Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants

Cell plate formation in tobacco root tips and synchronized dividing suspension cultured tobacco BY-2 cells was examined using cryofixation and immunocytochemical methods. Due to the much improved preservation of the cells, many new structural intermediates have been resolved, which has led to a new model of cell plate formation in higher plants. Our electron micrographs demonstrate that cell plate formation consists of the following stages: (1) the arrival of Golgi-derived vesicles in the equatorial plane, (2) the formation of thin (20 +/- 6 nm) tubes that grow out of individual vesicles and fuse with others giving rise to a continuous, interwoven, tubulo-vesicular network, (3) the consolidation of the tubulo-vesicular network into an interwoven smooth tubular network rich in callose and then into a fenestrated plate-like structure, (4) the formation of hundreds of finger-like projections at the margins of the cell plate that fuse with the parent cell membrane, and (5) cell plate maturation that includes closing of the plate fenestrae and cellulose synthesis. Although this is a temporal chain of events, a developing cell plate may be simultaneously involved in all of these stages because cell plate formation starts in the cell center and then progresses centrifugally towards the cell periphery. The "leading edge" of the expanding cell plate is associated with the phragmoplast microtubule domain that becomes concentrically displaced during this process. Thus, cell plate formation can be summarized into two phases: first the formation of a membrane network in association with the phragmoplast microtubule domain; second, cell wall assembly within this network after displacement of the microtubules. The phragmoplast microtubules end in a filamentous matrix that encompasses the delicate tubulo-vesicular networks but not the tubular networks and fenestrated plates. Clathrin-coated buds/vesicles and multivesicular bodies are also typical features of the network stages of cell plate formation, suggesting that excess membrane material may be recycled in a selective manner. Immunolabeling data indicate that callose is the predominant lumenal component of forming cell plates and that it forms a coat-like structure on the membrane surface. We postulate that callose both helps to mechanically stabilize the early delicate membrane networks of forming cell plates, and to create a spreading force that widens the tubules and converts them into plate-like structures. Cellulose is first detected in the late smooth tubular network stage and its appearance seems to coincide with the flattening and stiffening of the cell plate.     

Ultrastructure of vascular cambial cell cytokinesis in pine seedlings preserved by cryofixation and substitution

Summary.  Trees depend on the secondary vascular cambium to produce cells for new xylem and phloem. The fusiform cells of this lateral meristem are long and narrow, presenting special challenges for arranging the mitotic spindle and phragmoplast. Fusiform cambial cells of Pinus ponderosa and Pinus contorta were studied by cryofixation and cryosubstitution which preserved ultrastructure and phases of cytokinesis with a resolution not previously attained. Membranous structures including the plasma membrane, tonoplast, and those of other organelles were smooth and unbroken, indicating that they were preserved while the protoplasm was in a fully turgid state. Mitotic spindles separated daughter chromosomes diagonally across the radial width of the cells. The cell plate was initiated at an angle to the cell axis between the anaphase chromosomes by a microtubule array which organized vesicles at the phragmoplast midline. Within the phragmoplast, vesicles initially joined across thin tubular projections and then amalgamated into a tubulo-vesicular network. Axial expansion of the cell plate generated two opposing phragmoplasts connected by a thin, extended bridge of cell plate and cytoplasm that was oriented along the cell axis. In the cytoplasmic bridge trailing each phragmoplast, the callose-rich tubular network gradually consolidated into a fenestrated plate and then a complete cell wall. Where new membrane merged with old, the parent plasmalemma appeared to be loosened from the cell wall and the membranes joined via a short tubulo-vesicular network. These results have not been previously reported in cambial tissue, but the same phases of cytokinesis have been observed in cryofixed root tips and suspension-cultured cells of tobacco. Received February 11, 2002; accepted May 31, 2002; published online October 31, 2002 RID="*" ID="*" Correspondence and reprints: Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada. Abbreviations: CFS cryofixation and cryosubstitution; ER endoplasmic reticulum; HPF high-pressure freezing; PPB preprophase band.     

