Abstract: The motile proteins actin and myosin appear to be integral components ofplasmodesmata, the channels through plant cell walls that provide a direct linkbetween the cytoplasm of adjacent cells. Antibodies to both actin and myosinlabel the entire length of plasmodesmata in lower and higher plants. Incombination with ultrastructural evidence, we speculate that actin could bearranged as a spiral around the central strand of endoplasmic reticulum (ER)within each plasmodesma, and myosin may form the observed links betweenthe ER and the surrounding tube of plasma membrane that is also continuousfrom cell to cell. Inhibitor studies suggest that both proteins are important inmaintaining the structural integrity of plasmodesmata. Actin and myosindisruptors also affect intercellular transport in some species, implying thatactomyosin contraction may regulate transport either at the plasmodesma neckor by altering bulk flow from cell to cell. These findings suggest that, togetherwith other contractile proteins, actin and myosin playa key role in regulatingplasmodesma structure and intercellular transport in plants.
1. CELL-CELL COMMUNICATION VIAPLASMODESMATA
Plasmodesmata are plasma-membrane-lined channels that cross the cellwall, providing a pathway for transport between the cytoplasm ofneighbouring plant cells. They contain a central element of endoplasmicreticulum (ER) known as the desmotubule (Figs. 1a.b) which connects theER of neighbouring cells (Overall et aI., 1982). The structure and regulation
of these channels have been the subject of several recent reviews (Lucas etaI., 1993; Overall & Blackman 1996; McLean et aI., 1997; Ding, 1998, Dinget aI., 1999; Lucas, 1999; Overall, 1999; van Bel et aI., 1999). Until recently,plasmodesmata were considered to be simple conduits for the passivetransport of small molecules up to I kDa (Robards & Lucas, 1990).However, recent advances have shown that plasmodesmata are highlydynamic structures which under certain conditions, allow the intercellulartransport of much larger molecules (Lucas et aI., 1993; Ding, 1999; Lucas,1999). Despite these recent findings, we know little about the moleculararchitecture of plasmodesmata, nor the exact mechanism of transportthrough them, or how this transport is regulated.
Transport through plasmodesmata and its dynamic regulation might beexpected to involve motility or contraction generated by the cytoskeleton. Inthis chapter, we discuss the evidence showing that cytoskeletal proteins forman integral part of plasmodesma structure and that these proteins may beinvolved in intercellular transport or its regulation.
2. ACTIN AND MYOSIN ARE FOUND INPLASMODESMATA
Actin bundles labelled with the specific F-actin stain, rhodaminephalloidin, occasionally focus on pit fields in epidermal tissue, and faintstrands can be seen crossing cell walls that contain only individualplasmodesmata not concentrated in pit fields (White et aI., 1994). Pit fieldsthemselves are sometimes brightly stained by rhodamine-phalloidin (Fig. 2a)(Hush & Overall, 1992; White et aI., 1994). Antibodies to animal or plantactin label a single band at approximately 43 kDa on Western blots of wholeplant extracts (Metcalf et aI., 1980; White et aI., 1994; Blackman & Overall,1998). These anti-actin antibodies labeled plasmodesmata in young andmature cell walls of Hordeum (Figs. 2b,c), Nicotiana (White et aI., 1994),Zea (Reichelt et aI., 1999) and Chara (Figure 2d) (Blackman & Overall,1998) at the transmission electron microscopy (TEM) level.
28. Actin and Myosin in Plasmodesmata 499
Figure 1. Electron micrographs comparing plasmodesmata with actin and brush bordermicrovillae. (a-b) Longitudinal images of plasmodesmata showing the plasma membrane(PM), endoplasmic reticulum (ER) and desmotubule (arrow). The desmotubule is surroundedby a spiral of electron dense material (arrowheads). (a) Azolla root conventionally fixed in thepresence of tannic acid (from Overall et aI., 1982). Scale bar = 25 nm. (b) Freeze substituted
500 Overall, White Blackman and Radford
barley root. (R. G. White, unpublished) Scale bar = 50 nm. (c-d) Transverse sections ofplasmodesmata delimited by the plasma membrane and containing a desmotubule (arrow).The desmotubule is surrounded by a 'mottled layer' of electron -lucent particles which areparticularly clear in (c) (arrowhead). In (d-e), an electron-lucent cytoplasmic lumen is seenbetween the 'mottled layer' and the plasma membrane (PM). This region is traversed byelectron-dense spokes (arrowheads). (c) Azolla root (from Overall et aI., 1982). Scale bar = IOnm. (d) Barley root (S. 1. Brett and R. G. White, unpublished). Scale bar = IO nm. (e) Egeriadensa leaves (supplied by 1. E. Radford) . Scale bar = IO nm. (f) Cross section of a bundle ofnegatively stained actin filaments (from Maciver et aI., 1991). Scale bar = 20 nm. (g)Longitudinal section through a brush border microvillus. The core bundle of actin filaments(A) is connected laterally to the plasma membrane (PM) by myosin cross filaments (M). (h)Longitudinal section through a de-membranated brush border microvillus in which the crossfilaments retain their striped pattern . (g-h) From Matsudaira and Burgess (1982) . Scale bars =
50 nm. (i) Oblique section of plasmodesma with extracellular filaments (arrowheads) andparticles, fixed in the presence of tannic acid (from Badelt et aI., 1994). Scale bar =25 nm.
Since plasmodesmata were labeled along their entire length and at theneck where they open into the cytoplasm, we conclude that actin mostprobably lines the cytoplasmic lumen between the desmotubule andsurrounding plasma membrane, linking the cytoskeleton of adjacent cells.Actin integrity and antigenicity is compromised during preparation for TEM,and these results were obtained only after all measures to preserve actin(omission of osmium, minimal chemical fixation or freeze-fixation, lowtemperatures during polymerization) were taken.
