Fig. 3.
Continued ATP hydrolysis is required for IVA polymerization. (A) Kinetic analysis of IVA polymerization in the presence of ATP as measured by centrifugation in the absence of apyrase (●), when apyrase was added at the beginning of the experiment (■), or when apyrase was added after 4 h of incubation with ATP (▲). Symbols represent mean values of at least three independent measurements; error bars represent SEM. (B) Mixing experiment wherein WT IVA and IVA variants were labeled with a fluorophore [Alexa 488 (green) or Alexa 647 (red)] and incorporation of either variant into the growing polymer over time (indicated above in minutes) was observed using TIRF microscopy. The top and bottom of each panel represent the same field of view taken with the appropriate filter to detect the indicated fluorescent label. First row: WT IVA labeled with Alexa 488 mixed with an equimolar amount of WT IVA labeled with Alexa 647. Second and third rows: WT IVA, labeled with either Alexa 488 or Alexa 647, mixed with an equimolar amount of IVAB*, labeled with either Alexa 647 or Alexa 488, respectively. Fourth and fifth rows: Similar to rows 2 and 3, but WT IVA was mixed with IVAT*.