Cell-to-cell communication via plant endomembranes

Cell-to-cell communication was investigated in epidermal cells cut from stem internodal tissue of Nicotiana tabacum and Torenia fournieri. Fluorescently labelled peptides and dextrans were microinjected using iontophoresis into the cytoplasm andcortical endomembrane network of these cells. The microinjected endomembrane network was similar in location and structure to the endoplasmic reticulum (ER) as revealed by staining with 3, 3'-dihexyloxacarbocyanine iodide (DiOC(6)). No cell-to-cell movement of dextrans was observed following cytoplasmic injections but injection of dextrans into the endomembrane network resulted in rapid diffusion of the probes to neighbouring cells. It is proposed that the ER acts as a pathway for intercellular communication via the desmotubule through plasmodesmata.     

The 5′ Cap of Tobacco Mosaic Virus (TMV) Is Required for Virion Attachment to the Actin/Endoplasmic Reticulum Network During Early Infection

Almost nothing is known of the earliest stages of plant virus infections. To address this, we microinjected Cy3 (UTP)‐labelled tobacco mosaic virus (TMV) into living tobacco trichome cells. The Cy3‐virions were infectious, and the viral genome trafficked from cell to cell. However, neither the fluorescent vRNA pool nor the co‐injected green fluorescent protein (GFP) left the injected trichome, indicating that the synthesis of (unlabelled) progeny viral (v)RNA is required to initiate cell‐to‐cell movement, and that virus movement is not accompanied by passive plasmodesmatal gating. Cy3‐vRNA formed granules that became anchored to the motile cortical actin/endoplasmic reticulum (ER) network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actin‐dependent RNA movement. The 5′ methylguanosine cap was shown to be required for vRNA anchoring to the actin/ER. TMV vRNA lacking the 5′ cap failed to form granules and was degraded in the cytoplasm. Removal of the 3′ untranslated region or replicase both inhibited replication but did not prevent granule formation and movement. Dual‐labelled TMV virions in which the vRNA and the coat protein were highlighted with different fluorophores showed that both fluorescent signals were initially located on the same ER‐bound granules, indicating that TMV virions may become attached to the ER prior to uncoating of the viral genome.     

胞间连丝：共质体运输的动态通道

胞间连丝作为一种细胞质结构将相邻的细胞连系起来而形成植物的共质体。胞间连丝通过调控许多离子和分子的共质体运输而广泛地参与植物的生命活动。胞间连丝的主要构成部分是细胞质膜、连丝小管、以及位于二之间的环层细胞质。这三都很容易在电子显微镜下观察到。细胞骨架的成分（肌动蛋白和肌球蛋白）起到稳定胞间连丝的作用。同时,钙结合蛋白可能具有调节间连丝功能的作用。在胞间连丝里,环层细胞质为大多数溶质提供共质体运输的通道,而有些 共质体运输则可能是通过连丝小管的内腔、连丝小管的壳层、甚或是细胞质膜来实现的。共质体可以细分为数个区块,它们各自允许不同大小的分子（从低于1000到高于10000道尔顿）通过。从发生上看,胞间连丝可以是初生的,也可以是次生的。前是伴随着新细胞壁的形成则产生的,而后则是在已有的细胞壁上产生的。胞间连丝的动态性质还表现在它们的频率是处于变化之中,这是由于组织或植物整体的发育和生理状态决定的。虽然共质体运输的基本形式是扩散,但胞间连丝对于某些离子和分子却是选择性的。在病毒感染细胞时,病毒的移动蛋白作用于胞间连丝的受体蛋白,结果,胞间连丝被显地扩张（其机理尚不清楚）。于是,病毒的移动蛋白连同与之结合在一起的病毒基因组进入毗邻的健康细胞。一些植物源性的蛋白质也能够通过胞间连丝来运输;推测其方式类似于病毒的移动蛋白。有些植物蛋白质本身就是信号分子,它们调节分化和其他活动。与此相反,还有一些植物蛋白质的共质体运输并不是通过特异的方式来实现的。     

