Endosome fusion and microtubule-based dynamics in the early endocytic pathway of dictyostelium

Dictyostelium amoebae, like mammalian macrophages, take up fluid by macropinocytosis. The present study used fluorescent fluid-phase markers and GFP-labeled microtubules to visualize the uptake, dynamics, and fusion of early endosomes in Dictyostelium. Consecutive labeling with two fluorescent fluid-phase markers demonstrated that within the first few minutes after uptake, new macropinosomes underwent fusion with pre-existing endosomes. The fusing endosomes, which represent the mixing compartment, displayed extreme shape changes and rapid transport about the cell in association with microtubules. The great plasticity of endosomes at this stage of maturation was also evident by electron microscopy. The constant undulatory motion of microtubules was implemental in establishing contact with endosomes. Treatment of cells with agents that selectively disrupted either actin filaments or microtubules confirmed that endosome dynamics were microtubule based. Further maturation of endosomes led to loss of pleiomorphy in favor of a spherical shape, inability to fuse with new macropinosomes, and diminished motility.     

Cold exposure reveals two populations of microtubules in pulmonary endothelia

Microtubules are composed of α-tubulin and β-tubulin dimers. Microtubules yield tubulin dimers when exposed to cold, which reassemble spontaneously to form microtubule fibers at 37°C. However, mammalian neurons, glial cells, and fibroblasts have cold-stable microtubules. While studying the microtubule toxicity mechanisms of the exotoxin Y from Pseudomonas aeruginosa in pulmonary microvascular endothelial cells, we observed that some endothelial microtubules were very difficult to disassemble in the cold. As a consequence, we designed studies to test the hypothesis that microvascular endothelium has a population of cold-stable microtubules. Pulmonary microvascular endothelial cells and HeLa cells (control) were grown under regular cell culture conditions, followed by exposure to an ice-cold water bath and a microtubule extraction protocol. Polymerized microtubules were detected by immunofluorescence confocal microscopy and Western blot analyses. After cold exposure, immunofluorescence revealed that the majority of HeLa cell microtubules disassembled, whereas a smaller population of endothelial cell microtubules disassembled. Immunoblot analyses showed that microvascular endothelial cells express the microtubule cold-stabilizing protein N-STOP (neuronal stable tubule-only polypeptides), and that N-STOP binds to endothelial microtubules after cold exposure, but not if microtubules are disassembled with nocodazole before cold exposure. Hence, pulmonary endothelia have a population of cold-stable microtubules.     

A spiral array of microtubules in the fertilized sea urchin egg cortex examined by indirect immunofluorescence and electron microscopy

A transient spiral system of fibers in the cortex of fertilized eggs of the sea urchin Strongylocentrotus purpuratus was examined with indirect immunofluorescence microscopy and found to contain tubulin. Electron microscopy identified the tubulin-containing bands as bundles of up to 40 or more microtubules. These cortical microtubules, which are initially radial, form a spiral array about the time of pronuclear fusion. This basket-like structure, at a depth of 10–15 μm below the cell surface, reaches a peak of development about 45 min after fertilization and disappears before the streak stage at 70 min, in a division cycle of slightly more than 2 h. Possible functions of the cortical microtubules, which appear to be independent of the interphase asters, are discussed.     

Receptor-mediated transcytosis of follicle-stimulating hormone through the rat testicular microvasculature

Accumulated data suggest that endothelial cells express specific receptors for several peptide and (glyco)protein hormones that may transport hormones across the cell to be delivered to the interstitial fluid and tissue target cells. Surprisingly, very little information is available on the actual endothelial organelles involved in this cellular process. In the present study the transfer of follicle-stimulating hormone (FSH) through the endothelial barrier of rat testes was examined by analysing the binding and transport of gold-tagged recombinant human (rh)FSH under various conditions using electron microscopy. At 4 degrees C the probe bound specifically to the luminal surface of the endothelial cells without internalization. The use of 125I-rhFSH, which allows precise quantitation of the binding, confirmed the specificity of hormone interaction with the testicular microvasculature. At 37 degrees C the hormone was internalized via coated pits and vesicles into an extensive subluminal tubulo-vesicular compartment and was transported across the endothelium via a system of tubules and vesicles. Moreover, monoclonal antibodies against the FSH receptor ectodomain coupled to colloidal gold followed the same route. In contrast, a non specific, fluid-phase uptake via caveolae was observed for a major plasma protein - rat serum albumin and a fluid-phase tracer - peroxidase. These results suggest that FSH transcytosis across the testicular endothelial barrier is receptor-mediated and involves luminal uptake via coated pits/vesicles, sorting at the level of luminal early endosomes, and transcellular transport through transcytotic tubulo-vesicular organelles. Similar receptor-mediated pathways are likely to be involved in the physiological functioning of a number of other protein and peptide hormones that must translocate specifically from blood to the target cells.     

