Female genital mutilation: an overview

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4 degrees C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60-68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.     

The characterization of plasma membrane-bound tubulin of cauliflower using Triton X-114 fractionation

The cortical microtubules determine how cellulose microfibrils are deposited in the plant cell wall and are thus important for the control of cell expansion. To understand how microtubules can control microfibril deposition, the components that link the microtubules to the plasma membrane (PM) of plant cells must be isolated. To obtain information on the properties of the tubulin-membrane associations, cauliflower (Brassica oleracea) PM was subjected to Triton X-114 fractionation, and the distribution of alpha- and beta-tubulin was analyzed using immunoblotting. Approximately one-half of the PM-associated tubulin was solubilized by Triton X-114 and 10 to 15% of both alpha- and beta-tubulin was recovered in the detergent phase (indicative of hydrophobic properties) and 30 to 40% was recovered in the aqueous phase. The hydrophobic tubulin could be released from the membrane by high pH extraction with preserved hydrophobicity. A large part of the PM-associated tubulin was found in the Triton-insoluble fraction. When this insoluble material was extracted a second time, a substantial amount of hydrophobic tubulin was released if the salt concentration was increased, suggesting that the hydrophobic tubulin was linked to a high-salt-sensitive protein aggregate that probably includes other components of the cytoskeleton. The hydrophobicity of a fraction of PM-associated tubulin could reflect a direct or indirect interaction of this tubulin with the lipid bilayer or with an integral membrane protein and may represent the anchoring of the cortical microtubules to the PM, a key element in the regulation of cell expansion.     

Upregulation and redistribution of E-MAP-115 (epithelial microtubule-associated protein of 115 kDa) in terminally differentiating keratinocytes is coincident with the formation of intercellular contacts

Microtubules are involved in the positioning and movement of organelles and vesicles and therefore play fundamental roles in cell polarization and differentiation. Their organization and properties are cell-type specific and are controlled by microtubule-associated proteins (MAP). E-MAP-115 (epithelial microtubule-associated protein of 115 kDa) has been identified as a microtubule-stabilizing protein predominantly expressed in epithelial cells. We have used human skin and primary keratinocytes as a model to assess a putative function of E-MAP-115 in stabilizing and reorganizing the microtubule network during epithelial cell differentiation. Immunolabeling of skin sections indicated that E-MAP-115 is predominantly expressed in the suprabasal layers of the normal epidermis and, in agreement with this observation, is relatively abundant in squamous cell carcinomas but barely detectable in basal cell carcinomas. In primary keratinocytes whose terminal differentiation was induced by increasing the Ca2+ concentration of the medium, E-MAP-115 expression significantly increased during the first day, as observed by northern and western blot analysis. Parallel immunofluorescence studies showed an early redistribution of E-MAP-115 from microtubules with a paranuclear localization to cortical microtubules organized in spike-like bundles facing intercellular contacts. This phenomenon is transient and can be reversed by Ca2+ depletion. Treatment of cells with cytoskeleton-active drugs after raising the Ca2+ concentration indicated that E-MAP-115 is associated with a subset of stable microtubules and that the cortical localization of these microtubules is dependent on other microtubules but not on strong interactions with the actin cytoskeleton or the plasma membrane. The mechanism whereby E-MAP-115 would redistribute to and stabilize cortical microtubules used for the polarized transport of vesicles towards the plasma membrane, where important reorganizations take place upon stratification, is discussed.     

Dynein and dynactin are localized to astral microtubules and at cortical sites in mitotic epithelial cells

The mitotic spindle is often positioned in a characteristic location during development, for example to enable the proper segregation of developmental determinants [1,2]. When epithelial cells divide, the mitotic spindle is often positioned parallel to the plane of the epithelium, so that both daughter cells contribute to the epithelium [3]. The mechanisms by which mitotic spindles are positioned have not been characterized in great detail, but evidence is accumulating that in some systems the dynein-dynactin microtubule motor complex plays a role [4-6]. Dynein has yet not been localized to cortical sites where it could bind to microtubules and exert a force that might orient the mitotic spindle, however [7,8]. Here, we report that in mitotic polarized epithelial cells, the dynein-dynactin complex accumulates, from prometaphase onwards, along astral microtubules and at cortical spots, into which many of the astral microtubules dock. The spots are assembled at the lateral plasma membrane, in the region below the tight junctions. Their formation is inhibited by cytochalasin D, and under these conditions the spindles do not orient properly. This novel localization of the dynein-dynactin complex is consistent with a role for the complex in the positioning of the mitotic spindle. We also show that, during prophase, the motor complex colocalizes with the nuclear envelope, consistent with it having a role in separating the centrosomes that are associated with the nuclear envelope.     

The fine structure of a microplate-microtubule array, microfilaments and polyhedral body associated microtubules in several species of Anabaena

A microplate-microtubule array was observed in Anabaena sp. (B-378). This structure consists of an arched plate, about 8 nm thick, and various microtubules, 12 nm in diameter and 50 nm long, arranged in rows. The microtubules project at right angles from one side of the plate into the cytoplasm or towards the plasma membrane. Up to twelve microplate-microtubule arrays were observed in a single section of a cell. Microfilaments, about 2.8 nm in diameter and of undetermined length, were observed in four isolates of Anabaena. The microfilaments were always found in bundles, nwhich varied in size, up to 0.63 mum across and 0.91 mum long. Microtubules, 10 nm in diameter and about 150 nm in length, were observed associated with one facet of polyhedral bodies in 8 out of 20 isolates of Anabaena. The microtubules occurred in groups of up to 20 or more, and were always oriented with the long axis parallel to the facet of a polyhedral body. In cross section, the microtubules had an electron transparent lumen 5 nm wide and a wall 2.5 nm thick. These structures are compared to previously described microtubules and microfilaments.     

Cellulose Synthases and Synthesis in Arabidopsis

Plant cell walls are complex structures composed of high-molecular-weight polysaccharides,proteins,and lignins. Among the wall polysaccharides,cellulose,a hydrogen-bonded β-1,4-linked glucan microfibril,is the main load-bearing wall component and a key precursor for industrial applications. Cellulose is synthesized by large multi-meric cellulose synthase (CesA) complexes,tracking along cortical microtubules at the plasma membrane. The only known components of these complexes are the cellulose synthase proteins. Recent studies have identified tentative interaction partners for the CesAs and shown that the migratory patterns of the CesA complexes depend on phosphorylation status. These advances may become good platforms for expanding our knowledge about cellulose synthesis in the near future. In addition,our current understanding of cellulose chain polymerization in the context of the CesA complex is discussed.     