Caffeine inhibition of cytokinesis: Ultrastructure of cell plate formation/degradation

Summary Caffeine is a potent inhibitor of cell plate formation in dividing plant cells. Previous studies living cells reveal that the drug always permits the cell plate to arise and grow normally until about 80% complete, but then causes it to break down. In the present investigation we examine this formation/degradation cycle at the ultrastructure level. Our results show that during the formation phase the caffeine treated plate is indistinguishable from untreated controls. Phragmoplast microtubules arise and align in the interzone, Golgi vesicles are produced and aggregate in a line that defines the young cell plate, and considerable fusion of these vesicles occurs to form islands of plate material. However, under the influence of caffeine these islands do not fuse to form the enlarged lamellar expanses characteristic of maturing cell plates. Instead, the partially fused material reverts to small vesicles which appear to become resorbed by the cellular membrane systems. The resorption process continues leaving no evidence of the previously developing plate, although occasionally we observe a stub of fused vesicles attached to the parent wall. Following cell plate disintegration the reformed nuclei move close together and occupy the central region of the cell. These observations focus attention on the consolidation phase of cell plate formation as the one being maximally affected by caffeine.Dedicated to the memory of Professor Oswald Kiermayer     

Comparative effects of caffeine, its analogues and calcium deficiency on cytokinesis

The effects of caffeine, aminophylline, caffeic acid, and calcium deficiency on cytokinesis were studied by light and electron microscopy. All these treatments blocked cell plate formation, resulting in the formation of binucleate cells. The aggregation and organization of membranous vesicles at the ‘presumptive cell plate’ during these treatments appears similar to that of normal cells, but fusion of the vesicles is insufficient to form a complete cell plate. It is suggested that some aspect of membrane recognition and fusion is the process actually interfered with by these treatments. Greater numbers of binucleate cells and fewer partial cell plates were observed in cells treated with caffeine and aminophylline as compared with those exposed to caffeic acid or calcium deficiency, indicating that the latter treatments do not block cell plate formation as efficiently as the former.     

Electron tomographic analysis of post-meiotic cytokinesis during pollen development in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The mechanism of cell wall formation after male meiosis was studied in microsporocytes of Arabidopsis thaliana (L.) Heynh. by means of thin-section and immuno-electron microscopy and dual-axis electron tomography of high-pressure-frozen/freeze-substituted cells. The cellularization of four-nucleate microsporocytes involves a novel type of cell plate, called a post-meiotic-type cell plate. As in the syncytial endosperm, the microsporocyte cell plates assemble in association with mini-phragmoplasts. However, in contrast to the endosperm cell plates, post-meiotic type cell plates arise simultaneously across the entire division plane. Vesicles are transported along mini-phragmoplast microtubules by putative kinesin proteins and, prior to fusion, they become connected together by 24-nm-long linkers that resemble exocyst complexes. These vesicles fuse with each other to form wide tubules and wide tubular networks. In contrast to endosperm cell plates, the wide tubular networks in microsporocytes completely lack callose and do not appear to be constricted by dynamin rings. The most peripheral wide tubular networks begin to fuse with the plasma membrane before the more central cell plate assembly sites become integrated into a coherent cell plate. Fusion with the parental plasma membrane triggers callose synthesis and the wide tubular domains are converted into convoluted sheets. As the peripheral convoluted sheets accumulate callose and arabinogalactan proteins, they are converted into stub-like projections, which grow centripetally, i.e. toward the interior of the syncytium, fusing with the wide tubular networks already assembled in the division plane. We also demonstrate that the ribosome-excluding cell plate assembly matrix is delivered to the mini-phragmoplast with the first vesicles, and encompasses all the linked vesicles and intermediate stages in cell plate formation.Abbreviations AGP Arabinogalactan protein - MT Microtubule     

Three-dimensional analysis of syncytial-type cell plates during endosperm cellularization visualized by high resolution electron tomography

The three-dimensional architecture of syncytial-type cell plates in the endosperm of Arabidopsis has been analyzed at approximately 6-nm resolution by means of dual-axis high-voltage electron tomography of high-pressure frozen/freeze-substituted samples. Mini-phragmoplasts consisting of microtubule clusters assemble between sister and nonsister nuclei. Most Golgi-derived vesicles appear connected to these microtubules by two molecules that resemble kinesin-like motor proteins. These vesicles fuse with each other to form hourglass-shaped intermediates, which become wide (approximately 45 nm in diameter) tubules, the building blocks of wide tubular networks. New mini-phragmoplasts also are generated de novo around the margins of expanding wide tubular networks, giving rise to new foci of cell plate growth, which later become integrated into the main cell plate. Spiral-shaped rings of the dynamin-like protein ADL1A constrict but do not fission the wide tubules at irregular intervals. These rings appear to maintain the tubular geometry of the network. The wide tubular network matures into a convoluted fenestrated sheet in a process that involves increases of 45 and 130% in relative membrane surface area and volume, respectively. The proportionally larger increase in volume appears to reflect callose synthesis. Upon fusion with the parental plasma membrane, the convoluted fenestrated sheet is transformed into a planar fenestrated sheet. This transformation involves clathrin-coated vesicles that reduce the relative membrane surface area and volume by approximately 70%. A ribosome-excluding matrix encompasses the cell plate membranes from the fusion of the first vesicles until the onset of the planar fenestrated sheet formation. We postulate that this matrix contains the molecules that mediate cell plate assembly.     