Antibodies to animal and plant myosin label bands in whole plantextracts consistent with either complete myosin or its breakdown products(Parke et aI., 1986; Qiao et aI., 1994; Miller et aI., 1995; Blackman &Overall, 1998; Radford & White, 1998; Reichelt et aI., 1999). Althoughmyosin was extremely difficult to preserve intact for Western blot analysis(Radford & White, 1998), the antigenic sites targeted by the antibodies usedappear relatively robust, and plasmodesmata can be stained by bothimmunofluorescence and immuno-EM (Blackman & Overall, 1998; Radford& White, 1998; Reichelt et aI., 1999). Immunofluorescence images showintense labelling of pit fields (Figs. Ja-d) (Radford & White, 1998; Reicheltet aI., 1999) and faint labeling of single plasmodesmata (Radford and White,1998) by antibodies to animal or plant myosins. TEM images revealed thatantibodies against myosin labeled the entire length of plasmodesmata (Figs.3e-h) (Blackman & Overall, 1998; Radford & White , 1998; Reichelt et aI.,1999). Radford and White (1998) showed that the antibodies that labeledplasmodesmata also labeled the surface of the ER, strands of cytoplasm andother organelle surfaces in cells, in locations where myosin would beexpected (Grolig et aI., 1988; Qiao et aI., 1994; Miller et aI., 1995). Myosinantibodies also labeled the modified central cavities of matureplasmodesmata in Chara cell walls (Figure 3h), indicating that, as for actin,
28. Actin and Myosin in Plasmodesmata 501
the myosin cytoskeleton appears to be continuous from cell to cell in lowerand higher plants.
.~. ~
\
\.
\
.:
,,:...~
Figure 2. Localisation of actin to plasmodesmata. (a) Confocal laser scanning microscopeimages of inner epidermal peels of Hordeum vulgare stained with rhodamine-phalloidin,showing actin filaments and fluorescent pit fields (arrowheads). Scale bar = 5 urn (fromWhite et aI., 1994). (b-d) Electron micrographs showing labelling of plasmodesmata by antiactin antibodies. (b-e) Gold label (arrowheads) on plasmodesmata from Hordeum vulgare .Scale bars = 50 nm. (from White et aI., 1994). (d) Plasmodesmata branches (arrows) fromChara corallina. (L. M. Blackman, unpublished) Scale bar = 100 nm.
502
h
Overall, White Blackman and Radford
--.. ••• •
•.-.-
-Figure 3. Immunolocalisation of myosin to plasmodesmata. (a-d) Adjacent optical sections ofa cell wall of Zea mays immunofluorescently labelled with anti-myosin antibodies, showingfluorescence traversing the wall at pit-fields (arrow). Scale bar = 3 um. (e-h) Immuno-EM ofanti-myosin antibodies on plasmodesmata. (e-t) Z. mays plasmodesmata (arrows). Scale bar =50 nm (a-f from Radford & White, 1998). (g) Immature plasmodesmata from Characora/lina. Scale bar = 100 nm. (h) Mature plasmodesmata from Chara cora/lina showinglabelled middle cavity (Blackman, unpublished). Scale bar = 100 nm.
28. Actin and Myosin in Plasmodesmata 503
Fifteen families of myosins have been identified so far (Titus & Gilbert,1999) but only some of these have been identified in plants (Knight andKendrick-Jones, 1993; Kinkema & Schiefelbein, 1994; Kinkema et al., 1994;Yamamoto et al., 1999). Interestingly, the antibodies raised against the tailregion of a specific plant myosin VIII labeled only the plasma membraneand plasmodesmata but no other structures in the cells (Reichelt et al., 1999).In addition, the antibody used to localize myosin in Chara plasmodesmata(Blackman & Overall, 1998) probably recognizes myosin V (Plazinski et al.,1997). Since different types of myosin are involved in different functions(Baker & Titus, 1998), the identification of the types of myosin inplasmodesmata will be critical in determining the function of actin andmyosin in cell-to-cell communication.
3. ARRANGEMENT OF ACTIN AND MYOSIN INPLASMODESMATA
The large size of primary and secondary antibodies , each of which isabout 8 nm (Roth, 1982), means that immuno-EM does not have sufficientresolution to localize actin or myosin to specific structures seen inultrastructural images of plasmodesmata. Therefore, the exact location andarrangement of actin and myosin can only be speculated upon.
One approach to this speculation is to compare the staining patterns anddimensions of components of the plasmodesmata with that for actin andmyosin. Surrounding the desmotubule is a 'mottled layer' which may abutdirectly onto the plasma membrane (Figs. 1a,c) or there may be an electronlucent region between this 'mottled layer' and the cell membrane (Figs. 1b,d,e). This electron-lucent region may contain electron-dense spokes(Burgess, 1971; Tilney et al., 1991; Ding et al., 1992; Schulz, 1995; Cook etal., 1997) connecting the 'mottled layer' to the cell membrane (Figs. 1d,e).These images have been variously interpreted (for review see van Bel,1999), but one interpretation outlined in Overall (1999) is that the 'mottledlayer' consists of negatively stained electron lucent particles arranged in aspiral (Fig. 1b). This spiral is connected to the cell membrane via electrondense spokes traversing the electron-lucent region or cytoplasmic lumen.
Actin filaments that have been fixed in the presence of tannic acid appearin cross section as electron-lucent particles (Fig. If) (Maciver et al., 1991).These particles are of similar dimensions and staining patterns to theelectron-lucent particles in the 'mottled layer' of plasmodesmata in tissuefixed in the presence of tannic acid (compare Figs. 1c and 1f). Thenegatively stained actin in Figure 1f is a little larger than a pure actinfilament as it has been co-polymerised with alpha actinin and actaphorin.
504 Overall, White Blackman and Radford
This suggests that the spiral of electron-lucent particles around thedesmotubule is actin. In transverse sections of plasmodesmata, there isusually only one site per plasmodesma labeled with the anti-actin antibody(White et aI, 1994). Presumably, only one antigenic site was at the surface ofthe section, as would be expected if it were in a spiral arrangement.
Similarly, the electron dense spokes in the plasmodesmata are alsocomparable in appearance and dimensions to brush border myosin I (BBM)in epithelial microvilli (compare Figs. ld,e and 19). Here, actin is bundled toform a stable structural element in the microvillus, and around this bundle isa 'finger' of cell membrane which is anchored to the actin via regularlyspaced BBM molecules (Fig. l g) (Matsudaira & Burgess, 1982). When themembrane has been removed, the myosin retains a transverse striping patternaround the core of actin (Fig. l h), reminiscent of the patterning around thedesmotubule (Fig. 1b). The electron-dense spokes in plasmodesmata maywell be myosin.
