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

Chris R. L. Thompson^* and Mark S. Bretscher

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
^* Present address: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

View larger version (41K): [in a new window]   Fig. 1. Southern blots of genomic DNA establish homologous gene replacement with Com6 site included. (A) Structure of the transforming DNA used. (B) Arrangement of restriction sites around the nsfA gene in Ax2 and in a homologous recombinant (HR) containing the Com6 site (marked by A). Restriction sites: P (PstI); H (HindIII); A (AflII); E (EcoRI); B (BamHI). The probe covers bases 1700-2860. (C) Blot of Ax2, ts1, ts2 and C6 (a pC6-derived clone) cut with B+H. (D) As in C, but cut with P and E±A. The main band at 2120 in Ax2 is insensitive to A; the HR lines are sensitive, showing that they each contain Com6. Additional bands are partial cleavages.

View larger version (34K): [in a new window]   Fig. 2. Endocytosis by Ax2 and ts mutants after a 20 minutes preincubation at 22°C or 28°C. (A,B) Fluid phase uptake measured with FITC-dextran with cells harvested from bacterial plates (A) or held overnight in axenic medium at 5x106 cells/ml (B). (C) Cell surface uptake measured with FM1-43. (D) Phagocytosis of fluorescent beads. Each experiment was carried out at least five times, all with similar results; a representative example of each experiment is shown.

View larger version (96K): [in a new window]   Fig. 3. Behaviour of ts2 cells during temperature shift. Cells from the growth zone on a bacterial plate were allowed to attach to a coverslip, rinsed and placed above a temperature-controlled chamber at 22°C and observed (top two left-hand panels). The temperature was then raised to 28°C (top two right-hand panels and lower panels); the cells became rounder by 10 minutes and stopped migrating at about 20 minutes.

View larger version (83K): [in a new window]   Fig. 4. Capping of ConA receptors. Amoebae, preincubated either at 22°C or 28°C, were labelled with Fl-ConA for 5 minutes at the appropriate temperature, washed and incubated a further 5 minutes, fixed with formaldehyde and viewed according to Aguado-Velasco and Bretscher (Aguado-Velasco and Bretscher, 1997). The fraction of capped cells, measured as those with the fluorescence confined to one half of the cell surface, was: at 22°C, Ax2, 76±5%; ts1, 68±2%; ts2, 77±4%; and at 28°C, Ax2, 79±2%; ts1, 0%; ts2, 0%.

View larger version (52K): [in a new window]   Fig. 5. Actin polymerisation in sensitised amoebae. (A) F-actin levels in sensitised amoebae after addition of cAMP – known as the ‘cringe’ reaction (Hall et al., 1988). (B,C) ts2 amoebae held at 22°C (B) or 28°C (C), fixed with formaldehyde and stained with Alexa 488-phalloidin to reveal the intracellular distribution of F-actin. The levels of F-actin at 28°C, based on phalloidin binding (as in A) and normalised to that at 22°C, were measured as Ax2, 0.68; ts1, 0.57; ts2, 0.68.