The ultrastructure of yeast: cell wall structure and formation

Yeasts are unicellular eukaryotes, and are used widely as a model system in basic and applied fields of life science, medicine, and biotechnology. The ultrastructure of yeast cells was first studied in 1957 and the techniques used have advanced greatly in the 40 years since then; an overview of these methods is first presented in this review. The ultrastructure of budding and dimorphic yeast cells observed with a scanning electron microscope (SEM) and a transmission electron microscope (TEM) after thin sectioning and freeze-etching are then described, followed by discussion of the regeneration of the cell wall of Candida albicans protoplasts detected by cryosectioning. C. albicans protoplasts are regenerated to synthesize microfibrils on their surface. They are aggregated into thicker bundles which are intermeshed, forming a wide-meshed network of long fibrils. These microfibrillar structures are chains of beta-1,3-glucan which are broken down after treatment with beta-1,3-glucanase. Morphologically identical microfibrils are synthesized in vitro by a cell-free system in which the active cell membrane fraction as a source of beta-1,3-glucan synthetase and UDP glucose as the sole substrate are used. The diameter of an elemental fibril of beta-glucan is estimated to be 2.8 nm from the pattern of autocorrelation of the image obtained by computer processing. In contrast, in the presence of aculeacin A the formation of normal fibrillar nets or bundles is significantly inhibited, resulting in the occurrence of short fibrils. These electron microscopic data suggest that aculeacin A inhibits not only the synthesis of beta-1,3-glucan but the aggregation of microfibrils of this polysaccharide, allowing formation of the crystalline structure. On the basis of the cumulative data obtained from the electron microscopic studies, we are led to the assumption that de novo synthesized beta-glucan chains might initially form fine particles which are then transformed into thin fibrils with single to multiple strands which appear to be oriented parallel to each other so that they develop into fibrillar structures. This process of assembly of beta-glucan molecules leads to the development of a fibrous network within the regenerating Candida cell wall. Third, the mechanism of cell wall formation is shown by low-voltage (LV) SEM and TEM, using various techniques and computer graphics, of the regeneration system of Schizosaccharomyces pombe protoplasts: after 10 min of regeneration, the protoplasts begin to grow fibrillar substances of a beta-glucan nature, and a fibrillar network covers the surface of all protoplasts. The network is originally formed as fine particles on the protoplast surface and these are subsequently lengthened to microfibrils 2 nm thick. The microfibrils twist around each other and develop into 8 nm thick fibrils forming flat bundles 16 nm thick. Interfibrillar spaces are gradually filled with amorphous particles of an alpha-galactomannan nature and, finally, the complete cell wall is formed after 12 h. Treatment of reverting protoplasts with RuO4 provided clear TEM images of glucan fibrils with high electron density. The relationship between cell wall regeneration and intracellular organelles was examined by using serial thin sections stained with PATAg and computer-aided three-dimensional reconstruction. The secretory vesicles in a protoplast had increased markedly by 1.4, 3.4, and 5.8 times at 1.5, 3.0, and 5 h, respectively. Three-dimensional analysis indicates that Golgi apparatuses are located close together in the nucleus of the protoplast and are dispersed into the cytoplasm during the progress of cell wall formation.     

Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.     

Cytoskeletal changes during neurogenesis in cultures of avain neural crest cells

Neural crest cells are motile and mitotic, whereas their neuronal derivatives are terminally post-mitotic and consist of stationary cell body from which processes grow. The present study documents changes in the cytoskeleton that occur during neurogenesis in cultures of avain neural crest cells. The undifferentiated neural crest cells contain dense bundles of actin filaments throughout their cytoplasm, and a splayed array of microtubules attached to the centrosome. In newly differentiating neurons, the actin bundles are disrupted and most of the remaining actin filaments are reorganized into a cortical layer underlying the plasma membrane of the cell body and processes. Microtubules are more abundant in newly-differentiating neurons than in the undifferentiated cells, and individual microtubules can be seen dissociated from the centrosome. Neuron-specific beta-III tubulin appears in some crest cells prior to cessation of motility and cell division, and expression increases with total microtubule levels during neurogenesis. To investigate how these early cytoskeletal changes might contribute to alterations in morphology during neurogenesis, we have disrupted the cytoskeleton with pharmacologic agents. Microfilament disruption by cytochalasin immediately arrests the movement of neural crest cells and causes them to round-up, but does not significantly change the morphology of the immature neurons. Microtubule depolymerization by nocodazole slows the movement of undifferentiated cells and causes retraction of processes extended by the immature neurons. These results suggest that changes in the actin and microtubule arrays within neural crest cells govern distinct aspects of their morphogenesis into neurons.     

Throat disorders

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25-50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) approximately 40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.     

Compartmentalized vesicular traffic around the hair cell cuticular plate

Through thin-section and freeze-fracture electron microscopy, we identify structural correlates of an intense vesicular traffic in a narrow band of cytoplasm around the cuticular plate of the bullfrog vestibular hair cells. Myriads of coated and uncoated vesicles associated with longitudinally oriented microtubules populate the narrow cytoplasmic region between the cuticular plate and the actin network of the apical junctional belt. If microtubules in the sensory hair cells, like those in axons, are pathways for organelle transport, then the characteristic distribution of microtubules around the cuticular plate represents transport pathways across the apical region of the hair cells. This compartmentalized membrane traffic system appears to support an intense vesicular release and uptake along a band of apical plasma membrane near the cell border. Functions of this transport system may include membrane recycling as well as exocytotic and endocytotic exchange between the hair cell cytoplasm and the endolymphatic compartment.     

