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Cytoplasmic Microtubules (cytoplasmic + microtubule)
Selected AbstractsGlutamylated tubulin: Diversity of expression and distribution of isoformsCYTOSKELETON, Issue 1 2003Marie-Louise Kann Abstract Glutamylation of , and , tubulin isotypes is a major posttranslational modification giving rise to diversified isoforms occurring mainly in neurotubules, centrioles, and axonemes. Monoglutamylated tubulin isoforms can be differentially recognized by two mAbs, B3 and GT335, which both recognize either polyglutamylated isoforms. In the present study, immunoelectron microscopy and immunofluorescence analyses were performed with these two mAbs to determine the expression and distribution of glutamylated tubulin isoforms in selected biological models whose tubulin isotypes are characterized. In mouse spermatozoa, microtubules of the flagellum contain polyglutamylated isoforms except in the tip where only monoglutamylated isoforms are detected. In spermatids, only a subset of manchette microtubules contain monoglutamylated tubulin isoforms. Cytoplasmic microtubules of Sertoli cells are monoglutamylated. Mitotic and meiotic spindles of germ cells are monoglutamylated whereas the HeLa cell mitotic spindle is polyglutamylated. Three models of axonemes are demonstrated as a function of the degree and extent of tubulin glutamylation. In lung ciliated cells, axonemes are uniformly polyglutamylated. In sea urchin sperm and Chlamydomonas, flagellar microtubules are polyglutamylated in their proximal part and monoglutamylated in their distal part. In Paramecium, cilia are bi- or monoglutamylated only at their base. In all cells, centrioles or basal bodies are polyglutamylated. These new data emphasize the importance of glutamylation in all types of microtubules and strengthen the hypothesis of its role in the regulation of the intracellular traffic and flagellar motility. Cell Motil. Cytoskeleton 55:14,25, 2003. © 2003 Wiley-Liss, Inc. [source] Mutagenesis of ,-tubulin cysteine residues in Saccharomyces cerevisiae: Mutation of cysteine 354 results in cold-stable microtubulesCYTOSKELETON, Issue 2 2001Mohan L. Gupta Jr. Abstract Cysteine residues play important roles in the control of tubulin function. To determine which of the six cysteine residues in ,-tubulin are critical to tubulin function, we mutated the cysteines in Saccharomyces cerevisiae ,-tubulin individually to alanine and serine residues. Of the twelve mutations, only three produced significant effects: C12S, C354A, and C354S. The C12S mutation was lethal in the haploid, but the C12A mutation had no observable phenotype. Based on interactive views of the electron crystallographic structure of tubulin, we suggest that substitution of serine for cysteine at this position has a destabilizing effect on the interaction of tubulin with the exchangeable GTP. The two C354 mutations, although not lethal, produced dramatic effects on microtubules and cellular processes that require microtubules. The C354 mutant cells had decreased growth rates, a slowed mitosis, increased resistance to benomyl, and impaired nuclear migration and spindle assembly. The C354A mutation produced a more severe phenotype than the C354S mutation: the haploid cells had chromosome segregation defects, only 50% of cells in a culture were viable, and a significant percentage of the cells were misshapened. Cytoplasmic microtubules in the C354S and C354A cells were longer than in the control strain and spindle structures appeared shorter and thicker. Both cytoplasmic and spindle microtubules in the two C354 mutants were extremely stable to cold temperature. After 24 h at 4°C, the microtubules were still present and, in fact, very long and thick tubulin polymers had formed. Evidence exists to indicate that the C354 residue in mammalian tubulin is near the colchicine binding site and the electron crystal structure of tubulin places the residue at the interface between the ,- and ,-subunits. The sulfhydryl group is situated in a polar environment, which may explain why the alanine mutation is more severe than the serine mutation. When the C12S and the two C354 mutations were made in a diploid strain, the mutated tubulin was incorporated into microtubules and the resulting heterozygotes had phenotypes that were intermediate between those of the mutated haploids and the wild-type strains. The results suggest that the C12 and C354 residues play important roles in the structure and function of tubulin. Cell Motil. Cytoskeleton 49:67,77, 2001. © 2001 Wiley-Liss, Inc. [source] Evolution and persistence of the ciliumCYTOSKELETON, Issue 12 2007Peter Satir Abstract The origin of cilia, a fundamental eukaryotic organelle, not present in prokaryotes, poses many problems, including the origins of motility and sensory function, the origins of nine-fold symmetry, of basal bodies, and of transport and selective mechanisms involved in ciliogenesis. We propose the basis of ciliary origin to be a self-assembly RNA enveloped virus that contains unique tubulin and tektin precursors. The virus becomes the centriole and basal body, which would account for the self-assembly and self-replicative properties of these organelles, in contrast to previous proposals of spirochaete origin or endogenous differentiation, which do not readily account for the centriole or its properties. The viral envelope evolves into a sensory bud. The host cell supplies the transport machinery and molecular motors to construct the axoneme. Polymerization of cytoplasmic microtubules in the 9 + 0 axoneme completes the 9 + 2 pattern. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Cut1/separase C-terminus affects spindle pole body positioning in interphase of fission yeast: pointed nuclear formationGENES TO CELLS, Issue 11 2002Takahiro Nakamura Background: The separase-securin complex is required for anaphase. Separase activated by securin destruction cleaves the cohesin subunit Scc1/Rad21 enriched in kinetochores. Fission yeast Cut1/separase resides in interphase cytoplasm and mobilizes to the spindle and the spindle pole bodies (SPBs) in mitosis, while Cut2/securin remains in the nucleus from interphase to metaphase, and temporarily locates at the short spindle. Results: We here report a novel SPB-led dynamic nuclear movement in fission yeast, when the Cut1 C-terminal fragment is over-expressed. The tip of the pointed nucleus contained both SPB and centromeric DNA, and rapidly moved along the bundled cytoplasmic microtubules. The same pointed nucleus was produced when the human separase C-fragment was over-expressed. The pointed nuclear formation did not require the protease site of separase, but required the conserved C-terminus and a microtubule- and kinetochore-binding protein Mtc1/Alp14, a homologue of frog XMAP215 and budding yeast Stu2. The movement-inducing C-fragment should be cytoplasmic, as the pointed nucleus was abolished when the fragment contained the NLS (nuclear localization signal). Conclusions: Overproduced separase C-fragment abolishes correct SPB-positioning in interphase. Resulting pointed nuclear formation (alternatively called ,pigtail movement') requires cytoplasmic microtubules and Mtc1/Alp14. [source] Ultrastructure of the spermatid of Caprimulgus europaeus Linnaeus 1758, the European nightjar (Aves; Caprimulgidae), with phylogenetic implicationsJOURNAL OF MORPHOLOGY, Issue 10 2006Sandro Tripepi Abstract The sperm of Caprimulgus europaeus is typical of other nonpasserines in many respects. Features shared with Paleognathae and Galloanserae are the conical acrosome, shorter than the nucleus; the presence of a perforatorium and endonuclear canal; the presence of a proximal as well as distal centriole; the elongate midpiece with mitochondria grouped around a central axis (here maximally six mitochondria in ,10 tiers); and the presence of a fibrous or amorphous sheath around the principal piece of the axoneme. A major (apomorphic) difference from paleognaths and galloanserans is the short distal centriole, the midpiece being penetrated for most of its length by the axoneme and for only a very short proximal portion by the centriole. Nonpasserines differ from paleognaths in that the latter have a transversely ribbed fibrous sheath, whereas in nonpasserines it is amorphous, as in Caprimulgus, or absent. The absence of an annulus is an apomorphic feature of Caprimulgus, apodiform, psittaciform, gruiform, and passerine sperm, homoplastic in at least some of these. In contrast to passerines, in Caprimulgus the cytoplasmic microtubules in the spermatid are restricted to a transient longitudinal manchette. The structure of the spermatid and spermatozoon is consistent with placement of the Caprimulgidae near the Psittacidae, but is less supportive of close proximity to the Apodidae, from DNA,DNA hybridization and some other analyses. J. Morphol. © 2006 Wiley-Liss, Inc. [source] Alterations and reversibility in the chromatin, cytoskeleton and development of pig oocytes treated with roscovitineMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003Jyh-Cherng Ju Abstract Germinal vesicle (GV) breakdown in mammalian oocytes is regulated by the activation of maturation promoting factor (MPF). We investigated a specific cdc2 kinase inhibitor, roscovitine, to maintain pig oocytes in the GV stage. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 44 hr in NCSU#23 medium containing different levels of roscovitine (0, 10, 20, 30, 40, 50 ,M in Experiment 1 and 0, 40, 60, 80, 100, 120 ,M in Experiment 2). The COCs were cultured for another 44 hr after removal of the chemical. Twenty oocytes in each group were fixed at 44 hr for immunocytochemical labeling of the cytoskeleton and the rest (,20/group) were fixed at the end of 88 hr after culture. Results showed that the inhibition of the oocyte in the GV stage was not effective when 10,50 ,M (Experiment 1) of roscovitine were used (19,34%). When oocytes were released from the inhibitor, similar proportions (70,83%) of oocytes were observed in the MII or advanced stages among treatments. However, when higher concentrations of roscovitine were used (Experiment 2), significantly greater inhibitory effect was observed at the levels of 80,120 ,M with 83,91% oocytes being blocked in the GV stage when compared to the control (9%) and the 40,60 ,M (27,43%) groups (P,<,0.05). Although 15,21% of the oocytes showed abnormal MII morphology with aberrant meiotic spindles and/or formation of cytoplasmic microtubules, a substantial number of oocytes resumed meiosis and reached MII stage at 44 hr after removal of this chemical. In Experiment 3, different concentrations of roscovitine (0, 20, 40, and 80 ,M) were tested to examine the length of intervals (0, 11, 22, 33, and 44 hr) for an effective inhibition. Results showed that the inhibitory effect was significantly more prominent at 22 hr than that at 33 and 44 hr after roscovitine treatment in all treatment groups (P,<,0.05). This study demonstrated that roscovitine-treated oocytes resumed meiosis after removal of the inhibitor. This could provide flexibility for studying porcine oocyte development and embryo cloning and may have application in other species. Mol. Reprod. Dev. 64: 482,491, 2003. © 2003 Wiley-Liss, Inc. [source] | ||||||||||