Cell-to-cell movement of potato virus X revealed by micro-injection of a viral vector tagged with the β-glucuronidase gene

The replication and cell-to-cell movement of potato virus X (PVX) has been studied using a PVX vector construct which expressed the β-glucuronidase (GUS) reporter gene in infected cells. Nicotiana clevelandii leaf trichome cells were micro-injected with the PVX-GUS vector and histochemical staining was used to locate GUS activity. The distribution of GUS activity revealed that PVX had moved from the injected cell into other trichome cells and into the cells along the leaf margin. GUS activity was always restricted to the cells at the edge of the leaf suggesting that PVX was unable to move out of the marginal cells. At the infection front, scattered along the leaf margin, there were isolated groups of cells staining for GUS activity. The absence of GUS activity in the intervening cells suggests that PVX had moved through several cells without replicating within them. This latter observation is consistent with previously reported observations that viral movement proteins are capable of moving between cells.     

Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV)

Plasmodesmatal gating in epidermal cells of Nicotianatabacum was examined in expanding infection sites of tobacco mosaic virus (TMV) expressing a fusion between the viral movement protein and the green fluorescent protein (MP-GFP). The infection sites were circular in profile and within 3 days post-inoculation had developed a brightly fluorescent leading edge, giving them a characteristic ‘halo’ shape. Co-localization of MP-GFP with callose demonstrated that nearly all epidermal cell plasmodesmata were targeted with MP-GFP. The fusion protein was located in the centre of the plasmodesmal pore, between paired callose platelets. Increase in plasmodesmatal size exclusion limit, as determined by the passage of microinjected 10 kDa Texas Red dextran, was restricted predominantly to cells within the fluorescent halo, and was virtually absent from cells in the centre of the expanding infection site. The plasmodesmata of these cells, however, remained fluorescently labelled with MP-GFP. Injections outside the fluorescent infection site failed to show movement of dextran, while dextran injected into cells at the leading edge moved inwards towards the centre of the lesion but not outwards into cells lacking GFP. Leaf incisions through cells ahead of the infection front halted the advance of the virus, indicating that virus replication was absent in non-fluorescent cells outside the infection site. The data provide the first demonstration that within an expanding infection site plasmodesmatal gating is under temporal control.     

Intercellular translocation of molecules via plasmodesmata in the multiseriate filamentous brown alga,Halopteris congesta (Sphacelariales,Phaeophyceae)

Despite the high number of studies on the fine structure of brown algal cells, only limited information is available on the intercelluar transportation of molecules via plasmodesmata in brown algae. In this study, plasmodesmatal permeability of Halopteris congesta was examined by observing the translocation of microinjected fluorescent tracers of different molecular sizes. The tip region of H. congesta consists of a cylindrical apical cell, while the basal region is multiseriate. Fluorescein isothiocyanate‐dextran (FD; 3, 10, and 20 kDa) and recombinant green fluorescent protein (27 kDa) were injected into the apical cell and were observed to diffuse into the neighboring cells. FD of 40 kDa was detected only in the injected apical cell. The plasmodesmatal size exclusion limit was considered to be more than 20 kDa and less than 40 kDa. The extent of translocation of 3 and 10 kDa FD from the apical to neighboring cells 2 h postinjection was estimated based on the fluorescence intensity. It was suggested that the diffusing capacity of plasmodesmata varied according to molecular size. In order to examine acropetal and/or basipetal direction of molecular movement, 3 and 10 kDa FD were injected into the third cell from the apical cell. Successive observations indicated that the diffusion of fluorescence in the acropetal direction took longer than that in the basipetal direction. No ultrastructural difference in plasmodesmata was noted among the cross walls.     

