Organization Of Cytoplasmic Microtubules Research Articles

The fission yeast NDR kinase Orb6 and its signalling pathway MOR regulate cytoplasmic microtubule organization during the cell cycle.

Microtubule organization and reorganization during the cell cycle are achieved by regulation of the number, distribution and activity of microtubule-organizing centres (MTOCs). In fission yeast, the Mto1/2 complex determines the activity and distribution of cytoplasmic MTOCs. Upon mitosis, cytoplasmic microtubule nucleation ceases; inactivation of the Mto1/2 complex is triggered by Mto2 hyperphosphorylation. However, the protein kinase(s) that phosphorylates Mto2 remains elusive. Here we show that a conserved signalling network, called MOR (morphogenesis Orb6 network) in fission yeast, negatively regulates cytoplasmic MTOCs through Mto2 phosphorylation to ensure proper microtubule organization. Inactivation of Orb6 kinase, the most downstream MOR component, by attenuation of MOR signalling leads to reduced Mto2 phosphorylation, coincident with increased number of both Mto2 puncta and cytoplasmic microtubules. These defects cause the emergence of uncoordinated mitotic cells with cytoplasmic microtubules, resulting in reduced spindle assembly. Thus, the regulation of Mto2 by the MOR is crucial for cytoplasmic microtubule organization and contributes to reorganization of the microtubule cytoskeletons during the cell cycle.

Elucidating Differences in Early-Stage Centrosome Amplification in Primary and Immortalized Mouse Cells.

The centrosome is involved in cytoplasmic microtubule organization during interphase and in mitotic spindle assembly during cell division. Centrosome amplification (abnormal proliferation of centrosome number) has been observed in several types of cancer and in precancerous conditions. Therefore, it is important to elucidate the mechanism of centrosome amplification in order to understand the early stage of carcinogenesis. Primary cells could be used to better understand the early stage of carcinogenesis rather than immortalized cells, which tend to have various genetic and epigenetic changes. Previously, we demonstrated that a poly(ADP-ribose) polymerase (PARP) inhibitor, 3-aminobenzamide (3AB), which is known to be nontoxic and nonmutagenic, could induce centrosome amplification and chromosomal aneuploidy in CHO-K1 cells. In this study, we compared primary mouse embryonic fibroblasts (MEF) and immortalized MEF using 3AB. Although centrosome amplification was induced with 3AB treatment in immortalized MEF, a more potent PARP inhibitor, AG14361, was required for primary MEF. However, after centrosome amplification, neither 3AB in immortalized MEF nor AG14361 in primary MEF caused chromosomal aneuploidy, suggesting that further genetic and/or epigenetic change(s) are required to exhibit aneuploidy. The DNA-damaging agents doxorubicin and γ-irradiation can cause cancer and centrosome amplification in experimental animals. Although doxorubicin and γ-irradiation induced centrosome amplification and led to decreased p27Kip protein levels in immortalized MEF and primary MEF, the phosphorylation ratio of nucleophosmin (Thr199) increased in immortalized MEF, whereas it decreased in primary MEF. These results suggest that there exists a yet unidentified pathway, different from the nucleophosmin phosphorylation pathway, which can cause centrosome amplification in primary MEF.

Exploring prognostic markers for patients with acute myeloid leukemia based on cuproptosis related genes.

Acute myeloid leukemia (AML), a common form of acute leukemia, is due to tumor changes and clonal proliferation caused by genetic variants. Cuproptosis is a novel form of regulated cell death. This study aimed to explore the role of cuproptosis-related genes (CRGs) in AML. Initially, differentially expressed genes (DEGs) between AML samples and normal samples were obtained by differential analysis, which were further intersected with the cuproptosis score-related genes (CSRGs) acquired by weighted gene co-expression network analysis (WGCNA) to obtain cuproptosis score-related differentially expressed genes (CS-DEGs). Then, a risk model was constructed by Cox analysis and least absolute shrinkage and selection operator (LASSO) analysis. Finally, immune infiltration analysis was performed and the functions and pathways of model genes were explored by single sample gene set enrichment analysis (ssGSEA). Thirty-two CS-DEGs were obtained by overlapping 11,160 DEGs and 132 CSRGs. These 32 CS-DEGs were mainly enriched to cytoplasmic microtubule organization, RNA methylation, mTOR signaling pathway, and notch signaling pathway. Two model genes, PACS2 and NDUFV1, were finally screened for the construction of the risk model. In addition, PACS2 and NDUFV1 were significantly positively correlated with activated B cells, CD56dim natural killer (NK) cells, and negatively correlated with effector memory CD4 T cells and activated CD4 T cells. PACS2 gene was significantly enriched to inositol phosphate metabolism, histone modification, etc. NDUFV1 was mainly enriched to ncRNA metabolic process, 2-oxocarboxylic acid metabolism, and other pathways. A cuproptosis-related risk model consisting of PACS2 and NDUFV1 was built, which provided a new direction for the diagnosis and treatment of AML.

