Lewis H. Romer

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–α-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–α-actinin binding to the cytoplasmic domain of the β1 integrin subunit, but not to actin. Thus, α-actinin is essential for the binding of microfilaments to integrins in the FA. CALI-induced changes in α-actinin result in the breakage of that link and the subsequent retraction of the stress fibre.

Localization of Multiple Functional Domains on Human PECAM-1 (CD31) by Monoclonal Antibody Epitope Mapping

PECAM-1, a cell adhesion molecule of the immunoglobulin gene (Ig) superfamily, has been implicated in white cell transmigration, integrin activation on lymphocytes, and cell-cell adhesion. The purpose of this investigation was to identify specific regions of the PECAM-1 extracellular domain mediating these functions by identifying the location of epitopes of bioactive anti-PECAM-1 monoclonal antibodies. The binding regions of mAbs important in PECAM-1-mediated leukocyte transmigration (Hec 7.2 and 3D2) were mapped to N-terminal Ig-like domains. The epitopes of monoclonal antibodies that activated integrin function on lymphocytes were dispersed over the entire extracellular region, but those that had the strongest activating effect were preferentially localized to the N-terminus of the molecule. The binding regions of mAbs that blocked PECAM-1-mediated heterophilic L-cell aggregation were located either in Ig-like domain 2 (NIH31.4) or Ig-like domain 6 (4G6 and 1.2). Site-directed mutagenesis further pinpointed the epitope of the 4G6 mAb to a hexapeptide, CAVNEG, within Ig-like domain 6. These results demonstrate that PECAM-1 contains multiple functional domains. Regions within N-terminal Ig-like domains appear to be required for transmigration. In contrast, two distinct regions were implicated in L-cell mediated heterophilic aggregation.

Microinjection of protein tyrosine phosphatases into fibroblasts disrupts focal adhesions and stress fibers

Microinjection and scrape-loading have been used to load cells in culture with soluble protein tyrosine phosphatases (PTPs). The introduction of protein tyrosine phosphatases into cells caused a rapid (within 5 minutes) decrease in tyrosine phosphorylation of major tyrosine phosphorylated substrates, including the focal adhesion kinase and paxillin. This decrease was detected both by blotting whole cell lysates with anti-phosphotyrosine antibodies and visualizing the phosphotyrosine in focal adhesions by immunofluorescence microscopy. After 30 minutes, many of the cells injected with tyrosine phosphatases revealed disruption of focal adhesions and stress fibers. To determine whether this disruption was due to the dephosphorylation of FAK and its substrates in focal adhesions, we have compared the effects of protein tyrosine phosphatase microinjection with the effects of displacing FAK from focal adhesions by microinjection of a dominant negative FAK construct. Although both procedures resulted in a marked decrease in the level of phosphotyrosine in focal adhesions, disruption of focal adhesions and stress fibers only occurred in cells loaded with exogenous protein tyrosine phosphatases. These results lead us to conclude that although tyrosine phosphorylation regulates focal adhesion and stress fiber stability, this does not involve FAK nor does it appear to involve tyrosine-phosphorylated proteins within focal adhesions. The critical tyrosine phosphorylation event is upstream of focal adhesions, a likely target being in the Rho pathway that regulates the formation of stress fibers and focal adhesions.

PECAMI ASSOCIATION WITH TALIN IN HUMAN VASCULAR ENDOTHELIAL CELLS. |[bull]| 291

Our previous work indicated that cytoskeletal association may regulate the redistribution of PECAM1 on the surface of human endothelial cells after treatment with TNFα or IFNγ (Journal of Immunology 154:65826592, 1995). We have, therefore, investigated PECAM1 interactions with cytoskeletal proteins. Immunofluorescence studies indicated that PECAM1 colocalized with cytoskeletal proteins between Factin arrays and the plasma membrane in human umbilical vein endothelial cell (HUVEC) monolayers, Multiple interdigitations and irregularities in the intercellular appearance of PECAM1 were found to correspond with the configuration of the Factin at the cells perimeters. This pattern of intercellular localization appeared to correspond with staining for alphaactinin, filamin, talin, and vinculin. These cytoskeletal proteins and the actinbinding protein cortactin were all investigated by immunoprecipitation for evidence of biochemical interaction with PECAM1. AntiPECAM1 immunoprecipitates from whole cell lysates of confluent HUVEC monolayers were analyzed by SDSPAGE and immunoblotting. They were found to contain talin. Alphaactinin, cortactin, filamin, and vinculin were not reproducibly identified in these PECAM1 immunoprecipitates. The reverse analysis was also performed in each case. Immunoblot analyses of antialphaactinin, anticortactin, antifilamin, and antivinculin immunoprecipitates from HUVEC lysates were negative for PECAM1. PECAM1 did appear in antitalin immunoprecipitates from confluent, but not subconfluent, HUVEC monolayers. These experiments indicate that PECAM1 may associate with the cytoskeletal protein talin in normal human vascular endothelium. The role of talin in the regulation of the intercellular localization of PECAM1 is under investigation.