Mapping trajectories of pgp-1 membrane protein patches on surfaces of motile fibroblasts reveals a distinct boundary separating capping on the lamella and forward transport on the retracting tail

Abstract

Patches of aggregated membrane proteins on motile fibroblasts are transported from the surfaces of the leading lamella to a site just ahead of the nucleus in the phenomenon known as capping. A major cell surface glycoprotein, Pgp-1 (GP80), was tagged with a monoclonal IgG and then aggregated with fluorescent secondary antibodies. Correlated digitized fluorescence and phase-contrast microscopy were used to map the trajectories of fluorescent Pgp-1 patches located in various regions of the cell surface. The response of patches located in lamellar and nonlamellar regions to spontaneous retraction of the trailing cell margin, or tail was examined in detail. During capping, Pgp-1 patches accumulated at a morphologically distinct site on the cell surface, the ‘null border’, corresponding to the boundary between lamelloplasm and endoplasm and the posterior edge of the dorsal cortical F-actin sheath. Posterior to this site, gradual forward movement of patches accompanied the gradual narrowing phase of the trailing edge retraction that occurs prior to abrupt detachment of the tail, but patches did not actually accumulate at the null border. The rate of forward patch movement was generally greater at positions further behind the boundary. Patch movement correlated approximately with forward organelle movement in the trailing region of the cell. The boundary was also apparent during simultaneous capping and retraction when forward patch transport on the trailing edge and rearward transport of patches across the lamellar surface appeared to converge on the null border. Forward patch transport was strictly confined to regions behind the boundary while retrograde patch transport was confined to the lamellar region ahead of the boundary. Patches are thought to be linked to the cortical cytoskeleton and their transport is discussed in terms of the very different cortical cytoskeletal dynamics occurring in the leading and trailing edges of locomoting cells.