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Biomedical subjects

Carol Otey

Publications and source records attributed to Carol Otey.

7 recordsLinked to original sources
PubMed2007

Connexin 43 is localized with actin in tenocytes.

Varieties of cell-matrix or cell-cell adhesions are associated with the actin cytoskeleton. However, for gap junctions, which are both channels and adhesions, there has been little evidence for such an association. The purpose of this study was to determine if connexin 43 (Cx43) associates with actin and to determine if this association is altered under mechanical load in tenocytes, a mechanically sensitive cell. Avian tenocytes were subjected to multiple cyclic strain regimens and then fixed and examined immunohistochemically at various times poststrain to determine where Cx43 protein was localized within the cells in relation to actin filaments. Four percent of tenocytes had colocalization of actin filaments and Cx43, which was significantly increased with 5% cyclic strain. To confirm this phenomenon, human tenocytes and COS-7 cells were also subjected to cyclic strain and then fixed at the same times after strain. As with avian tenocytes, Cx43 was colocalized with actin in human tenocytes and COS-7 cells. The colocalization increased significantly after cyclic strain in human tenocytes but not in COS-7 cells. The lack of detectable changes in COS-7 cells may indicate that they are less mechanosensitive than tenocytes perhaps due to the less robust actin cytoskeleton seen in the COS-7 cells when compared to the tenocytes. Furthermore, inhibiting myosin II activity greatly reduced the immunohistochemically-detectable Cx43 on actin filaments. Connexins may associate with actin to stabilize gap junctions at the plasma membrane, ensuring that tenocytes remain coupled during periods of prolonged or intense mechanical loading.

Actin Cytoskeleton↗
PubMed2004

Simultaneous stretching and contraction of stress fibers in vivo.

To study the dynamics of stress fiber components in cultured fibroblasts, we expressed alpha-actinin and the myosin II regulatory myosin light chain (MLC) as fusion proteins with green fluorescent protein. Myosin activation was stimulated by treatment with calyculin A, a serine/threonine phosphatase inhibitor that elevates MLC phosphorylation, or with LPA, another agent that ultimately stimulates phosphorylation of MLC via a RhoA-mediated pathway. The resulting contraction caused stress fiber shortening and allowed observation of changes in the spacing of stress fiber components. We have observed that stress fibers, unlike muscle myofibrils, do not contract uniformly along their lengths. Although peripheral regions shortened, more central regions stretched. We detected higher levels of MLC and phosphorylated MLC in the peripheral region of stress fibers. Fluorescence recovery after photobleaching revealed more rapid exchange of myosin and alpha-actinin in the middle of stress fibers, compared with the periphery. Surprisingly, the widths of the myosin and alpha-actinin bands in stress fibers also varied in different regions. In the periphery, the banding patterns for both proteins were shorter, whereas in central regions, where stretching occurred, the bands were wider.

Actinin↗
PubMed2003

Internet-based image analysis quantifies contractile behavior of individual fibroblasts inside model tissue.

In a cell-populated collagen gel, intrinsic fiber structure visible in differential interference contrast images can provide markers for an in situ strain gauge to quantify cell-gel mechanics, while optical sections of fluorescent protein distribution capture cytoskeletal kinematics. Mechanics quantification can be derived automatically from timelapse differential interference contrast images using a Deformation Quantification and Analysis software package accessible online at http://dqa.web.cmu.edu. In our studies, fibroblast contractile machinery was observed to function entirely within pseudopods, while GFP-alpha-actinin concentrated in pseudopod tips and cortex. Complex strain patterns around individual cells showed instances of both elastic and inelastic strain transmission, suggesting a role in observed long-range alignment of cells.

3T3 Cells↗
PubMed2002

Regulation of focal complex composition and disassembly by the calcium-dependent protease calpain.