Upregulation of miR-181a-5p and miR-125b-2-3p in the Maternal Circulation of Fetuses with Rh-Negative Hemolytic Disease of the Fetus and Newborn Could Be Related to Dysfunction of Placental Function.

The transplacental transfer of maternal antibodies to the fetus is a critical mechanism for infant protection and perinatal disease. Hemolytic disease of the fetus and newborn (HDFN) is a representative fetal disease caused by transplacental transfer of maternal IgG antibodies. However, it is unclear whether placental-related miRNAs are expressed in Rh-HDFN. Through the investigation of the miR-181a-5p and miR-125b-2-3p levels in maternal plasma using qPCR, we found that both miR-181a-5p and miR-125b-2-3p were highly expressed in maternal plasma of newborns with Rh-HDFN compared with healthy controls, indicating the potential roles of these two miRNAs in Rh-HDFN. To demonstrate whether dysregulation of miR-125b-2-3p and miR-181a-5p contributes to Rh-HDFN development, we analyze the placental miRNA-/mRNA sequencing data (GSE73714) using weighted gene coexpression network analysis (WGCNA), miRNA target predictive databases, and DAVID (Database for Annotation, Visualization, and Integrated Discovery). The results showed that miR-125b-2-3p and miR-181a-5p could regulate several biological processes including cytoplasmic microtubule organization and angiogenesis. Moreover, core promoter sequence-specific DNA binding and protein binding were highly enriched molecular functions, indicating the potential roles of transcriptional regulation. Further pathway enrichment showed that miR-181a-5p and miR-125b-2-3p could regulate several biological pathways that were closely related to placental function, including the FoxO signaling pathway, focal adhesion, mTOR signaling pathway, and central carbon metabolism in cancer. In conclusion, the present results first revealed miRNA expression in the maternal circulation of newborns with Rh-HDFN, which could be caused by dysfunction of the placenta.

Bacterial community composition in breast cancer subtypes.

e13063 Background: Breast cancer is a heterogeneous disease with multiple molecular subtypes and prognostic outcomes. Among these subtypes, Luminal-A is enriched with estrogen receptor and has the best clinical prognosis, whereas triple-negative breast cancer (TNBC) remains an aggressive subtype with poor prognosis. Changes in the diversity of breast microbiome have been associated with breast cancer initiation and progression. We hypothesized that Luminal-A and TNBC subtypes will have differences in both bacterial community composition as well as differences in microbial genomes and pathways. Methods: 6 Luminal-A and 6 TNBC FPPE tissue blocks were processed for both 16S V4 rRNA amplicon sequencing and shotgun metagenomics sequencing. 16S sequencing data was processed using mothur (v1.44.3) and reads were assigned taxonomy against the SILVA 16S rRNA database (v132). Shotgun sequencing was processed using the EBI-Metagenomic (v5) pipeline; bacterial identification was performed using the Kraken pipeline. Results: Shannon alpha-diversity or species enrichment, was significantly higher in Luminal-A compared to TNBC (nonparametric, P = 0.008) and their microbiome composition was significantly different by beta-diversity analysis (bray-curtis dissimilarity, PERMANOVA, P = 0.014). Bacteroides and Sphingomonadaceae species were significantly higher in abundance in Luminal-A, compared to Pseudomonas and Neisseriaceae species abundance in TNBC. Results were validated by shotgun sequencing. Luminal-A and TNBC bacterial metabolic pathways were not statistically different overall (PERMANOVA, P = 0.312). Pathways associated with Luminal-A included Response to Wounding (GO: 0009611), tRNA modification (GO: 0006400), and Cytoplasmic Microtubule Organization (GO: 0031122). Pathways associated with TNBC included troponin complex (GO: 0005861), rRNA processing (GO: 0006364), and CDC73 PAF1 (GO: 0016593). Conclusions: Using both shotgun and amplicon sequencing, this is the first analysis to show that Luminal-A and TNBC subtypes have statistically significant differences in bacterial species composition. Luminal-A had higher species diversity and abundance of Sphingomonadaceae, which are associated with healthy breast tissue and better clinical outcomes. TNBC had lower species diversity and abundance of Pseudomonas and Neisseriaceae, which have been associated with tumor survival and cell growth. Understanding the role of microbiota and its complex interaction with tumor microenvironment could help identify new treatment targets in breast cancer subtypes, and warrants further investigations.

