Inhibition of dynamin with dynasore and a dominant-negative mutant.

<p>Dynasore-pretreated and non-pretreated HeLa cells were incubated with (A) 5 nM diphtheria toxin for 180 min or (B) 4 pM toxin B for 75 min, prior to microscopical analysis of the cell morphology. (C) HeLa cells preincubated with dynasore or with solvent only were treated with 4 pM toxin B for 75 min, 1.5 nM toxin A for 105 min, 4 nM lethal toxin for 75 min or 1.5 nM α-toxin for 60 min. The percentage of rounded cells was quantified and data are given +/− SD (n = 3) and from a minimum of 200 cells in total. (D) Dynasore-pretreated HT-29 cells and untreated cells were intoxicated with 40 pM toxin. After onset of cell rounding (150 min), glucosylation status of Rac1 in cell lysates was analyzed with antibodies recognizing either only unmodified Rac1 (α-Rac1_non-Glc) or all Rac1 (α-Rac1_total). (E) HeLa cells expressing dominant-negative dynamin (HA-dynamin_K44A) for 24 h or mock-transfected cells were intoxicated with 4 pM toxin B for 75 min and cell morphology analyzed microscopically. (F) Selective expression of HA-dynamin_K44A in plasmid-transfected HeLa cells, but not in mock-transfected cells, was detected in cell lysates with an anti-HA antibody. Antibody detection of the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (α-GAPDH) served as a control for equal loading of lysate samples.