An ultra-structural study of the gills of Echinus esculentus

The nerve growth factor protein (NGF) induces organization of in vitro preformed microtubules (MTs) to form multiple arrays. These complexes, in cross-section, appear to be formed by clusters of single tubules in parallel in close contact with center distance similar to the diameter of a microtubule. In the absence of NGF, on the contrary, microtubules are far apart from each other. Addition of vinblastine to NGF-MT complexes does not result in the usual reorganization of microtubules into paracrystalline arrays, noticeable when the growth factor is not present. The vinblastine treated NGF-MT complexes have several round-shaped elements along the tubules and at their endings which, at higher magnification, appear to be formed by closely packed, ring-like structures. These 'boutons' are postulated to represent the point of resistance to the depolymerizing action of the vinca alkaloid. These findings are discussed in relation to a similar antagonistic effect observed in vivo between NGF and vinblastine and to the possible involvement of microtubules in the mechanism of action of the growth factor.     

A novel direct interaction of endoplasmic reticulum with microtubules

The positioning and dynamics of organelles in eukaryotic cells critically depend on membrane-cytoskeleton interactions. Motor proteins play an important role in the directed movement of organelle membranes along microtubules, but the basic mechanism by which membranes stably interact with the microtubule cytoskeleton is largely unknown. Here we report that p63, an integral membrane protein of the reticular subdomain of the rough endoplasmic reticulum (ER), binds microtubules in vivo and in vitro. Overexpression of p63 in cell culture led to a striking rearrangement of the ER and to concomitant bundling of microtubules along the altered ER. Mutational analysis of the cytoplasmic domain of p63 revealed two determinants responsible for these changes: an ER rearrangement determinant near the N-terminus and a central microtubule-binding region. The two determinants function independently of one another as indicated by deletion experiments. A peptide corresponding to the cytoplasmic tail of p63 promoted microtubule polymerization in vitro. p63 is the first identified integral membrane protein that can link a membrane organelle directly to microtubules. By doing so, it may contribute to the positioning of the ER along microtubules.     

Calcium determines the supramolecular organization of fibrillin-rich microfibrils

Microfibrils are ubiquitous fibrillin-rich polymers that are thought to provide long-range elasticity to extracellular matrices, including the zonular filaments of mammalian eyes. X-ray diffraction of hydrated bovine zonular filaments demonstrated meridional diffraction peaks indexing on a fundamental axial periodicity (D) of approximately 56 nm. A Ca2+-induced reversible change in the intensities of the meridional Bragg peaks indicated that supramolecular rearrangements occurred in response to altered concentrations of free Ca2+. In the presence of Ca2+, the dominant diffracting subspecies were microfibrils aligned in an axial 0.33-D stagger. The removal of Ca2+ caused an enhanced regularity in molecular spacing of individual microfibrils, and the contribution from microfibrils not involved in staggered arrays became more dominant. Scanning transmission electron microscopy of isolated microfibrils revealed that Ca2+ removal or addition caused significant, reversible changes in microfibril mass distribution and periodicity. These results were consistent with evidence from x-ray diffraction. Simulated meridional x-ray diffraction profiles and analyses of isolated Ca2+-containing, staggered microfibrillar arrays were used to interpret the effects of Ca2+. These observations highlight the importance of Ca2+ to microfibrils and microfibrillar arrays in vivo.     

Regulation of cytoskeleton by myelin components: studies on shiverer oligodendrocytes carrying an Mbp transgene

