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Figure 3


Fig. 3. Temperature-sensitive NSF cells can be polarized by cyclic-AMP gradients. Each panel shows temperature sensitive NSF cells (HM1067) expressing the appropriate GFP fusion proteins to act as reporters for cell polarization, were made aggregation-competent and stimulated with cAMP from a micropipette, at either the permissive temperature of 20°C (shown only for ABD-GFP) or the restrictive temperature of 28°C. Reporters: CRAC, reports PtdIns(3,4,5)P3 in the membrane; PTEN, normally bound to the membrane but repelled from the leading edge; ABD, binds to F-actin, thus reporting sites of actin polymerization. Wild-type cells behaved in a similar way at both temperatures, and did not round up at 28°C. When the pipette was moved to a new position (white circle) the image shown is the first taken at the new location; time in seconds after introducing the pipette is shown in each image; bar, 10 µm. See Movies 4-6 in supplementary material.





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