F-actin is required for spindle anchoring and rotation in Xenopus oocytes: a re-examination of the effects of cytochalasin B on oocyte maturation

Zygote ◽  
1995 ◽  
Vol 3 (1) ◽  
pp. 17-26 ◽  
Author(s):  
David Lynn Gard ◽  
Byeong-Jik Cha ◽  
Amy Diane Roeder
Keyword(s):  
Cytochalasin B ◽  
Meiotic Spindle ◽  
The Novel ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Rhodamine Phalloidin ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Meiotic Spindles ◽  
Bipolar Spindles

SummaryWe used confocal immunofluorescence microscopy to examine spindle migration, morphology and orientation during the maturation of Xenopus oocytes, in the presence or absence of cytochalasin B (CB), an inhibitor of actin assembly. Treatment with CB during maturation (10–50 μg/ml beginning 0–3h prior to addition of progesterone) disrupted the normal organisation of the novel MTOC and transient microtubule array (MTOC-TMA complex) that serves as the immediate precursor of the first meiotic spindle, suggesting that F-actin plays an important role in the assembly or maintenance of this complex. However, CB treatment did not block translocation of the MTOC-TMA complex to the oocyte cortex, suggesting that MTOC-TMA translocation is not dependent on an actin-based mechanism. Bipolar spindles were observed in CB-treated oocytes fixed during both M1 and M2. However, rotation of the M1 and M2 spindles into an orientation orthogonal to the oocyte surface was inhibited by CB. Rhodamine-phalloidin revealed a concentration of F-actin at the site of M1 spindle attachment, further suggesting that cortical actin is required for anchoring and rotation of the meiotic spindles. Finally, the incidence of M1 monasters was significantly increased in CB-treated oocytes, suggesting that interactions between the nascent M1 spindle and cortex are dependent on F-actin.

Maize meiotic spindles assemble around chromatin and do not require paired chromosomes

1998 ◽  
Vol 111 (23) ◽  
pp. 3507-3515 ◽  
Author(s):  
A. Chan ◽  
W.Z. Cande
Keyword(s):  
Nuclear Envelope ◽  
Higher Plants ◽  
Metaphase Plate ◽  
Meiotic Spindle ◽  
Wild Type ◽  
Homologous Chromosomes ◽  
Nuclear Envelope Breakdown ◽  
Meiotic Spindles ◽  
Meiotic Mutations ◽  
Bipolar Spindles

To understand how the meiotic spindle is formed and maintained in higher plants, we studied the organization of microtubule arrays in wild-type maize meiocytes and three maize meiotic mutants, desynaptic1 (dsy1), desynaptic2 (dsy2), and absence of first division (afd). All three meiotic mutations have abnormal chromosome pairing and produce univalents by diakinesis. Using these three mutants, we investigated how the absence of paired homologous chromosomes affects the assembly and maintenance of the meiotic spindle. Before nuclear envelope breakdown, in wild-type meiocytes, there were no bipolar microtubule arrays. Instead, these structures formed after nuclear envelope breakdown and were associated with the chromosomes. The presence of univalent chromosomes in dsy1, dsy2, and afd meiocytes and of unpaired sister chromatids in the afd meiocytes did not affect the formation of bipolar spindles. However, alignment of chromosomes on the metaphase plate and subsequent anaphase chromosome segregation were perturbed. We propose a model for spindle formation in maize meiocytes in which microtubules initially appear around the chromosomes during prometaphase and then the microtubules self-organize. However, this process does not require paired kinetochores to establish spindle bipolarity.

scholarly journals Adipocytes support cAMP-dependent translocation of aquaporin-2 from intracellular sites distinct from the insulin-responsive GLUT4 storage compartment

2006 ◽  
Vol 290 (5) ◽  
pp. F985-F994 ◽  
Author(s):  
Giuseppe Procino ◽  
Donne Bennett Caces ◽  
Giovanna Valenti ◽  
Jeffrey E. Pessin
Keyword(s):  
Plasma Membrane ◽  
Three Dimensional ◽  
Dominant Negative ◽  
Cortical Actin ◽  
Aquaporin 2 ◽  
Confocal Immunofluorescence Microscopy ◽  
Dominant Negative Form ◽  
Confocal Immunofluorescence ◽  
Hormone Sensitive ◽  
Insulin Responsiveness

