The overall aim of the next 5 years of this long standing grant is to learn how cells migrate on or through extracellular matrix (ECM) and to decipher the rules governing epithelial-mesenchymal transformation in ECM. I. Mesenchymal cell migration. We seek to prove the theory that the actin cortex is fixed to ECM substrate during cell migration and the myosin-rich endoplasm, containing cell organelles, microtubules, and intermediate filaments, slides past membrane-bound actin into newly forming leading edge. (1) The actin cortex and outer surface of the plasmalemma will be labeled in the living fibroblast by appropriate markers, such as fluorescent phalloidin and antibodies to ECM receptors, and their locations monitored by external reference markers during forward movement. (2) We will seek additional evidence for the fixed cortex theory by labelling the endoskeleton and watching it move in relation to the cell surface. II. Epithelial-mesenchymal transformation in collagen gels. (1) We wish to prove that the fixed cortex motility mechanism is operating during epithelial-mesenchymal transformation. We will show that new actin front end is continuously forming when a mesenchymal cell leaves an epithelium, and that part of the trailing end remains attached to former epithelial neighbors in patches. We will use polarized virus budding and labeled ECM receptors to see if new membrane is being added to front end. (2) We will use in situ hybridization to determine changes in ECM and cytoskeletal gene transcription during epithelial-mesenchymal transformation. We expect vimentin mRNA to appear before cells elongate, actin mRNA to move to the front end as the cells elongate and type I collagen to be activated after the cell is bipolar. III. Epithelial-mesenchymal transformation in the embryo. (1) We will analyze ECM and ECM receptors of the early embryo and correlate their production with the onset of neural crest and sclerotome formation. We will use in situ hybridization to localize their mRNAs. (2) We will use carboxyfluorescein to discover novel locations of epithelial-mesenchymal transformation during embryonic tissue remodeling. The immediate health relevance is to our understanding of normal development, but the work could give insights into the causes of congenital anomalies and abnormal tissue organization, as for example in cataracts, cleft palate, and cancer.
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