Microtubular organization in flat epitheloid and stellate process-bearing astrocytes in culture

Microtubules and microfilament patterns in cultured astrocytes were revealed by using indirect immunofluorescent microscopy in conjunction with anti-tubulin immune serum and anti-actin immunoglobulins respectively. In flat epitheloid astroglial cells (either polygonal or elongated) colchicine-sensitive immunofluorescent fibres, which correspond to bundles of microtubules, extend from the perinuclear cytoplasm into the cell periphery by running for long distances through the different focal planes. These patterns of organization differ markedly from the patterns of organization of microfilaments which are arranged in fibres parallel to each other and often oriented along the cell boundary. In response to the combined treatments of serum withdrawal and administration of dBcAMP, flat epitheloid astrocytes adopt a morphology similar to that of the mature astrocytes in situ in the CNS, that is of stellate process-bearing cells. This is prevented or is reverted by the administration of colchicine at the appropriate times. There are strong suggestions indicating that during cell processes formation the microtubular network is reorganized and microtubules assembled into dense bundles which are oriented along the axis of the cell processes. In view of these results, we suggest that, in contrast to microfilaments, microtubules are not determinant for the maintenance of cellular shape in elongated or polygonal flat epitheloid astroglial cells but they are required for both the formation and maintenance of processes in stellate astrocytes.     

Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein-280–deficient Melanoma Cell Line

The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.     

PD大鼠胼胝体和扣带胶质原纤维酸性蛋白的表达

目的观察不同病程帕金森病(PD)大鼠胼胝体和扣带胶质原纤维酸性蛋白(GFAP)的表达。方法大鼠右侧前脑内侧束注射六羟基多巴胺制备PD模型。大鼠分为正常对照组,2周、4周和6周模型组。以酪氨酸羟化酶(TH)和GFAP抗体免疫组化阳性分别显示黑质的多巴胺能神经元、胼胝体与扣带处的星形胶质细胞。结果 2、4、6周模型组右侧黑质TH阳性细胞数较正常对照组均显著降低,而2、4、6周模型组之间无显著差异。正常对照组和4周模型组胼胝体和扣带处星形胶质细胞呈未活化状态,两组之间的GFAP表达强度和细胞密度均无显著差异。2周和6周模型组两部位星形胶质细胞呈活化状态,其表达强度和细胞密度均显著高于正常对照组和4周模型组。结论急性完全损伤PD模型大鼠注射侧胼胝体和扣带处GFAP表达随病程呈增高、降低和增高趋势。     

Development of a differentiated microtubule structure: formation of the chicken erythrocyte marginal band in vivo

The microtubules of mature nucleated erythrocytes are organized into a marginal band that is confined to a single plane at the periphery and that contains essentially the same number of microtubule profiles in each individual cell. Developing erythrocytes can be isolated in homogeneous and synchronously developing populations from chicken embryos. For these reasons, these cells offer a particularly accessible system for study of the pathway leading to a specific microtubule structure in a normal, terminally differentiated animal cell. Along this developmental course, striking changes occur in the properties of the microtubules. Between the postmitotic cell and the formation of the band, a novel arrangement is found: bundles of laterally associated microtubules in each cell, coursing through the cytoplasm but not confined to the periphery. The microtubule organizing centers evident at early stages disappear by the time the band forms. The microtubules in early cells are readily depolymerized by drugs, but that drug sensitivity is lost in the mature cells. The microtubule arrangement of mature cells is faithfully recapitulated after reversible depolymerization, while that of the immature cells is not. Finally, as the band forms, the microtubules and microfilaments increasingly become coaligned. In sum, the microtubules of immature cells have many properties in common with those of cultured cells, but during maturation those properties change. The results suggest that lateral interactions become increasingly important in stabilizing and organizing the microtubules. The properties of marginal band microtubules, and comparable properties of axonal microtubules, may reflect differences between the requirements for cytoskeletal structures of cycling cells and terminally differentiated cells.     

Irradiation in vitro discriminates between different O-2A progenitor cell subpopulations in the perinatal central nervous system of rats

The effects of X irradiation on oligodendrocyte-type-2-astrocyte (O-2A) progenitor cells derived from different regions of the perinatal central nervous system (CNS) of rats were investigated in vitro. The O-2A progenitor cells can differentiate into either oligodendrocytes or type-2 astrocytes. The depletion of these cells could lead to demyelination, seen as a delayed reaction after irradiation of the CNS in vivo. To quantify cell survival, O-2A progenitor cells were grown on monolayers of type-1 astrocytes. Monolayers of type-1 astrocytes stimulate O-2A progenitor cells to divide. O-2A progenitor cells were irradiated in vitro and clonogenic cell survival was measured. The O-2A progenitor cells derived from perinatal optic nerve were quite radiosensitive in contrast to O-2A progenitor cells derived from perinatal spinal cord and perinatal corpus callosum. Furthermore, O-2A progenitor cells derived from the optic nerve formed smaller colonies, with most colonies showing early differentiation into oligodendrocytes. In contrast, more than half of the colonies derived from corpus callosum did not show any differentiation after 2 weeks in vitro and kept growing. These differences support the view that perinatal O-2A progenitor cells derived from the optic nerve are committed progenitor cells while the O-2A progenitor cells derived from the perinatal corpus callosum and the perinatal spinal cord have more stem cell properties.     

