scholarly journals Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos.

1990 ◽  
Vol 111 (6) ◽  
pp. 2815-2828 ◽  
Author(s):  
Y Hiraoka ◽  
D A Agard ◽  
J W Sedat
Keyword(s):  
Drosophila Melanogaster ◽  
Nuclear Envelope ◽  
Temporal Dynamics ◽  
Three Dimensional ◽  
Metaphase Plate ◽  
Time Lapse ◽  
Three Dimensions ◽  
Nuclear Envelope Breakdown ◽  
High Resolution Analysis ◽  
Nuclear Structures

The spatial and temporal dynamics of diploid chromosome organization, microtubule arrangement, and the state of the nuclear envelope have been analyzed in syncytial blastoderm embryos of Drosophila melanogaster during the transition from prophase to metaphase, by three-dimensional optical sectioning microscopy. Time-lapse, three-dimensional data recorded in living embryos revealed that congression of chromosomes (the process whereby chromosomes move to form the metaphase plate) at prometaphase occurs as a wave, starting at the top of the nucleus near the embryo surface and proceeding through the nucleus to the bottom. The time-lapse analysis was augmented by a high-resolution analysis of fixed embryos where it was possible to unambiguously trace the three-dimensional paths of individual chromosomes. In prophase, the centromeres were found to be clustered at the top of the nucleus while the telomeres were situated at the bottom of the nucleus or towards the embryo interior. This polarized centromere-telomere orientation, perpendicular to the embryo surface, was preserved during the process of prometaphase chromosome congression. Correspondingly, breakdown of the nuclear envelope started at the top of the nucleus with the mitotic spindle being formed at the positions of the partial breakdown of the nuclear envelope. Our observation provide an example in which nuclear structures are spatially organized and their functions are locally and coordinately controlled in three dimensions.

scholarly journals Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lillian K Fritz-Laylin ◽  
Megan Riel-Mehan ◽  
Bi-Chang Chen ◽  
Samuel J Lord ◽  
Thomas D Goddard ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Light Sheet ◽  
Time Lapse ◽  
Three Dimensions ◽  
Cortical Actin ◽  
Linear Arrangement ◽  
Collagen Matrices ◽  
Flat Surfaces ◽  
Actin Networks ◽  
Light Sheet Microscopy

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

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 Budding yeast chromatin is dispersed in a crowded nucleoplasm in vivo

2016 ◽  
Vol 27 (21) ◽  
pp. 3357-3368 ◽  
Author(s):  
Chen Chen ◽  
Hong Hwa Lim ◽  
Jian Shi ◽  
Sachiko Tamura ◽  
Kazuhiro Maeshima ◽  
...  
Keyword(s):  
Budding Yeast ◽  
Three Dimensional ◽  
Chromatin Organization ◽  
Three Dimensions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nuclear Structures ◽  
Three Dimensional Models ◽  
Electron Cryotomography ◽  
Fundamental Units

Chromatin organization has an important role in the regulation of eukaryotic systems. Although recent studies have refined the three-dimensional models of chromatin organization with high resolution at the genome sequence level, little is known about how the most fundamental units of chromatin—nucleosomes—are positioned in three dimensions in vivo. Here we use electron cryotomography to study chromatin organization in the budding yeast Saccharomyces cerevisiae. Direct visualization of yeast nuclear densities shows no evidence of 30-nm fibers. Aside from preribosomes and spindle microtubules, few nuclear structures are larger than a tetranucleosome. Yeast chromatin does not form compact structures in interphase or mitosis and is consistent with being in an “open” configuration that is conducive to high levels of transcription. From our study and those of others, we propose that yeast can regulate its transcription using local nucleosome–nucleosome associations.

scholarly journals Direct cell lineage analysis in Drosophila melanogaster by time-lapse, three-dimensional optical microscopy of living embryos.

