Depolymerization dynamics of individual filaments of bacterial cytoskeletal protein FtsZ

Abstract

We report observation and analysis of the depolymerization filaments of the bacterial cytoskeletal protein FtsZ (filament temperature-sensitive Z) formed on a mica surface. At low concentration, proteins adsorbed on the surface polymerize forming curved filaments that close into rings that remain stable for some time before opening irreversibly and fully depolymerizing. The distribution of ring lifetimes (T ) as a function of length (N), shows that the rate of ring aperture correlates with filament length. If this ring lifetime is expressed as a bond survival time, (T b ≡ NT), this correlation is abolished, indicating that these rupture events occur randomly and independently at each monomer interface. After rings open irreversibly, depolymerization of the remaining filaments is fast, but can be slowed down and followed using a nonhydrolyzing GTP analogue. The histogram of depolymerization velocities of individual filaments has an asymmetric distribution that can be fit with a computer model that assumes two rupture rates, a slow one similar to the one observed for ring aperture, affecting monomers in the central part of the filaments, and a faster one affecting monomers closer to the open ends. From the quantitative analysis, we conclude that the depolymerization rate is affected both by nucleotide hydrolysis rate and by its exchange along the filament, that all monomer interfaces are equally competent for hydrolysis, although depolymerization is faster at the open ends than in central filament regions, and that all monomer-monomer interactions, regardless of the nucleotide present, can adopt a curved configuration.bacterial division | atomic force microscopy | computer simulations | GTP hydrolysis B acterial cytoskeletal protein FtsZ (filamenting temperaturesensitive Z) assembles into dynamic polymers on the inner side of the bacterial cytoplasmic membrane in the presence of GTP. Its role in cell division is twofold: recruiting the rest of the proteins involved in the assembly of the septal ring and contributing to generate the force needed for cell rupture. Recent in vitro experiments with artificially membrane-bound FtsZ reconstituted in model systems support the suggestion that FtsZ is capable of generating a contractile force without the need of any other proteins (1, 2). Several theoretical models trying to explain the molecular mechanism by which the GTP-dependent self-assembling polymerization process generates a contractile force have been recently proposed (3-12), but the debate is still open. Existing controversy over the relative contribution of filament curvature and lateral interactions in the constriction process is difficult to resolve until new experimental data determine how GTP hydrolysis is associated to monomer exchange within filaments.FtsZ is known to polymerize above a critical concentration in the presence of GTP (13). Bulk experiments in solution using a fluorescence signal to follow polymerization (14) have provided indirect evidence of monomer exchange, annealing, and depolymerizatio...

FtsZ filament capping by MciZ, a developmental regulator of bacterial division

“…5A), we postulate that blocking annealing may be an important pathway by which MciZ reduces filament size. Fragmentation and annealing of FtsZ filaments have been observed for filaments adsorbed to mica or supported lipid bilayers (49)(50)(51), and also in solution (43), although the rates of these reactions are yet to be precisely determined. Annealing may be particularly important in vivo, when the filaments are concentrated on the membrane at the center of the cell (5, 52).…”

Defining the rate-limiting processes of bacterial cytokinesis

“…However, Z-ring discontinuity has been observed in many bacterial species (19,37,48,(51)(52)(53)(54), and incomplete Z-rings have been shown to lead to cell wall indentation (110), suggesting that ring completion is not required for Z-ring function. We suggest that the continuous and discontinuous organizations reflect different dynamic states of the Z-ring modulated by FtsZ's GTPase activity, which is known to promote the fracture of long FtsZ polymers (111) and to modulate FtsZ turnover dynamics (82). Active GTP hydrolysis may thus serve to constantly remodel the Z-ring, generating a discontinuous, clustered organization that can reorganize promptly in response to cellular cues.…”

A model of membrane contraction predicting initiation and completion of bacterial cell division

“…We do not directly account for the potential of membranebound polymers to reanneal after fragmentation. However, the increased likelihood that a GTP hydrolysis/dissociation event occurring towards either end of the FtsZ polymer results in permanent loss of subunits from the polymer, compared to a GTP hydrolysis/dissociation event in the centre of the polymer, as found by Mateos-Gil et al 22 , is accounted for in the model. By calculation of the number of binding sites available to the polymer fragments produced on dissociation, smaller polymers are less likely to be bound to an anchor and so are more likely to move back to the soluble fraction with loss from the Z-ring.…”

“…10 Surovtsev et al 14 assumed that upon GTP hydrolysis, the GDP-bound subunit dissociates from the intact polymers on either side instantaneously. We use the equivalent rate equation given by 22 The significance of the change in the model is that we now account for the time spent by FtsZ subunits within polymers in the GDP-bound state following hydrolysis, prior to dissociation.…”