Development of capping ability during differentiation of HL-60 human promyelocytic leukemia cells

Developmental changes in cell surface and cytoskeletal elements have been studied in human promyelocytic leukemia cells (line HL-60) which differentiate into functionally mature myeloid cells when grown in dimethyl sulfoxide (DMSO)-supplemented medium. Both differentiated and undifferentiated HL-60 cells bind fluorescent concanavalin A (F-Con A) in a diffuse pattern over the entire cell surface. As with normal neutrophils, pretreatment of the differentiated HL-60 cells with colchicine before incubation with Con A causes the formation of large cytoplasmic protrusions over which the lectin associates into a cap. On the other hand, similarly treated undifferentiated HL-60 cells do not form the cytoplasmic protuberances and are unable to cap the Con A. Transmission electron microscopy reveals that the number and distribution of microtubules and microfilaments change during differentiation. Thus, developing myeloid cells undergo important alterations in the structure and function of the cytoskeleton as they differentiate into mature phagocytes.     

Effect of colchicine on rat small intestinal absorptive cells. I Formation of basolateral microvillus borders

Treatment of rats with colchicine (0.5 mg/100 g of body weight) for more than 3 hr causes formation of microvillus borders along lateral and basal surfaces of absorptive cells in the small intestine. Morphologically, these strongly resemble the apical brush border inclusive of the terminal-web region. Formation of basolateral microvilli is restricted to mature absorptive cells. At 6 hr after administration of colchicine, 3.47% (+/- 1.94%) of the basolateral cell surfaces exhibit "implantation" of microvillus borders. The results show that colchicine induces formation of surface differentiations at lateral and basal surface regions that are restricted to the apical cell surface in controls. Redistribution of constituents of the plasma membrane from apical to basolateral membrane portions, as well as rearrangement in the organization of microfilaments can be considered to underlie formation of basolateral microvillus borders. From the antimicrotubular effect of colchicine it may be deduced that microtubules exert a regulative function in the formation of surface differentiations on absorptive cells of the small intestine and in the maintenance of the polarity of the cells.     

Characterization of Glial Populations in the Aging and Remyelinating Mouse Corpus Callosum

Cells in the white matter of the adult brain have a characteristic distribution pattern in which several cells are contiguously connected to each other, making a linear array (LA) resembling pearls-on-a-string parallel to the axon axis. We have been interested in how this pattern of cell distribution changes during aging and remyelination after demyelination. In the present study, with a multiplex staining method, semi-quantitative analysis of the localization of oligodendrocyte lineage cells (oligodendrocyte progenitors, premyelinating oligodendrocytes, and mature oligodendrocytes), astrocytes, and microglia in 8-week-old (young adult) and 32-week-old (aged) corpus callosum showed that young adult cells still include immature oligodendrocytes and that LAs contain a higher proportion of microglia than isolated cells. In aged mice, premyelinating oligodendrocytes were decreased, but microglia continued to be present in the LAs. These results suggest that the presence of microglia is important for the characteristic cell localization pattern of LAs. In a cuprizone-induced demyelination model, we observed re-formation of LAs after completion of cuprizone treatment, concurrent with remyelination. These re-formed LAs again contained more microglia than the isolated cells. This finding supports the hypothesis that microglia contribute to the formation and maintenance of LAs. In addition, regardless of the distribution of cells (LAs or isolated cells), astrocytes were found to be more abundant than in the normal corpus callosum at 24 weeks after cuprizone treatment when remyelination is completed. This suggests that astrocytes are involved in maintaining the functions of remyelinated white matter.