The Centriole's Role in Miscarriages.

Centrioles are subcellular organelles essential for normal cell function and development; they form the cell’s centrosome (a major cytoplasmic microtubule organization center) and cilium (a sensory and motile hair-like cellular extension). Centrioles with evolutionarily conserved characteristics are found in most animal cell types but are absent in egg cells and exhibit unexpectedly high structural, compositional, and functional diversity in sperm cells. As a result, the centriole’s precise role in fertility and early embryo development is unclear. The centrioles are found in the spermatozoan neck, a strategic location connecting two central functional units: the tail, which propels the sperm to the egg and the head, which holds the paternal genetic material. The spermatozoan neck is an ideal site for evolutionary innovation as it can control tail movement pre-fertilization and the male pronucleus’ behavior post-fertilization. We propose that human, bovine, and most other mammals–which exhibit ancestral centriole-dependent reproduction and two spermatozoan centrioles, where one canonical centriole is maintained, and one atypical centriole is formed–adapted extensive species-specific centriolar features. As a result, these centrioles have a high post-fertilization malfunction rate, resulting in aneuploidy, and miscarriages. In contrast, house mice evolved centriole-independent reproduction, losing the spermatozoan centrioles and overcoming a mechanism that causes miscarriages.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

The Centrosome as the Main Integrator of Endothelial Cell Functional Activity.

The centrosome is an intracellular structure of the animal cell responsible for organization of cytoplasmic microtubules. According to modern concepts, the centrosome is a very important integral element of the living cell whose functions are not limited to its ability to polymerize microtubules. The centrosome localization in the geometric center of the interphase cell, the high concentration of various regulatory proteins in this area, the centrosome-organized radial system of microtubules for intracellular transport by motor proteins, the centrosome involvement in the perception of external signals and their transmission - all these features make this cellular structure a unique regulation and distribution center managing dynamic morphology of the animal cell. In conjunction with the tissue-specific features of the centrosome structure, this suggests the direct involvement of the centrosome in execution of cell functions. This review discusses the involvement of the centrosome in the vital activity of endothelial cells, as well as its possible participation in the implementation of barrier function, the major function of endothelium.

Identification of human mutations in TRAF3IP1 in patients with nephronophthisis and retinal degeneration

Nephronophthisis (NPH) is an autosomal recessive inherited cystic kidney disorder. It represents the most frequent genetic cause of end-stage renal disease in the first three decades of life. NPH is characterized by the dysfunction of sensory cilia which explains the complexity of the NPH phenotype. It can be associated with retinitis pigmentosa (Senior-Loken syndrome), mental retardation and ataxia (Joubert syndrome), skeletal anomalies (Jeune syndrome), or situs inversus. To date, recessive mutations causing NPH have been identified in more than eighteen different genes (NPHP1-NPHP18). Their gene products localize at the primary cilia-centrosome complex, along the cilium as intraflagellar transport proteins and are important in signaling pathways downstream of cilia including Wnt signaling, Shh signaling and the DNA damaged response pathway. Using whole and targeted exome sequencing, we identified novel protein altering mutations in TRAF3IP1 in patients presenting with NPH, retinitis pigmentosa, skeletal defects of the pelvis, hexadactyly and hepatic fibrosis. TRAF3IP1 encodes IFT54 which is involved in the anterograde transport along the primary cilia. Besides its known function in cilia we demonstrate that TRAF3IP1 act as a key regulator of cytoplasmic microtubule organization. Mass spectrometry analyses as well as pull-down experiments demonstrated that mutations in TRAF3IP1 lead to an altered binding to actin and microtubule associated proteins. Immunofluorescence stainings using patient fibroblasts as wells as mIMCD3 TRAF3IP1 knock-down cells confirmed the observed defects in microtubule organization. Furthermore, sphere formation assays as well as the pronephros of elipsa zebrafish embryos showed defects in epithelialization. Altogether our findings demonstrate that NPH causing mutations of TRAF3IP1 affect both ciliary and non-ciliary functions of TRAF3IP1 which can provide an explanation for kidney tubules morphogenesis defects as well as the other disease phenotypes e.g. retinal, skeletal and hepatic defects.

