Organization and Dynamics of Growing Microtubule Plus Ends during Early Mitosis

A stable cell line expressing EB1-green fluorescent protein was used to image growing microtubule plus ends at the G2/M transition. By late prophase growing ends no longer extend to the cell periphery and were not uniformly distributed around each centrosome. Growing ends were much more abundant in the area surrounding the nuclear envelope, and microtubules growing around the nucleus were 1.5 fold longer than those growing in the opposite direction. The growth of longer ends toward the nucleus did not result from a localized faster growth rate, because this rate was ∼11 μm/min in all directions from the centrosome. Rather, microtubule ends growing toward the nucleus seemed stabilized by dynein/dynactin associated with the nuclear envelope. Injection of p50 into late prophase cells removed dynein from the nuclear envelope, reduced the density of growing ends near the nuclear envelope and resulted in a uniform distribution of growing ends from each centrosome. We suggest that the cell cycle-dependent binding of dynein/dynactin to the nuclear envelope locally stabilizes growing microtubules. Both dynein and microtubules would then be in a position to participate in nuclear envelope breakdown, as described in recent studies.

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Cell Cycle-dependent Dynamics and Regulation of Mitotic Kinesins in Drosophila S2 Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0118 ◽

2005 ◽

Vol 16 (8) ◽

pp. 3896-3907 ◽

Cited By ~ 103

Author(s):

Gohta Goshima ◽

Ronald D. Vale

Keyword(s):

Cell Cycle ◽

Nuclear Export ◽

Fluorescent Protein ◽

Active Form ◽

Nuclear Export Signal ◽

Nuclear Envelope Breakdown ◽

Spindle Poles ◽

Cycle Dependent ◽

Green Fluorescent ◽

And Function

Constructing a mitotic spindle requires the coordinated actions of several kinesin motor proteins. Here, we have visualized the dynamics of five green fluorescent protein (GFP)-tagged mitotic kinesins (class 5, 6, 8, 13, and 14) in live Drosophila Schneider cell line (S2), after first demonstrating that the GFP-tag does not interfere with the mitotic functions of these kinesins using an RNA interference (RNAi)-based rescue strategy. Class 8 (Klp67A) and class 14 (Ncd) kinesin are sequestered in an active form in the nucleus during interphase and engage their microtubule targets upon nuclear envelope breakdown (NEB). Relocalization of Klp67A to the cytoplasm using a nuclear export signal resulted in the disassembly of the interphase microtubule array, providing support for the hypothesis that this kinesin class possesses microtubule-destabilizing activity. The interactions of Kinesin-5 (Klp61F) and -6 (Pavarotti) with microtubules, on the other hand, are activated and inactivated by Cdc2 phosphorylation, respectively, as shown by examining localization after mutating Cdc2 consensus sites. The actions of microtubule-destabilizing kinesins (class 8 and 13 [Klp10A]) seem to be controlled by cell cycle-dependent changes in their localizations. Klp10A, concentrated on microtubule plus ends in interphase and prophase, relocalizes to centromeres and spindle poles upon NEB and remains at these sites throughout anaphase. Consistent with this localization, RNAi analysis showed that this kinesin contributes to chromosome-to-pole movement during anaphase A. Klp67A also becomes kinetochore associated upon NEB, but the majority of the population relocalizes to the central spindle by the timing of anaphase A onset, consistent with our RNAi result showing no effect of depleting this motor on anaphase A. These results reveal a diverse spectrum of regulatory mechanisms for controlling the localization and function of five mitotic kinesins at different stages of the cell cycle.

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Activity of the GR in G2 and Mitosis

Molecular Endocrinology ◽

10.1210/mend.16.6.0842 ◽

2002 ◽

Vol 16 (6) ◽

pp. 1352-1366 ◽

Cited By ~ 17

Author(s):

G. Alexander Abel ◽

Gabriela M. Wochnik ◽

Joëlle Rüegg ◽

Audrey Rouyer ◽

Florian Holsboer ◽

...

