Figure 2

CD-BODIPY binds free-barbed-end actin protomers in vitro. (A) Concentration dependency of CD-BODIPY fluorescence in polymerised actin solutions (heavy dashed line, filled circles). The drug's dose dependent fluorescence in fixed cells (HEK293) is also shown (filled squares, fine dashed line). All data are normalised to fluorescence maxima at 1μM CD-BODIPY. (B) Measurement of the concentration dependent effect of LatA on CD-BODIPY fluorescence (50 nM) in the presence of polymerised actin. Data points are normalised to the fluorescence measured in unpolymerised samples containing 10μM (ATP. actin). Note that in the absence of LatA, CD-BODIPY fluorescence in the presence of polymerised actin is three-fold higher than that measured in unpolymerised actin solutions. (C) (i) fluorescence of 50 nM CD-BODIPY in the presence of increasing [actin] in the presence of ATP (circles); (ii) corresponding FITC-actin fluorescence (squares) measured from the same sample fields. Fluorescence data are normalised to the average signals measured in unpolymerised conditions ([actin]) = 8.8μM). Graph (iii) shows the ratio of normalised CD-BODIPY fluorescence to normalized FITC-actin fluorescence. The border between the two shaded areas indicating conditions favouring short (light grey shading) or long (dark grey) actin polymers defines the critical concentration expected for actin polymerisation in the presence of 50 nM CD [17]. In all graphs vertical bars show S.E.M. from triplicate samples.