Analysis of zeins' ER retention in Xenopus oocytes

Zeins, maize storage proteins, are retained in the endoplasmic reticulum (ER) during the intracellular protein targeting process. Hydrophobic interaction has been postulated as the driving force of zeins' aggregation and retention in the ER. Recently, a class of zein (the 27K zein) has been proposed to facilitate zeins' ER retention by anchoring to the ER membrane. This study investigated the significance of the two proposed mechanisms toward zeins' ER retention using Xenopus oocyte. Following injection of the total or 27K zein mRNA, zein's movement within the ER was analyzed based upon the extent of diffusion to the non-injected oocyte half. This study indicates that the total zeins freely move within the lumen of the ER, thus, suggesting that the intermolecular aggregation, leading to insolubility and exclusion from the ER lumenal fluid, may not be essential for zeins' ER retention. This study also suggests that the 27K zein may not facilitate zeins' ER retention by virtue of an anchor to the ER membrane based on its free movement in the ER. Free movement of the total and 27K zeins, under conditions where zein aggregates should form, necessitates a reevaluation of the mechanisms responsible for zein polypeptides' ER retention and protein body formation.     

Distribution and structure of the plasmodesmata in mesophyll and bundle-sheath cells of Zea mays L.

In leaf blades of Zea mays L. plasmodesmata between mesophyll cells are aggregated in numerous thickened portions of the walls. The plasmodesmata are unbranched and all are characterized by the presence of electron-dense structures, called sphincters by us, near both ends of the plasmodesmatal canal. The sphincters surround the desmotubule and occlude the cytoplasmic annulus where they occur. Plasmodesmata between mesophyll and bundle-sheath cells are aggregated in primary pit-fields and are constricted by a wide suberin lamella on the sheath-cell side of the wall. Each plasmodesma contains a sphincter on the mesophyll-cell side of the wall. The outer tangential and radial walls of the sheath cells exhibit a continuous suberin lamella. However, on the inner tangential wall only the sites of plasmodesmatal aggregates are consistently suberized. Apparently the movement of photosynthetic intermediates between mesophyll and sheath cells is restricted largely or entirely to the plasmodesmata (symplastic pathway) and transpirational water movement to the cell walls (apoplastic pathway).Abbreviation ER endoplasmic reticulum     

Evidence that actin filaments are involved in controlling the permeability of plasmodesmata in tobacco mesophyll

The role of actin filaments in regulating plasmodesmal transport has been studied by microinjection experiments in mesophyll cells of tobacco (Nicotiana tabacum L. cv. Samsun). When fluorescent dextrans of various molecular sizes were each co-injected with specific actin filament perturbants cytochalasin D (CD) or profilin into these cells, dextrans up to 20 kilodalton (kDa) moved from the injected cell into surrounding cells within 3–5 min. In contrast, when such dextrans were injected alone or co-injected with phalloidin into the mesophyll cells, they remained in the injected cells. Phalloidin co-injection slowed down or even inhibited CD- or profilin-elicited dextran cell-to-cell movement. Dextrans of 40 kDa or larger were unable to move out of the injected cell in the presence of CD or profilin. These data suggest that actin filaments may participate in the regulation of plasmodesmal transport by controlling the permeability of plasmodesmata.     

