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 Cell adaptive response to extracellular matrix density is controlled by ICAP-1–dependent β1-integrin affinity

2008 ◽  
Vol 180 (2) ◽  
pp. 427-441 ◽  
Author(s):  
Angélique Millon-Frémillon ◽  
Daniel Bouvard ◽  
Alexei Grichine ◽  
Sandra Manet-Dupé ◽  
Marc R. Block ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Β1 Integrin ◽  
Embryonic Fibroblasts ◽  
Matrix Surface ◽  
Extracellular Microenvironment ◽  
And Migration ◽  
Integrated Response ◽  
Insight Into

Cell migration is an integrated process requiring the continuous coordinated assembly and disassembly of adhesion structures. How cells orchestrate adhesion turnover is only partially understood. We provide evidence for a novel mechanistic insight into focal adhesion (FA) dynamics by demonstrating that integrin cytoplasmic domain–associated protein 1 (ICAP-1) slows down FA assembly. Live cell imaging, which was performed in both Icap-1–deficient mouse embryonic fibroblasts and cells expressing active β1 integrin, shows that the integrin high affinity state favored by talin is antagonistically controlled by ICAP-1. This affinity switch results in modulation in the speed of FA assembly and, consequently, of cell spreading and migration. Unexpectedly, the ICAP-1–dependent decrease in integrin affinity allows cell sensing of matrix surface density, suggesting that integrin conformational changes are important in mechanotransduction. Our results clarify the function of ICAP-1 in cell adhesion and highlight the central role it plays in the cell's integrated response to the extracellular microenvironment.

scholarly journals Supramolecular Structures of the Dictyostelium Lamin NE81

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 162
Author(s):  
Marianne Grafe ◽  
Petros Batsios ◽  
Irene Meyer ◽  
Daria Lisin ◽  
Otto Baumann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Model Organism ◽  
Super Resolution ◽  
Nuclear Lamina ◽  
Stimulated Emission ◽  
Structural Level ◽  
Animal Cells ◽  
Nuclear Lamins

Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.

scholarly journals LRRK2 mediates tubulation and vesicle sorting from membrane damaged lysosomes

2020 ◽  
Author(s):  
Luis Bonet-Ponce ◽  
Alexandra Beilina ◽  
Chad D. Williamson ◽  
Eric Lindberg ◽  
Jillian H. Kluss ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Leucine Rich Repeat ◽  
Biological Functions ◽  
New Process ◽  
Sporadic Parkinson’S Disease

ABSTRACTMutations in the leucine rich repeat kinase 2 (LRRK2) gene are a cause of familial and sporadic Parkinson’s disease (PD). Nonetheless, the biological functions of LRRK2 remain incompletely understood. Here, we observed that LRRK2 is recruited to lysosomes that have a ruptured membrane. Using unbiased proteomics, we observed that LRRK2 is able to recruit the motor adaptor protein JIP4 to permeabilized lysosomes in a kinase-dependent manner through the phosphorylation of RAB35 and RAB10. Super-resolution live cell imaging microscopy and FIB-SEM revealed that once at the lysosomal membrane, JIP4 promotes the formation of LAMP1-negative lysosomal tubules that release membranous content from ruptured lysosomes. Released vesicular structures are able to interact with other lysosomes. Thus, we described a new process that uses lysosomal tubulation to release vesicular structures from permeabilized lysosomes. LRRK2 orchestrates this process that we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2) that, given the central role of the lysosome in PD, is likely to be disease relevant.

scholarly journals EB1 binding restricts STIM1 translocation to ER–PM junctions and regulates store-operated Ca2+ entry

2018 ◽  
Vol 217 (6) ◽  
pp. 2047-2058 ◽  
Author(s):  
Chi-Lun Chang ◽  
Yu-Ju Chen ◽  
Carlo Giovanni Quintanilla ◽  
Ting-Sung Hsieh ◽  
Jen Liou
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Cellular Functions ◽  
Binding Ability ◽  
Trapping Mechanism ◽  
Spatiotemporal Regulation

The endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER–plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) after ER Ca2+ depletion. STIM1 also interacts with EB1 and dynamically tracks microtubule (MT) plus ends. Nevertheless, the role of STIM1–EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with a synthetic construct approach, we found that EB1 binding constitutes a trapping mechanism restricting STIM1 targeting to ER–PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. By trapping STIM1 molecules at dynamic contacts between the ER and MT plus ends, EB1 binding delayed STIM1 translocation to ER–PM junctions during ER Ca2+ depletion and prevented excess SOCE and ER Ca2+ overload. Our study suggests that STIM1–EB1 interaction shapes the kinetics and amplitude of local SOCE in cellular regions with growing MTs and contributes to spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.