There are other filamentous structures associated with plasmodesmatathat could also comprise elements of the cytoskeleton. For example, there isan electron dense connection between the plasma membrane and theendoplasmic reticulum around the neck region of plasmodesmata (Overall etal., 1982) and a variety of electron-dense filaments and particles surroundingthe plasmodesmata in material treated with tannic acid (Fig. 1i) (Badelt etal., 1994). The position of these structures suggests that they could beinvolved in contraction to regulate intercellular transport pathways so thatthe involvement of actin or myosin in these structures is also a possibility.
4. ROLE OF ACTIN AND MYOSIN INMAINTENANCE OF STRUCTURE OFPLASMODESMA
One possible role for actin and myosin in plasmodesmata could besimilar to that of actin and its associated BBM in the intestinal epithelium ofanimal cells (Matsudaira & Burgess, 1982), that is, maintenance of structure.Nevertheless, this myosin retains some dynamic behaviour in that calcium,and other regulatory molecules, can alter the attachment to actin and thetightness of binding to the plasma membrane (for review see Hammer,1994). There are several other animal myosins whose main role in cells is toprovide structural integrity, such as ninaC (Hicks et al., 1996), and similarly,both actin and myosin may serve to maintain plasmodesma structure. Plantmyosins are generally small compared to the large, motile myosin II ofanimal cells, and some of these may well have a primarily structural role, asis postulated for the unconventional small myosin VIII (Reichelt et ai.,1999).
28. Actin and Myosin in Plasmodesmata 505
A structural role is further supported by the effects of the actin-disrupter,cytochalasin D, which caused plasmodesmata in the fern, Nephrolepisexultata , to open wide at their necks and to lose their extracellular structures,although little effect was seen in plasmodesmata from higher plants (Whiteet aI., 1994). One functional approach to assessing structural integrity is todetermine the largest size of molecules that can freely move throughplasmodesmata, termed the size exclusion limit (SEL). Ding et al. (1996)showed that in tobacco mesophyll cells, application or injection of the actinantagonists, cytochalasin D or profilin, increased the SEL of plasmodesmata,pointing to a role for actin in maintaining structural integrity. Furtherevidence for the role of actin and myosin in maintaining structure andcontrol over the SEL comes from studies in which ATP levels have beenexperimentally altered, leading to a change in the SEL (Reid & Overall,1992; Tucker, 1993; Cleland et aI., 1994). A myosin antagonist, 2,3butanedione monoxime, caused plasmodesma closure rather than opening, inZea mays and Allium cepa (Radford & White, 1998). Tilney et al. (1991) andTurner et al. (1994) observed drastic loss of structure in plasmodesmatatreated with proteases, emphasizing the role of proteins in maintenance ofplasmodesma structure.
5. ROLE OF ACTIN AND MYOSIN ININTERCELLULAR TRANSPORT
Intercellular transport is thought to occur mainly through the cytoplasmiclumen between the desmotubule and the plasma membrane, but the ERlumen and membrane are also possible routes (Overall, 1999). Actin andmyosin may be involved in orchestrating or regulating this transport throughplasmodesmata. Certainly, components necessary for a functionalactomyosin system, such as adenosine triphosphate (ATP) and calcium,appear to be involved. ATPase activity has been localized at plasmodesmata(van Steveninck, 1976; Belitser et aI., 1982; Franceschi & Lucas, 1982;Nougarede et aI., 1985; Chauhan et aI., 1991). In barley roots, this ATPaseactivity has been shown to be calcium activated (Belitser et aI., 1982).Higher than normal intracellular levels of calcium will halt intercellularmovement (Erwee & Goodwin, 1983, Tucker, 1990; Lew, 1994; HoldawayClarke et aI., 2000).
5.1 Non-selective transport in the cytoplasmic lumen
Transport through plasmodesmata is defined as non-selective whensubstances below the normal SEL pass without requiring any special
506 Overall, White Blackman and Radford
transport mechanism or modulation of the plasmodesma structure (forreview see Schulz, 1999). The non-selective transport can vary betweenspecies, tissue types and developmental stages. For example, while the usualSEL for plasmodesmata is in the order of 1 kDa (Robards & Lucas, 1990),free green fluorescent protein (GFP), which is a 27 kDa protein (Imlau et aI.,1999: Oparka et aI., 1999), and GFP fusion proteins as large as 50 kDa(Oparka et aI., 1999), can move from cell to cell in sink tissue.
Transport may take place by diffusion through the cytoplasmic lumen inthe plasmodesmata. In Chara, for example, the cell-to-cell movement ofradioactive chloride (Bostrom & Walker, 1975) and rubidium (Ding &Tazawa, 1989) occurs by diffusion. Disruption of actin filaments bycytochalasin E in Chara does not inhibit the cell-to-cell diffusion ofrubidium (Ding & Tazawa, 1989).
However, it is also possible that there is an actomyosin-powered bulkflow through the plasmodesmata, essentially allowing for cytoplasmicstreaming to be continuous between adjacent cells. In support of this idea isthat movement of carbon isotopes in Nitella flexilis (Zawadzki & Fensom,1986a,b) and N. translucens occurs by an active mechanism (Dale et aI.,1983). Carbon transport rates in Chara and N. translucens show a polarity(Dale et aI., 1983; Ding et aI., 1991) which is not explained by differences inphotosynthetic activity (Ding et aI., 1991). Treatments which decrease theATP concentration inhibit both cytoplasmic streaming and cell-to-celltransport in N. flexilis (Zawadzki & Fensom, 1986a) and Chara (Reid &Overall, 1992). The disruption of streaming in N. translucens by Nethy1ma1eimide, which inhibits the action of myosin, also stops cell-to-cellmovement of radioactive carbon tracers (Dale et aI., 1983). Cell-to-celltransport in S. purpurea is inhibited by the secondary messengers IP2 andIP3 (Tucker, 1988). Interestingly, the injection of CaBAPTA, a calciumloaded calcium chelator, stops cytoplasmic streaming in adjacent cells andcell-to-cell transport (Tucker, 1990). These findings predict that actin andmyosin may be involved in the intercellular transport in some species.
