Fig 1.
Nucleation and growth of FtsZ filaments into rings on SLBs.
(A) Representative snapshots from a time-lapse experiment displaying different stages of ring formation. Images were taken every 10 s using TIRF illumination (YFP channel). Frames correspond to the times (min) indicated after the addition of GTP and Mg2+. (B) Polar clockwise growth of a single FtsZ from a nucleation point. Growth seems to occur stepwise and depend on the accessibility of small filaments nearby (panel 1 and 2). Lower local protein density (white arrow) correlates with a higher flexibility of the polymer (panels 7–12). Breakage occurs primarily in trailing regions (15). After about 3 min, a primitive ring made of 3 distinct short filaments is exhibited (17). (C) Directional filament gliding via treadmilling. Fragmentation or depolymerization destabilizes the trailing (“older”) edge as shown in the kymograph (d-labeled white line). Images in (B) and (C) were taken every 2 s, and the scale bar represents 500 nm. Further details are under “Results.” Movies reconstructed from the whole collection of images can be found in supporting information S2 and S3 Movies. GTP, guanosine triphosphate; SLB, supported lipid bilayer; TIRF, total internal reflection fluorescence; YFP, yellow fluorescent protein.
Fig 2.
Dependence of FtsZ-YFP-mts vortex formation on protein surface concentration.
(A) Time dependence of the average fluorescence intensity of FtsZ-YFP-mts on the bilayer upon 4 mM GTP and 5 mM Mg2+ addition, as measured by TIRFM, at 0.2 (blue line) and 0.5 (red line) μM protein concentration. The gray area marks the intensity when first closed rings are observed, which is approximately the same for both protein concentrations. After closed rings have formed, the further accumulation of protein at the surface is strongly concentration dependent. The dashed line represents the phase in which clearly discernible, locally stable dynamic vortices are observed. While at 0.2 μM, the system reaches this regime after an elapsed time of 45 min (time point 3); at 0.5 μM, it only takes approximately 20 min (time point 2). (B) Representative images of the experiment shown in panel (A). Frames were taken at elapsed times in minutes. Right: Ring size distributions at time points 2 and 3, indicated in panel (A), with average diameters of 0.94 +/− 0.16 μm, N = 140, and 0.98 +/− 0.14 μm, N = 128, respectively. Size distributions of rings are similar since both correspond to the same protein surface density (approximately 880 A.U.) Further details are under “Results.” A.U., arbitrary units; GTP, guanosine triphosphate; mts, membrane-targeting sequence; TIRFM, total internal reflection fluorescence microscope; YFP, yellow fluorescent protein.
Fig 3.
Free Mg2+ regulates protein surface concentration and thus self-organization of membrane-targeted GTP-FtsZ.
Representative snapshots showing TIRFM images of FtsZ-YFP-mts (0.2 μM) polymers on the bilayer taken 2–3 min after the addition of 0.04 mM GTP in the presence of 1 mM and 5 mM free Mg2+ concentrations, respectively. Next to each image, the mean fluorescence intensity, proportional to the FtsZ-YFP-mts density on the membrane, is shown (average of 3 experiments). The protein network observed at 5 mM free Mg2+ correlates with a high FtsZ-YFP-mts density regime, at least 3-fold larger (approximately 1,500 A.U.) than required for ring formation (approximately 500 A.U.) The scale bar represents 5 μM. A.U., arbitrary units; GTP, guanosine triphosphate; mts, membrane-targeting sequence; TIRFM, total internal reflection fluorescence microscope; YFP, yellow fluorescent protein.
Fig 4.
Steady-state treadmilling and chirality of FtsZ vortices: Dependence on GTPase activity and location of the mts.
(A-C) Left panels: Representative snapshots of the rings formed upon addition of GTP (4 mM) and Mg2+ (5 mM) by (A) FtsZ-YFP-mts, whose mts is located at the C-terminus of FtsZ; (B) mts-H-FtsZ-YFP, with mts located at the N-terminus of FtsZ; and (C) FtsZ*[T108A]-YFP-mts, a variant of FtsZ-mts with diminished GTPase activity. Right panels: Kymograph analysis showing (A) a positive slope that corresponds to the apparent clockwise rotation time of the selected ring (red circle); (B) a negative slope that corresponds to an apparent counterclockwise rotation, indicating that the position of the mts determines the chirality of the apparent rotation; (C) no apparent slope corresponding to static rings, suggesting that the apparent rotation in (A) and (B) is mediated by GTP hydrolysis. (D) Velocity distributions for FtsZ-YFP-mts (red) and mts-H-FtsZ-YFP (blue) with mean rotational speed values of 34 nm/s and 25 nm/s, respectively. Further details under Materials and methods and Results. GTP, guanosine triphosphate; mts, membrane-targeting sequence; YFP, yellow fluorescent protein.
Fig 5.
Residence times of single membrane-targeted FtsZ molecules at the bilayer, dependent on nucleotide and free Mg2+.
(A) Overlaid images of FtsZ-YFP-mts structures (yellow channel) incubated with GFP-Booster-Atto647N (nanobody) (single molecules: red channel) in the presence of GTP (a, b), GDP (c, d), and indicated free Mg2+ concentration. The protein concentration in all cases was around 0.2 μM. (B) Mean residence times of FtsZ-YFP-mts were calculated using an exponential fit of the cumulative residence time distribution. Mean residence times were measured for different GTP and Mg2+ conditions. Further details under Materials and methods and Results. GDP, guanosine diphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; mts, membrane-targeting sequence; YFP, yellow fluorescent protein.
Fig 6.
(A) FtsZ-YFP-mts ring formation is dependent on GTP and protein surface concentration.
At low protein concentration and no GTP and Mg2+, FtsZ transiently binds to the membrane without forming visible structures. With GTP, dynamic chiral rings are formed as a function of time. Once stable swirls are built, they exhibit a mean velocity of 34 nm/s and a turnover time of short fragments of 11.5 s. From the velocity and the turnover time, the average length of the protofilaments can be estimated to 390 nm. However, ring formation is only observed at intermediate protein density regimes. At high protein density, a parallel network of filaments (nematic phase) is observed. (B) To guarantee chirality, attachment needs to have a perpendicular component to both the ring curvature and filament polarity to have a preferential binding face. In this case, the mts interacts with the flat surface on opposite sides of the FtsZ filament, so curvature is also in the opposite direction. (C) We here suggest that an intrinsic helical FtsZ shape, characterized by a radius and a pitch, can alternatively explain previous FtsZ-induced inwards/outwards deformations in the following way: Due to the intrinsic pitch, the growing filament would either pull up (left) or push down (right) the surface. On the contrary, if the surface is not deformable (SLB), the filament would experience a strain, get destabilized, and eventually break upon growth. GDP, guanosine diphosphate; GTP, guanosine triphosphate; GUV, giant unilamellar vesicles; mts, membrane-targeting sequence; SLB, supported lipid bilayer; YFP, yellow fluorescent protein.