Fine structure of plasmodesmata in mature leaves of sugarcane

The fine structure of plasmodesmata in vascular bundles and contiguous tissues of mature leaf blades of sugarcane (Saccharum interspecific hybrid L62–96) was studied with the transmission electron microscope. Tissues were fixed in glutaraldehyde, with and without the addition of tannic acid, and postfixed in OsO₄. The results indicate that the fine structure of plasmodesmata in sugarcane differs among various cell combinations in a cell-specific manner, but that three basic structural variations can be recognized among plasmodesmata in the mature leaf: 1) Plasmodesmata between mesophyll cells. These plasmodesmata possess amorphous, electron-opaque structures, termed sphincters, that extend from plasma membrane to desmotubule near the orifices of the plasmodesmata. The cytoplasmic sleeve is filled by the sphincters where they occur; elsewhere it is open and entirely free of particulate or spokelike components. The desmotubule is tightly constricted and has no lumen within the sphincters, but between the sphincters it is a convoluted tubule with an open lumen. 2) Plasmodesmata that traverse the walls of chlorenchymatous bundle-sheath cells and mestome-sheath cells. In addition to the presence of sphincters, these plasmodesmata are modified by the presence of suberin lamellae in the walls. Although the plasmodesmata are quite narrow and the lumens of the desmotubules are constricted where they traverse the suberin lamellae, the cytoplasmic sleeves are still discernible and appear to contain substructural components there. 3) Plasmodesmata between parenchymatous cells of the vascular bundles. These plasmodesmata strongly resemble those found in the roots of Azolla, in that their desmotubules are closed for their entire length and their cytoplasmic sleeves appear to contain substructural components for their entire length. The structural variations exhibited by the plasmodesmata of the sugarcane leaf are compared with those proposed for a widely-adopted model of plasmodesmatal structure.Abbreviation ER endoplasmic reticulum This study was supported by National Science Foundation grants DCB 87-01116 and DCB 90-01759 to R.F.E. and a University of Wisconsin-Madison Dean's Fellowship to K. R.-B. We also thank Claudia Lipke and Kandis Elliot for photographic and artistic assistance, respectively.     

Intercellular communication inAzolla roots: I. Ultrastructure of plasmodesmata

Summary A model is proposed for the structure of the plasmodesmata ofAzolla root primordia, based on micrographs obtained by a combination of fixation in glutaraldehyde/p-formaldehyde/tannic acid/ferric chloride, digestion of cell walls and the use of stereo pairs. Unlike the model for plasmodesmatal structure proposed byRobards (1971), the desmotubule is depicted as a virtually closed cylindrical bilayer providing little or no open pathway for transport. In this respect it is similar to the model ofLópez-Sáez et al. (1966). An analysis of the molecular packing of types of lipids found in endoplasmic reticulum (of which the desmotubule is an extension) indicates that the model is geometrically feasible. Details cannot be discerned with accuracy, but material, possibly particulate, occupies much of the space between desmotubule and plasma membrane, the cytoplasmic lumen being reduced to inter-particle spaces of cross-sectional area comparable to that of the bore in a gap junction connexon. Implications for intercellular transport are discussed.     

Changes in macromolecular movement accompany organogenesis in thin cell layers of <Emphasis Type="Italic">Torenia fournieri</Emphasis>

A range of fluorescently labelled probes of increasing molecular weight was used to monitor diffusion via the symplast in regenerating thin cell layer (TCL) explants of Torenia fournieri. An increase in intercellular movement of these molecules was associated with the earliest stages of vegetative shoot regeneration, with the movement of a 10 kDa dextran (FD 10000) observed between epidermal cells prior to the appearance of the first cell divisions. A low frequency of dextran movement in thin cell layers maintained under non-regenerating conditions was also observed, indicating a possible wound induced increase in intercellular movement. Dextran movement between epidermal cells reached a peak by day 4 of culture and then declined as cell division centres (CDCs) formed, became meristematic regions and finally emerged as adventitious shoots. Within CDCs, testing with small fluorescent probes (CF: carboxyfluorescein, mw 376 Da and F(Glu)₃: fluorescein-triglutamic acid, mw 799 Da) revealed a mosaic of cell isolation and regions of maintained symplastic linkage. Within shoots, surface cells of the presumptive apical meristem permitted the intercellular movement of 10 kDa dextrans but epidermal cells of the surrounding leaf primordia did not permit dextran movement. In some cases, intercellular movement of CF was maintained within leaf primordia. Symplastic movement of labelled dextrans during regeneration in Torenia thin cell layers represents a significant increase in the basal size exclusion limit (SEL) of this tissue and reveals the potential for intercellular trafficking of developmentally related endogenous macromolecules.     