1989 ◽  
Vol 109 (2) ◽  
pp. 505-516 ◽  
Author(s):  
J S Minden ◽  
D A Agard ◽  
J W Sedat ◽  
B M Alberts
Keyword(s):  
Drosophila Melanogaster ◽  
Three Dimensional ◽  
Cell Lineage ◽  
Time Lapse ◽  
Small Region ◽  
Early Embryo ◽  
Nuclear Cycle ◽  
Cell Position ◽  
History Of ◽  
Direct Cell

One of the first signs of cell differentiation in the Drosophila melanogaster embryo occurs 3 h after fertilization, when discrete groups of cells enter their fourteenth mitosis in a spatially and temporally patterned manner creating mitotic domains (Foe, V. E. and G. M. Odell, 1989, Am. Zool. 29:617-652). To determine whether cell residency in a mitotic domain is determined solely by cell position in this early embryo, or whether cell lineage also has a role, we have developed a technique for directly analyzing the behavior of nuclei in living embryos. By microinjecting fluorescently labeled histones into the syncytial embryo, the movements and divisions of each nucleus were recorded without perturbing development by using a microscope equipped with a high resolution, charge-coupled device. Two types of developmental maps were generated from three-dimensional time-lapse recordings: one traced the lineage history of each nucleus from nuclear cycle 11 through nuclear cycle 14 in a small region of the embryo; the other recorded nuclear fate according to the timing and pattern of the 14th nuclear division. By comparing these lineage and fate maps for two embryos, we conclude that, at least for the examined area, the pattern of mitotic domain formation in Drosophila is determined by the position of each cell, with no effect of cell lineage.

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 Ordered Kinetochore Assembly in the Human-Pathogenic Basidiomycetous Yeast Cryptococcus neoformans

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Lukasz Kozubowski ◽  
Vikas Yadav ◽  
Gautam Chatterjee ◽  
Shreyas Sridhar ◽  
Masashi Yamaguchi ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Cryptococcus Neoformans ◽  
Budding Yeast ◽  
Genetic Material ◽  
Time Lapse ◽  
Content Type ◽  
Nuclear Envelope Breakdown ◽  
Kinetochore Assembly ◽  
Transmission Electron ◽  
Partial Opening

ABSTRACT Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes. IMPORTANCE Successful propagation of genetic material in progeny is essential for the survival of any organism. A proper kinetochore-microtubule interaction is crucial for high-fidelity chromosome segregation. An error in this process can lead to loss or gain of chromosomes, a common feature of most solid cancers. Several proteins assemble on centromere DNA to form a kinetochore. However, significant differences in the process of kinetochore assembly exist between unicellular yeasts and multicellular metaozoa. Here, we examined the key events that lead to formation of a proper kinetochore in a basidiomycetous budding yeast, Cryptococcus neoformans. We found that, during the progression of the cell cycle, nonclustered centromeres gradually clustered and kinetochores assembled in an ordered manner concomitant with partial opening of the nuclear envelope in this organism. These events have higher similarity to mitotic events of metazoans than to those previously described in other yeasts.

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 Mitotic checkpoint protein Mad1 is required for early Nup153 recruitment to chromatin and nuclear envelope integrity

2020 ◽  
Author(s):  
Ikram Mossaid ◽  
Guillaume Chatel ◽  
Valérie Martinelli ◽  
Marcela Vaz ◽  
Birthe Fahrenkrog
Keyword(s):  
Nuclear Envelope ◽  
Three Dimensional ◽  
Time Lapse ◽  
Mitotic Checkpoint ◽  
Membrane Curvature ◽  
Nuclear Pore ◽  
Structured Illumination ◽  
Electron Microscopic ◽  
Checkpoint Protein ◽  
Mitotic Checkpoint Protein

AbstractThe nucleoporin Nup153 is a multifunctional protein and the mitotic checkpoint protein Mad1one of its many binding partners. The functional relevance of their interaction has remained elusive. Here, we have further dissected Nup153’s and Mad1’s interface and functional interplay. By in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is prerequisite for the Nup153-Mad1 interaction. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of Nup153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, Nup153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of the membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between the inner and outer nuclear membranes. Nup153 depletion, but not of Mad1, caused defects in interphase NPC assembly with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Together our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE reformation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.SummaryThe mitotic checkpoint protein is required for Nup153 recruitment to anaphase chromatin and in turn post-mitotic, but not interphase nuclear pore complex assembly.

scholarly journals Mitotic checkpoint protein Mad1 is required for early Nup153 recruitment to chromatin and nuclear envelope integrity