Aquaporin-2 (AQP2), when expressed in fully differentiated 3T3-L1 adipocytes, displays cAMP-dependent plasma membrane translocation in a manner similar to its behavior in renal epithelial cells. The translocation of AQP2 required phosphorylation at serine 256, as the expression of AQP2/S256D was constitutively plasma membrane localized, whereas AQP2/S256A was refractory to forskolin stimulation. Unlike GLUT4, this property is not inhibited by depolymerization of cortical actin. In addition, coexpression with the dominant negative form of TC10 (TC10/T31N) or inhibition of phosphatidylinositol 3-kinase did not abrogate the cAMP-mediated response. Under basal conditions, AQP2 is localized in both the perinuclear region and in punctate vesicles scattered within the periphery of the cell. Two- and three-dimensional confocal immunofluorescence microscopy demonstrated that the adipocyte AQP2 cAMP-responsive compartment was distinct from the GLUT4 insulin-responsive compartment. Consistent with this conclusion, insulin was an effective stimulator of GLUT4 translocation but had no effect on AQP2. Conversely, forskolin induced AQP2 translocation but not GLUT4. Colocalization studies with the early endosomal marker EEA1 and transferrin receptor suggested that the AQP2 compartment is mostly distinct from endosomal vesicles. Interestingly, however, the peripheral AQP2 vesicles significantly overlapped vesicle-associated membrane protein-2, underscoring the role of the latter in hormone-regulated exocytosis. To acquire insulin responsiveness following biosynthesis, GLUT4 undergoes a slow sorting step that requires 6–9 h. In contrast, AQP2 rapidly acquires forskolin responsiveness (3 h following biosynthesis) and directly enters the cAMP-regulated compartment without transiting the plasma membrane. Together, these data demonstrate that adipocytes display two different intracellular sorting mechanisms that direct distinct hormone-sensitive partitioning of GLUT4 and AQP2.

25 COMPARISON BETWEEN CHEMICALLY ASSISTED, CHEMICALLY INDUCED AND MECHANICAL ENUCLEATION OF MOUSE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 113
Author(s):  
N. Costa-Borges ◽  
S. González ◽  
J. Santaló ◽  
E. Ibáñez
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Cumulus Cell ◽  
Meiotic Spindle ◽  
Immunofluorescence Analysis ◽  
Mouse Oocytes ◽  
Polar Bodies ◽  
Chemically Induced ◽  
Spindle Microtubules ◽  
Bipolar Spindles