Cytoplasmic microtubule organization in fission yeast

During the cell cycle of the fission yeast Schizosaccharomyces pombe, striking changes in the organization of the cytoplasmic microtubule cytoskeleton take place. These may serve as a model for understanding the different modes of microtubule organization that are often characteristic of differentiated higher eukaryotic cells. In the last few years, considerable progress has been made in our understanding of the organization and behaviour of fission yeast cytoplasmic microtubules, not only in the identification of the genes and proteins involved but also in the physiological analysis of function using fluorescently-tagged proteins in vivo. In this review we discuss the state of our knowledge in three areas: microtubule nucleation, regulation of microtubule dynamics and the organization and polarity of microtubule bundles. Advances in these areas provide a solid framework for a more detailed understanding of cytoplasmic microtubule organization.

Mto2p, a Novel Fission Yeast Protein Required for Cytoplasmic Microtubule Organization and Anchoring of the Cytokinetic Actin Ring

Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires gamma-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Delta cells fail to establish a stable EMTOC and localize gamma-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Delta cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Delta cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis.

Higher order structure of the PCM adjacent to the centriole.

The centrosome is the major microtubule organizing center in most animal cells. This cytoplasmic organelle consists of two components : a mature centriole (or a pair of centrioles) and a mass of pericentriolar material (PCM). The PCM has been described as either a cloud of material that encases the entire centriole or as a cluster of proteins divided into two subsets, one that adheres to the lateral surface of the centriole and another that extends outward from this region as a cloud of material. In contrast to these protein distribution patterns, we demonstrated in a previous study that a subset of proteins present within the PCM is integrated together to form a tube (PCM tube) with an open and closed end that is duplicated in concert with centrosome duplication. The present study was undertaken to determine if this tubular conformation represents proteins that are confined to the surface of the centriole or if it represents a subset of proteins within the cloud of material that extends outward from the centriole. We document that : (1) the PCM tube represents a portion of the PCM directly associated with the centriole; (2) the PCM tube has a specific and reproducible relationship to the polar structure of the centriole; (3) the tube is a site of cytoplasmic microtubule organization, and has a structure that influences the initial pattern of microtubule assembly within the juxta-centriolar region; and (4) the PCM tube has a structural relationship with respect to the centriole, which allows the simultaneous expression of centriole- and PCM-based functions (e.g., ciliogenesis and cytoplasmic microtubule organization). Based on these findings, we propose a new model of the PCM at the centriole. This model highlights the role played by the proximal end of the centriole in the nucleation and organization of centriole-associated PCM, and indicates that the centrosome has an overall polarity in the region of the centriole.

Depletion of a polo-like kinase in Candida albicans activates cyclase-dependent hyphal-like growth.

Morphogenesis in the fungal pathogen Candida albicans is an important virulence-determining factor, as a dimorphic switch between yeast and hyphal growth forms can increase pathogenesis. We identified CaCDC5, a cell cycle regulatory polo-like kinase (PLK) in C. albicans and demonstrate that shutting off its expression induced cell cycle defects and dramatic changes in morphology. Cells lacking CaCdc5p were blocked early in nuclear division with very short spindles and unseparated chromatin. GFP-tagged CaCdc5p localized to unseparated spindle pole bodies, the spindle, and chromatin, consistent with a role in spindle elongation at an earlier point in the cell cycle than that described for the homologue Cdc5p in yeast. Strikingly, the cell cycle defects were accompanied by the formation of hyphal-like filaments under yeast growth conditions. Filament growth was determinate, as the filaments started to die after 24 h. The filaments resembled serum-induced hyphae with respect to morphology, organization of cytoplasmic microtubules, localization of nuclei, and expression of hyphal-specific components. Filament formation required CaCDC35, but not EFG1 or CPH1. Similar defects in spindle elongation and a corresponding induction of filaments occurred when yeast cells were exposed to hydroxyurea. Because CaCdc5p does not appear to act as a direct repressor of hyphal growth, the data suggest that a target of CaCdc5p function is associated with hyphal-like development. Thus, an internal, cell cycle-related cue can activate hyphal regulatory networks in Candida.