2020 ◽  
Vol 133 (21) ◽  
pp. jcs249243
Author(s):  
Ikram Mossaid ◽  
Guillaume Chatel ◽  
Valérie Martinelli ◽  
Marcela Vaz ◽  
Birthe Fahrenkrog
Keyword(s):  
Nuclear Envelope ◽  
Three Dimensional ◽  
Time Lapse ◽  
Mitotic Checkpoint ◽  
Membrane Curvature ◽  
Nuclear Pore ◽  
Structured Illumination ◽  
Electron Microscopic ◽  
Checkpoint Protein ◽  
Mitotic Checkpoint Protein

ABSTRACTNucleoporin Nup153 is a multifunctional protein and a known binding partner of mitotic checkpoint protein Mad1 (also known as MAD1L1). The functional relevance of their interaction has remained elusive. Here, we have further dissected the interface and functional interplay of Nup153 and Mad1. Using in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is a prerequisite for the Nup153–Mad1 association. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of Nup153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, Nup153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between inner and outer nuclear membranes. Nup153 depletion, but not Mad1 depletion, caused defects in interphase NPC assembly, with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Taken together, our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE re-formation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.

Changes in the metaphase transit times and the pattern of sister chromatid separation in stamen hair cells of Tradescantia after treatment with protein phosphatase inhibitors

1992 ◽  
Vol 102 (4) ◽  
pp. 691-715 ◽  
Author(s):  
S.M. Wolniak ◽  
P.M. Larsen
Keyword(s):  
Nuclear Envelope ◽  
Transit Time ◽  
Okadaic Acid ◽  
Hair Cells ◽  
Protein Phosphatase ◽  
Metaphase Plate ◽  
Spindle Pole ◽  
Phosphatase Inhibitors ◽  
Nuclear Envelope Breakdown ◽  
Transit Times

Stamen hair cells from the spiderwort plant, Tradescantia virginiana, exhibit remarkably predictable metaphase transit times, making them uniquely suitable for temporal studies on mitotic regulation. In this study, we describe two kinds of experiments that test whether protein phosphatase activity is a necessary prerequisite for entry into anaphase in living, mitotic cells. We treated cells at specific points during prophase, prometaphase and metaphase with the broad-spectrum protein phosphatase inhibitor, alpha-naphthyl phosphate (administered by microinjection), or with the naturally occurring, potent phosphatase inhibitors okadaic acid, microcystin-LR or microcystin-RR (administered by perfusion), and we have observed changes in the metaphase transit time that are primarily dependent on the time of initial exposure to the inhibitor. Maximal extensions of the metaphase transit time result from alpha-naphthyl phosphate microinjections initiated in mid-metaphase, 10–20 min after nuclear envelope breakdown. Perfusions with okadaic acid started during a specific interval in mid-metaphase, 15–20 min after nuclear envelope breakdown, resulted in a statistically significant extension of the metaphase transit time. Perfusions with either microcystin-LR or microcystin-RR initiated 15–26 min after nuclear envelope breakdown extended the metaphase transit times significantly. Treatments of cells with okadaic acid or with either of the microcystins initiated outside this mid-metaphase interval either were without effect or, alternatively, resulted in a significant shortening of the metaphase transit time. In addition to their effects on the timing of anaphase onset, treatments with these protein phosphatase inhibitors also resulted in a remarkable change in the way in which these cells enter anaphase. Sister chromatid separation in stamen hair cells typically requires only 5 seconds, but after treatment with any of these inhibitors some, but not all, of the chromatids split apart at anaphase onset. Those that split begin to migrate toward the spindle pole regions, while those that fail to split remain at the metaphase plate. Later, more of the paired chromatids split apart and begin moving toward the spindle pole regions. Those that fail to separate remain at the metaphase plate. This process can be repeated several times before all of the chromatids have separated. Thus, entry into anaphase becomes extremely asynchronous, and as much as 30 min can transpire between the centromeric separation of the first and last chromosomes. Some of the chromosomes complete their anaphase movements before others have even split apart at the metaphase plate. Asynchronous separation did not result in a permanent segregation anomaly.(ABSTRACT TRUNCATED AT 400 WORDS)

Sign in / Sign up

Export Citation Format

Share Document