Keyword(s):

Cell Cycle ◽

Fluorescent Protein ◽

Chromatin Condensation ◽

Tyrosine Aminotransferase ◽

Cycle Phase ◽

M Cell ◽

Tumor Virus ◽

Mmtv Promoter ◽

Cycle Dependent ◽

Green Fluorescent

Abstract To elucidate the mechanisms mediating the reported transient physiological glucocorticoid resistance in G2/M cell cycle phase, we sought to establish a model system of glucocorticoid-resistant cells in G2. We synchronized various cell lines in G2 to measure dexamethasone (DEX)-induced transactivation of either two endogenous promoters (rat tyrosine aminotransferase and mouse metallothionein I) or the mouse mammary tumor virus (MMTV) promoter stably or transiently transfected. To circumvent the need for synchronization drugs, we stably transfected an MMTV-driven green fluorescent protein to directly correlate DEX-induced transactivation with the cell cycle position for each cell of an asynchronous population using flow cytometry. Surprisingly, all promoters tested were DEX-inducible in G2. Even in mitotic cells, only the stably transfected MMTV promoter was repressed, whereas the same promoter transiently transfected was inducible. The use of Hoechst 33342 for synchronization in previous studies probably caused a misinterpretation, because we detected interference of this drug with GR-dependent transcription independent of the cell cycle. Finally, GR activated a simple promoter in G2, excluding a functional effect of cell cycle-dependent phosphorylation of GR, as implied previously. We conclude that GR itself is fully functional throughout the entire cell cycle, but GR responsiveness is repressed in mitosis due to chromatin condensation rather than to specific modification of GR.

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A New Model for Nuclear Envelope Breakdown

Molecular Biology of the Cell ◽

10.1091/mbc.12.2.503 ◽

2001 ◽

Vol 12 (2) ◽

pp. 503-510 ◽

Cited By ~ 73

Author(s):

Mark Terasaki ◽

Paul Campagnola ◽

Melissa M. Rolls ◽

Pascal A. Stein ◽

Jan Ellenberg ◽

...

Keyword(s):

Nuclear Envelope ◽

Fluorescent Protein ◽

Three Dimensional ◽

Nuclear Pore ◽

Permeability Barrier ◽

Second Phase ◽

New Model ◽

Nuclear Envelope Breakdown ◽

Two Phases ◽

Green Fluorescent

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

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Dynamics of the Endoplasmic Reticulum and Golgi Apparatus during Early Sea Urchin Development

Molecular Biology of the Cell ◽

10.1091/mbc.11.3.897 ◽

2000 ◽

Vol 11 (3) ◽

pp. 897-914 ◽

Cited By ~ 89

Author(s):

Mark Terasaki

Keyword(s):

Endoplasmic Reticulum ◽

Nuclear Envelope ◽

Sea Urchin ◽

Fluorescent Protein ◽

Time Lapse ◽

Permeability Barrier ◽

Cell Cycles ◽

Nuclear Envelope Breakdown ◽

Quantitative Measurements ◽

Green Fluorescent

The endoplasmic reticulum (ER) and Golgi were labeled by green fluorescent protein chimeras and observed by time-lapse confocal microscopy during the rapid cell cycles of sea urchin embryos. The ER undergoes a cyclical microtubule-dependent accumulation at the mitotic poles and by photobleaching experiments remains continuous through the cell cycle. Finger-like indentations of the nuclear envelope near the mitotic poles appear 2–3 min before the permeability barrier of the nuclear envelope begins to change. This permeability change in turn is ∼30 s before nuclear envelope breakdown. During interphase, there are many scattered, disconnected Golgi stacks throughout the cytoplasm, which appear as 1- to 2-μm fluorescent spots. The number of Golgi spots begins to decline soon after nuclear envelope breakdown, reaches a minimum soon after cytokinesis, and then rapidly increases. At higher magnification, smaller spots are seen, along with increased fluorescence in the ER. Quantitative measurements, along with nocodazole and photobleaching experiments, are consistent with a redistribution of some of the Golgi to the ER during mitosis. The scattered Golgi coalesce into a single large aggregate during the interphase after the ninth embryonic cleavage; this is likely to be preparatory for secretion of the hatching enzyme during the following cleavage cycle.

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Role for Arf3p in Development of Polarity, but Not Endocytosis, inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e03-01-0013 ◽

2003 ◽

Vol 14 (9) ◽

pp. 3834-3847 ◽

Cited By ~ 28

Author(s):

Chun-Fang Huang ◽

Ya-Wen Liu ◽

Luh Tung ◽

Chiou-Hong Lin ◽

Fang-Jen S. Lee

Keyword(s):

