scholarly journals Closed mitosis requires local disassembly of the nuclear envelope

2019 ◽  
Author(s):  
Gautam Dey ◽  
Siân Culley ◽  
Scott Curran ◽  
Ricardo Henriques ◽  
Wanda Kukulski ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Electron Tomography ◽  
Classical Model ◽  
Nuclear Fission ◽  
Nuclear Compartment ◽  
Nuclear Compartments ◽  
Intact Nucleus ◽  
Narrow Bridge

At the end of mitosis, eukaryotic cells must segregate both copies of their replicated genome into two new nuclear compartments (1). They do this either by first dismantling and later reassembling the nuclear envelope in a so called “open mitosis”, or by reshaping an intact nucleus and then dividing into two in a “closed mitosis” (2, 3). However, while mitosis has been studied in a wide variety of eukaryotes for over a century (4), it is not known how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment (5). In studying this problem in the fission yeast Schizosaccharomyces pombe, a classical model for closed mitosis (5), we use genetics, live cell imaging and electron tomography to show that nuclear fission is achieved via local disassembly of the nuclear envelope (NE) within the narrow bridge that links segregating daughter nuclei. In doing so, we identify a novel inner NE-localised protein Les1 that restricts the process of local NE breakdown (local NEB) to the bridge midzone and prevents the leakage of material from daughter nuclei. The mechanics of local NEB in a closed mitosis closely mirror those of NEB in open mitosis (3), revealing an unexpectedly deep conservation of nuclear remodelling mechanisms across diverse eukaryotes.

scholarly journals Cdc42 GTPase regulates ESCRTs in nuclear envelope sealing and ER remodeling

2020 ◽  
Vol 219 (8) ◽  
Author(s):  
Michelle Seiko Lu ◽  
David G. Drubin
Keyword(s):  
Cell Migration ◽  
Genetic Analysis ◽  
Nuclear Envelope ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Molecular Switches ◽  
Small Gtpases ◽  
Cytoskeletal Remodeling ◽  
Cell Divisions ◽  
Rho Family

Small GTPases of the Rho family are binary molecular switches that regulate a variety of processes including cell migration and oriented cell divisions. Known Cdc42 effectors include proteins involved in cytoskeletal remodeling and kinase-dependent transcription induction, but none are involved in the maintenance of nuclear envelope integrity or ER morphology. Maintenance of nuclear envelope integrity requires the EndoSomal Complexes Required for Transport (ESCRT) proteins, but how they are regulated in this process remains unknown. Here, we show by live-cell imaging a novel Cdc42 localization with ESCRT proteins at sites of nuclear envelope and ER fission and, by genetic analysis of cdc42 mutant yeast, uncover a unique Cdc42 function in regulation of ESCRT proteins at the nuclear envelope and sites of ER tubule fission. Our findings implicate Cdc42 in nuclear envelope sealing and ER remodeling, where it regulates ESCRT disassembly to maintain nuclear envelope integrity and proper ER architecture.

scholarly journals Nucleoplasmic Lamin C Rapidly Accumulates at Sites of Nuclear Envelope Rupture with BAF and cGAS

2022 ◽  
Author(s):  
Yohei Kono ◽  
Stephen A. Adam ◽  
Karen Reddy ◽  
Yixian Zheng ◽  
Ohad Medalia ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Nuclear Lamina ◽  
Structural Components ◽  
Cell Nuclei ◽  
Embryonic Fibroblasts ◽  
Nuclear Lamins ◽  
Laser Microirradiation

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that lamin C but not the other lamin isoforms rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The immunoglobulin-like fold domain and the NLS were required for the recruitment from the nucleoplasm to the rupture sites with the Barrier-to-autointegration factor (BAF). The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP (cGAMP) synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF and cGAS concertedly accumulate at sites of NE rupture for repair.

scholarly journals Development for Dynamic Live Cell Imaging by Cryo-Electron Tomography and Stem

2013 ◽  
Vol 104 (2) ◽  
pp. 354a
Author(s):  
Atsuko H. Iwane ◽  
Ruriko Ogawa ◽  
Rina Nagai ◽  
Akihiro Kawamoto ◽  
Kazuhiro Aoyama
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Electron Tomography ◽  
Live Cell ◽  
Cryo Electron Tomography

scholarly journals C. elegans chromosomes connect to centrosomes by anchoring into the spindle network

2016 ◽  
Author(s):  
Stefanie Redemann ◽  
Johannes Baumgart ◽  
Norbert Lindow ◽  
Sebastian Fürthauer ◽  
Ehssan Nazockdast ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Mitotic Spindle ◽  
Live Cell Imaging ◽  
Large Scale ◽  
Cell Imaging ◽  
Electron Tomography ◽  
Mitotic Spindles ◽  
C Elegans ◽  
Microtubule Growth ◽  
Segregation Of Chromosomes

AbstractThe mitotic spindle ensures the faithful segregation of chromosomes. To discover the nature of the crucial centrosome-to-chromosome connection during mitosis, we combined the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging. Using tomography, we reconstructed the positions of all microtubules in 3D, and identified their plus- and minus-ends. We classified them as kinetochore (KMTs), spindle (SMTs), or astral microtubules (AMTs) according to their positions, and quantified distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs are directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.

