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

2000 ◽  
Vol 11 (3) ◽  
pp. 897-914 ◽  
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.

scholarly journals A New Model for Nuclear Envelope Breakdown

2001 ◽  
Vol 12 (2) ◽  
pp. 503-510 ◽  
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.

scholarly journals Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

2006 ◽  
Vol 17 (7) ◽  
pp. 3009-3020 ◽  
Author(s):  
Johan-Owen De Craene ◽  
Jeff Coleman ◽  
Paula Estrada de Martin ◽  
Marc Pypaert ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Cortex ◽  
Proteomic Screen ◽  
Wide Range ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Hydrophilic Loop ◽  
Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

scholarly journals The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

2001 ◽  
Vol 152 (2) ◽  
pp. 385-400 ◽  
Author(s):  
Patrick Heun ◽  
Thierry Laroche ◽  
M.K. Raghuraman ◽  
Susan M. Gasser
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Structure ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Yeast Cells ◽  
G1 Phase ◽  
Nuclear Pore ◽  
Origin Firing ◽  
Green Fluorescent

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

scholarly journals Vaccinia Virus DNA Replication Occurs in Endoplasmic Reticulum-enclosed Cytoplasmic Mini-Nuclei

2001 ◽  
Vol 12 (7) ◽  
pp. 2031-2046 ◽  
Author(s):  
Nina Tolonen ◽  
Laura Doglio ◽  
Sibylle Schleich ◽  
Jacomine Krijnse Locker
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Nuclear Envelope Assembly ◽  
Green Fluorescent Protein Tagging ◽  
Green Fluorescent

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ∼45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [3H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.

scholarly journals Time-Lapse Video Microscopy Analysis Reveals Astral Microtubule Detachment in the Yeast Spindle Pole Mutantcnm67

2000 ◽  
Vol 11 (4) ◽  
pp. 1197-1211 ◽  
Author(s):  
Dominic Hoepfner ◽  
Arndt Brachat ◽  
Peter Philippsen
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Attachment Site ◽  
Time Lapse ◽  
Spindle Pole ◽  
Cell Cycles ◽  
Astral Microtubule ◽  
Normal Behavior ◽  
Green Fluorescent ◽  
Diploid Cells

Saccharomyces cerevisiae cnm67Δ cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein–labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the γ-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Δ cells Spc72–γ-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Δ cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.

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

2011 ◽  
Vol 435 (3) ◽  
pp. 661-667 ◽  
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.

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

2007 ◽  
Vol 178 (4) ◽  
pp. 595-610 ◽  
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.

Lithium blocks cell cycle transitions in the first cell cycles of sea urchin embryos, an effect rescued by myo-inositol

Development ◽  
1997 ◽  
Vol 124 (6) ◽  
pp. 1099-1107 ◽  
Author(s):  
A. Becchetti ◽  
M. Whitaker
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Sea Water ◽  
Cleavage Stage ◽  
Teratogenic Effects ◽  
Cell Cycles ◽  
Lytechinus Pictus ◽  
Nuclear Envelope Breakdown ◽  
Phosphoinositide Pathway

Lithium is a classical inhibitor of the phosphoinositide pathway and is teratogenic. We report the effects of lithium on the first cell cycles of sea urchin (Lytechinus pictus) embryos. Embryos cultured in 400 mM lithium chloride sea water showed marked delay to the cell cycle and a tendency to arrest prior to nuclear envelope breakdown, at metaphase and at cytokinesis. After removal of lithium, the block was reversed and embryos developed to form normal late blastulae. The lithium-induced block was also reversed by myo- but not epi-inositol, indicating that lithium was acting via the phosphoinositide pathway. Lithium microinjection before fertilization caused arrest prior to nuclear envelope breakdown at much lower concentrations (3-5 mM). Co-injection of myo-inositol prevented the block. Microinjection of 1–2 mM lithium led to block at the cleavage stage. This was also reversed by coinjection of myo-inositol. Embryos blocked by lithium microinjection proceeded rapidly into mitosis after photolysis of caged inositol 1,4,5-trisphosphate. These data demonstrate that a patent phosphoinositide signalling pathway is essential for the proper timing of cell cycle transitions and offer a possible explanation for lithium's teratogenic effects.

scholarly journals Organization and Dynamics of Growing Microtubule Plus Ends during Early Mitosis

2003 ◽  
Vol 14 (3) ◽  
pp. 916-925 ◽  
Author(s):  
Michelle Piehl ◽  
Lynne Cassimeris
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Stable Cell Line ◽  
Cell Periphery ◽  
Late Prophase ◽  
Nuclear Envelope Breakdown ◽  
Cycle Dependent ◽  
Green Fluorescent ◽  
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.

scholarly journals Changes in Organization of the Endoplasmic Reticulum duringXenopus Oocyte Maturation and Activation

2001 ◽  
Vol 12 (4) ◽  
pp. 1103-1116 ◽  
Author(s):  
Mark Terasaki ◽  
Linda L. Runft ◽  
Arthur R. Hand
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Polar Body ◽  
Meiotic Spindle ◽  
Body Formation ◽  
Nuclear Envelope Breakdown ◽  
First Polar Body ◽  
Polar Body Formation ◽  
Green Fluorescent ◽  
Small Clusters

The organization of the endoplasmic reticulum (ER) in the cortex ofXenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releaseability of Ca2+ by IP3. The clusters dispersed during the Ca2+ wave at activation. Possible relationships of ER structure and Ca2+ regulation are discussed.

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