Lipid order and molecular assemblies in the plasma membrane of eukaryotic cells

2009 ◽  
Vol 37 (5) ◽  
pp. 1056-1060 ◽  
Author(s):  
Marek Cebecauer ◽  
Dylan M. Owen ◽  
Anna Markiewicz ◽  
Anthony I. Magee
Keyword(s):  
Plasma Membrane ◽  
Physical Properties ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Eukaryotic Cells ◽  
Dynamic Nature ◽  
Membrane Components ◽  
Technological Improvements

Multimolecular assemblies on the plasma membrane exhibit dynamic nature and are often generated during the activation of eukaryotic cells. The role of lipids and their physical properties in helping to control the existence of these structures is discussed. Technological improvements for live cell imaging of membrane components are also reviewed.

scholarly journals A Mutant Defective in Sexual Development Produces Aseptate Ascogonia

2010 ◽  
Vol 9 (12) ◽  
pp. 1856-1866 ◽  
Author(s):  
Sandra Bloemendal ◽  
Kathryn M. Lord ◽  
Christine Rech ◽  
Birgit Hoff ◽  
Ines Engh ◽  
...  
Keyword(s):  
Sexual Development ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Sexual Cycle ◽  
Sequence Analyses ◽  
Gene Encoding ◽  
Content Type ◽  
Domain Of Unknown Function ◽  
Sterile Mutant

ABSTRACT The transition from the vegetative to the sexual cycle in filamentous ascomycetes is initiated with the formation of ascogonia. Here, we describe a novel type of sterile mutant from Sordaria macrospora with a defect in ascogonial septum formation. This mutant, named pro22, produces only small, defective protoperithecia and carries a point mutation in a gene encoding a protein that is highly conserved throughout eukaryotes. Sequence analyses revealed three putative transmembrane domains and a C-terminal domain of unknown function. Live-cell imaging showed that PRO22 is predominantly localized in the dynamic tubular and vesicular vacuolar network of the peripheral colony region close to growing hyphal tips and in ascogonia; it is absent from the large spherical vacuoles in the vegetative hyphae of the subperipheral region of the colony. This points to a specific role of PRO22 in the tubular and vesicular vacuolar network, and the loss of intercalary septation in ascogonia suggests that PRO22 functions during the initiation of sexual development.

scholarly journals EB1 binding provides a diffusion trap mechanism regulating STIM1 localization and Ca2+ signaling

2017 ◽  
Author(s):  
Chi-Lun Chang ◽  
Yu-Ju Chen ◽  
Jen Liou
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Time Lapse ◽  
Cellular Functions ◽  
Binding Ability ◽  
Spatiotemporal Regulation ◽  
Time Lapse Imaging

AbstractThe endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER-plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion. STIM1 also directly interacts with end binding protein 1 (EB1) at microtubule (MT) plus-ends and resembles comet-like structures during time-lapse imaging. Nevertheless, the role of STIM1-EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with pharmacological perturbation and a reconstitution approach, we revealed that EB1 binding constitutes a diffusion trap mechanism restricting STIM1 targeting to ER-PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. EB1 binding delayed the translocation of STIM1 oligomers to ER-PM junctions and recaptured STIM1 to prevent excess SOCE and ER Ca2+ overload. Thus, the counterbalance of EB1 binding and PM targeting of STIM1 shapes the kinetics and amplitude of local SOCE in regions with growing MTs, and contributes to precise spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.SummarySTIM1 activates store-operated Ca2+ entry (SOCE) by translocating to endoplasmic reticulum-plasma membrane junctions. Chang et al. revealed that STIM1 localization and SOCE are regulated by a diffusion trap mechanism mediated by STIM1 binding to EB1 at growing microtubule ends.

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 Brain and Breast Cancer Cells with PTEN Loss of Function Demonstrate Enhanced Durotaxis and RHOB Dependent Amoeboid Migration Using 3D Printed Scaffolds and Aligned Microfiber Tracts

2021 ◽  
Author(s):  
Annalena Wieland ◽  
Pamela L. Strissel ◽  
Hannah Schorle ◽  
Ezgi Bakirci ◽  
Dieter Janzen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Morphology ◽  
Live Cell Imaging ◽  
Essential Role ◽  
Cell Imaging ◽  
Loss Of Function ◽  
Pten Loss ◽  
3D Printed ◽  
And Migration

Abstract Background: Glioblastoma multiforme (GBM) and triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with very poor patient outcomes. GBM uses axons and vessels as migratory cues to disseminate, however it is not known, if TNBC shares the same behavior. There is a need to understand brain tumor cell spreading and if GBM and TNBC have similar migration properties involving the signaling pathway RHOB-ROCK-PTEN. We tested for durotaxis, adherence and migration of GBM and TNBC using live-cell imaging and performed molecular analyses on three-dimensional (3D) structures.Methods: Aligned 3D printed scaffolds and microfibers were designed to mimic brain axon tracts and vessels for migration. GBM and TNBC cell lines, each with opposing PTEN genotypes, were analysed with RHO, ROCK and PTEN inhibitors and rescuing PTEN function using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while SEM, confocal microscopy and cell tracking addressed cell morphology. Results: GBM and TNBC with homozygote PTEN loss of function and RHOB high expression were amoeboid shaped and demonstrated enhanced durotaxis, adhesion and migration on 3D microfibers, in contrast to PTEN wildtype GBM and TNBC showing elongated cells and low RHOB. RNA-sequencing exhibited that RHOB was significantly the highest expressed gene in GBM PTEN loss of function cells. Pathway inhibitors and PTEN rescue of function verified an essential role of RHOB-ROCK-PTEN signaling for durotaxis, adhesion, migration, cell morphology and plasticity using 3D printed microfibers. Conclusions: This study validates a significant role of a PTEN genotype for cellular properties including durotaxis, adhesion and migration. GBM and TNBC cells with PTEN loss of function have a greater affinity for stiffer brain structures promoting metastasis. We propose the significance of PTEN and RHOB in cellular oncology not only for primary tumors, but also for metastasizing tumors, where RHOB inhibitors could play an essential role for improved therapy.

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