scholarly journals Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 449
Author(s):  
Melanie B. Horev ◽  
Yishaia Zabary ◽  
Revital Zarka ◽  
Simona Sorrentino ◽  
Ohad Medalia ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Platelet Adhesion ◽  
Cell Imaging ◽  
Electron Tomography ◽  
Collagen Iv ◽  
Short Term ◽  
Functionalized Surfaces ◽  
Cryo Electron Tomography ◽  
Coated Surfaces ◽  
Adhesion Process

Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet’s periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.

scholarly journals Torsin ATPases are required to complete nuclear pore complex biogenesis in interphase

2019 ◽  
Author(s):  
Anthony J. Rampello ◽  
Ethan Laudermilch ◽  
Nidhi Vishnoi ◽  
Sarah M. Prohet ◽  
Lin Shao ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Nuclear Pore ◽  
Ubiquitin Conjugation ◽  
Bleb Formation ◽  
Kinetics Of

AbstractNuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based approach to identify MLF2 as a luminal component of the bleb. Using an MLF2-based live cell imaging platform, we demonstrate that NE blebbing occurs rapidly and synchronously immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its dependence on POM121, and a reduction of mature NPCs in Torsin deficient cells lead us to conclude that the hallmark phenotype of Torsin manipulation represents the accumulation of stalled NPC assembly intermediates.

scholarly journals Torsin ATPase deficiency leads to defects in nuclear pore biogenesis and sequestration of MLF2

2020 ◽  
Vol 219 (6) ◽  
Author(s):  
Anthony J. Rampello ◽  
Ethan Laudermilch ◽  
Nidhi Vishnoi ◽  
Sarah M. Prophet ◽  
Lin Shao ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Myeloid Leukemia ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Nuclear Pore ◽  
Ubiquitin Conjugation ◽  
Bleb Formation ◽  
Kinetics Of

Nuclear envelope herniations (blebs) containing FG-nucleoporins and ubiquitin are the phenotypic hallmark of Torsin ATPase manipulation. Both the dynamics of blebbing and the connection to nuclear pore biogenesis remain poorly understood. We employ a proteomics-based approach to identify myeloid leukemia factor 2 (MLF2) as a luminal component of the bleb. Using an MLF2-based live-cell imaging platform, we demonstrate that nuclear envelope blebbing occurs rapidly and synchronously immediately after nuclear envelope reformation during mitosis. Bleb formation is independent of ubiquitin conjugation within the bleb, but strictly dependent on POM121, a transmembrane nucleoporin essential for interphase nuclear pore biogenesis. Nup358, a late marker for interphase nuclear pore complex (NPC) biogenesis, is underrepresented relative to FG-nucleoporins in nuclear envelopes of Torsin-deficient cells. The kinetics of bleb formation, its dependence on POM121, and a reduction of mature NPCs in Torsin-deficient cells lead us to conclude that the hallmark phenotype of Torsin manipulation represents aberrant NPC intermediates.

scholarly journals Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia

2020 ◽  
Vol 220 (1) ◽  
Author(s):  
Landi Sun ◽  
Lihong Cui ◽  
Zhen Liu ◽  
Qixuan Wang ◽  
Zhaoyu Xue ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Electron Tomography ◽  
3D Structure ◽  
Functional Roles ◽  
Microtubule Severing ◽  
Structure And Dynamics ◽  
Sensory Cilia ◽  
Potential Factors ◽  
Neuronal Type

Mechanoreceptor cells develop a specialized cytoskeleton that plays structural and sensory roles at the site of mechanotransduction. However, little is known about how the cytoskeleton is organized and formed. Using electron tomography and live-cell imaging, we resolve the 3D structure and dynamics of the microtubule-based cytoskeleton in fly campaniform mechanosensory cilia. Investigating the formation of the cytoskeleton, we find that katanin p60-like 1 (kat-60L1), a neuronal type of microtubule-severing enzyme, serves two functions. First, it amplifies the mass of microtubules to form the dense microtubule arrays inside the sensory cilia. Second, it generates short microtubules that are required to build the nanoscopic cytoskeleton at the mechanotransduction site. Additional analyses further reveal the functional roles of Patronin and other potential factors in the local regulatory network. In all, our results characterize the specialized cytoskeleton in fly external mechanosensory cilia at near-molecular resolution and provide mechanistic insights into how it is formed.

scholarly journals 2PS043 Development for dynamic Live Cell imaging by cryo-electron tomography(The 50th Annual Meeting of the Biophysical Society of Japan)

2012 ◽  
Vol 52 (supplement) ◽  
pp. S117
Author(s):  
Atsuko H. Iwane ◽  
Ruriko Ogawa ◽  
Rina Nagai ◽  
Akihiro Kawamoto ◽  
Kazuhiro Aoyama
Keyword(s):  
Annual Meeting ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Electron Tomography ◽  
Live Cell ◽  
Cryo Electron Tomography ◽  
Biophysical Society
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