Cell polarity and locomotion, as well as endocytosis, depend on NSF.
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 degrees C, they round up and cease to chemotax, move or cap ConA receptors. They fully recover when returned to 22 degrees 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 degrees 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.
Thompson
Chris R L
CR
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Bretscher
Mark S
MS
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D016764:Cell Polarity / Q000502:physiology*; D017931:DNA Primers; D004023:Dictyostelium / Q000235:genetics / Q000502:physiology; D004705:Endocytosis / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D052066:N-Ethylmaleimide-Sensitive Proteins; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D017931:DNA Primers; D033921:Vesicular Transport Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins