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.

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 Bub1 mediates cell death in response to chromosome missegregation and acts to suppress spontaneous tumorigenesis

2007 ◽  
Vol 179 (2) ◽  
pp. 255-267 ◽  
Author(s):  
Karthik Jeganathan ◽  
Liviu Malureanu ◽  
Darren J. Baker ◽  
Susan C. Abraham ◽  
Jan M. van Deursen
Keyword(s):  
Cell Death ◽  
Chromosome Segregation ◽  
Physiological Role ◽  
Mitotic Checkpoint ◽  
Critical Threshold ◽  
Wild Type ◽  
Checkpoint Protein ◽  
Chromosome Missegregation ◽  
Mitotic Checkpoint Protein

The physiological role of the mitotic checkpoint protein Bub1 is unknown. To study this role, we generated a series of mutant mice with a gradient of reduced Bub1 expression using wild-type, hypomorphic, and knockout alleles. Bub1 hypomorphic mice are viable, fertile, and overtly normal despite weakened mitotic checkpoint activity and high percentages of aneuploid cells. Bub1 haploinsufficient mice, which have a milder reduction in Bub1 protein than Bub1 hypomorphic mice, also exhibit reduced checkpoint activity and increased aneuploidy, but to a lesser extent. Although cells from Bub1 hypomorphic and haploinsufficient mice have similar rates of chromosome missegregation, cell death after an aberrant separation decreases dramatically with declining Bub1 levels. Importantly, Bub1 hypomorphic mice are highly susceptible to spontaneous tumors, whereas Bub1 haploinsufficient mice are not. These findings demonstrate that loss of Bub1 below a critical threshold drives spontaneous tumorigenesis and suggest that in addition to ensuring proper chromosome segregation, Bub1 is important for mediating cell death when chromosomes missegregate.

scholarly journals Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components.

1990 ◽  
Vol 110 (4) ◽  
pp. 883-894 ◽  
Author(s):  
R Reichelt ◽  
A Holzenburg ◽  
E L Buhle ◽  
M Jarnik ◽  
A Engel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Nuclear Envelope ◽  
Three Dimensional ◽  
Structural Features ◽  
Nuclear Pore ◽  
Xenopus Laevis Oocyte ◽  
Three Dimensional Model ◽  
Transmission Electron ◽  
Freeze Dried

Nuclear pore complexes (NPCs) prepared from Xenopus laevis oocyte nuclear envelopes were studied in "intact" form (i.e., unexposed to detergent) and after detergent treatment by a combination of conventional transmission electron microscopy (CTEM) and quantitative scanning transmission electron microscopy (STEM). In correlation-averaged CTEM pictures of negatively stained intact NPCs and of distinct NPC components (i.e., "rings," "spoke" complexes, and "plug-spoke" complexes), several fine structural features arranged with octagonal symmetry about a central axis could reproducibly be identified. STEM micrographs of unstained/freeze-dried intact NPCs as well as of their components yielded comparable but less distinct features. Mass determination by STEM revealed the following molecular masses: intact NPC with plug, 124 +/- 11 MD; intact NPC without plug, 112 +/- 11 MD; heavy ring, 32 +/- 5 MD; light ring, 21 +/- 4 MD; plug-spoke complex, 66 +/- 8 MD; and spoke complex, 52 +/- 3 MD. Based on these combined CTEM and STEM data, a three-dimensional model of the NPC exhibiting eightfold centrosymmetry about an axis perpendicular to the plane of the nuclear envelope but asymmetric along this axis is proposed. This structural polarity of the NPC across the nuclear envelope is in accord with its well-documented functional polarity facilitating mediated nucleocytoplasmic exchange of molecules and particles.

scholarly journals The Vertebrate Mitotic Checkpoint Protein BUBR1 Is an Unusual Pseudokinase

2012 ◽  
Vol 22 (6) ◽  
pp. 1321-1329 ◽  
Author(s):  
Saskia J.E. Suijkerbuijk ◽  
Teunis J.P. van Dam ◽  
G. Elif Karagöz ◽  
Eleonore von Castelmur ◽  
Nina C. Hubner ◽  
...  
Keyword(s):  
Mitotic Checkpoint ◽  
Checkpoint Protein ◽  
Mitotic Checkpoint Protein

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 Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes

2020 ◽  
Author(s):  
Jiji Chen ◽  
Hideki Sasaki ◽  
Hoyin Lai ◽  
Yijun Su ◽  
Jiamin Liu ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Network Performance ◽  
Resolution Enhancement ◽  
Three Dimensional ◽  
Ground Truth ◽  
Time Lapse ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Attention Networks

Abstract We demonstrate residual channel attention networks (RCAN) for restoring and enhancing volumetric time-lapse (4D) fluorescence microscopy data. First, we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy 4D super-resolution data, enabling image capture over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables class-leading resolution enhancement, superior to other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling ~2.5-fold lateral resolution enhancement using stimulated emission depletion (STED) microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy ground truth, achieving improvements of ~1.4-fold laterally and ~3.4-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluating and further enhancing network performance.

The nuclear pore complex: three-dimensional surface structure revealed by field emission, in-lens scanning electron microscopy, with underlying structure uncovered by proteolysis

1993 ◽  
Vol 106 (1) ◽  
pp. 261-274 ◽  
Author(s):  
M.W. Goldberg ◽  
T.D. Allen
Keyword(s):  
Nuclear Pore Complex ◽  
Three Dimensional ◽  
Underlying Structure ◽  
Nuclear Pore ◽  
Electron Microscopic ◽  
Biologically Relevant ◽  
Fine Grained ◽  
Outer Periphery ◽  
Electron Microscopes ◽  
Scanning Electron

The structure of the nuclear pore complex (NPC) has been previously studied by many different electron microscopic techniques. Recently, scanning electron microscopes have been developed that can visualise biologically relevant structural detail at the same level of resolution as transmission electron microscopes and have been used to study NPC structure. We have used such an instrument to visualise directly the structure of both cytoplasmic and nucleoplasmic surfaces of the NPC of manually isolated amphibian oocyte nuclear envelopes that have been spread, fixed, critical point dried and coated with a thin fine-grained film of chromium or tantalum. We present images that directly show features of the NPC that are visible at each surface, including coaxial rings, cytoplasmic particles, plug/spoke complexes and the nucleoplasmic basket or fishtrap. Some cytoplasmic particles are rod-shaped or possibly “T”-shaped, can be quite long structures extending into the cytoplasm and may be joined to the coaxial ring at a position between each subunit. Both coaxial rings, which are proud of the membranes, can be exposed by light proteolytic digestion, revealing eight equal subunits each of which may be bipartite. We have determined that the nucleoplasmic filaments that make up the baskets are attached to the outer periphery of the coaxial ring at a position between each of its subunits. These filaments extend into the nucleoplasm and insert at the distal end to the smaller basket ring. The space left between adjacent basket filaments would exclude particles bigger than about 25 nm, which is consistent with the exclusion limit previously found for NPC-transported molecules.

The human mitotic checkpoint protein BubR1 regulates chromosome–spindle attachments

2004 ◽  
Vol 7 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Michael A. Lampson ◽  
Tarun M. Kapoor
Keyword(s):  
Mitotic Checkpoint ◽  
Checkpoint Protein ◽  
Mitotic Checkpoint Protein
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