A final possibility is that since the actomyosin system is contractile,perhaps it could play a role in pumping materials through plasmodesmata,maybe even against a concentration gradient. If pumping were co-ordinatedat each end, a peristaltic type of contraction could force cytoplasmic contentsfrom one cell to the next. There is some evidence for asymmetry in theelectrical resistance of plasmodesmata (Overall & Gunning, 1982), but dyeinjection studies have, so far, provided no evidence for asymmetric transportof material from one tissue or cell to the next.
28. Actin and Myosin in Plasmodesmata
5.2 Selective transport in the cytoplasmic lumen
507
Transport of large molecules through plasmodesmata, made possible onlyby modulation of the structure of plasmodesmata or a specific transportmechanism, is defined as selective ( for review see Schulz, 1999).
One such example may occur in the plasmodesmata between companioncells and sieve elements in the phloem. These plasmodesmata appear toallow the movement of specific proteins into the enucleate sieve element thathave no protein synthesis machinery (Fisher et aI, 1992). Up to 200 differentproteins, with molecular sizes between 10 and 200 kDa, including actin andprofilin (Schobert et aI., 1998), have been found in phloem exudates (Fisheret aI., 1992; Sakuth et al., 1993; Nakamura et aI., 1993; Balachandran et aI.,1997). The sucrose-transporter protein, SUTl , and SUTl mRNA are alsofound in the sieve element, indicating that both the protein and mRNAundergo cell-to-cell transport into the sieve element (Kuhn et aI., 1997).
Complex molecular interactions occur between viruses andplasmodesmata during cell-to-cell (for review see Reichel et aI., 1999; alsosee McLean & Zambryski, this volume) and long-distance movement ofplant viruses (for review see Carrington et aI., 1996; Gilbertson & Lucas,1996). Viruses such as tobacco mosaic virus (TMV) temporarily modifyplasmodesmata (Oparka et aI., 1997) and move from cell to cell in anuncapsulated form. Essential for cell to cell movement of TMV is a virusencoded 30 kDa movement protein (MP) (Deom et al., 1987). The MPincreases the SEL (Citovsky & Zambryski, 1991), binds to the TMV mRNA(Citovsky et aI., 1990) and chaperones unfolded viral RNA through theenlarged plasmodesma (Citovsky et aI., 1992). Recent findings show thatthese functions are not restricted to viral MPs. For example, a plant proteinwhich can modify plasmodesmata and transport sense and anti-sense mRNAhas been identified in Cucurbita maxima (Xoconostile-Cazares et aI., 1999).Interestingly, the TMV MP also co-localises with actin filaments (McLean etaI., 1995) and microtubules (Heinlein et al., 1995; McLean et aI., 1995),indicating a role for the cytoskeleton in cell-to-cell transport (also seeMcLean & Zambryski, this volume).
During development, regulation of the permeability of plasmodesmataoccurs, creating symplastic domains (Goodwin & Lyndon, 1983; Duckett etaI., 1994; Rinnie et al., 1998; Gisel et aI., 1999) and presumably triggeringorgan development in a similar manner to that seen in animal systems(Kalimi & Lo, 1988). Also critical to normal development is non-cellautonomous action of some transcription factors (for review see Jackson &Hake, 1997). The shoot meristem consists of three distinct layers, theepidermal layer, the subepidermal layer and the inner core (Szymkowiak &Sussex, 1992; Carpenter & Coen, 1995). Through the use of chimeras, it hasbeen shown that signals from one layer can affect the development of the
508 Overall, White Blackman and Radford
other layers (Szymkowiak & Sussex, 1992). For example, the 45 kDatranscription factor, KNOTTED1, is found in all layers of the meristem,however the mRNA is only ever found in the inner core of cells (Jackson etaI., 1994). Microinjected fluorescently labelled KNOTTEDI protein hasrecently been shown to increase the SEL of plasmodesmata and catalyse itsown movement, and that of the KNOTTEDl mRNA, through theplasmodesmata (Lucas et aI., 1995; Mezitt & Lucas, 1996). Other examplesof non-cell automonous acting transcription factors include the Antirrhinumfloral homeotic genes FLORICAULA (Carpenter & Coen, 1995; Hantke etaI., 1995), DEFICIENS and GLOBOSA, the Arabidopsis trichome patterninggene, TRiPTYCHON (Schnittger et aI., 1999) and LIGULELESS-l frommaize (Becraft et aI., 1990). Central to our understanding of plantdevelopment will be the exploration of plasmodesmata function andregulation.
We propose that actin and myosin may be directly responsible for thespecific movement of viral and plant proteins and mRNA. In yeast (Long etaI., 1997; Takizawa et aI., 1997) and Caenorhabditis elegans (Guo &Kemphues, 1996), actin is directly responsible for the correct distribution ofmRNA and hence for subsequent developmental events .
5.3 Transport via the endoplasmic reticulum
In general, the ER component of plasmodesmata, as viewed in TEMimages, is tightly constricted and is only rarely seen dilated (for example , seeFig. l e, Overall & Blackman, 1996). Most studies have assumed thatintercellular transport would not occur through this compartment (Gunning& Overall, 1983). However, a recent micro-injection study has shown thatthe ER lumen can provide another intercellular transport pathway .Fluorescent probes up to 3 kDa injected into the ER of tobacco and 10 kDainto Torenia stem epidermal cells can move into the ER and nucleus of theadjacent cell (Cantrill et aI., 1999). In contrast , probes of 3 kDa and abovewill not move into the adjacent cell if the injection is cytoplasmic. Inaddition, Gamalei et aI., (1994) have shown that a contraction of theER/plasmodesmata system of the intermediary cells is associated withdecreased phloem loading at low temperatures.
Studies using fluorescent lipid and phospholipid analogs have shown thatthe ER membrane can also form a dynamic pathway through plasmodesmata(Grabski et aI., 1993). In addition, fusion proteins of GFP and MPs fromTMV (Mas & Beachy, 1998; Reichel & Beachy, 1998) and alfalfa mosaicvirus (AMY) (Huang & Zhang, 1999) localize with the ER. The biochemicalstudies of the MPs of AMY and TMY indicate that they behave as integralmembrane proteins (Moore et aI., 1992; Schaad et al., 1997; Huang &
28. Actin and Myosin in Plasmodesmata 509
Zhang, 1999). Perhaps the ER membrane provides a pathway forintercellular movement of these proteins.