Chemically-assisted (AE) and chemically induced (IE) enucleation using demecolcine (DEM) or nocodazole (NOC) have proven to be technically simple procedures to prepare developmentally competent cytoplasts for nuclear transfer (NT) in different species. In this study, we analyzed AE and IE in mouse oocytes in terms of enucleation efficiency, amount of cytoplasmic volume removed and distribution of spindle-associated γ-tubulin after enucleation, and spindle morphology after cytoplast reconstruction by NT. Results were compared to the standard mechanical enucleation (ME) method. Outbred CD-1 and hybrid B6CBAF1 oocytes were collected at 13 to 16 h post-hCG. In AE experiments, oocytes were treated with either 0.4 μg mL–1 DEM or 0.3 μg mL–1 NOC in KSOM for 30 min. Protrusions induced in CD-1 (92.2%, n = 695) and B6CBAF1 (83.3%, n = 370) oocytes were aspirated by piezo-actuated micromanipulation, in H-KSOM with 2.5 μg mL–1 cytochalasin B and 0.05 m sucrose. In IE experiments, oocytes were preactivated with 7% ethanol for 5 min and treated with DEM or NOC in calcium-free KSOM containing 10 mm strontium. At 90 min postactivation (p.a.), completely- and partially-extruded second polar bodies (PBs) were mechanically aspirated. Enucleation efficiencies were higher than 90% both for AE (90.8%, n = 509 CD-1; 90.4%, n = 260 B6CBAF1) and IE methods (90.3%, n = 167 CD-1; 92.9%, n = 197 B6CBAF1), though they were significantly lower than those obtained for ME in nontreated CD-1 (98.4%; n = 126) or B6CBAF1 (100%, n = 498) oocytes. The amount of cytoplasmic volume removed in CD-1 oocytes was smaller in AE than in ME (2.1%, n = 35 and 3.9%, n = 30, respectively). In B6CBAF1 oocytes, used to compare IE (5.4%, n = 60) and ME (4.9%, n = 41), no differences were found. Volumes were calculated using the CellA software on images of cytoplasts and karyoplasts taken after enucleation. Even though both AE and IE methods avoided the removal of the oocyte spindle microtubules, spindle-associated γ-tubulin was eliminated from the cytoplasts generated by all 3 enucleation procedures, as confirmed by immunofluorescence analysis of the cytoplasts and the complementary karyoplasts produced. Finally, spindle morphology was examined in enucleated oocytes reconstructed by NT with a cumulus cell nucleus. Cytoplasts prepared by NOC-AE or NOC-IE displayed morphologically normal bipolar spindles by 2 h post-NT or 18 to 20 h post-activation (hpa), respectively, similar to cytoplasts prepared by ME. However, when DEM was used, microtubule repolymerization was slower and bipolar spindles could not be observed until 4 h post-NT (AE) or 22 to 24 hpa (IE). In conclusion, although enucleation rates are slightly higher for ME, AE and IE protocols allow oocyte enucleation without removal of the meiotic spindle, and a very small cytoplasm volume is eliminated during AE. Treatments with NOC and DEM are reversible, and cytoplasts produced by AE and IE can form morphologically normal spindles after NT, similar to those of cytoplasts produced by ME. MEC BIO 2006-11792; 2005-SGR00437; Portuguese FCT.

scholarly journals XMAP230 is required for normal spindle assembly in vivo and in vitro

1999 ◽  
Vol 112 (23) ◽  
pp. 4337-4346 ◽  
Author(s):  
B. Cha ◽  
L. Cassimeris ◽  
D.L. Gard
Keyword(s):  
Spindle Assembly ◽  
Chromosome Loss ◽  
Mitotic Spindles ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
M Phase ◽  
Spindle Microtubules ◽  
Bipolar Spindles

XMAP230 is a high molecular mass microtubule-associated protein isolated from Xenopus oocytes and eggs, and has been recently shown to be a homolog of mammalian MAP4. Confocal immunofluorescence microscopy revealed that XMAP230 is associated with microtubules throughout the cell cycle of early Xenopus embryos. During interphase XMAP230 is associated with the radial arrays of microtubules and midbodies remaining from the previous division. During mitosis, XMAP230 is associated with both astral microtubules and microtubules of the central spindle. Microinjection of affinity-purified anti-XMAP230 antibody into blastomeres severely disrupted the assembly of mitotic spindles during the rapid cleavage cycles of early development. Both monopolar half spindles and bipolar spindles were assembled from XMAP230-depleted extracts in vitro. However, spindles assembled in XMAP230-depleted extracts exhibited a reduction in spindle width, reduced microtubule density, chromosome loss, and reduced acetylation of spindle MTs. Similar defects were observed in the spindles assembled in XMAP230-depleted extracts that had been cycled through interphase. Depletion of XMAP230 had no effect on the pole-to-pole length of spindles, and depletion of XMAP230 from both interphase and M-phase extracts had no effect on the rate of microtubule elongation. From these results, we conclude that XMAP230 plays an important role in normal spindle assembly, primarily by acting to stabilize spindle microtubules, and that the observed defects in spindle assembly may result from enhanced microtubule dynamics in XMAP230-depleted extracts.

scholarly journals Visualization and Molecular Analysis of Actin Assembly in Living Cells

1998 ◽  
Vol 143 (7) ◽  
pp. 1919-1930 ◽  
Author(s):  
Dorothy A. Schafer ◽  
Matthew D. Welch ◽  
Laura M. Machesky ◽  
Paul C. Bridgman ◽  
Shelley M. Meyer ◽  
...  
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cell ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lim Kinase ◽  
Permeabilized Cells ◽  
Capping Protein ◽  
Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

scholarly journals Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation.