     

Tissue slices from living root caps as a model system in which to study cytodifferentiation of polar cells

Tissue slices of living root caps of cress (Lepidium sativum L.), two to three cell layers in thickness, were prepared by a microsurgical procedure. The viability, cellular structures and cytoplasmic movement of the cells were examined in the light microscope. Nuclei, amyloplasts, vacuoles and endoplasmic reticulum were identified and their positions confirmed after fixation and observation of the same cells in the electron microscope. The distribution of microtubules was shown by immunocytochemistry. During germination, microtubules appear first at the distal edges of the statocytes, while in mature statocytes a distal domain of criss-crossed microtubules could be distinguished from a proximal domain with transversally oriented microtubules. Microfilaments in young statocytes form a nuclear enclosure; in mature statocytes bundles of microfilaments fan out into the cell cortex. The transition from statocytes to secretion cells is accompanied by a more pronounced cortical network of microfilaments, while the nucleus-associated microfilaments remain visible. It is suggested that these microfilaments play a role in the positioning of the nucleus and the translocation of endoplasmic reticulum.Abbreviations ER endoplasmic reticulum - MF microfilament - MT microtubule     

Role of Cytoskeletal Elements in the Recruitment of Cx43-GFP and Cx26-YFP into Gap Junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gapjunctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.     

Role of Cytoskeletal Elements in the Recruitment of Cx43-GFP and Cx26-YFP into Gap Junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gapjunctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.     

Development of midline glial populations at the corticoseptal boundary

Three midline glial populations are found at the corticoseptal boundary: the glial wedge (GW), glia within the indusium griseum (IGG), and the midline zipper glia (MG). Two of these glial populations are involved in axonal guidance at the cortical midline, specifically development of the corpus callosum. Here we investigate the phenotypic and molecular characteristics of each population and determine whether they are generated at the same developmental stage. We find that the GW is derived from the radial glial scaffold of the cortex. GW cells initially have long radial processes that extend from the ventricular surface to the pial surface, but by E15 loose their pial attachment and extend only part of the way to the pial surface. Later in development the radial morphology of cells within the GW is replaced by multipolar astrocytes, providing supportive evidence that radial glia can transform into astrocytes. IGG and MG do not have a radial morphology and do not label with the radial glial markers, Nestin and RC2. We conclude that the GW and IGG have different morphological and molecular characteristics and are born at different stages of development. IGG and MG have many phenotypic and molecular characteristics in common, indicating that they may represent a common population of glia that becomes spatially distinct by the formation of the corpus callosum.     

Cyclic amp and cell morphology in cultured fibroblasts. Effects on cell shape, microfilament and microtubule distribution, and orientation to substratum

The change in shape of 3T3 and L929 cells due to Bt2cAMP treatment is accompanied by altered intracellular distribution of microfilaments and microtubules. Bt2cAMP added to cells in low density culture causes (a) microfilaments to accumulate in bundles near the plasma membrane, mainly at the cell periphery, and (b) microtubules to accumulate beneath these microfilament bundles. In narrow cell processes that form characteristically in Bt2cAMP-treated L cells, microtubules accumulate in parallel arrays near the center of these processes. A new simple method for evaluating the relative distance of the cell from its underlying substratum is desribed. In normal medium, 3T3 cells attach to their substratum near the nucleus and at the tips of cell processes, bridging irregularities in the plastic surface. With Bt2cAMP treatment, attachment occurs at the cell edge and at many isolated points under the cytoplasm, and the cells conform more closely to irregularities of the underlying substratum. A model of the mechanism by which cAMP modulates cell shape is presented.     

Nuclear control of neurite induction in neuroblastoma cells

Induction of neurite formation in neuroblastoma cells by dibutyryl cyclic 3':5'-AMP (db-cAMP) or prostaglandin EI (PGE1) was enhanced after enucleation. Cells selected for resistance to db-cAMP were induced to form neurites by db-cAMP or PGE1 only after, but not before enucleation. Inhibition of protein synthesis inhibited neurite induction in nucleated, but not in enucleated cells, and enucleated cells were less sensitive to inhibition of neurite formation by concanavalin A (ConA). Colchicine, vinblastine and cytochalasin B (CB), compounds that interfere with the assembly of microtubules and microfilaments, inhibited induction in both types of cells. It is suggested that enucleation removes a nuclear inhibitor of neurite induction by db-cAMP and PGE1, and that neurite induction in nucleated cells requires that cAMP activates the assembly of microtubules and microfilaments and inactivates the nuclear inhibitor.     

Effects of tubulozole on the amoeboflagellate transformation in Physarum polycephalum.

The Amoeboflagellate Transformation (AFT) of Physarum polycephalum involves rapid changes in the cytoskeleton, cell shape and cell motility. Use of pharmacologic agents to probe the role of cytoskeletal elements in the AFT are impeded because the transforming cells are very sensitive to such commonly-used drug solvents as DMSO. The anti-microtubule agent tubulozole is found to disrupt, rapidly and transiently, the AFT, inhibiting flagella formation, cell elongation and the arrangement of microtubules and microfilaments. Cells recover quickly, possibly due to precipitation of the drug; the reappearance of normal arrays of microfilaments and cytoplasmic microtubules lags behind flagella formation.     

Oligoprogenitor Cells Derived from Spermatogonia Stem Cells Improve Remyelination in Demyelination Model

Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells.     