Expression pattern of DMAP-85 during Drosophila embryonic development.

Microtubule-associated proteins (MAPs) play major regulatory roles on the organization and integrity of the cytoskeletal network. Previously, we identified DMAP-85, a Drosophila MAP that promotes tubulin polymerization in vitro. In this work, we examine the distribution of DMAP-85 and its association pattern with microtubules at embryonic stages. Immunoblots revealed that DMAP-85 was present throughout embryogenesis, but it was most abundant in stages 6-9. Immunofluorescence studies showed that DMAP-85 was associated with sub-populations of stable microtubules during embryo cellularization, and after gastrulation with interphase microtubule arrays. At late embryonic stages, it was preferentially found in the ventral nerve cord, co-localizing with axonal microtubules. These observations are in agreement with previous reports on DMAP-85 functions, suggesting that DMAP-85 might be required for the stabilization and organization of cytoplasmic microtubules during embryonic development.

Alanine-scanning mutagenesis of Aspergillus gamma-tubulin yields diverse and novel phenotypes.

We have created 41 clustered charged-to-alanine scanning mutations of the mipA, gamma-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of gamma-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that gamma-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of gamma-tubulin and these data give a good initial indication of the functionally important regions of the molecule.

An insertional mutant of Chlamydomonas reinhardtii with defective microtubule positioning.

cmu1-1 is a new mutation of Chlamydomonas reinhardtii that causes a change in cell shape due to an alteration of cytoplasmic microtubule organization. cmu1 mutant cells were first identified based on their altered cell shape. Unlike wild-type cells, which are ellipsoid, cmu1 cells tend to be either round or egg-shaped with the flagella extending from the narrow end of the cell. Electron microscopic comparison of mutant and wild-type cells indicated that microtubule distribution was altered in the mutant cells. Immunofluorescence microscopy using anti-beta-tubulin antibodies revealed that, in wild-type cells, microtubules arise from the anterior end of the cell in the region of the basal bodies, pass posteriorly subjacent to the plasma membrane, and terminate near the posterior end of the cell. In mutant cells, the microtubules also arise from the basal body region but then become disarrayed. They frequently curl back anteriorly or wrap around the equator of the cell; some microtubules also extend completely to the posterior end of the cell, then turn back toward the anterior end. No changes in the basal body region were detected by electron microscopy. Some cmu1 cells had multiple nuclei or an aberrant number of flagella, both of which may be due to defects in cell division, a process dependent upon microtubules. Thus, cmu1-1, which was generated by insertional mutagenesis and is tagged, appears to encode a protein that plays an essential role in the spatial organization of cytoplasmic microtubules involved in both interphase and mitotic functions.

Induction of tyrosine aminotransferase of primary cultured rat hepatocytes depends on the organization of microtubules.

We investigated the relationship between the expression of tyrosine aminotransferase (TAT) and cytoskeletal systems of cultured rat hepatocytes by using serum-free culture conditions and changing three factors: 1) the concentration of calcium, 2) the dish-coating material, and 3) the cell-plating density. In hepatocytes in low-calcium medium, induction of TAT by dexamethasone and glucagon was maintained, although cell-cell adhesion was lost. Hepatocytes on Matrigel formed a nonspreading, spherical shape that provided them with the full extent of TAT activity without cell-cell adhesion. Hepatocytes plated on collagen at low cell density spread and changed shape, and the induction of TAT activity was markedly reduced. By using confocal laser-scanning microscopy, we analyzed the three-dimensional organization of cytoplasmic microtubules of hepatocytes maintaining the ability of TAT induction. Hepatocytes plated on collagen at low cell density possessed the radial filamentous structure of cytoplasmic microtubules. When the spherical shape of hepatocytes was maintained by cultivating cells on Matrigel, a ring-like structure of cytoplasmic microtubules beneath the plasma membrane was dominant. Moreover, the induction of TAT activity of hepatocytes in a standard culture system was strongly inhibited by the addition of 1 microM colchicine. These studies suggest that the organization of cytoplasmic microtubules may participate in the shape-related regulation of cell function.

