Cell polarity and locomotion, as well as endocytosis, depend on NSF

Abstract

NEM-sensitive factor (NSF) is an essential protein required during membrane transport. We replaced part of the endogenous D. discoideum NSF gene (nsfA) by a PCR-mutagenised library and isolated 11 mutants temperature-sensitive (ts) for growth. Two of these have been studied in detail. As expected, both are ts for FITC-dextran uptake by macropinocytosis, for internalising their surface membrane (monitored with FM1-43) and for phagocytosis. However, after 10-20 minutes at 28°C, they round up and cease to chemotax, move or cap ConA receptors. They fully recover when returned to 22°C. These cells carry out a normal ‘cringe’ reaction in response to cAMP, indicating that the actin cytoskeleton and this signal transduction pathway are still functional at 28°C. The behaviour of these mutants shows that NSF-catalysed processes are required not only for the different endocytic cycles but also for the maintenance of cell polarity. As cell locomotion depends on a cell having a polarity, the mutants stop moving at high temperature. A tentative model is proposed to explain the surprising link between membrane recycling and cell polarity revealed here.