Protein Transport ◽

Fluorescent Protein ◽

Fluid Phase ◽

Cell Periphery ◽

Dependent Manner ◽

Vesicular Membrane ◽

A Cell ◽

Cycle Dependent ◽

Green Fluorescent ◽

Gtpase Cycle

ADP-ribosylation factors (ARFs) are ubiquitous regulators of virtually every step of vesicular membrane traffic. Yeast Arf3p, which is most similar to mammalian ARF6, is not essential for cell viability and not required for endoplasmic reticulum-to-Golgi protein transport. Although mammalian ARF6 has been implicated in the regulation of early endocytic transport, we found that Arf3p was not required for fluid-phase, membrane internalization, or mating-type receptor-mediated endocytosis. Arf3p was partially localized to the cell periphery, but was not detected on endocytic structures. The nucleotide-binding, N-terminal region, and N-terminal myristate of Arf3p are important for its proper localization. C-Terminally green fluorescent protein-tagged Arf3, expressed from the endogenous promoter, exhibited a polarized localization to the cell periphery and buds, in a cell cycle-dependent manner. Arf3-GFP achieved its proper localization during polarity growth through an actin-independent pathway. Both haploid and homologous diploid arf3 mutants exhibit a random budding defect, and the overexpression of the GTP-bound form Arf3p(Q71L) or GDP-binding defective Arf3p(T31N) mutant interfered with budding-site selection. We conclude that the GTPase cycle of Arf3p is likely to be important for the function of Arf3p in polarizing growth of the emerging bud and/or an unidentified vesicular trafficking pathway.

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Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent

Development ◽

10.1242/dev.121.10.3259 ◽

1995 ◽

Vol 121 (10) ◽

pp. 3259-3266 ◽

Cited By ~ 24

Author(s):

K.T. Jones ◽

J. Carroll ◽

J.A. Merriman ◽

D.G. Whittingham ◽

T. Kono

Keyword(s):

Cell Cycle ◽

Nuclear Envelope ◽

Dependent Manner ◽

Nuclear Envelope Breakdown ◽

Mouse Oocytes ◽

A Cell ◽

Mature Mouse ◽

Cycle Dependent ◽

Stage 1

Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca2+ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca2+ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca2+ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4–5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca2+ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca2+ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca2+ transients in oocytes is modulated in a cell cycle-dependent manner.

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Two proteins that cycle asynchronously between centrosomes and nuclear structures: Drosophila CP60 and CP190

Journal of Cell Science ◽

10.1242/jcs.110.14.1573 ◽

1997 ◽

Vol 110 (14) ◽

pp. 1573-1583 ◽

Cited By ~ 3

Author(s):

K. Oegema ◽

W.F. Marshall ◽

J.W. Sedat ◽

B.M. Alberts

Keyword(s):

Cell Cycle ◽

Nuclear Envelope ◽

Wide Field ◽

Dependent Manner ◽

Independent Manner ◽

Nuclear Envelope Breakdown ◽

Nuclear Structures ◽

Dynamic Structures ◽

Cycle Dependent

Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two Drosophila proteins of unknown function that shuttle between centrosomes and nuclei in a cell cycle-dependent manner. These two proteins are associated in vitro, and localize to centrosomes in a microtubule independent manner. We injected fluorescently labeled, bacterially expressed CP190 and CP60 into living Drosophila embryos and followed their behavior during the rapid syncytial blastoderm divisions (nuclear cycles 10–13). Using quantitative 3-D wide-field fluorescence microscopy, we show that CP190 and CP60 cycle between nuclei and centrosomes asynchronously with the accumulation of CP190 leading that of CP60 both at centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately following nuclear envelope breakdown, CP190 localizes to centrosomes where it remains until telophase, thereafter accumulating in reforming nuclei. Unlike CP190, CP60 accumulates at centrosomes primarily during anaphase, where it remains into early interphase. During nuclear cycles 10 and 11, CP60 accumulates in nuclei simultaneous with nuclear envelope breakdown, suggesting that CP60 binds to an unknown nuclear structure that persists into mitosis. During nuclear cycles 12 and 13, CP60 accumulates gradually in nuclei during interphase, reaching peak levels just before nuclear envelope breakdown. Once in the nucleus, both CP190 and CP60 appear to form fibrous intranuclear networks that remain coherent even after nuclear envelope breakdown. The CP190 and CP60 networks do not co-localize extensively with each other or with DNA. This work provides direct evidence, in living cells, of a coherent protein network that may represent a nuclear skeleton.