Organization of the cytoskeleton in early Drosophila embryos

The cytoskeleton of early, non-cellularized Drosophila embryos has been examined by indirect immunofluorescence techniques, using whole mounts to visualize the cortical cytoplasm and sections to visualize the interior. Before the completion of outward nuclear migration at nuclear cycle 10, both actin filaments and microtubules are concentrated in a uniform surface layer a few micrometers deep, while a network of microtubules surrounds each of the nuclei in the embryo interior. These two filament-rich regions in the early embryo correspond to special regions of cytoplasm that tend to exclude cytoplasmic particles in light micrographs of histological sections. After the nuclei in the interior migrate to the cell surface and form the syncytial blastoderm, each nucleus is seen to be surrounded by its own domain of filament-rich cytoplasm, into which the cytoskeletal proteins of the original surface layer have presumably been incorporated. At interphase, the microtubules seem to be organized from the centrosome directly above each nucleus, extending to a depth of at least 40 microns throughout the cortical region of cytoplasm (the periplasm). During this stage of the cell cycle, there is also an actin "cap" underlying the plasma membrane immediately above each nucleus. As each nucleus enters mitosis, the centrosome splits and the microtubules are rearranged to form a mitotic spindle. The actin underlying the plasma membrane spreads out, and closely spaced adjacent spindles become separated by transient membrane furrows that are associated with a continuous actin filament-rich layer. Thus, each nucleus in the syncytial blastoderm is surrounded by its own individualized region of the cytoplasm, despite the fact that it shares a single cytoplasmic compartment with thousands of other nuclei.     

Fluid-Phase Markers in the Basolateral Endocytic Pathway Accumulate in Response to the Actin Assembly-promoting Drug Jasplakinolide

To investigate the role of filamentous actin in the endocytic pathway, we used the cell-permeant drug Jasplakinolide (JAS) to polymerize actin in intact polarized Madin–Darby canine kidney (MDCK) cells. The uptake and accumulation of the fluid-phase markers fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP) were followed in JAS-treated or untreated cells with confocal fluorescence microscopy, biochemical assays, and electron microscopy. Pretreatment with JAS increased the uptake and accumulation of fluid-phase markers in MDCK cells. JAS increased endocytosis in a polarized manner, with a marked effect on fluid-phase uptake from the basolateral surface but not from the apical surface of polarized MDCK cells. The early uptake of FITC-dextran and HRP was increased more than twofold in JAS-treated cells. At later times, FITC-dextran and HRP accumulated in clustered endosomes in the basal and middle regions of JAS-treated cells. The large accumulated endosomes were similar to late endosomes but they were not colabeled for other late endosome markers, such as rab7 or mannose-6-phosphate receptor. JAS altered transport in the endocytic pathway at a later stage than the microtubule-dependent step affected by nocodazole. JAS also had a notable effect on cell morphology, inducing membrane bunching at the apical pole of MDCK cells. Although other studies have implicated actin in endocytosis at the apical cell surface, our results provide novel evidence that filamentous actin is also involved in the endocytosis of fluid-phase markers from the basolateral membrane of polarized cells.     