1992 ◽  
Vol 118 (3) ◽  
pp. 561-571 ◽  
Author(s):  
S Chowdhury ◽  
K W Smith ◽  
M C Gustin
Keyword(s):  
Osmotic Stress ◽  
Actin Filament ◽  
Physiological Process ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rhodamine Phalloidin ◽  
Diploid Cells

In the yeast Saccharomyces cerevisiae, actin filaments function to direct cell growth to the emerging bud. Yeast has a single essential actin gene, ACT1. Diploid cells containing a single copy of ACT1 are osmosensitive (Osms), i.e., they fail to grow in high osmolarity media (D. Shortle, unpublished observations cited by Novick, P., and D. Botstein. 1985. Cell. 40:415-426). This phenotype suggests that an underlying physiological process involving actin is osmosensitive. Here, we demonstrate that this physiological process is a rapid and reversible change in actin filament organization in cells exposed to osmotic stress. Filamentous actin was stained using rhodamine phalloidin. Increasing external osmolarity caused a rapid loss of actin filament cables, followed by a slower redistribution of cortical actin filament patches. In the recovery phase, cables and patches were restored to their original levels and locations. Strains containing an act1-1 mutation are both Osms and temperature-sensitive (Ts) (Novick and Botstein, 1985). To identify genes whose products functionally interact with actin in cellular responses to osmotic stress, we have isolated extragenic suppressors which revert only the Osms but not the Ts phenotype of an act1-1 mutant. These suppressors identify three genes, RAH1-RAH3. Morphological and genetic properties of a dominant suppressor mutation suggest that the product of the wild-type allele, RAH3+, is an actin-binding protein that interacts with actin to allow reassembly of the cytoskeleton following osmotic stress.

Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 699-709 ◽  
Author(s):  
M.M. Schroeder ◽  
D.L. Gard
Keyword(s):  
Cell Cycle ◽  
Cortical Microtubule ◽  
Cortical Microtubules ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Vegetal Cortex ◽  
M Phase ◽  
Tubulin Antibodies ◽  
Microtubule Networks ◽  
Sperm Aster

Anti-tubulin antibodies and confocal immunofluorescence microscopy were used to examine the organization and regulation of cytoplasmic and cortical microtubules during the first cell cycle of fertilized Xenopus eggs. Appearance of microtubules in the egg cortex temporally coincided with the outgrowth of the sperm aster. Microtubules of the sperm aster first reached the animal cortex at 0.25, (times normalized to first cleavage), forming a radially organized array of cortical microtubules. A disordered network of microtubules was apparent in the vegetal cortex as early as 0.35. Cortical microtubule networks of both animal and vegetal hemispheres were reorganized at times corresponding to the cortical rotation responsible for specification of the dorsal-ventral (D-V) axis. Optical sections suggest that the cortical microtubules are continuous with the microtubules of the sperm aster in fertilized eggs, or an extensive activation aster in activated eggs. Neither assembly and organization, nor disassembly of the cortical microtubules coincided with MPF activation during mitosis. However, cycloheximide or 6-dimethylaminopurine, which arrest fertilized eggs at interphase, blocked cortical microtubule disassembly. Injection of p13, a protein that specifically inhibits MPF activation, delayed or inhibited cortical microtubule breakdown. In contrast, eggs injected with cyc delta 90, a truncated cyclin that arrest eggs in M-phase, showed normal microtubule disassembly. Finally, injection of partially purified MPF into cycloheximide-arrested eggs induced cortical microtubule breakdown. These results suggest that, despite a lack of temporal coincidence, breakdown of the cortical microtubules is dependent on the activation of MPF.

scholarly journals RNA polymerase II transcription is concentrated outside replication domains throughout S-phase