Oligodendrocytes from the shiverer mutant mouse are missing most of the myelin basic protein (Mbp) gene. In axon-free cultures, they produce membrane sheets with abnormally assembled microtubule and actin-based structures. This suggests that an Mbp gene product may have an important role in regulating the organization and stability of the wild-type oligodendrocyte cytoskeleton. We now present evidence extending these observations, using cultured oligodendrocytes that carry both the shiverer mutation and the Mbp1 transgene which partially corrects their deficit. Shiverer oligodendrocytes that carry one dose of the Mbp1 transgene abnormally express MBP along major cytoskeletal vein-like structures in processes and sheets. Shiverer oligodendrocytes that carry two doses of the Mbp1 transgene contain two types of membrane sheet regions, i.e. regions filled with aberrant punctate foci of MBP, and regions with normal domains of MBP. Immunocytochemical staining data show that the distribution of cytoskeleton and associated 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) is dependent upon how MBP is organized. Bundling of actin filaments occurs only around MBP domains, and the colocalization of CNPase along microtubular structures also appears to be regulated by MBP domains in sheets. Multinucleated oligodendrocytes are observed, a likely result of the inability of dividing pro-oligodendrocytes to bundle actin filaments. In addition, the ability of MBP to mediate extracellular signals that modulate cytoskeleton appears to be dependent upon MBP's organization. Transduction of the galactocerebroside signaling pathway, which results in the destabilization of microtubules but not actin filaments, occurs only in sheets containing MBP domains. The distribution of MBP, however, does not affect the myelin/oligodendrocyte-specific protein signaling pathway, which results in growth of microtubular structures and extensive destabilization of the actin cytoskeleton.     

Porous channels in the cuticle of the head-arrester system in dragon/damselflies (Insecta:Odonata)

The ultrastructure of the porous channels (PC) of the postcervical sclerite (SPC), which provides additional head fixation to the neck in adult odonates, was studied using TEM and high resolution SEM microscopy. Single chitin-protein microfibrils, about 0.14 micron thick, are arranged into channels with cylinder-like shapes. The axial rod of the chitin fiber (0.04 micron thick) is located in the center of the cylinder. The orientation of the axial rods was three-dimensionally demonstrated after dissolving the protein cover with NaOH. The PCs are arranged vertically to the surface and pass from the epidermal cells through all the cuticular layers to the surface of the cuticle. In the exo- and endocuticle, the PCs are usually oval in cross-section and about 0.3 micron thick. In the endocuticle, the cross-sectional area of the PCs varies widely, from 0.01-0.15 micron2. The shape of the PC is determined by the macromolecular organization of the chitin-protein microfibrils: the long axis of the channel is orientated parallel to the axis of the preferred orientation of the cuticular microfibrils. The microfibrils tend to follow the line of the channel very closely. In fractures orientated perpendicular to the surface, the PC resembles a ribbon-like construction, which was clearly demonstrated by casts. The strongly parallel orientation of PCs in the deep layers of the cuticle changes within the microtrichia (MT), and they begin to be curved. Numerous PCs pass through the microtrichium, and most of them end on its side wall. PCs usually contain channel filaments about 0.09 micron thick. Usually, a single channel contained one filament, but channels located in the deep layers of the endocuticle have from one to five single filaments. The filaments were observed in the intact cuticle and in the cuticle enzymatically treated with chitinase, while in the cuticle treated with NaOH filaments were absent. The porous channel system of the odonate arrester is interpreted as a device transporting adhesive excretions from the epidermal cells to the cuticular surface.     

Wide-spectrum antibiotic activity of synthetic, amphipathic peptides

Transbilayer phosphatidylethanolamine (PtdEtn) movements in the plasma membrane of Saccharomyces cerevisiae are regulated by an ATP-dependent, protein-mediated process(es). To examine whether this process is influenced by the actin cytoskeleton, we have studied the PtdEtn translocation in S. cerevisiae cells after treatment with microfilament disrupting and microtubule-disrupting agents. PtdEtn translocation was studied by measuring the external PtdEtn levels, using fluorescamine as the external membrane probe, in the ATP-depleted, ATP-depleted and repleted, and N-ethylmaleimide-treated cells. The microfilaments and microtubules were disrupted by treatment with various cytochalasins and colchicine (or benomyl) respectively PtdEtn translocation became abnormal in the cytochalasin-treated cells but not in cells that were treated with microtubule-disrupting agents, such as colchicine or benomyl. These results have been interpreted to suggest that the actin cytoskeleton is involved in regulating the PtdEtn translocase activity in the yeast cell plasma membrane.     