These intercellular movements via the lumen and membrane of the ER,may be simply diffusive. However, it is possible that there is some selectivefiltering within the ER lumen. Indeed, an antibody against calreticulin, acalcium-sequestering protein found specifically in the ER lumen, is localisedto plasmodesmata, whereas antibodies against two other ER proteins, theHDEL retention signal and BiP (immunoglobulin binding protein), did notlocalize to plasmodesmata (Baluska et aI., 1999).
It is a tantalizing possibility that the ER may actually move throughplasmodesmata. In the cytoplasm, actin (Goosen-de Roo et al., 1983; Quaderet aI., 1987; Lichtscheidl & Uri, 1990) and myosin (Grolig et al., 1988; Qiaoet al., 1994), which are intimately associated with the ER, act together toprovide constant rearrangement and movement of the ER (Grolig et aI.,1988; Kachar & Reese, 1988; Knebel et aI., 1990). If ER does move throughplasmodesmata, which way would it travel? Is all of the actin in aplasmodesma oriented parallel, and is the orientation within a cell wallaligned? Perhaps the recently characterized myosin VI, which movescontrary to other myosins (Wells et aI., 1999) would allow for bidirectionaltransport . Before we can answer these questions, we need to determinewhether actin or myosin antagonists affect this ER trafficking. In addition,intercellular movement of the ER would need to be reconciled with thesuggestion that ER can be divided into a number of domains, including thatwithin the plasmodesmata (Staehelin, 1997).
6. OTHER COMPONENTS OF THECYTOSKELETON AT PLASMODESMATA
Centrin has been localised to the cytoplasmic opening of individualplasmodesmata in a number of species and is particularly prominent in theforming cell plate when primary plasmodesmata are forming in the new cellwall (Blackman et aI., 1999). Centrin may be responsible for regulation atthe neck region, since it contracts in response to calcium and relaxes inresponse to ATP. This work correlates with several studies showing effectsof ATP on cell-to-cell transport . At present, it is unclear how this mechanismwould be coordinated with a structural or functional role for actin andmyosin.
Interestingly, tubulin was found in protein extracts of walls containingplasmodesmata and not in extracts from walls without plasmodesmata fromChara (Blackman & Overall, 1998). It may be that microtubules areinvolved in bringing macromolecules such as viruses (Heinlein et al., 1995;McLean et aI., 1995) to plasmodesmata where actin and myosin are involved
510 Overall, White Blackman and Radford
in the movement of molecules through plasmodesmata. Such systems, wheremicrotubules provide long-distance transport and actin provides short-rangemovement, have been identified in the transport of vesicles in animal cells(Langford, 1995; Allan & Schroer, 1999).
7. CONCLUDING REMARKS
We conclude that, although the evidence is only beginning to emerge,actin, myosin and their associated regulatory and structural proteins, willprove to be essential components of many types of plasmodesmata. It nowremains to clarify what role these proteins play in the operation ofplasmodesmata.
REFERENCES
Allan VJ and Schroer TA (1999) Membrane motors. CUffOpin Cell Bioi 11: 476-482Badelt K, White RG, Overall RL and Vesk M (1994) Ultrastructural specializations of the cell
wall sleeve around plasmodesmata. Amer 1 Bot 81: 1422-1427Baker JP and Titus MA (1998) Myosins: Matching functions with motors. CUff Opin Cell
Bioi 10: 80-86Balachandran S, Xiang Y, Schobert C, Thompson GA and Lucas Wl (1997) Phloem sap
proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell tocell through plasmodesmata. Proc Natl Acad Sci USA 94: 14150-14155
Baluska F, Samaj J, Napier R and Volkmann D (1999) Maize calreticulin localizespreferentially to plasmodesmata in root apex. Plant 1 19: 481-488
Becraft PW, Bongard-Pierce DK, Sylvester AW, Poethig RS, Freeling M, Becraft PW andFreeling M (1990) The liguleless-I gene acts tissue specifically in maize leafdevelopment.Dev Bioi 141: 220-232
Belitser NV, Zaalishvili GV and Sytnianskaja NP (1982) Ca2+-binding sites and Ca2+-ATPaseactivity in barley root tip cells. Protoplasma III: 63-78
Blackman LM and Overall RL (1998) Immunolocalization of the cytoskeleton toplasmodesmata ofChara cora/lina. Plant 114: 733-741
Blackman LM, Harper lDl and Overall RL (1999) Localization of a centrin-like protein tohigher plant plasmodesmata. Eur 1 Cell BioI 78: 297-304
Bostrom TE and Walker NA (1975) Intercellular transport in plants. I. The rate of transport ofchloride and the electric resistance. 1 Exp Bot 95: 767-782
Burgess 1 (1971) Observations on structure and differentiation in plasmodesmata.Protoplasma 73: 83-95
Cantrill LC, Overall RL and Goodwin PB (1999) Cell-to-cell communication via plantendomembranes. Cell Bioi Int 23: in press
Carpenter R and Coen ES (1995) Transposon induced chimeras show that floricaula, ameristem identity gene, acts non-autonomously between cell layers. Development 121: 1926
Carrington lC, Kasschau KD, Mahajan SK and Schaad MC (1996) Cell-to-cell and longdistance transport of viruses in plants. Plant Cell 8: 1669-1681
28. Actin and Myosin in Plasmodesmata 511
Chauhan E, Cowan DS and Hall JL (1991) Cytochemical localization of plasma membraneATPase activity in plant cells. Protoplasma 165: 27-36
Citovsky V, Knorr D, Schuster G and Zambryski P (1990) The P30 movement protein oftobacco mosaic virus is a single-strand nucleic acid binding protein. Cell 60: 637-647
Citovsky V, Wong ML, Shaw AL, Prasad BVV and Zambryski P (1992) Visualization andcharacterisation of tobacco mosaic virus movement protein binding to single-strandednucleic acids. Plant Cell 4: 397-411