Role of Epac1, an exchange factor for Rap GTPases, in endothelial microtubule dynamics and barrier function

Rap1 GTPase activation by its cAMP responsive nucleotide exchange factor Epac present in endothelial cells increases endothelial cell barrier function with an associated increase in cortical actin. Here, Epac1 was shown to be responsible for these actin changes and to colocalize with microtubules in human umbilical vein endothelial cells. Importantly, Epac activation with a cAMP analogue, 8-pCPT-2'O-Me-cAMP resulted in a net increase in the length of microtubules. This did not require cell-cell interactions or Rap GTPase activation, and it was attributed to microtubule growth as assessed by time-lapse microscopy of human umbilical vein endothelial cell expressing fluorophore-linked microtubule plus-end marker end-binding protein 3. An intact microtubule network was required for Epac-mediated changes in cortical actin and barrier enhancement, but it was not required for Rap activation. Finally, Epac activation reversed microtubule-dependent increases in vascular permeability induced by tumor necrosis factor-alpha and transforming growth factor-beta. Thus, Epac can directly promote microtubule growth in endothelial cells. This, together with Rap activation leads to an increase in cortical actin, which has functional significance for vascular permeability.     

Pollen mitosis in the slipper orchid Cypripedium fasciculatum

Pollen mitosis in the slipper orchid Cypripedium fasciculatum was studied using correlated methods of immunofluorescence and transmission electron microscopy. Unlike the more highly evolved orchids, the cypripedioid orchids shed pollen as monosulcate monads. Prior to pollen mitosis, the microspore nucleus migrates to a proximal position opposite the aperture, as is typical of monocotyledons. There is no distinct generative pole microtubule system (GPMS) like that recently reported in development of pollen polarity in the vandoid moth orchid Phalaenopsis. Instead, microtubules in early prophase are concentrated around the nucleus and extend into the cytoplasm toward the future generative pole. Once the nucleus has migrated to the continuous surface opposite the aperture, microtubules surround the nucleus evenly and show no tendency to be more concentrated in the generative domain. The mitotic spindle, which develops from the perinuclear microtubules, is asymmetrically placed in the microspore and is cone-shaped. The generative pole is broad and closely appressed to the continuous spore surface, while the vegetative pole is pointed and located in the interior of the microspore. As the chromosomes move poleward, microtubules proliferate in the interzone and a phragmoplast develops. The phragmoplast expands in a hemispherical path beyond the interzone following an array of microtubules that radiates from the generative nucleus. Data from this study indicate that evolution of pollen in orchids includes a shift in location of the generative cell from proximal to distal and the evolution of a GPMS, in addition in the well-known trend toward increased pollen aggregation and loss of exine.     

Dose-dependent effect of nocodazole on endothelial cell cytoskeleton

The endothelium lining the inner surface of blood vessels fulfils an important barrier function and specifically, it controls vascular membrane permeability as well as nutrient and metabolite exchange in circulating blood and tissue fluids. Disturbances in vascular endothelium barrier function (vascular endothelium dysfunction) are coupled to cytoskeleton rearrangements, actomyosin contractility, and as a consequence, formation of paracellular gaps between endothelial cells. Microtubules constitute the first effector link in the reaction cascade resulting in vascular endothelium dysfunction. Increased vascular permeability associated with many human diseases is also manifested as a side effect in anticancer mitosis-blocking therapy. The aim of this study was to examine the possibility of preventing side effects of mitostatic drugs in patients with vascular endothelium dysfunction and to establish effective doses able to disrupt the microtubular network without interfering with the endothelial barrier function. Previously, it was found that the population of endothelial cell microtubules is heterogeneous. Along with dynamic microtubules, cell cytoplasm contains a certain amount of post-translationally modified microtubules that are less active and less susceptible to external influences than dynamic microtubules. We have shown that the area occupied with stable microtubules is relatively large (approx. one third of the total cell area). We assume that it can account for a higher resistance of the endothelial monolayer to factors responsible for vascular endothelium dysfunction. This hypothesis was validated in this study, in which nocodazole was used to induce vascular endothelium dysfunction in lung endothelial cells. The effect of nocodazole on endothelial cell cytoskeleton was found to be dose-dependent. Nocodazole in micromolar concentrations not only irreversibly changed the barrier function, but also upset the viability of endothelial cells and induced their death. Nanomolar concentrations of nocodazole also increased the permeability of the endothelial monolayer; this effect was reversible at the drug concentration ranging from 100 to 200 nM. At 100 nM, nocodazole induced partial disruption of the microtubule network near the cell margin without any appreciable effect on acetylated microtubules and actin filaments. At 200 nM, nocodazole exerted a pronounced effect on the system of dynamic (but not acetylated) microtubules and increased the population of actin filaments in the central region of the cell. Our data suggest that disruption of peripheral microtubules triggers a cascade of reactions culminating in endothelial barrier dysfunction; however, the existence of a large population of microtubules resistant to nanomolar concentrations of the drug provides higher viability of endothelial cells and restores their functional activity.     

