The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

Integrins are receptors that play a critical role in important cellular processes, such as cell migration, cell adhesion, and cell proliferation. Integrin function has been most extensively studied in focal adhesions, the attachments formed in cultured cells when integrins are activated upon ligand binding. The body-wall muscle attachments of C. elegans are in vivo homologs of focal adhesions, and therefore provide a means to genetically dissect integrin adhesion and signaling functions within an intact, developing embryo. We use the following complementary approaches:

1. Genetic screens: To identify the genes involved in adhesion structure assembly, we have screened for a new class of C. elegans mutants that arrest during development because they fail to make functional muscles. Current efforts are focused on a subset of mutants that fail to make normal muscle attachments. Some of these genes have been molecularly isolated, and they all code for proteins that have been implicated in integrin-mediated adhesions. To date these include the genes coding for integrin subunits, an ECM protein that is a potential ligand for integrin, and a few adaptor proteins that link integrin to the cytoskeleton.

2. Molecular isolation of genes: Two of the genes identified in the above screens that we have recently molecularly isolated are pat-4 and pat-6, which code for the worm homologue of integrin-linked kinase (ILK) and actopaxin, respectively. Both ILK and actopaxin are critical components of focal adhesions, and are involved in integrin signaling in cell adhesions. We are using our pat-4 and pat-6 null mutants to investigate the function of these two proteins in vivo. Other genes like pat-4 and pat-6 were discovered in our genetic screens; they remain to be molecularly isolated.

3. Genetic dissection of muscle assembly: Using the mutants recovered in our screens in combination with a collection of antibodies to C. elegans muscle proteins and several functional fluorescently tagged muscle proteins (transgenically expressed fusions with the Green Fluorescent Protein reporter), we are able to determine which proteins are assembled properly when a particular component is eliminated by mutation. To date, our results are consistent with a model in which the muscle attachment structures assemble from the membrane by step-wise addition of components in a proximal to distal order. We will extend this analysis as we isolate additional genes and examine the localization of the proteins that they encode.

4. Analysis of integrin function: We are systematically testing the functions of integrin domains in vivo through site-directed mutagenesis followed by expression of the altered genes in transgenic worms. Two current areas of focus are: 1) the role of integrin activation, which occurs in response to ligand binding, in the proper positioning of muscle cells in developing embryos, and 2) identification of integrin domains required for muscle-specific functions.

By exploiting the highly ordered structure of body-wall muscle, and the multitude of genetic, cellular, and molecular approaches available in C. elegans, we hope to provide new insights into membrane-ECM interactions and the mechanisms of their downstream effects on cell differentiation.