Citovsky V and Zambryski P (1991) How do plant virus nucleic acids move throughintercellular connections? BioEssays 13: 373-379
Cleland RE, Fujiwara T and Lucas WJ (1994) Plasmodesmal mediated cell to cell transport inwheat roots is modulated by anaerobic stress. Protoplasma 178: 81-85
Cook ME, Graham LE, Botha CEJ and Lavin CA (1997) Comparative ultrastructure ofplasmodesmata of Chara and selected bryophytes: Toward an elucidation of theevolutionary origin of plant plasmodesmata. Amer J Bot 84: 1169-1178
Dale N, Lunn G and Fensom DS (1983) Rates of axial transport of IIC and 14C in Characeancells: Faster than visible streaming? J Exp Bot 34: 130-143
Deom CM, Oliver MJ and Beachy RN (1987) The 30-kilodalton gene product of tobaccomosaic virus potentiates virus movement. Science 237: 389-393
Ding B (1998) Intercellular protein trafficking through plasmodesmata. Plant Mol Bioi 38:279-310
Ding B, Itaya A and Woo Y.M (1999) Plasmodesmata and cell-to-cell communication inplants. Int Rev Cytol 190: 251-252
Ding B, Kwon M.-O and Warnberg L (1996) Evidence that actin filaments are involved incontrolling the permeability ofplasmodesmata in tobacco mesophyll. Plant J 10: 157-164
Ding B, Turgeon R and Parthasarathy M.V (1992) Substructure of freeze-substitutedplasmodesmata. Protoplasma 169: 28-41
Ding D-Q, Amino S, Mimura T, Nagata T and Tazawa M (1991) Intercellular transport andsubcellular distribution of photoassimilates in Chara corallina. J Exp Bot 42: 1392-1398
Ding D-Q and Tazawa M (1989) Influence of cytoplasmic streaming and turgor pressuregradient on transnodal transport of rubidium and electrical conductance in Characorallina . Plant Cell Physiol 30: 739-748
Duckett CM, Oparka KJ, Prior DAM, Dolan L and Roberts K (1994) Dye-coupling in the rootepidermis ofArabidopsis is progressively reduced during development. Development 120:3247-3255
Erwee MG, and Goodwin PB (1983) Characterisation of Egeria densa Planch. leaf symplast.Inhibition of the intercellular movement of fluorescent probes by group II ions. Planta 158:320-328
Fisher DB, Wu Y and Ku MSB (1992) Turnover of soluble proteins in the wheat sieve tube.Plant Physiol 100: 1433-1441
Franceschi VR and Lucas WJ (1982) The relationship between the charasome to chlorideuptake in Chara coral/ina : Physiological and histochemical investigations. Planta 154:525-537
Gamalei YV, van Bel AJE, Pakhomova M, V and Sjutkina AV (1994) Effects of temperatureon the conformation of the endoplasmic reticulum and on starch accumulation in leaveswith the symplasmic minor-vein configuration. Planta 194: 443-453
Gilbertson RL and Lucas WJ (1996) How do viruses traffic on the 'vascular highway'?Trends Plant Sci I : 260-268
Gisel A, Barella S, Hempel FD and Zambryski PC (1999) Temporal and spatial regulation ofsymplastic trafficking during development in Arabidopsis thaliana apices. Development126: 1879-1889
Goodwin PB and Lyndon RF (1983) Synchronisation of cell division during transition toflowering in Silene apices not due to increased symplast permeability. Protoplasma 116:219-222
512 Overall, White Blackman and Radford
Goosen-de Roo L, Burggraaf PD and Libbenga KR (1983) Microtubule bundles associatedwith tubular endoplasmic reticulum in fusiform cel1s in the active cambial zone ofFraxinus excelsior L. Protoplasma 116: 204-208
Grabski S, de Feijter AW and Schindler M (1993) Endoplasmic reticulum forms a dynamiccontinuum for lipid diffusion between contiguous soybean root cel1s. Plant Cel1 5: 25-38
Grolig F, Williamson RE, Parke J, Miller C and Anderton BH (1988) Myosin and Ca2+_
sensitive streaming in the alga Chara : Detection of two polypeptides reacting with amonoclonal anti-myosin and their localization in the streaming endoplasm. Eur J Cel1 Bioi47: 22-31
Gunning BES and Overal1 RL (1983) Plasmodesmata and cel1-to-cel1 transport in plants.BioScience 33: 260-265
Guo S and Kemphues KJ (1996) A non-muscle myosin required for embryonic polarity inCaenorhabditis elegans. Nature 382: 455-458
Hammer JA (1994) The structure and function of unconventional myosins: A review. JMuscle Res Cel1 Motil 15: 1-10
Hantke SS, Carpenter R and Coen ES (1995) Expression offloricaula in single cel1 layers ofpericlinal chimeras activates downstream homeotic genes in all layers of floral meristems.Development 121: 27-35
Heinlein M, Epel BL, Padgett HS and Beachy RN (1995) Interaction of tobamovirusmovement proteins with the plant cytoskeleton. Science 270: 1983-1985
Hicks JL Liu XR and Williams DS (1996) Role of the ninaC proteins in photoreceptor cel1structure-ultrastructure of ninaC deletion mutants and binding to actin filaments. Cel1Motil Cytoskel35: 367-379
Holdaway-Clarke TL, Walker NA, Hepler PK and Overal1 RL (2000) Physiologicalelevations in cytoplasmic free calcium by cold or ion injection result in transient closure ofhigher plant plasmodesmata. Planta 210: 329-335
Huang M and Zhang L (1999) Association of the movement protein of alfalfa mosaic viruswith the endoplasmic reticulum and its trafficking in epidermal cells of onion bulb scales.Mol Plant Microbe Interact 12: 680-690
Hush JM and Overall RL (1992) Re-orientation of cortical F-actin is not necessary for woundinduced microtubule re-orientation and cell polarity establishment. Protoplasma 169: 97106
Imlau A, Truernit E and Sauer N (1999 ) Cel1-to-cel1 and long-distance trafficking of thegreen fluorescent protein in the phloem and symplastic unloading of the protein into sinktissues. Plant Cel1 11: 309-322