Actin-dependent fluid-phase endocytosis in inner cortex cells of maize root apices

The fluorescent dye Lucifer Yellow (LY) is a well-known and widely-used marker for fluid-phase endocytosis. In this paper, both light and electron microscopy revealed that LY was internalized into transition zone cells of the inner cortex of intact maize root apices. The internalized LY was localized within tubulo-vesicular compartments invaginating from the plasma membrane at actomyosin-enriched pit-fields and individual plasmodesmata, as well as within adjacent small peripheral vacuoles. The internalization of LY was blocked by pretreating the roots with the F-actin depolymerizing drug latrunculin B, but not with the F-actin stabilizer jasplakinolide. F-actin enriched plasmodesmata and pit-fields of the inner cortex also contain abundant plant-specific unconventional class VIII myosin(s). In addition, 2,3 butanedione monoxime, a general inhibitor of myosin ATPases, partially inhibited the uptake of LY into cells of the inner cortex. Conversely, loss of microtubules did not inhibit fluid-phase endocytosis of LY into these cells. In conclusion, specialized actin- and myosin VIII-enriched membrane domains perform a tissue-specific form of fluid-phase endocytosis in maize root apices. The possible physiological relevance of this process is discussed.     

Rapid flow-induced responses in endothelial cells

Endothelial cells alter their morphology, growth rate, and metabolism in response to fluid shear stress. To study rapid flow-induced responses in the 3D endothelial cell morphology and calcium distribution, coupled fluorescence microscopy with optical sectioning, digital imaging, and numerical deconvolution techniques have been utilized. Results demonstrate that within the first minutes of flow application nuclear calcium is increasing. In the same time frame whole cell height and nuclear height are reduced by about 1 microm. Whole cell height changes may facilitate reduction of shear stress gradients on the luminal surface, whereas nuclear structural changes may be important for modulating endothelial growth rate and metabolism. To study the role of the cytoskeleton in these responses, endothelial cells have been treated with specific disrupters (acrylamide, cytochalasin D, and colchicine) of each of the cytoskeleton elements (intermediate filaments, microfilaments, and microtubules, respectively). None of these compounds had any effect on the shear-induced calcium response. Cytochalasin D and acrylamide did not affect the shear-induced nuclear morphology changes. Colchicine, however, completely abrogated the response, indicating that microtubules may be implicated in force transmission from the plasma membrane to the nucleus. A pedagogical model based on tensegrity theory principles is presented that is consistent with the results on the 3D endothelial morphology.     

The microtubule system in endothelial barrier dysfunction: disassembly of peripheral microtubules and microtubules reorganization in internal cytoplasm

Endothelial cell barrier dysfunction is often associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction and finally gap formation. At present time the role of microtubules in endothelial cell barrier regulation is not fully understood, however a number of observations allow to assume that microtubules reaction is the extremely important part in development of endothelial dysfunction. These observations have been forced us to examine the role of microtubule system reorganization in endothelial cell barrier regulation. In quiescent endothelial cells microtubule density is the highest in the centrosome region and insignificant near the cell margin. The analysis of microtubules distribution after specific antibodies staining using the method of measurement of their fluorescence intensity has shown that in control endothelial cells the reduction of fluorescence intensity from the cell center to its periphery is described by the equation of an exponential regression. The hormone agent, thrombin (25 nM), causes rapid increase of endothelial cell barrier permeability accompanied by fast decrease in quantity of peripheral microtubules and reorganization of microtubule system in internal cytoplasm of endothelial cells (the decrease of fluorescence intensity is described by the equation of linear regress already through 10 min after the beginning of the treatment). Both effects are reversible -- through 60 min after the beginning of the treatment the microtubule network does not differ from normal one, so the microtubule system is capable to adapt for influence of a natural regulator thrombin. The microtubules reaction develops more quickly, than reorganization of the actin filaments system, which responsible for the subsequent changes in the cell shape during barrier dysfunction. Apparently, the microtubules are the first part in a circuit of the reactions leading to the pulmonary endothelial cell barrier compromise.     

Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy

Direct visualization of the fluid-phase/ordered-phase domain structure in mica-supported bilayers composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine mixtures is performed with atomic force microscopy. The system studied is a double bilayer supported on a mica surface in which the top bilayer (which is not in direct contact with the mica) is visualized as a function of temperature. Because the top bilayer is not as restricted by the interactions with the surface as single supported bilayers, its behavior is more similar to a free-standing bilayer. Intriguing straight-edged anisotropic fluid-phase domains were observed in the fluid-phase/ordered-phase coexistence temperature range, which resemble the fluid-phase/ordered-phase domain patterns observed in giant unilamellar vesicles composed of such phospholipid mixtures. With the high resolution provided by atomic force microscopy, we investigated the origin of these anisotropic lipid domain patterns, and found that ripple phase formation is directly responsible for the anisotropic nature of these domains. The nucleation and growth of fluid-phase domains are found to be directed by the presence of ripples. In particular, the fluid-phase domains elongate parallel to the ripples. The results show that ripple phase formation may have implications for domain formation in biological systems.     

Microtubule regulation of cell surface receptor topography during granulosa cell differentiation

Abstract. A possible role for cytoplasmic microtubules in modulating lectin binding site topography has been examined during the hormone-directed differentiation of rat ovarian granulosa cells in vitro. Indirect immunofluorescence staining with anti-tubulin antibodies indicates that undifferentiated cultured granulosa cells contain a network of microtubules which radiate from the cell center to the cell periphery. Cultures induced to differentiate by a three day treatment with 1 μg/ml prolactin exhibit a marginal distribution of microtubules and a centrally-located primary cilium. Prolactin enhances the incidence of granulosa cells containing a primary colium from 9% in undifferentiated cultures to 53% in hormone-treated cultures. The pattern of lectin binding site redistribution induced by Concanavalin A (Con A) is also modified by prolactin treatment. In contrast to undifferentiated cells, which randomly endocytose fluorescein Con A, granulosa cells exposed to prolactin respond to fluorescein Con A by forming central surface caps to a greater extent (75%) than undifferentiated controls (25%). Double label fluorescence microscopy and transmission electron microscopy on Con A labeled cells show that caps form at central cell surface sites which contain the primary cilium. Disruption of cytoplasmic microtubules by colchicine, in undifferentiated granulosa cells, results in the formation of cell surface caps upon Con A addition. These data suggest that cytoplasmic microtubules modulate the topography of lectin bindings sites which is subject to hormonal control during the in vitro differentiation of ovarian granulosa cells.     

Microtubule regulation of cell surface receptor topography during granulosa cell differentiation

A possible role for cytoplasmic microtubules in modulating lectin binding site topography has been examined during the hormone-directed differentiation of rat ovarian granulosa cells in vitro. Indirect immunofluorescence staining with anti-tubulin antibodies indicates that undifferentiated cultured granulosa cells contain a network of microtubules which radiate from the cell center to the cell periphery. Cultures induced to differentiate by a three day treatment with 1 microgram/ml prolactin exhibit a marginal distribution of microtubules and a centrally-located primary cilium. Prolactin enhances the incidence of granulosa cells containing a primary cilium from 9% in undifferentiated cultures to 53% in hormone-treated cultures. The pattern of lectin binding site redistribution induced by Concanavalin A (Con A) is also modified by prolactin treatment. In contrast to undifferentiated cells, which randomly endocytose fluorescein Con A, granulosa cells exposed to prolactin respond to fluorescein Con A by forming central surface caps to a greater extent (75%) than undifferentiated controls (25%). Double label fluorescence microscopy and transmission electron microscopy on Con A labeled cells show that caps form at central cell surface sites which contain the primary cilium. Disruption of cytoplasmic microtubules by colchicine, in undifferentiated granulosa cells, results in the formation of cell surface caps upon Con A addition. These data suggest that cytoplasmic microtubules modulate the topography of lectin bindings sites which is subject to hormonal control during the in vitro differentiation of ovarian granulosa cells.     

Astral Microtubule Dynamics in Yeast: A Microtubule-based Searching Mechanism for Spindle Orientation and Nuclear Migration into the Bud

Localization of dynein–green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at 0.3–1.5 μm/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.     