Cell migration requires the regulated and dynamic turnover of adhesive complexes. We have previously demonstrated that the calcium-dependent protease, calpain, regulates the organization of adhesive complexes and cell detachment during cell migration. Evidence is now provided that inhibiting calpain through over-expression of the endogenous inhibitor of calpain, calpastatin, and pharmacological inhibitors results in an inhibition of adhesive complex disassembly with stabilization of GFP-vinculin and GFP/RFP-zyxin at the cell periphery. Calpain was also required for the microtubule-mediated turnover of adhesive complex sites after nocodazole wash-out, suggesting that calpain may mediate focal complex disassembly downstream of microtubules. Using dual imaging of RFP-zyxin and GFP-alpha-actinin, we observed a temporal and spatial relationship between alpha-actinin localization to focal contacts and the subsequent disassembly or translocation of RFP-zyxin containing focal complexes in areas of cell retraction. Calpain inhibition disrupted alpha-actinin localization to zyxin-containing focal contacts and focal complex disassembly or translocation to the cell center. In addition, disrupting alpha-actinin localization to focal complexes through expression of the alpha-actinin rod domain, but not the head domain, resulted in inhibition of focal adhesion disassembly similar to calpain inhibition. Our studies suggest a novel mechanism of action whereby calpain may modulate alpha-actinin localization into focal complexes and their subsequent disassembly or translocation.

Actinin↗
PubMed2002

Microfilament-dependent movement of the beta3 integrin subunit within focal contacts of endothelial cells.

To gain insight into the dynamic properties of focal contacts, we induced expression of green fluorescent protein-tagged beta3 integrin (GFP-beta3) and actinin-1 (GFP-actinin-1) in endothelial cells. Both tagged proteins localize with alpha(v)beta3 integrin in focal contacts distributed towards the periphery of transfected cells. Labeled focal contacts migrate at about 0.1 mm/min in stationary live endothelial cells. We compared beta3 integrin and actinin-1 dynamics in focal contacts by using fluorescence recovery after photobleaching. Recovery of signal in bleached focal contacts that have incorporated actinin-1 is rapid and occurs within less than 4 min. This recovery is energy-dependent. In contrast, recovery of bleached focal contacts that contain GFP-beta3 integrin takes longer than 30 min. Yet, when a narrow stripe of fluorescence is bleached across a beta3 integrin-labeled focal contact, recovery is complete within 16 min. The latter recovery is energy-dependent and is blocked not only by actin-filament disrupting drugs but also by a myosin light chain kinase inhibitor. Thus, integrins are not immobile when incorporated into focal contacts, as some have suggested. We propose that integrins are mobile within the confines of focal contacts and that this mobility is supported by an actin-associated molecular motor.

Actin Cytoskeleton↗
PubMed2002

Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins.

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique used to selectively inactivate proteins within cells. Here, we demonstrate that GFP can be used as a CALI reagent to locally inactivate proteins in living cells. We show that focused laser irradiation of EGFP-alpha-actinin expressed in Swiss 3T3 fibroblasts results in the detachment of stress fibres from focal adhesions (FAs), whereas the integrity of FAs, as determined by interference reflection microscopy (IRM), is preserved. Moreover, consistent with a function for focal adhesion kinase (FAK) in FA signalling and not FA structure, laser irradiation of EGFP-FAK did not cause either visible FA damage or stress fibre detachment, although in vitro CALI of isolated EGFP-FAK decreased its kinase activity, but not its binding to paxillin. These data indicate that CALI of specific FA components may be used to precisely dissect the functional significance of individual proteins required for the maintenance of this cytoskeletal structure. In vitro CALI experiments also demonstrated a reduction of EGFP-alpha-actinin binding to the cytoplasmic domain of the beta(1) integrin subunit, but not to actin. Thus, alpha-actinin is essential for the binding of microfilaments to integrins in the FA. CALI-induced changes in alpha-actinin result in the breakage of that link and the subsequent retraction of the stress fibre.

3T3 Cells↗