Electron microscopic study of rapid morphogenetic processes in embryonic tissue explants of amphibia]

Submicroscopic rearrangements are described. They serve as the bases of rapid (up to 20 min) changes in the form of the common frog neurula explants: formation of filamentous layer under "naked" surface, appearance of lobopodia on "naked" surface, their "flow", cell polarization and submersion. In all these processes an active part appears to be played by microtubules and microfilaments the bundles of which are always oriented along the long axes of active cells or the directions of passive mechanical tensions. In the cells which are not yet polarized the microtubules form under the surface adjacent to the already polarized cell. This may be considered as one of the chains of cooperative cell polarization.     

Histone deacetylase expression in white matter oligodendrocytes after stroke

Histone deacetylases (HDACs) constitute a super-family of enzymes grouped into four major classes (Class I–IV) that deacetylate histone tails leading to chromatin condensation and gene repression. Whether stroke-induced oligodendrogenesis is related to the expression of individual HDACs in the oligodendrocyte lineage has not been investigated. We found that 2 days after stroke, oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes (OLGs) were substantially reduced in the peri-infarct corpus callosum, whereas at 7 days after stroke, a robust increase in OPCs and OLGs was observed. Ischemic brains isolated from rats sacrificed 7 days after stroke were used to test levels of individual members of Class I (1 and 2) and Class II (4 and 5) HDACs in white matter oligodendrocytes during stroke-induced oligodendrogenesis. Double immunohistochemistry analysis revealed that stroke substantially increased the number of NG2+ OPCs with nuclear HDAC1 and HDAC2 immunoreactivity and cytoplasmic HDAC4 which were associated with augmentation of proliferating OPCs, as determined by BrdU and Ki67 double reactive cells after stroke. A decrease in HDAC1 and an increase in HDAC2 immunoreactivity were detected in mature adenomatous polyposis coli (APC) positive OLGs, which paralleled an increase in newly generated BrdU positive OLGs in the peri-infarct corpus callosum. Concurrently, stroke substantially decreased the acetylation levels of histones H3 and H4 in both OPCs and OLGs. Taken together, these findings demonstrate that stroke induces distinct profiles of Class I and Class II HDACs in white matter OPCs and OLGs, suggesting that the individual members of Class I and II HDACs play divergent roles in the regulation of OPC proliferation and differentiation during brain repair after stroke.     

Role of cytoskeletal elements in the recruitment of Cx43-GFP and Cx26-YFP into gap junctions

Cytoskeletal elements may be important in connexin transport to the cell surface, cell surface gap junction plaque formation and/or gap junction internalization. In this study, fluorescence recovery after photobleaching was used to examine the role of microfilaments and microtubules in the recruitment and coalescence of green fluorescent protein-tagged Cx43 (Cx43-GFP) or yellow fluorescent tagged-Cx26 (Cx26-YFP) into gap junctions in NRK cells. In untreated cells, both Cx26-YFP and Cx43-GFP were recruited into gap junctions within photobleached areas of cell-cell contact within 2 hrs. However, disruption of microfilaments with cytochalasin B inhibited the recruitment and assembly of both Cx26-YFP and Cx43-GFP into gap junctions within photobleached areas. Surprisingly, disruption of microtubules with nocodazole inhibited the recruitment of Cx43-GFP into gap junctions but had limited effect on the transport and clustering of Cx26-YFP into gap junctions within the photobleached regions of cell-cell contact. These results suggest that the recruitment of Cx43-GFP and Cx26-YFP to the cell surface or their lateral clustering into gap junctions plaques is dependent in part on the presence of intact actin microfilaments while Cx43-GFP was more dependent on intact microtubules than Cx26-YFP.     

Fine structure of shark leucocytes during chemotactic migration

It has previously been demonstrated that peripheral blood leucocytes of the nurse shark (Ginglymostoma cirrhatum) are capable of in vitro chemotaxis. In the present study we have identified the chemotactic cells as the shark granulocyte and monocyte-macrophage. Chemotaxis assays were set up in blind well chambers. Cells migrating into the filter in a chemotaxis assay were identified morphologically by transmission electron microscopy. Migration, in response to LPS-activated rat serum, was accompanied by change in cell shape and reorganization of the cytoskeleton. Ultrastructural changes included formation of lamellipodia with cell polarization, submembranous accumulation of microfilaments at points of plasma membrane contact with filter surfaces, densification of the membrane at these points of engagement, appearance of bundles of intermediate filaments extending into the protruded lamellipodia, and presence of oriented microtubules in the cytoplasma (cellular cortex). The phagocytic and chemotactic properties of the shark granulocyte and monocyte-macrophage points to their being structural and functional equivalents of the mammalian neutrophil and macrophage.