The herbicide dichlobenil disrupts cell plate formation: immunogold characterization

Summary We have utilized light and transmission electron microscopy and immunocytochemistry to examine onion roots treated with the herbicide dichlobenil (2,6-dichlorobenzonitrile; DCB), a purported disrupter of cellulose biosynthesis. The most salient effect of DCB is observed on cell plate formation, the process that gives rise to new cell walls. In the presence of DCB, cell plates develop normally up to the tubular network stage. They are the result of fusion of Golgi-derived vesicles and the accumulation of callose and the first strands of cellulose. The DCB-treated cell plates retain the reticulate and malleable nature of the tubular network/early fenestrated plate stage of cell plate formation, but fail to display signs of the stiffening and straightening associated with an accumulation of cellulose. Instead, the malleable cell plates in the DCB-treated cells retain a wavy architecture, accumulate pockets of electron opaque material, and produce plasmodesmata in abnormal orientations. Immunocytochemical investigations of the abnormal cell plates formed after DCB treatment show 20-fold increase in the level of callose labelling found in the control cell plates. Xyloglucans and rhamnogalacturonans can be detected in the partially-formed cell plates, with the labelling density of xyloglucan 4–5 times greater than in the control cell plates and that of the rhamnogalacturonans being similar to the controls. These data support the hypothesis that DCB inhibits cellulose biosynthesis as a primary mechanism of action, and that in the absence of cellulose synthesis the cell plates fail to mature and to give rise to new cross walls.Abbreviations DCB dichlorobenzonitrile - PGA/RGI polygalacturonic acid/rhamnogalacturonan I     

Caffeine inhibits callose deposition in the cell plate and the depolymerization of microtubules in the central region of the phragmoplast

Treatment of tobacco BY-2 cells with 10 mM caffeine that was started after the cells had entered the mitotic phase did not completely inhibit the deposition of callose in the cell plate and allowed the centrifugal redistribution of phragmoplast microtubules. On the other hand, when treatment with caffeine was started before the cells entered the mitotic phase, the deposition of callose was completely inhibited and the redistribution of phragmoplast microtubules was also inhibited. As the inhibition of redistribution of phragmoplast microtubules seems to be caused by the inhibition of depolymerization of microtubules at the central region of the phragmoplast, these results strongly suggest that the deposition of callose in the cell plate is tightly linked with the depolymerization of phragmoplast microtubules. Callose deposition was observed in phragmoplasts isolated from caffeine-treated cells as well as in those isolated from non-caffeine-treated cells, and caffeine did not inhibit callose synthesis in isolated phragmoplast, indicating that caffeine neither inhibits the accumulation of callose synthase at the equatorial regions of the phragmoplast nor arrests callose synthase itself.     

Syncytial-type cell plates: a novel kind of cell plate involved in endosperm cellularization of Arabidopsis

Cell wall formation in the syncytial endosperm of Arabidopsis was studied by using high-pressure-frozen/freeze-substituted developing seeds and immunocytochemical techniques. The endosperm cellularization process begins at the late globular embryo stage with the synchronous organization of small clusters of oppositely oriented microtubules ( approximately 10 microtubules in each set) into phragmoplast-like structures termed mini-phragmoplasts between both sister and nonsister nuclei. These mini-phragmoplasts produce a novel kind of cell plate, the syncytial-type cell plate, from Golgi-derived vesicles approximately 63 nm in diameter, which fuse by way of hourglass-shaped intermediates into wide ( approximately 45 nm in diameter) tubules. These wide tubules quickly become coated and surrounded by a ribosome-excluding matrix; as they grow, they branch and fuse with each other to form wide tubular networks. The mini-phragmoplasts formed between a given pair of nuclei produce aligned tubular networks that grow centrifugally until they merge into a coherent wide tubular network with the mini-phragmoplasts positioned along the network margins. The individual wide tubular networks expand laterally until they meet and eventually fuse with each other at the sites of the future cell corners. Transformation of the wide tubular networks into noncoated, thin ( approximately 27 nm in diameter) tubular networks begins at multiple sites and coincides with the appearance of clathrin-coated budding structures. After fusion with the syncytial cell wall, the thin tubular networks are converted into fenestrated sheets and cell walls. Immunolabeling experiments show that the cell plates and cell walls of the endosperm differ from those of the embryo and maternal tissue in two features: their xyloglucans lack terminal fucose residues on the side chain, and callose persists in the cell walls after the cell plates fuse with the parental plasma membrane. The lack of terminal fucose residues on xyloglucans suggests that these cell wall matrix molecules serve both structural and storage functions.     