Signalling via plasmodesmata—the neglected pathway

This review considers recent studies on the role of plasmodesmata in the conduction of small solutes and signalling molecules between plant cells. The substructure of plasmodesmata is described in relation to the potential pathways available for symplastic signalling between cells. At least two discrete pathways are available for transport through plasmodesmata, the cytoplasmic sleeve between the desmotubule and the plasmalemma, and the endoplasmic reticulum which connects contiguous cells via the central desmotubule. This latter pathway has been shown recently to function as a dynamic continuum for the movement of lipids and lipid-signalling molecules between plant cells. The role of plasmodesmata in the conduction of hormones and electrical signals is also considered, as is the potential for movement of macromolecular signalling molecules via the symplast. The factors which regulate plasmodesmatal conductance and the significance of symplast 'domains' are discussed in relation to the control of movement of signalling molecules in the symplast.     

Surface interactions of Fusarium graminearum on barley

The filamentous fungus Fusarium graminearum, a devastating pathogen of barley (Hordeum vulgare L.), produces mycotoxins that pose a health hazard. To investigate the surface interactions of F. graminearum on barley, we focused on barley florets, as the most important infection site leading to grain contamination. The fungus interacted with silica‐accumulating cells (trichomes and silica/cork cell pairs) on the host surface. We identified variation in trichome‐type cells between two‐row and six‐row barley, and in the role of specific epidermal cells in the ingress of F. graminearum into barley florets. Prickle‐type trichomes functioned to trap conidia and were sites of fungal penetration. Infections of more mature florets supported the spread of hyphae into the vascular bundles, whereas younger florets did not show this spread. These differences related directly to the timing and location of increases in silica content during maturation. Focal accumulation of cellulose in infected paleae of two‐row and six‐row barley indicated that the response is in part linked to trichome type. Overall, silica‐accumulating epidermal cells had an expanded role in barley, serving to trap conidia, provide sites for fungal ingress and initiate resistance responses, suggesting a role for silica in pathogen establishment.     

Cell-cell interactions in the process of differentiation of thyroid epithelial cells into follicles: a study by microinjection and fluorescence microscopy on in vitro reconstituted thyroid follicles

Thyroid cells, cultured in the presence of thyroid stimulating hormone, reorganized within 36-48 hr into follicular structures, the in vitro reconstituted thyroid follicles or RTF. By microinjection of fluorescent probes either into the neoformed intrafollicular lumen (IL) or into cells forming the follicles, we have studied the development and some functional properties of cell-cell contacts involved in a) the formation of the thyroid follicular lumen and b) the communication between thyrocytes within the follicle. The probes were compounds of either low (Lucifer Yellow: LY) or high molecular weight (Dextran labeled with fluorescein: FITC-Dextran and Cascade Blue conjugated to bovine serum albumin: CB-BSA). LY microinjected into IL of 2-9-day-old RTF was seen to label circular spaces with a diameter ranging from 10 to 100 microns. The cells delimiting the IL remained unlabeled. The fluorescent dye remained concentrated in IL for up to 24 hr. FITC-Dextran or CB-BSA microinjected into IL behaved as LY; the probes were restrained into the lumen. A 2 hr incubation of RTF with iodide induced alterations of the structure of IL; an effect mediated by an organic form of actively trapped iodide. A 15-30 min incubation of RTF in a low CA2+ medium caused the opening of IL visualized by the progressive decrease of the fluorescence of probes preinjected into the lumenal space. The same but more rapid effect was obtained by microinjection of EGTA into the IL. The low Ca2(+)-dependent opening of IL was also demonstrated by the release into the medium of thyroglobulin present in IL. Microinjection of LY in a cell involved in the follicle structure led to the rapid labeling of the other cells forming the follicle but LY did not penetrate the IL. Unlike LY, the distribution of FITC-Dextran or CB-BSA injected into cells delimiting the lumen was restricted to the microinjected cells. Alterations of medium or intralumenal Ca2+ concentration which caused the opening of IL did not affect the cell-to-cell transfer of LY. By using fluorescent probe microinjection, we show that the in vitro thyrocyte histiotypic differentiation leads to the reconstitution of functional intercellular junctions: tight junctions insuring the tightness of the neoformed lumen and gap junctions mediating the cell-to-cell exchange of small molecules. The structure of the thyroid follicles appears to be under the control of both extracellular and intralumenal Ca2+ concentrations.     