Jackson D and Hake S (1997) Morphogenesis on the move: Cel1-to-cel1 trafficking of plantregulatory proteins. CUIT Opin Gen Dev 7: 495-500
Jackson D, Veit B and Hake S (1994) Expression of maize KNOTTED] related homeoboxgenes in shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot.Development 120: 405-413
Kachar B and Reese TS (1988) The mechanism of cytoplasmic streaming in Characean algalcel1s: Sliding of endoplasmic reticulum along actin filaments. J Cell Biol 106: 1545-1552
Kalimi GH and Lo CW (1988) Communication compartments in the gastrulating mouseembryo. J Cel1 BioI 107: 241-255
Kinkema M and Schiefelbein J (1994) A myosin from a higher plant has structural similaritiesto class V myosins. J Mol Bioi 239: 591-597
Kinkema M, Wang H and Schiefelbein J (1994) Molecular analysis of the myosin gene familyin Arabidopsis thaliana . Plant Mol BioI 26: 1139-1153
Knebel W, Quader H and Schnepf E (1990) Mobile and immobile endoplasmic reticulum inonion bulb epidermis cel1s: Short- and long-term observations with a confocal laserscanning microscope. Eur J Cel1 BioI 52: 328-340
Knight AE and Kendrick-Jones J (1993) A myosin-like protein from a higher plant. J MolBioI 231: 148-154
28. Actin and Myosin in Plasmodesmata 513
Kuhn C, Franceschi VR, Schulz A, Lemoine R and Frommer WB (1997) Macromoleculartrafficking indicated by localization and turnover of sucrose transporters in enucleate sieveelements. Science 275 : 1298-1300
Langford GM (1995) Actin- and microtubule-dependent organelle motors: interrelationshipsbetween the two motility systems. Curr Opin Cell BioI 7: 82-88
Lew RR (1994) Regulation of electrical coupling between Arabidopsis root hairs . Planta 193:67-73
Lichtscheidl IK and UrI WG (1990) Organisation and dynamics of cortical endoplasmicreticulum in inner epidermal cells of onion bulb scales . Protoplasma 157: 203-215
Long RM, Singer RH, Meng XH, Gonzalez I, Nasmyth K and Jansen RP (1997) Mating typeswitching in yeast controlled by asymmetric localization of ASH] mRNA. Science 277 :383-387
Lucas WJ (1999) Plasmodesmata and the cell-to-cell transport of proteins and nucleoproteincomplexes. J Exp Bot 50: 979-987
Lucas WJ, Bouchepillon S, Jackson DP, Nguyen L, Baker L, Ding B and Hake S (1995)Selective trafficking of KNOTTEDI homeodomain protein and its mRNA throughplasmodesmata Science 270 : 1980-1983
Lucas WJ, Ding B and van der Schoot C (1993) Plasmodesmata and the supracellular natureof plants. New Phytol 125: 435-476
McLean BG, Hempel FE and Zambryski PC (1997) Plant intercellular communication viaplasmodesmata. Plant Cell 9: 1043-1054
McLean BG, Zupan J and Zambryski PC (1995) Tobacco mosaic virus movement proteinassociates with the cytoskeleton in tobacco cells . Plant Cell 7: 2101-2114
Maciver SK, Wachsstock DH, Schwarz WH and Pollard TO (1991) The actin filamentsevering protein actophorin promotes the formation of rigid bundles of actin filamentscrosslinked with u-actinin. J Cell BioI 115: 1621-1628
Mas P and Beachy RN (1998) Distribution of TMV movement protein in single livingprotoplasts immobilized in agarose. Plant J 15: 835-842
Matsudaira PT and Burgess DR (1982) Organization of the cross-filaments in intestinalmicrovilli. J Cell BioI 92: 657-664
Metfcalf TN, Szabo LJ, Schubert KR and Wang JL (1980) Immunochemical identification ofan actin-like protein from soybean seedlings. Nature 285 : 171-172
Mezitt LA and Lucas WJ (1996) Plasmodesmal cell-to-cell transport of proteins and nucleicacids. Plant Mol Bioi 32: 251-273
Miller DD, Scordilis SP and Hepler PK (1995) Identification and localization of three classesof myosins in pollen tubes of Lilium longiflorum and Nicotiana alata. J Cell Sci 108:2549-2563
Moore PJ, Fenczik CA, Deom CM and Beachy RN (1992) Developmental changes inplasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaicvirus. Protoplasma 170: 115-127
Nakamura S, Hayashi H, Mori S and Chino M (1993) Protein phosphorylation in the sievetubes of rice plants. Plant Cell Physiol34: 927-933
Nougarede A, Landre P, Rembur J and Niebla Hernandez M (1985) Des variations d'activitesde la 5'-nucleotidase et de l'adenylate-cyclase sont-elles des composantes de la leveed'inhibition du bourgeon cotyledonaire du pois? Can J Bot 63: 309-323
Oparka KJ, Roberts AG, Boevink P, Santa Cruz S, Roberts L, Pradel KS, Imlau A, KotlizkyG, Sauer N and Epel B (1999) Simple but not branched plasmodesmata allow thenonspecific trafficking ofproteins in developing tobacco leaves. Cell 97: 743-754
Oparka KJ, Prior DAM, Santa Cruz S, Padgett HS and Beachy RN (1997) Gating ofepidermal plasmodesmata is restricted to the leading edge of expanding infection sites oftobacco mosaic virus (TMV). Plant J 12: 781-789
514 Overall, White Blackman and Radford
Overall RL (1999) Substructure of plasmodesmata. In: van Bel AlE and van Kesteren WJP(eds) Plasmodesmata: Structure, Function, Role in Cell Communication. Berlin: SpringerVerlag, pp 129-148
Overall RL and Blackman LM (1996) A model of the macromolecular structure ofplasmodesmata. Trends Plant Sci 1: 307-311
Overall RL and Gunning BES (1982) Intercellular communication in Azolla roots: II.Electrical coupling. Protoplasma Ill : 151-160
Overall RL, Wolfe J and Gunning BES (1982) Intercellular communication in Azolla roots. I.Ultrastructure ofplasmodesmata. Protoplasma Ill : 134-150
Parke J, Miller C and Anderton BH (1986) Higher plant myosin heavy-chain using amonoclonal antibody. Eur J Cell Bioi 41: 9-13
Plazinski J, Elliott J, Hurley UA, Burch J, Arioli T and Williamson RE (1997) Myosins fromangiosperms ferns and algae. Amplification of gene fragments with versatile PCR primersand detection of protein products with a monoclonal antibody to a conserved head epitope.Protoplasma 196: 78-86