The timely deposition of callose is essential for cytokinesis in Arabidopsis

The primary plant cell wall is laid down over a brief period of time during cytokinesis. Initially, a membrane network forms at the equator of a dividing cell. The cross-wall is then assembled and remodeled within this membrane compartment. Callose is the predominant luminal component of the nascent cross-wall or cell plate, but is not a component of intact mature cell walls, which are composed primarily of cellulose, pectins and xyloglucans. Widely accepted models postulate that callose comprises a transient, rapid spreading force for the expansion of membrane networks during cytokinesis. In this study, we clone and characterize an Arabidopsis gene, MASSUE / AtGSL8 , which encodes a putative callose synthase. massue mutants are seedling-lethal and have a striking cytokinesis-defective phenotype. Callose deposition was delayed in the cell plates of massue mutants. Mutant cells were occasionally bi- or multi-nucleate, with cell-wall stubs, and we frequently observed gaps at the junction between cross-walls and parental cell walls. The results suggest that the timely deposition of callose is essential for the completion of plant cytokinesis. Surprisingly, confocal analysis revealed that the cell-plate membrane compartment forms and expands, seemingly as far as the parental wall, prior to the appearance of callose. We discuss the possibility that callose may be required to establish a lasting connection between the nascent cross-wall and the parental cell wall.     

Isolation of a gene encoding Arabidopsis membrane-associated acyl-CoA binding protein and immunolocalization of its gene product

Until recently, only cytosolic acyl-CoA binding proteins (ACBPs) have been characterized. The isolation of an Arabidopsis thaliana cDNA encoding a novel membrane-associated ACBP that accumulates in developing seeds, designated ACBP1, has provided evidence for the existence of membrane-associated forms of ACBPs (Chye, 1998, Plant Mol. Biol. 38, 827-838). We now report on the isolation of its corresponding gene from an A. thaliana Columbia genomic library using the ACBP1 cDNA as a hybridization probe. Nucleotide sequence analysis of Arabidopsis ACBP1 showed that its promoter lacks a TATA box, resembling the promoters of rat, Drosophila and human genes encoding cytosolic ACBP and suggesting that it is a housekeeping gene. We show by Western blot analysis that ACBP1 expression in developing seeds coincides with lipid deposition and that homologues of membrane-associated ACBP1 exist in other plants. Using light microscopy, we show that ACBP1 is strongly expressed in the embryo at the cotyledons, hypocotyl, procambium of the axis and in most peripheral cells of the cotyledons and hypocotyl. Immunogold labelling localized ACBP1 to vesicles, to the plasma membrane especially at epidermal cells of heart, torpedo and cotyledonary stage embryos, and to the cell wall of the outer integument cells at the seed coat. Our results suggest that ACBP1 is involved in intermembrane lipid transport from the ER via vesicles to the plasma membrane where it could maintain a membrane-associated acyl pool; its immunolocalization to the cell wall of outer integument cells at the seed coat suggests a role in cuticle and cutin formation.     