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Nuclear envelope dynamics during plant cell division suggest common mechanisms between kingdoms

Biochemical Journal ◽

10.1042/bj20101769 ◽

2011 ◽

Vol 435 (3) ◽

pp. 661-667 ◽

Cited By ~ 26

Author(s):

Katja Graumann ◽

David E. Evans

Keyword(s):

Nuclear Envelope ◽

Fluorescent Protein ◽

Cell Plate ◽

Lamin B ◽

Nuclear Envelope Breakdown ◽

Plant Cell Division ◽

Lamin B Receptor ◽

Close Proximity ◽

Green Fluorescent ◽

Membrane Components

Behaviour of the NE (nuclear envelope) during open mitosis has been explored extensively in metazoans, but lack of native markers has limited similar investigations in plants. In the present study, carried out using living synchronized tobacco BY-2 suspension cultures, the non-functional NE marker LBR (lamin B receptor)–GFP (green fluorescent protein) and two native, functional NE proteins, AtSUN1 [Arapidopsis thaliana SUN (Sad1/UNC84) 1] and AtSUN2, we provide evidence that the ER (endoplasmic reticulum)-retention theory for NE membranes is applicable in plants. We also observe two apparently unique plant features: location of the NE-membrane components in close proximity to chromatin throughout division, and spatially distinct reformation of the NE commencing at the chromatin surface facing the spindle poles and concluding at the surface facing the cell plate. Mobility of the proteins was investigated in the interphase NE, during NE breakdown and reformation, in the spindle membranes and the cell plate. A role for AtSUN2 in nuclear envelope breakdown is suggested.

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An in vitro nuclear disassembly system reveals a role for the RanGTPase system and microtubule-dependent steps in nuclear envelope breakdown

The Journal of Cell Biology ◽

10.1083/jcb.200703002 ◽

2007 ◽

Vol 178 (4) ◽

pp. 595-610 ◽

Cited By ~ 67

Author(s):

Petra Mühlhäusser ◽

Ulrike Kutay

Keyword(s):

Nuclear Envelope ◽

Fluorescent Protein ◽

Dynamic Rupture ◽

Vitro Assay ◽

Nuclear Envelope Breakdown ◽

Egg Extracts ◽

Guanosine Triphosphatase ◽

Green Fluorescent

During prophase, vertebrate cells disassemble their nuclear envelope (NE) in the process of NE breakdown (NEBD). We have established an in vitro assay that uses mitotic Xenopus laevis egg extracts and semipermeabilized somatic cells bearing a green fluorescent protein–tagged NE marker to study the molecular requirements underlying the dynamic changes of the NE during NEBD by live microscopy. We applied our in vitro system to analyze the role of the Ran guanosine triphosphatase (GTPase) system in NEBD. Our study shows that high levels of RanGTP affect the dynamics of late steps of NEBD in vitro. Also, inhibition of RanGTP production by RanT24N blocks the dynamic rupture of nuclei, suggesting that the local generation of RanGTP around chromatin may serve as a spatial cue in NEBD. Furthermore, the microtubule-depolymerizing drug nocodazole interferes with late steps of nuclear disassembly in vitro. High resolution live cell imaging reveals that microtubules are involved in the completion of NEBD in vivo by facilitating the efficient removal of membranes from chromatin.

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Microtubule Organization Requires Cell Cycle-dependent Nucleation at Dispersed Cytoplasmic Sites: Polar and Perinuclear Microtubule Organizing Centers in the Plant Pathogen Ustilago maydis

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0513 ◽

2003 ◽

Vol 14 (2) ◽

pp. 642-657 ◽

Cited By ~ 80

Author(s):

Anne Straube ◽

Marianne Brill ◽

Berl R. Oakley ◽

Tetsuya Horio ◽

Gero Steinberg

Keyword(s):

Cell Cycle ◽

Fluorescent Protein ◽

Spindle Pole Body ◽

Spindle Pole ◽

Microtubule Organization ◽

Microtubule Organizing Centers ◽

Nucleation Sites ◽

Cycle Dependent ◽

Green Fluorescent

Growth of most eukaryotic cells requires directed transport along microtubules (MTs) that are nucleated at nuclear-associated microtubule organizing centers (MTOCs), such as the centrosome and the fungal spindle pole body (SPB). Herein, we show that the pathogenic fungusUstilago maydis uses different MT nucleation sites to rearrange MTs during the cell cycle. In vivo observation of green fluorescent protein-MTs and MT plus-ends, tagged by a fluorescent EB1 homologue, provided evidence for antipolar MT orientation and dispersed cytoplasmic MT nucleating centers in unbudded cells. On budding γ-tubulin containing MTOCs formed at the bud neck, and MTs reorganized with >85% of all minus-ends being focused toward the growth region. Experimentally induced lateral budding resulted in MTs that curved out of the bud, again supporting the notion that polar growth requires polar MT nucleation. Depletion or overexpression of Tub2, the γ-tubulin from U. maydis, affected MT number in interphase cells. The SPB was inactive in G2 phase but continuously recruited γ-tubulin until it started to nucleate mitotic MTs. Taken together, our data suggest that MT reorganization in U. maydis depends on cell cycle-specific nucleation at dispersed cytoplasmic sites, at a polar MTOC and the SPB.

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