Figure 4.

FtsZ and ΔCTL polymers assemble distinct large-scale superstructures on SLBs. A. Schematic depicting the two-inlet flow cell used for rapid initiation of polymerization and depolymerization. During flow, the protein channel side is depleted of GTP and FtsZ is predominantly monomeric. Immediately after flow is stopped, GTP diffuses into the protein channel side initiating FtsZ (6% Alexa488 labeled) polymerization and recruitment to the membrane by copolymerizing with FtsZ-MTS, enabling visualization by TIRFM. B-E. Kymographs and corresponding fluorescence intensity vs time plots during periods of flow and no flow in the two-inlet flow cell. In the protein side, during flow, 2 μM FtsZ (6% Alexa488 labeled) and 2 μM FtsZ-MTS (unlabeled) (B, C) or 2 μM ΔCTL (6% Alexa488 labeled) and 2 μM ΔCTL-MTS (unlabeled) (D, E) is introduced at the flow rate of 5 μL minute⁻¹. Simultaneously, in the GTP side, 2 mM GTP is introduced at the same flow rate of 5 μL minute⁻¹. Time-lapse TIRF movies corresponding to the kymographs in B-E were obtained at 10× magnification. B. and D. represent kymographs and intensity plots corresponding to the first flow/stop cycle (flow up to 25 μL at 5 μL minute⁻¹ for each channel into a fresh flow cell and then no flow to allow mixing), C. and E. correspond to subsequent flow-stop cycle (flow up to 15 μL at 5 μL minute⁻¹ for each channel into the flow cell in B or D following steady state and then no flow to allow mixing). Scale bar = 100 μm in spatial axis (vertical) and 2 min in temporal axis (horizontal) of kymograph, asterisks of different colors correspond to intensity plots of the same color denoting regions within the flow cell at varying distances perpendicular to the laminar boundary (and the direction of flow). F. Line plots along axis perpendicular to the direction of flow at steady state following re-initiation of assembly (after flow) at the indicated time points corresponding to kymographs in C. (FtsZ/FtsZ-MTS) and E. (ΔCTL/ΔCTL-MTS). Reaction buffer contains 50 mM HEPES pH 8.0, 0.1 mM EDTA, 10 mM MgCl2, 300 mM KCl, 1% glycerol, 0.1 mg mL⁻¹ casein (blocking agent), 0.5 mg mL⁻¹ ascorbate.