Effect of caffeine and reduced temperature (20 degrees C) on the organization of the pre-Golgi and the Golgi stack membranes

In the present study we have dissected the transport pathways between the ER and the Golgi complex using a recently introduced (Kuismanen, E., J. Jantti, V. Makiranta, and M. Sariola. 1992. J. Cell Sci. 102:505- 513) inhibition of transport by caffeine at 20 degrees C. Recovery of the Golgi complex from brefeldin A (BFA) treatment was inhibited by caffeine at reduced temperature (20 degrees C) suggesting that caffeine inhibits the membrane traffic between the ER and the Golgi complex. Caffeine at 20 degrees C did not inhibit the BFA-induced retrograde movement of the Golgi membranes. Further, incubation of the cells in 10 mM caffeine at 20 degrees C had profound effects on the distribution and the organization of the pre-Golgi and the Golgi stack membranes. Caffeine treatment at 20 degrees C resulted in a selective and reversible translocation of the pre- and cis-Golgi marker protein (p58) to the periphery of the cell. This caffeine-induced effect on the Golgi complex was different from that induced by BFA, since mannosidase II, a Golgi stack marker, remained perinuclearly located and the Golgi stack coat protein, beta-COP, was not detached from Golgi membranes in the presence of 10 mM caffeine at 20 degrees C. Electron microscopic analysis showed that, in the presence of caffeine at 20 degrees C, the morphology of the Golgi stack was altered and accumulation of numerous small vesicles in the Golgi region was observed. The results in the present study suggest that caffeine at reduced temperature (20 degrees C) reveals a functional interface between the pre-Golgi and the Golgi stack.     

Cortical microtubules mark the mucilage secretion domain of the plasma membrane in Arabidopsis seed coat cells

During their differentiation Arabidopsis thaliana seed coat cells undergo a brief but intense period of secretory activity that leads to dramatic morphological changes. Pectic mucilage is secreted to one domain of the plasma membrane and accumulates under the primary cell wall in a ring-shaped moat around an anticlinal cytoplasmic column. Using cryofixation/transmission electron microscopy and immunofluorescence, the cytoskeletal architecture of seed coat cells was explored, with emphasis on its organization, function and the large amount of pectin secretion at 7 days post-anthesis. The specific domain of the plasma membrane where mucilage secretion is targeted was lined by abundant cortical microtubules while the rest of the cortical cytoplasm contained few microtubules. Actin microfilaments, in contrast, were evenly distributed around the cell. Disruption of the microtubules in the temperature-sensitive mor1-1 mutant affected the eventual release of mucilage from mature seeds but did not appear to alter the targeted secretion of vesicles to the mucilage pocket, the shape of seed coat cells or their secondary cell wall deposition. The concentration of cortical microtubules at the site of high vesicle secretion in the seed coat may utilize the same mechanisms required for the formation of preprophase bands or the bands of microtubules associated with spiral secondary cell wall thickening during protoxylem development.     

New 3-substituted xanthines : Potent inhibitors of cell plate formation

Caffeine and some related compounds are known to inhibit cell plate formation (CPF), but little is known about their mechanism of action. In this connection, the effects of xanthine derivatives on CPF in Tradescantia stamen hair cells were examined. Caffeine (1,3,7-trimethylxanthine) and 1,3-dimethyl-xanthine were more effective inhibitors of CPF than 1-methyl- or 1,7-dimethyl-xanthine. Four compounds with a substituent at the three position of 1,7-dimethyl-xanthine were found to be more effective than caffeine as inhibitors of CPF. Among them, 1,7-dimethyl-3-(3-ethylbutyl) xanthine was the most effective and the concentration required for inhibition of CBF was one tenth of that of caffeine. Breakdown of cell plates could be partially reversed using guanosine or adenosine, but reversibility was not detected using cytokinins.     

Ultrastructural Observations on CTC-Induced Callose Formation in Riella helicophylla

The Ca²⁺-chelator CTC binds to a specific site on both outer surfaces of all non-meristematic cells of the unistratose thallus of Riella, known to be rich in anionic wall components and calcium, and induces there the deposition of callose. Structural changes in this region during prolonged CTC treatment have been followed by light and transmission electron microscopy. With fluorescence microscopy punctate structures can be detected after 10 min, which upon longer incubation in CTC develop into large vesicular bodies, surrounded by a circular structure. The aniline blue-derived fluorescence intensity of these structures is highest in cells of the extension growth zone. At the ultrastructural level a mosaic of numerous smooth-surfaced vesicles, presumably containing callose, initially appears subjacent to the plasma membrane. These vesicles swell and fuse with each other, forming ultimately a circular fusion profile with the plasma membrane. This complex of callose-forming vesicles is thought to develop from elements of the partially coated reticulum (PCR), based on the presence of coated vesiculation profiles on the callose vesicles and numerous aggregates of coated vesicles in their immediate vicinity. After 30 min in CTC osmiophilic particles appear around these callose vesicles and at the cytoplasmic face of mitochondria. They are later (after 60 min) deposited in the periplasmic space between wall and plasma membrane and are also released into the surrounding medium. As judged by their reaction with FeCl₃, the osmiophilic particles appear to be phenolic in nature. We propose that upon binding of CTC a local increase of cytoplasmic calcium triggers callose synthesis in PCR-like compartments beneath the plasma membrane. However it remains to be shown as to why callose is synthesized exclusively in these intracellular compartments and not at the plasma membrane.     