Nonspecific depolarization of the plasma membrane potential induces cytoskeletal modifications of bovine corneal endothelial cells in culture

Modifications in the cell membrane potential have been suggested to affect signaling mechanisms participating in diverse cellular processes, many of which involve structural cellular alterations. In order to contribute some evidence in this respect, we explored the effects of several depolarizing procedures on the structure and monolayer organization of bovine corneal endothelial cells in culture. Visually confluent cell monolayers were incubated with or without the depolarizing agent, either in a saline solution or in culture medium for up to 30 min. Membrane potential was monitored by fluorescence microscopy using oxonol V. Fluorescent probes were employed for F-actin, microtubules, and vinculin. Depolarization of the plasma membrane, achieved via the incorporation of gramicidin D into confluent endothelial cells or by modifications of the extracellular saline composition, provoked an increment of oxonol fluorescence and changes in cell morphology, consisting mainly of modifications in the cytoskeletal organization. In some areas, noticeable intercellular spaces appear. The cytoskeleton modifications mainly consist of a marked redistribution of F-actin and microtubules, with accompanying changes in vinculin localization. The results suggest that the depolarization of the plasma membrane potential may participate in mechanisms involved in cytoskeleton organization and monolayer continuity in corneal endothelial cells in culture.     

Pollen development in orchids

Summary Cytokinesis in microsporocytes of moth orchids is unusual in that it occurs simultaneously after meiosis, the cytoplasm does not infurrow in the division planes, and cell plates are deposited in association with centrifugal expansion of phragmoplasts. Microtubules radiating from the nuclear envelopes appear to be of fundamental importance in establishment of division planes. Primary interzonal spindles develop between sister nuclei and interaction of radial microtubules triggers development of secondary interzonal spindles between non-sister nuclei. From three to six or more phragmoplasts, depending upon the arrangement of nuclei in the coenocyte, develop from these postmeiotic arrays. The phragmoplasts consist of co-aligned microtubules and F-actin organized into bundles that are broad proximal to the mid-plane and taper distally. Ultrastructure of the phragmoplast/cell plate reveals that abundant ER is associated with vesicle aggregation and coalescence. Cell plates are deposited in association with phragmoplasts as they expand centrifugally to join the parental wall and/or fuse with one another in the interior of the cell.Abbreviations CLSM confocal laser scanning microscope/microscopy - FITC flnorescein isothiocyanate - PPB preprophase band of microtubules - TEM transmission electron microscope/microscopy     

The interphase microtubule cytoskeleton of five different phenotypes of microvessel endothelial cell cultures derived from bovine corpus luteum.

The interphase microtubule cytoskeleton of five different microvessel endothelial cell cultures, recently established from bovine corpus luteum, was analysed using anti-tubulin immunofluorescence. An antibody against acetylated microtubules detected four cell types each of which possessed a single cilia. The length of the cilia were up to 10 microns for cell types 1 and 2. Ciliary stubs had a length of up to 0.37 microns in cell types 4 and 5. Cilia were missing in cell type 3. Long and short cilia were located in the perinuclear region from where cytoplasmic microtubules radiated. Cell type 3 displayed straight microtubules rather than the wavy path seen in the other cell types. The amount of tyrosinated microtubules visualized by a specific antibody was consistently higher than that of posttranslationally acetylated microtubules. The latter were more apparent in cell types 4 and 5 than in the other cell types. We conclude: Differences in the cytoplasmic microtubule inventory of each microvessel endothelial cell type points at individual functions maintained in culture.     

Mitosis and microtubule organizational changes in rice root-tip cells

The pattern of change of the microtubule cytoskeleton of the root-tip cells of rice during mitosis was studied using immunofluorescence technic and confocal laser scanning microscopy. All the major stages of ceil division including preprophase, prophase, metaphase, anaphase and telophase were observed. The most significant finding was that in the preprophase cells microtubules radiating from the nuclear surface to the cortex were frequently seen. During development these microtubules became closely associated with the preprophase band and prophase spindie indicating that the microtubules radiating from the nuclear surface, the preprophase band and the prophazc spindle were structurally and functionally closely related to each other. Granule-like anchorage sites for the radiating microtubules at the muclear surface were often seen and the possibility that these gramle-like anchorage sites might represent the microtubule organizing centres was discussed.