CONTROL OF STABLE LAMELLIPODIA FORMATION BY EXPRESSION OF CELL-SURFACE BETA-1,4-GALACTOSYLTRANSFERASE CYTOPLASMIC DOMAINS

Mesenchymal cell migration on basal lamina is mediated, in part, by th e binding of cell surface beta 1,4-galactosyltransferase (GalTase) to specific N-linked oligosaccharides in the E8 domain of laminin. On mig rating cells, surface GalTase is anchored to the cytoskeleton; when Ga lTase is prevented from associating with the cytoskeleton, lamellipodi a formation and subsequent migration are inhibited. To define better t he involvement of GalTase-cytoskeleton interactions in cell motility, we examined the lamellipodia formation, polarity and migratory behavio r of stably transfected 3T3 fibroblasts expressing increased or decrea sed levels of GalTase capable of interacting with the cytoskeleton, In itially, the motile behavior of individual cells was quantified in the absence of exogenous stimuli. Cells that overexpress GalTase binding sites for the cytoskeleton changed their polarity more frequently and translocated more erratically than did control cells when assayed on l aminin substrata. These differences were not observed, however, when c ells were plated on fibronectin, which does not contain binding sites for surface GalTase. GalTase-transfected cells were also assayed for t heir ability to polarize in response to a specific stimulus. In this c ase, the ability of a cell to reorient towards a gradient of platelet- derived growth factor was found to be directly proportional to the amo unt of GalTase associated with the cytoskeleton. Differences in respon se to platelet-derived growth factor were not due to differences in gr owth factor binding. Indirect immunofluorescence showed that altering the level of GalTase did not affect the ventrally distributed pool of GalTase stably associated with the cytoskeleton; however, stress fiber formation was inhibited. Thus, increasing surface GalTase binding sit es for the cytoskeleton leads to erratic, multipolar behavior in the a bsence of any vectorial stimulus, but the ability to form a functional lamellipodium in response to a stimulus is dependent upon the amount of surface GalTase associated with the cytoskeleton. Apparently, cells are able to regulate cytoskeletal assembly and lamellipodial stabilit y by altering the expression and/or affinity of appropriate matrix rec eptors, such as GalTase, and their corresponding binding sites in the cytoskeleton.