1994 ◽  
Vol 107 (6) ◽  
pp. 1449-1456 ◽  
Author(s):  
D.G. Wansink ◽  
E.E. Manders ◽  
I. van der Kraan ◽  
J.A. Aten ◽  
R. van Driel ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Spatial Relationship ◽  
S Phase ◽  
Double Labelling ◽  
Confocal Immunofluorescence Microscopy ◽  
Nuclear Domain ◽  
Confocal Immunofluorescence ◽  
Nuclear Domains ◽  
Transcription And Replication

Transcription and replication are, like many other nuclear functions and components, concentrated in nuclear domains. Transcription domains and replication domains may play an important role in the coordination of gene expression and gene duplication in S-phase. We have investigated the spatial relationship between transcription and replication in S-phase nuclei after fluorescent labelling of nascent RNA and nascent DNA, using confocal immunofluorescence microscopy. Permeabilized human bladder carcinoma cells were labelled with 5-bromouridine 5′-triphosphate and digoxigenin-11-deoxyuridine 5′-triphosphate to visualize sites of RNA synthesis and DNA synthesis, respectively. Transcription by RNA polymerase II was localized in several hundreds of domains scattered throughout the nucleoplasm in all stages of S-phase. This distribution resembled that of nascent DNA in early S-phase. In contrast, replication patterns in late S-phase consisted of fewer, larger replication domains. In double-labelling experiments we found that transcription domains did not colocalize with replication domains in late S-phase nuclei. This is in agreement with the notion that late replicating DNA is generally not actively transcribed. Also in early S-phase nuclei, transcription domains and replication domains did not colocalize. We conclude that nuclear domains exist, large enough to be resolved by light microscopy, that are characterized by a high activity of either transcription or replication, but never both at the same time. This probably means that as soon as the DNA in a nuclear domain is being replicated, transcription of that DNA essentially stops until replication in the entire domain is completed.

scholarly journals Involvement of ATP-dependentPseudomonasExotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

1998 ◽  
Vol 9 (2) ◽  
pp. 387-402 ◽  
Author(s):  
Mériem Alami ◽  
Marie-Pierre Taupiac ◽  
Hubert Reggio ◽  
Alain Bienvenüe ◽  
Bruno Beaumelle
Keyword(s):  
Atp Hydrolysis ◽  
Active Form ◽  
Brefeldin A ◽  
Recycling Endosomes ◽  
Confocal Immunofluorescence Microscopy ◽  
Weak Bases ◽  
Confocal Immunofluorescence ◽  
A Cell ◽  
Endocytic Vesicles ◽  
Cytosol Ph

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal125I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect125I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when125I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired125I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.

Role of microtubules and microtubule organizing centers on meiotic chromosome elimination in Sciara ocellaris

1997 ◽  
Vol 110 (6) ◽  
pp. 721-730 ◽  
Author(s):  
M.R. Esteban ◽  
M.C. Campos ◽  
A.L. Perondini ◽  
C. Goday
Keyword(s):  
Meiotic Chromosome ◽  
Chromosome Elimination ◽  
Male Meiosis ◽  
Meiotic Spindle ◽  
Monopolar Spindle ◽  
Meiotic Spindles ◽  
Cytoplasmic Microtubules ◽  
Spindle Microtubules ◽  
Meiosis I

Spindle formation and chromosome elimination during male meiosis in Sciara ocellaris (Diptera, Sciaridae) has been studied by immunofluorescence techniques. During meiosis I a monopolar spindle is formed from a single polar complex (centrosome-like structure). This single centrosomal structure persists during meiosis II and is responsible for the non-disjunction of the maternal X chromatids. During meiosis I and II non-spindle microtubules are assembled in the cytoplasmic bud regions of the spermatocytes. The chromosomes undergoing elimination during both meiotic divisions are segregated to the bud region where they associate with bundles of microtubules. The presence and distribution of centrosomal antigens in S. ocellaris meiotic spindles and bud regions has been investigated using different antibodies. gamma-Tubulin and centrin are present in the bud as well as in the single polar complex of first meiotic spindle. The results suggest that spermatocyte bud regions contain microtubule-organizing centres (MTOCs) that nucleate cytoplasmic microtubules that are involved in capturing chromosomes in the bud regions. The distribution of actin and myosin in the spermatocytes during meiosis is also reported.

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