Induction of tyrosine aminotransferase of primary cultured rat hepatocytes depends on the organization of microtubules

We investigated the relationship between the expression of tyrosine aminotransferase (TAT) and cytoskeletal systems of cultured rat hepatocytes by using serum-free culture conditions and changing three factors: (1) the concentration of calcium, (2) the dish-coating material, and (3) the cell-plating density. In hepatocytes in low-calcium medium, induction of TAT by dexamethasone and glucagon was maintained, although cell-cell adhesion was lost. Hepatocytes on Matrigel formed a nonspreading, spherical shape that provided them with the full extent of TAT activity without cell-cell adhesion. Hepatocytes plated on collagen at low cell density spread and changed shape, and the induction of TAT activity was markedly reduced. By using confocal laser-scanning microscopy, we analyzed the three-dimensional organization of cytoplasmic microtubules of hepatocytes maintaining the ability of TAT induction. Hepatocytes plated on collagen at low cell density possessed the radial filamentous structure of cytoplasmic microtubules. When the spherical shape of hepatocytes was maintained by cultivating cells on Matrigel, a ring-like structure of cytoplasmic micotubules beneath the plasma membrane was dominant. Moreover, the induction of TAT activity of hepatocytes in a standard culture system was strongly inhibited by the addition of 1 μM colchicine. These studies suggest that the organization of cytoplasmic microtubules may participate in the shape-related regulation of cell function. J. Cell. Physiol. 175:41–49, 1998. © 1998 Wiley-Liss, Inc.

The Organization and Animal–Vegetal Asymmetry of Cytokeratin Filaments in Stage VIXenopusOocytes Is Dependent upon F-Actin and Microtubules

Confocal immunofluorescence microscopy with anti-cytokeratin antibodies revealed a continuous and polarized network of cytokeratin (CK) filaments in the cortex of stage VIXenopusoocytes. In the animal cortex, CK filaments formed a dense meshwork that both was thicker and exhibited a finer mesh than the network of CK filaments previously observed in the vegetal cortex (Klymkowskyet al.,1987). CK filaments first appeared in association with germinal vesicle (GV) and mitochondrial mass (MM) of oocytes in early mid stage I, indicating that CK filaments are the last of the three cytoskeletal networks to be assembled. By late stage I, CK filaments formed complex networks surrounding the GV, surrounding and penetrating the MM, and linking these networks to a meshwork of CK filaments in the oocyte cortex. During stage III–early IV, CK filaments formed a highly interconnected, apparently unpolarized, radial array linking the perinuclear and cortical CK filament networks. Polarization of the CK filament network was observed during mid stage IV–stage V, as first the animal, then the vegetal CK filament networks adopted the organization characteristic of stage VI oocytes. Treatment of stage VI oocytes with cytochalasin B disrupted the organization of both cortical and cytoplasmic CK filaments, releasing CK filaments from the oocyte cortex and inducing formation of numerous cytoplasmic CK filament aggregates. CB also disrupted the organization of cytoplasmic microtubules (MTs) in stage VI oocytes. Disassembly of oocyte MTs with nocodazole resulted in loss of the characteristic A–V polarity of the cortical CK filament network. In contrast, disruption of cytoplasmic CK filaments by microinjection of anti-CK antibodies had no apparent effect on cytoplasmic or MT organization. We propose a model in which the organization and polarization of the cortical network of CK filaments in stage VIXenopusoocytes are dependent upon a hierarchy of interactions with actin filaments and microtubules.

Microtubule organization during zoosporogenesis inAllomyces macrogynus

The microtubule cytoskeleton and cytoplasmic organization ofAllomyces macrogynus during zoosporogenesis was studied using light and electron microscopy. Indirect immunofluorescence methods revealed that the microtubule cytoskeleton progressed through three distinct stages of cytoplasmic distribution during zoospore development. During the first 10 minutes of zoosporogenesis, nuclei were strictly located in the periphery of the cytoplasm, and their associated centrosomes were positioned immediately adjacent to the plasma membrane. Microtubules emanated from centrosomes into the surrounding cytoplasm. Within 20 to 30 min after the induction of zoosporangial cleavage, nuclei migrated to new positions throughout the sporangial cytoplasm and microtubule arrays were primarily organized at and emanated from nuclear surfaces. During the final stage of zoosporogenesis, nuclear envelope-associated microtubules were not observed. Instead, primary organization of cytoplasmic microtubules returned to centrosomes (i.e., basal bodies) and flagella formation was evident. The MPM-2 antibody, which recognizes phosphorylated epitopes of several proteins associated with microtubule nucleation, stained centrosome regions throughout zoosporogenesis but did not stain nuclear envelopes.