A cell surface display fluorescent biosensor for measuring MMP14 activity in real-time.

Despite numerous recent advances in imaging technologies, one continuing challenge for cell biologists and microscopists is the visualization and measurement of endogenous proteins as they function within living cells. Achieving this goal will provide a tool that investigators can use to associate cellular outcomes with the behavior and activity of many well-studied target proteins. Here, we describe the development of a plasmid-based fluorescent biosensor engineered to measure the location and activity of matrix metalloprotease-14 (MMP14). The biosensor design uses fluorogen-activating protein technology coupled with a MMP14-selective protease sequence to generate a binary, "switch-on" fluorescence reporter capable of measuring MMP14 location, activity, and temporal dynamics. The MMP14-fluorogen activating protein biosensor approach is applicable to both short and long-term imaging modalities and contains an adaptable module that can be used to study many membrane-bound proteases. This MMP14 biosensor promises to serve as a tool for the advancement of a broad range of investigations targeting MMP14 activity during cell migration in health and disease.

Shroom2 regulates contractility to control endothelial morphogenesis.

The intrinsic contractile, migratory, and adhesive properties of endothelial cells are central determinants in the formation of vascular networks seen in vertebrate organisms. Because Shroom2 (Shrm2) is expressed within the endothelium, is localized to cortical actin and cell-cell adhesions, and contains a conserved Rho kinase (Rock) binding domain, we hypothesized that Shrm2 may participate in the regulation of endothelial cell behavior during vascular morphogenesis. Consistent with this hypothesis, depletion of Shrm2 results in elevated branching and sprouting angiogenic behavior of endothelial cells. This is recapitulated in human umbilical vein endothelial cells and in a vasculogenesis assay in which differentiated embryonic stem cells depleted for Shrm2 form a more highly branched endothelial network. Further analyses indicate that the altered behavior observed following Shrm2 depletion is due to aberrant cell contractility, as evidenced by decreased stress fiber organization and collagen contraction with an increase in cellular migration. Because Shrm2 directly interacts with Rock, and Shrm2 knockdown results in the loss of Rock and activated myosin II from sites of cell-cell adhesion, we conclude that Shrm2 facilitates the formation of a contractile network within endothelial cells, the loss of which leads to an increase in endothelial sprouting, migration, and angiogenesis.

Identification of functional domains in sarcoglycans essential for their interaction and plasma membrane targeting.

Mutations in sarcoglycans have been reported to cause autosomal-recessive limb-girdle muscular dystrophies. In skeletal and cardiac muscle, sarcoglycans are assembled into a complex on the sarcolemma from four subunits (alpha, beta, gamma, delta). In this report, we present a detailed structural analysis of sarcoglycans using deletion study, limited proteolysis and co-immunoprecipitation. Our results indicate that the extracellular regions of sarcoglycans consist of distinctive functional domains connected by proteinase K-sensitive sites. The N-terminal half domains are required for sarcoglycan interaction. The C-terminal half domains of beta-, gamma- and delta-sarcoglycan consist of a cysteine-rich motif and a previously unrecognized conserved sequence, both of which are essential for plasma membrane localization. Using a heterologous expression system, we demonstrate that missense sarcoglycan mutations affect sarcoglycan complex assembly and/or localization to the cell surface. Our data suggest that the formation of a stable complex is necessary but not sufficient for plasma membrane targeting. Finally, we provide evidence that the beta/delta-sarcoglycan core can associate with the C-terminus of dystrophin. Our results therefore generate important information on the structure of the sarcoglycan complex and the molecular mechanisms underlying the effects of various sarcoglycan mutations in muscular dystrophies.