benthamiana leaf chloroplasts(Natesan et al. 2009). In addition to chloroplasts, these myosinXI‐tail fusions also colocalized with stromules—extendedtubules of plastid membranes (Natesan et al. 2009;Sattarzadeh et al. 2009). Taken together, colocalization ofmyosins with chloroplasts has been demonstrated in bothfixed and live cells, by several groups.

In addition, chloroplast distribution was affected by virus‐induced gene silencing (VIGS) of myosin XI in tobacco plants

4 Buchnik et al.


(Sattarzadeh et al. 2009). The latter finding was supported byexperiments in which known myosin inhibitors were shown toinhibit chloroplast movement. 2,3‐Butanedione monoxime(BDM), a myosin ATPase inhibitor, and 1‐(5‐Iodonaphthalene‐1‐sulfonyl) homopiperazine (ML‐7), a myosin‐light‐chain kinaseinhibitor, were shown to inhibit bundle‐sheath chloroplastreposition after centrifugation of Eleusine coracana plants(Kobayashi et al. 2009). These compounds, as well as N‐ethylmaleimide (NEM) which inhibits the actin‐mediatedATPase activity of myosin, were able to inhibit chloroplastmovement in A. thaliana leaves (Paves and Truve 2007).However, the specificity of these drugs has been questioned(McCurdy 1999; Seki et al. 2003).

Furthermore, dominant‐negative tail domains of myosinsXI‐F, XI‐K, XI‐2, and VIII‐B from N. benthamiana failed to inhibitlight‐avoidance or accumulation movement of chloroplasts inN. benthaniana leaves (Avisar et al. 2008), and double, triple,and quadruple A. thalianamutants for myosin XI (Prokhnevskyet al. 2008; Peremyslov et al. 2010) showed normal avoidanceand accumulation movement of chloroplasts (Suetsuguet al. 2010).

Sowhich of the 13myosin XI and fourmyosin VIII variants, ifany, are involved in chloroplast movement? Myosins XI‐A–Eand J and ATM2 exhibit high expression specifically in pollen,and lower expression in leaves (Peremyslov et al. 2011).However, their roles, and those of myosins XI‐G, H, ATM1, andVIII‐A in chloroplast movement are unknown. Of note, tailfusions of these myosins to GFP did not prove chloroplastlocalization (Avisar et al. 2009), except the full‐length GFP–ATM1, which did (Haraguchi et al. 2014). Myosin VIII was shownto localize to the plasmamembrane (Golomb et al. 2008; Avisaret al. 2009), and detailed in vitro thermodynamic studies ofmyosin VIII head have revealed that it displays a relatively slowto moderate ATPase cycle, with a slow calculated motility rate(Haraguchi et al. 2014). Based on these findings and oncomparisons with other classes of myosins with similarcharacteristics (Henn and De La Cruz 2005; Bloemink andGeeves 2011) it was suggested that myosin VIII is adapted tofunction as a tension sensor and/or tension generator(Haraguchi et al. 2014). Therefore, it is intriguing to assumethat myosin VIII could participate in anchoring cp‐actin to theplasma membrane. In addition, because unconventionalmyosins have been shown to regulate actin dynamics(Soldati 2003), it could be hypothesized that myosin XI familymembers have a role in cp‐actin remodeling. These activitiesmight be specific to plant type, cell type, and/or developmentalstage, which might explain the contradictory data accumulat-ed to date from different cells and plant types.

THE ROLE OF MYOSINS IN NUCLEARMOTILITYThere are three different cellular scenarios for plant nucleusmigration: (i) for cell division, the nucleus is positioned at thesite of the pending cytokinesis (Smith 2001); (ii) during tipgrowth of root hairs, for example, the nucleus is positioned at aconstant distance from the tip (Ketelaar et al. 2002; VanBruaene et al. 2003); (iii) following strong light exposure nucleiare repositioned to cell edges in order to avoid damage (Higaet al. 2014b). The contribution of microtubules and actin to

these scenarios of nuclear migration differ from species tospecies (reviewed in Takagi et al. 2011).

Myosin staining on the nuclear envelopewas shown by theuse of heterologous antibodies (Heslop‐Harrison and Heslop‐Harrison 1989). Later, a GFP fusion with the tail of myosin XI‐Iwas shown to localize to the nuclear envelope (Avisaret al. 2009). In addition, the PAL domain of myosins XI‐1, XI‐B, XI‐C/E, XI‐F, XI‐G, XI‐I, and XI‐K exhibited nuclear envelopelocalization (Sattarzadeh et al. 2013). Interestingly, meannucleus sphericity was reduced in xi‐2 and xik but not in xi‐1plants (Ojangu et al. 2012), suggesting some function ofthe two former myosins in nuclear shape. It was recentlyfound that a myosin XI‐I mutant allele (kaku1) has sphericalnuclei instead of elongated ones, and that these nuclei movemore slowly in root cells than the wild type (Tamuraet al. 2013). Curiously, normal nucleus movement wasobserved in pollen tubes of this mutant. In addition, whilenuclear movement during dark adaptation was hampered inthe kaku1 mutant, light‐induced movement was normal inmesophyll cells (Tamura et al. 2013). An interaction of myosinXI‐I with the nuclear envelope was found, which is mediatedby the nuclear envelope proteins WIT1 and WIT2 (Tamuraet al. 2013). Of note, nuclear movement is not solelydependent on myosin XI‐I, and myosin XI‐I might havefunctions other than nuclear migration. Recently it wasshown that nuclear migration in response to light depends onplastid attachment and the short‐actin‐filament machinery(Higa et al. 2014a). Additionally, MyoB7 the myosin XI‐Iinteracting protein was found to localize to vesicle‐likestructures not associated with the nucleus (Peremyslovet al. 2013). Taken together, unlike in the case of organellesand myosin XIK, there is agreement between colocalizationand functional studies regarding the role of myosin XI‐I in themovement of nuclei.

CONCLUSIONSWhile it is well established that members of the plant myosin XIfamily are responsible for the dynamic cytoplasmic streaming,and for organelles and vesicles motility, which in turn, areimportant for polar growth, it is not yet clear how, or even ifmyosins attach to each motile organelle. The discovery ofadditional myosin‐interacting proteins will shed more light ontheir mode of action.

ACKNOWLEDGEMENTSES acknowledges a grant from the Israeli Science Foundation(ISF) 401/09.

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Role of plant myosins in motile organelles: Is a direct interaction required?

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