Ramanujam Srinivasan

The bacterial cell division protein FtsZ assembles into cytoplasmic rings in fission yeast

During cytokinesis, most bacteria assemble a ring-like structure that is composed of the tubulin homolog FtsZ. The mechanisms regulating assembly and organization of FtsZ molecules into rings are not fully understood. Here, we express bacterial FtsZ in the fission yeast Schizosaccharomyces pombe and find that FtsZ filaments assemble into cytoplasmic rings. Investigation of the Escherichia coli FtsZ revealed that ring assembly occurred by a process of closure and/or spooling of linear bundles. We conclude that FtsZ rings can assemble in the absence of all other bacterial cytokinetic proteins and that the process might involve hydrolysis of FtsZ-bound GTP and lateral associations between FtsZ filaments.

Cylindrical cellular geometry ensures fidelity of division site placement in fission yeast

Summary Successful cytokinesis requires proper assembly of the contractile actomyosin ring, its stable positioning on the cell surface and proper constriction. Over the years, many of the key molecular components and regulators of the assembly and positioning of the actomyosin ring have been elucidated. Here we show that cell geometry and mechanics play a crucial role in the stable positioning and uniform constriction of the contractile ring. Contractile rings that assemble in locally spherical regions of cells are unstable and slip towards the poles. By contrast, actomyosin rings that assemble on locally cylindrical portions of the cell under the same conditions do not slip, but uniformly constrict the cell surface. The stability of the rings and the dynamics of ring slippage can be described by a simple mechanical model. Using fluorescence imaging, we verify some of the quantitative predictions of the model. Our study reveals an intimate interplay between geometry and actomyosin dynamics, which are likely to apply in a variety of cellular contexts.

Filament Formation of the Escherichia coli Actin-Related Protein, MreB, in Fission Yeast

Proteins structurally related to eukaryotic actins have recently been identified in several prokaryotic organisms. These actin-like proteins (MreB and ParM) and the deviant Walker A ATPase (SopA) play a key role in DNA segregation and assemble into polymers in vitro and in vivo. MreB also plays a role in cellular morphogenesis. Whereas the dynamic properties of eukaryotic actins have been extensively characterized, those of bacterial actins are only beginning to emerge. We have established the fission yeast Schizosaccharomyces pombe as a cellular model for the functional analysis of the Escherichia coli actin-related protein MreB. We show that MreB organizes into linear bundles that grow in a symmetrically bidirectional manner at 0.46 +/- 0.03 microm/min, with new monomers and/or oligomers being added along the entire length of the bundle. Organization of linear arrays was dependent on the ATPase activity of MreB, and their alignment along the cellular long axis was achieved by sliding along the cortex of the cylindrical part of the cell. The cell ends appeared to provide a physical barrier for bundle elongation. These experiments provide new insights into the mechanism of assembly and organization of the bacterial actin cytoskeleton.

Comparing contractile apparatus-driven cytokinesis mechanisms across kingdoms†

Cytokinesis is the final stage of the cell cycle during which a cell physically divides into two daughters through the assembly of new membranes (and cell wall in some cases) between the forming daughters. New membrane assembly can either proceed centripetally behind a contractile apparatus, as in the case of prokaryotes, archaea, fungi, and animals or expand centrifugally, as in the case of higher plants. In this article, we compare the mechanisms of cytokinesis in diverse organisms dividing through the use of a contractile apparatus. While an actomyosin ring participates in cytokinesis in almost all centripetally dividing eukaryotes, the majority of bacteria and archaea (except Crenarchaea) divide using a ring composed of the tubulin‐related protein FtsZ. Curiously, despite molecular conservation of the division machinery components, division site placement and its cell cycle regulation occur by a variety of unrelated mechanisms even among organisms from the same kingdom. While molecular motors and cytoskeletal polymer dynamics contribute to force generation during eukaryotic cytokinesis, cytoskeletal polymer dynamics alone appears to be sufficient for force generation during prokaryotic cytokinesis. Intriguingly, there are life forms on this planet that appear to lack molecules currently known to participate in cytokinesis and how these cells perform cytokinesis remains a mystery waiting to be unravelled.

Novel actin-like filament structure from Clostridium tetani

Background: Alp12 is a novel plasmid-encoded actin-like protein from Clostridium tetani. Results: Alp12 forms dynamically unstable filaments with an open helical cylinder structure composed of four protofilaments. Conclusion: Specialized prokaryotic filament systems have evolved to execute a single function in comparison with the general multitasking force provider, double-stranded F-actin. Significance: Repetitive Alp12 polymerization cycles may be incorporated into nanomachines. Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines.

Bacterial cell division protein FtsZ is a specific substrate for the AAA family protease FtsH

The role of AAA (ATPases Associated to a variety of cellular Activities) family protease FtsH in bacterial cell division is not known, although mutations in ftsH were found to inhibit cell growth and division (1, 6, 13). Overexpression of heterologous FtsH in Escherichia coli results in the formation of multinucleate ®lamentous cells due to the abolition of cell septation (8). Further, independent studies on FtsH (15) and FtsZ (2), which is the key regulator of bacterial cell division, have shown that FtsH protease and FtsZ protein are localized to the mid-cell site during septation. FtsZ protein is the prokaryotic homologue of tubulin (5, 10, 12), possessing GTP-dependent polymerization activity (4, 11). igni®cantly, the AAA family ATPase member katanin disassembles tubulin polymers in an ATP-dependent manner (7). Based on these observations, we reasoned that an interaction similar to that between katanin and tubulin might hold true for FtsH and FtsZ in prokaryotes as well. To verify this hypothesis, we examined whether the FtsH protease of Escherichia coli

Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation

(FtsH_{Ec})

The Nitrosopumilus maritimus CdvB, but Not FtsZ, Assembles into Polymers

could degrade FtsZ of E. coli

In vitro polymerization of Mycobacterium leprae FtsZ OR Mycobacterium tuberculosis FtsZ is revived or abolished, respectively, by reciprocal mutation of a single residue

(FtsH_{Ec})

Determining the most probable natural resting orientation of sector shaped parts

in vitro.