The Endoplasmic Spreading Mechanism of Fibroblasts: Showcasing the Integrated Cytoskeleton
Cell motility is an essential process that depends on a coherent, cross-linked cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. In culture, a common feature of cells is the coherent movement of the endoplasmic reticulum and membranous organelles to...
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ndltd-columbia.edu-oai-academiccommons.columbia.edu-10.7916-D82805RB2019-05-09T15:14:24ZThe Endoplasmic Spreading Mechanism of Fibroblasts: Showcasing the Integrated CytoskeletonLynch, Christopher D.2012ThesesCytologyBiophysicsMolecular biologyCell motility is an essential process that depends on a coherent, cross-linked cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. In culture, a common feature of cells is the coherent movement of the endoplasmic reticulum and membranous organelles toward the periphery during substrate adhesion and spreading. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB-/- mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that leads to increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA-/- MEFs, but not FlnB-/- MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus I suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions. I also report that treatment with the calpain inhibitor N-[N-(N-Acetyl-L-leucyl)-L-leucyl]- L-norleucine (ALLN) restores endoplasmic spreading and focal adhesion (FA) maturation in the absence of Fln. Further, expression of calpain-uncleavable talin, but not full-length talin, also rescues endoplasmic spreading in Fln-depleted cells and indicates a crucial role for stable, mature FAs in endoplasmic spreading. Because FA maturation involves the vimentin intermediate filament (vIF) network, I also examined the role of vIFs in endoplasmic spreading. Wild-type cells expressing a dominant-negative vimentin variant incapable of vIF polymerization exhibit deficient endoplasmic spreading as well as defects in FA maturation. ALLN treatment restores FA maturation despite the lack of vIFs, but does not restore endoplasmic spreading. Consistent with a role for vIFs in endoplasmic spreading, adhesive structures do not contain vIFs when the endoplasm does not spread. Fln-depleted cells also exhibit a microtubule-dependent mistargeting of vIFs. Thus, I propose a model in which cellular force generation and interaction of vIFs with mature FAs are required for endoplasmic spreading. Additionally, I discuss future lines of investigation concerning the role of FlnA in the endoplasmic spreading mechanism as well as mechanosensitive functions of FlnA. Finally, I speculate on a potential application of endoplasmic spreading deficiencies as hallmarks of metastatic breast cancer.Englishhttps://doi.org/10.7916/D82805RB |
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NDLTD |
language |
English |
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Cytology Biophysics Molecular biology |
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Cytology Biophysics Molecular biology Lynch, Christopher D. The Endoplasmic Spreading Mechanism of Fibroblasts: Showcasing the Integrated Cytoskeleton |
description |
Cell motility is an essential process that depends on a coherent, cross-linked cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. In culture, a common feature of cells is the coherent movement of the endoplasmic reticulum and membranous organelles toward the periphery during substrate adhesion and spreading. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB-/- mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that leads to increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA-/- MEFs, but not FlnB-/- MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus I suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions. I also report that treatment with the calpain inhibitor N-[N-(N-Acetyl-L-leucyl)-L-leucyl]- L-norleucine (ALLN) restores endoplasmic spreading and focal adhesion (FA) maturation in the absence of Fln. Further, expression of calpain-uncleavable talin, but not full-length talin, also rescues endoplasmic spreading in Fln-depleted cells and indicates a crucial role for stable, mature FAs in endoplasmic spreading. Because FA maturation involves the vimentin intermediate filament (vIF) network, I also examined the role of vIFs in endoplasmic spreading. Wild-type cells expressing a dominant-negative vimentin variant incapable of vIF polymerization exhibit deficient endoplasmic spreading as well as defects in FA maturation. ALLN treatment restores FA maturation despite the lack of vIFs, but does not restore endoplasmic spreading. Consistent with a role for vIFs in endoplasmic spreading, adhesive structures do not contain vIFs when the endoplasm does not spread. Fln-depleted cells also exhibit a microtubule-dependent mistargeting of vIFs. Thus, I propose a model in which cellular force generation and interaction of vIFs with mature FAs are required for endoplasmic spreading. Additionally, I discuss future lines of investigation concerning the role of FlnA in the endoplasmic spreading mechanism as well as mechanosensitive functions of FlnA. Finally, I speculate on a potential application of endoplasmic spreading deficiencies as hallmarks of metastatic breast cancer. |
author |
Lynch, Christopher D. |
author_facet |
Lynch, Christopher D. |
author_sort |
Lynch, Christopher D. |
title |
The Endoplasmic Spreading Mechanism of Fibroblasts: Showcasing the Integrated Cytoskeleton |
title_short |
The Endoplasmic Spreading Mechanism of Fibroblasts: Showcasing the Integrated Cytoskeleton |
title_full |
The Endoplasmic Spreading Mechanism of Fibroblasts: Showcasing the Integrated Cytoskeleton |
title_fullStr |
The Endoplasmic Spreading Mechanism of Fibroblasts: Showcasing the Integrated Cytoskeleton |
title_full_unstemmed |
The Endoplasmic Spreading Mechanism of Fibroblasts: Showcasing the Integrated Cytoskeleton |
title_sort |
endoplasmic spreading mechanism of fibroblasts: showcasing the integrated cytoskeleton |
publishDate |
2012 |
url |
https://doi.org/10.7916/D82805RB |
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AT lynchchristopherd theendoplasmicspreadingmechanismoffibroblastsshowcasingtheintegratedcytoskeleton AT lynchchristopherd endoplasmicspreadingmechanismoffibroblastsshowcasingtheintegratedcytoskeleton |
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