The ER-Membrane Transport System Is Critical for Intercellular Trafficking of the NSm Movement Protein and Tomato Spotted Wilt Tospovirus

Plant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall, forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking. Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network, NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected. We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly, TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant, and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical, cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV.     

The sugar is sIRVed: sorting Glut4 and its fellow travelers

Translocation of Glut4 to the plasma membrane of fat and skeletal muscle cells is mediated by specialized insulin‐responsive vesicles (IRVs), whose protein composition consists primarily of glucose transporter isoform 4 (Glut4), insulin‐responsive amino peptidase (IRAP), sortilin, lipoprotein receptor‐related protein 1 (LRP1) and v‐SNAREs. How can these proteins find each other in the cell and form functional vesicles after endocytosis from the plasma membrane? We are proposing a model according to which the IRV component proteins are internalized into sorting endosomes and are delivered to the IRV donor compartment(s), recycling endosomes and/or the trans‐Golgi network (TGN), by cellugyrin‐positive transport vesicles. The cytoplasmic tails of Glut4, IRAP, LRP1 and sortilin play an important targeting role in this process. Once these proteins arrive in the donor compartment, they interact with each other via their lumenal domains. This facilitates clustering of the IRV proteins into an oligomeric complex, which can then be distributed from the donor membranes to the IRV as a single entity with the help of adaptors, such as Golgi‐localized, gamma‐adaptin ear‐containing, ARF‐binding (GGA).      

The endoplasmic reticulum membrane is permeable to small molecules

The lumen of the endoplasmic reticulum (ER) differs from the cytosol in its content of ions and other small molecules, but it is unclear whether the ER membrane is as impermeable as other membranes in the cell. Here, we have tested the permeability of the ER membrane to small, nonphysiological molecules. We report that isolated ER vesicles allow different chemical modification reagents to pass from the outside into the lumen with little hindrance. In permeabilized cells, the ER membrane allows the passage of a small, charged modification reagent that is unable to cross the plasma membrane or the lysosomal and trans-Golgi membranes. A larger polar reagent of approximately 5 kDa is unable to pass through the ER membrane. Permeation of the small molecules is passive because it occurs at low temperature in the absence of energy. These data indicate that the ER membrane is significantly more leaky than other cellular membranes, a property that may be required for protein folding and other functions of the ER.     

Cytoplasmic force gradient in migrating adhesive cells

Amoeboid movement is believed to involve a pressure gradient along the cell length, with contractions in the posterior region driving cytoplasmic streaming forward. However, a parallel mechanism has yet to be demonstrated in migrating adhesive cells. To probe the distribution of intracellular forces, we microinjected high molecular weight linear polyacrylamide (PAA) as a passive force sensor into migrating NIH3T3 fibroblasts. Injected PAA appeared as amorphous aggregates that underwent shape change and directional movement in response to differential forces exerted by the surrounding environment. PAA injected into the posterior region moved toward the front, whereas PAA in the anterior region never moved to the posterior region. This preferential forward movement was observed only in migrating cells with a defined polarity. Disruption of myosin II activity by blebbistatin inhibited the forward translocation of PAA while cell migration persisted in a disorganized fashion. These results suggest a myosin II-dependent force gradient in migrating cells, possibly as a result of differential cortical contractions between the anterior and posterior regions. This gradient may be responsible for the forward transport of cellular components and for maintaining the directionality during cell migration.