Constitutive transport between the trans-Golgi network and the plasma membrane according to the maturation model. A hypothesis

Here we examine the application of the cisternal/carrier maturation model to describe transport of cargo proteins from the Golgi apparatus to the plasma membrane. Interpretation of the available evidence in the light of carrier maturation suggests that the transport intermediates between these stations are large pleiomorphic carriers formed by maturation of the trans-Golgi compartment, rather than vesicles, as would be postulated by the vesicular shuttle model. Mature carriers move along microtubules towards the plasma membrane via a microtubule/(kinesin)-based motor system. The maturation and vesicular transport models are compared in terms of consistency with the available literature.     

A microtubule-associated protein in maize is expressed during phytochrome-induced cell elongation

Plants can adapt their shape to environmental stimuli. This response is mediated by the reorganization of cortical microtubules, a unique element of the cytoskeleton. However, the molecular base of this response has remained obscure so far. In an attempt to solve this problem, signal-dependent changes in the pattern of microtubule-binding proteins were analysed during coleoptile elongation in maize, that is, under the control of the plant photoreceptor phytochrome. Two putative MAPs of 100 kDa (P100) and 50 kDa apparent molecular weights were identified in cytosolic extracts from non-elongating and elongating cells. Both proteins co-assembled with endogenous tubulin, bound to neurotubules and were immunologically related to the neural MAP tau: the P100 protein, depending on the physiological situation, was manifest as a double band and was always found to be heat-stable. In contrast, the 50 kDa MAP was heat-stable only for particular tissues and physiological treatments. The P100 protein was present in all tissues, however in a reduced amount in elongating coleoptiles. The 50 kDa MAP was expressed exclusively upon induction of phytochrome-dependent cell elongation. As shown by immunofluorescence double-staining, an epitope shared by both proteins colocalized with cortical microtubules in situ, but exclusively in elongating cells. In non-elongating cells, only the nuclei were stained. Partially purified nuclei from elongating cells were enriched in P100, whereas the 50 kDa MAP became enriched in a partially purified plasma membrane fraction.     

Meiotic cells monitor the status of the interhomolog recombination complex

Cortical granule exocytosis is important for the block to polyspermy at fertilization in the eggs of most vertebrates and many invertebrates. Cortical granules are poised at the cell surface and exocytose in response to sperm stimulation. Following exocytosis, the cortical granule contents modify the extracellular environment of the egg, the major result of which is to block additional sperm binding. Here we show that proteins homologous to members of the SNARE hypothesis-a molecular model designed to explain the trafficking, docking, and exocytosis of vesicles in the secretory compartment-are present in eggs at the right time and place to be involved in the regulation of cortical granule exocytosis. Using polymerase chain reaction (PCR) screens we have found homologues of synaptobrevin/VAMP, syntaxin, synaptotagmin, and rab3. Antibodies generated to fusion proteins or to synthetic peptides encoded by the cloned cDNAs were used in an immunofluorescence assay to show that each of the cognate proteins are present in the cortex of the egg. A synaptobrevin/VAMP homologue appears to be specifically associated with the membrane of cortical granules before fertilization and, following cortical granule exocytosis, is incorporated into the plasma membrane of the zygote. A rab3 homologue is also associated with cortical granules specifically but, following fertilization, the protein reassociates with different, yet undefined, vesicles throughout the cytoplasm of the zygote. Homologues of synaptotagmin and syntaxin are also present at the egg cortex but, in contrast to rab3 and VAMP, appear to be associated with the plasma membrane. Following fertilization, syntaxin and tagmin remain associated with the plasma membrane and are more readily immunolabeled, presumably due to an increased accessibility of the antibodies to the target protein domains. We also show by immunoblotting experiments that the cognate proteins are of the sizes predicted for these homologues. These results suggest that at least some steps in the biology of cortical granules may be mediated by SNARE homologues, and this finding, along with the unique biology of cortical granules, should facilitate examination of specific events of the fertilization reaction and the mechanism of stimulus-dependent exocytosis.