Cytokinesis in microspore mother cells of Impatiens sultani

Summary Cytokinesis in Impatiens sultani microspore mother cells is simultaneous. It starts with the formation of small ingrowths of the surrounding callosic wall. Next, an incomplete cell plate is formed by fusion of small dictyosome vesicles. The cell plate consists of a network of anastomosing tubules and sacs. Aggregates of fusing vesicles are associated with bundles of microtubules, which are oriented perpendicular to the plane of the future cell walls. In the sacculate parts of the cell plate, some callose is deposited, while the associated microtubules disappear. The cell walls ultimately develop by enlargement of the previously formed wall ingrowths, which successively incorporate the elements of the cell plate. The enlargement and thickening of the walls is not accompanied by a further fusion and incorporation of dictyosome vesicles.     

In vitro prooxidant effect of caffeine

Caffeine is a methylxanthine compound that acts as a stimulant in humans. It is the most widely consumed behaviourally active substance in the western world and it affects calcium influx and release in living cells. Caffeine is present in various substances such as tea, coffee and some medications. This article is focused on the impact of caffeine on oxidation. Caffeine promoted the peroxidation of linoleic acid emulsions used by 32.5, 48.9, and 54.3%, respectively, at 15, 30 and 45 microg/mL concentrations. Standard antioxidants such as alpha-tocopherol and trolox, a water-soluble analogue of tocopherol, inhibited 76.2 and 93.2% peroxidation of linoleic acid emulsion at 45 microg/mL concentration. Also, PC(50) value (caffeine concentration that produced a 50% increase in linoleic acid peroxidation) of caffeine was found to be 185 microM.     

Somatic cytokinesis and pollen maturation in Arabidopsis depend on TPLATE, which has domains similar to coat proteins

TPLATE was previously identified as a potential cytokinesis protein targeted to the cell plate. Disruption of TPLATE in Arabidopsis thaliana leads to the production of shriveled pollen unable to germinate. Vesicular compartmentalization of the mature pollen is dramatically altered, and large callose deposits accumulate near the intine cell wall layer. Green fluorescent protein (GFP)-tagged TPLATE expression under the control of the pollen promoter Lat52 complements the phenotype. Downregulation of TPLATE in Arabidopsis seedlings and tobacco (Nicotiana tabacum) BY-2 suspension cells results in crooked cell walls and cell plates that fail to insert into the mother wall. Besides accumulating at the cell plate, GFP-fused TPLATE is temporally targeted to a narrow zone at the cell cortex where the cell plate connects to the mother wall. TPLATE-GFP also localizes to subcellular structures that accumulate at the pollen tube exit site in germinating pollen. Ectopic callose depositions observed in mutant pollen also occur in RNA interference plants, suggesting that TPLATE is implicated in cell wall modification. TPLATE contains domains similar to adaptin and beta-COP coat proteins. These data suggest that TPLATE functions in vesicle-trafficking events required for site-specific cell wall modifications during pollen germination and for anchoring of the cell plate to the mother wall at the correct cortical position.     

Auxilin facilitates membrane traffic in the early secretory pathway

Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER–Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.     

Effect of caffeine on kinetics of accumulation and release of Ca2+ by vesicles of the sarcoplasmic reticulum of skeletal muscle

The action of caffeine was studied on the heavy sarcoplasmic reticulum fraction enriched by vesicles derived from terminal cisterns. Caffeine lowers the ATP-dependent accumulation of Ca2+ by vesicles and enhances the first rapid phase of the Ci2+ release from vesicles. The action of caffeine was transient, reversed, Ca2+-dependent. The data obtained suggest that the reduction of ATP-dependent calcium accumulation and enhancement of calcium release by caffeine are mediated by the mechanism of Ca2+-induced Ca2+ release and support the view that caffeine may regulate the equilibrium between open and closed states of Ca2+-channel by increasing the affinity of Ca2+-receptor site of the channel.