Qiao L, Jablonsky PP, Elliott J and Williamson RE (1994) A 170 kDa polypeptide from mungbean shares multiple epitopes with rabbit skeletal myosin and binds ADP-agarose. CellBioi Int 18: 1035-1047
Quader H, Hofmann A and Schnepf E (1987) Shape and movement of the endoplasmicreticulum in onion bulb epidermis cells: possible involvement of actin. Eur J Cell Bioi 44:17-26
Radford JE and White R.G (1998) Localization of a myosin-like protein to plasmodesmata.Plant J 14: 743-750
Reichel C and Beachy RN (1998) Tobacco mosaic virus infection induces severemorphological changes of the endoplasmic reticulum. Proc Nat! Acad Sci USA 95: 1116911174
Reichel C, Mas P and Beachy RN (1999) The role of the ER and cytoskeleton in plant viraltrafficking. Trends Plant Sci 4: 458-462
Reichelt S, Knight AE, Hodge TP, Baluska F, Samaj J, Volkmann D and Kendrick-Jones J(1999) Characterization of the unconventional myosin VIII in plant cells and itslocalization oat the post-cytokinetic cell wall. Plant J 19: 555-567
Reid RJ Overall RL (1992) Intercellular communication in Chara: Factors affectingtransnodal electrical resistance and solute fluxes. Plant Cell Environ 15: 507-517
Rinne PLH and van der Schoot C (1998) Symplasmic fields in the tunica of the shoot apicalmeristem coordinate morphogenetic events. Development 125: 1477-1485
Robards AW and Lucas WJ (1990) Plasmodesmata. Annu Rev Plant Physiol Plant Mol Bioi41: 369-419
Roth J (1982) The protein A-gold (pAg) technique: A quantitative approach for antigenlocalization on thin sections. In: Bullock GR and Perutz P (eds) Techniques inImmunocytochemistry. New York: Academic Press, pp 15-57
Sakuth T, Schobert C, Pecsvaradi A, Eichholz A, Komor E and Orlich G (1993) Specificproteins in the sieve-tube exudate of Ricinus communis L. seedlings: Separation,characterization and in-vivo labelling. Planta 191: 207-13.
Schaad MC, Jensen PE and Carrington JC (1997) Formation of plant RNA virus replicationcomplexes on membranes: Role of an endoplasmic reticulum-targeted viral protein.EMBO J 16: 4049-4059
Schnittger A, Folkers U, Schwab B, JUrgens G and Hulskamp M (1999) Generation of aspacing pattern: The role of TRIPTYCHON in trichome patterning in Arabidopsis. PlantCell 11: 1105-1116
Schobert C, Baker L, Szederkenyi J, Grossmann P, Komor E, Hayashi H, Chino M and LucasWJ (1998) Identification of immunologically related proteins in sieve-tube exudatecollected from monocotyledonous and dicotyledonous plants. Planta 206: 245-252
28. Actin and Myosin in Plasmodesmata 515
Schulz A (1995) Plasmodesmal widening accompanies the short-term increase in symplasmicphloem unloading in pea root tips under osmotic stress. Protoplasma 188: 22-37
Schulz A (1999) Physiological control of plasmodesmal gating. In: van Bel AlE and vanKesteren WJP (eds) Plasmodesmata: Structure, Function, Role in Cell Communication .Berlin: Springer-Verlag, pp 173-204
Staehelin LA (1997) The plant ER: A dynamic organelle composed of a large number ofdiscrete functional domains. Plant J 11: 1151-1165
Szymkowiak EJ and Sussex 1M (1992) The internal meristem layer (L3) determines floralmeristem size and carpel number in tomato periclinal chimeras. Plant Cell 4: 1089-1100
Takizawa PA, Sil A, Swedlow JR, Herskowitz I and Vale RD (1997) Actin-dependentlocalization of an RNA encoding a cell-fate determinant in yeast. Nature 389: 90-93
Tilney LG, Cooke TJ, Connelly PS and Tilney MS (1991) The structure of plasmodesmata asrevealed by plasmolysis detergent extraction and protease digestion. J Cell Bioi 112: 739747
Titus MA and Gilbert SP (1999) The diversity of molecular motors: An overview. Cell MolLife Sci 56: 181-183
Tucker EB (1988) Inositol bisphosphate and inositol trisphosphate inhibit cell-to-cell passageof carboxyfluorescein in staminal hairs ofSetcreasea purpurea. Planta 174: 358-363
Tucker EB (1990) Calcium-loaded 1,2-bis(2-aminophenoxy)ethane -N,N,N',N'-tetraaceticacid blocks cell-to-cell diffusion of carboxyfluorescein in staminal hairs of Setcreaseapurpurea. Planta 182: 34-38
Turner A, Wells B and Roberts K (1994) Plasmodesmata of maize root tips: Structure andcomposition. J Cell Sci 107: 3351-3361
van Bel AlE, Giinther S and van Kesteren WJP (1999) Plasmodesmata a maze of questions .In: van Bel AlE and van Kesteren WJP (eds) Plasmodesmata: Structure, Function, Role inCell Communication. Berlin: Springer-Verlag, pp 1-26
van Steveninck FM (1976) Cytochemical evidence for ion transport through plasmodesmata.In: Gunning BES and Robards AW (eds) Intercellular Communication in Plants: Studieson Plasmodesmata. Berlin : Springer-Verlag, pp 131-147
Wells AL, Lin AW, Chen L-Q, Safer D, Cain SM, Hasson T, Carragher BI, Milligan RA andSweeney HL (1999) Myosin VI is an actin-based motor that moves backwards. Nature40 I: 505·508
White RG, Badelt K, Overall RL and Vesk M (1994) Actin associated with plasmodesmata.Protoplasma 180: 169-184
Xoconostle-Cazares B, Yu X, Ruiz-Medrano R, Wang HL, Monzer J, Yoo BC, McFarlandKC, Franceschi VR and Lucas WJ (1999) Plant paralog to viral movement protein thatpotentiates transport ofmRNA into the phloem. Science 283: 94-98
Yamamoto K, Hamada S and Kashiyama T (1999) Myosins from plants. Cell Mol Life Sci56: 227-232
Zawadzki T and Fensom DS (1986a) Transnodal transport of 14C in Nitellaflexilis. I. Tandemcells without applied pressure gradients . J Exp Bot 37: 1341-1352
Zawadzki T and Fensom DS (1986b) Transnodal transport of 14C in Nitella flexilis . II.Tandem cells with applied pressure gradients . J Exp Bot 37: 1353-1363
