Dynamic Instability in a DNA-Segregating Prokaryotic Actin Homolog

Garner, Ethan C.; Campbell, Christopher S.; Mullins, R. Dyche

doi:10.1126/science.1101313

Cited by 253 publications

(403 citation statements)

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“…MreB has a Cc of 3 nM and displays relatively normal ATPase rates (Esue et al, 2005). ParM, a bacterial actinlike protein involved in DNA segregation, also has a very rapid polymerization rate, some 300 times that of vertebrate actin, yet it displays dynamic instability driven by ATP hydrolysis (Garner et al, 2004). Bacterial actin homologues are not phylogenetically similar to parasite actins, thus it is not surprising that TgACT1 does not display these extreme behaviors.…”

Section: Discussionmentioning

confidence: 99%

“…Bacterial actin-like proteins such as ParM are highly sensitive to ATP and show dynamic instability due to high turnover of nucleotide (Garner et al, 2004). This feature leads to rapid polymerization that often overshoots the plateau followed by rapid disassembly (Garner et al, 2004).…”

Section: Polymerization Kinetics Of Tgact1mentioning

confidence: 99%

“…This feature leads to rapid polymerization that often overshoots the plateau followed by rapid disassembly (Garner et al, 2004). To determine the sensitivity of TgACT1 to nucleotide, we monitored polymerization over time in 1.0 mM ATP versus ADP.…”

Section: Polymerization Kinetics Of Tgact1mentioning

confidence: 99%

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Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the ParasiteToxoplasma gondii

Sahoo

Beatty

Heuser

et al. 2006

MBoC

118

138

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Toxoplasma is a protozoan parasite in the phylum Apicomplexa, which contains a number of medically important parasites that rely on a highly unusual form of motility termed gliding to actively penetrate their host cells. Parasite actin filaments regulate gliding motility, yet paradoxically filamentous actin is rarely detected in these parasites. To investigate the kinetics of this unusual parasite actin, we expressed TgACT1 in baculovirus and purified it to homogeneity. Biochemical analysis showed that Toxoplasma actin (TgACT1) rapidly polymerized into filaments at a critical concentration that was 3-4-fold lower than conventional actins, yet it failed to copolymerize with mammalian actin. Electron microscopic analysis revealed that TgACT1 filaments were 10 times shorter and less stable than rabbit actin. Phylogenetic comparison of actins revealed a limited number of apicomplexan-specific residues that likely govern the unusual behavior of parasite actin. Molecular modeling identified several key alterations that affect interactions between monomers and that are predicted to destabilize filaments. Our findings suggest that conserved molecular differences in parasite actin favor rapid cycles of assembly and disassembly that govern the unusual form of gliding motility utilized by apicomplexans.

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Section: Discussionmentioning

confidence: 99%

Section: Polymerization Kinetics Of Tgact1mentioning

confidence: 99%

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Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the ParasiteToxoplasma gondii

Sahoo

Beatty

Heuser

et al. 2006

MBoC

118

138

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“…Although the cryo-EM reconstruction is from the wild-type ParM with the nonhydrolyzable ATP analog AMP-PNP, we have also done reconstructions from negatively stained images using the slowly hydrolyzable ATP analog ATP-γS and using ATP with the E148A mutant of ParM that does not hydrolyze ATP 26 . At a resolution of ~24 Å, there were no major differences among these negativestain reconstructions.…”

Section: Domain-domain Motions In the Actin Superfamilymentioning

confidence: 99%

The structure of bacterial ParM filaments

Orlova

Garner

Galkin

et al. 2007

Nat Struct Mol Biol

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Bacterial ParM is a homolog of eukaryotic actin and is involved in moving plasmids so that they segregate properly during cell division. Using cryo-EM and three-dimensional reconstruction, we show that ParM filaments have a different structure from F-actin, with very different subunitsubunit interfaces. These interfaces result in the helical handedness of the ParM filament being opposite to that of F-actin. Like F-actin, ParM filaments have a variable twist, and we show that this involves domain-domain rotations within the ParM subunit. The present results yield new insights into polymorphisms within F-actin, as well as the evolution of polymer families.ParM is a bacterial actin homolog involved in plasmid segregation 1,2 . The structures of both ParM and MreB 3 , another bacterial actin homolog, as well as FtsZ 4 , a tubulin homolog, show that the eukaryotic cytoskeleton is similar to a prokaryotic cytoskeleton that has only recently been studied. Although the higher-order structure of FtsZ is still unknown, it has been assumed that both ParM and MreB assemble into filaments with an arrangement of subunits very similar to that found in F-actin. However, a crystal structure of monomeric ParM has unexpectedly shown that its main differences from actin are in regions expected to be involved in the filamentous subunit-subunit interface 2 . A previous study 2 has used EM of negatively stained ParM filaments to generate a low-resolution three-dimensional reconstruction, which suggests that the ParM filament is very similar to F-actin.We have used negative staining, cryo-EM and quick-freeze/deep-etch EM to examine filaments formed by the ParM protein from Escherichia coli. The question that we intended to answer was how similar to F-actin the ParM filaments appear at higher resolution. We show that the ParM filaments are substantially different from F-actin, with a very different subunit-subunit interface. Because of the very different interface, the helical handedness of the ParM filaments is opposite to that of F-actin. RESULTS Variable twist in ParM filamentsEM shows that ParM forms long filaments when incubated with the nonhydrolyzable ATP analog AMP-PNP (Fig. 1). We analyzed both negatively stained (Fig. 1a) and frozenhydrated ParM filaments (Fig. 1b), but took advantage of the iterative helical real-space reconstruction (IHRSR) method 5 to surmount the problems posed by flexible and disordered filaments. In this method, rather than individual filaments being treated as ideal helices with a uniform structure, short segments are analyzed, classified and reconstructed separately. One of the most obvious forms of disorder revealed by such a method is the presence of variable twist in the ParM filaments, even greater than what has been shown for F-actin 6-8 . The variability in average twist angles found in unstained frozen-hydrated ParM segments (28,886 segments, each segment ~480 Å long) is shown in Figure 2a. This variability arises from different segments within the same filaments, rather than different ...

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“…ParR induces ATPase activity of ParM, which abolished affinity between the two proteins-new ATP-bound ParM can insert between ParR/parS and the filament thus extends while keeping close contact to the plasmid. In vitro, ParM filaments can undergo growth as well as spontaneous shrinkage at both ends, thus paralleling tubulin's dynamic instability [Garner et al, 2004]. ParR and ParM are indeed sufficient to push parS containing plasmids apart in vitro , so ParM powers a simple bacterial mitotic-like machinery.…”

Section: Mreb Parm and Other Bacterial Actin-like Proteinsmentioning

confidence: 99%

Dynamics of bacterial cytoskeletal elements

Graumann

2009

Cell Motil. Cytoskeleton

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Bacterial cytoskeletal elements are involved in an astonishing spectrum of cellular functions, from cell shape determination to cell division, plasmid segregation, the positioning of membrane-associated proteins and membrane structures, and other aspects of bacterial physiology. Interestingly, these functions are not necessarily conserved, neither between different bacterial species nor between bacteria and eukaryotic cells. The flexibility of cytoskeletal elements in performing different tasks is amazing and emphasises their very early development during evolution. This review focuses on the dynamics of cytoskeletal elements from bacteria. Cell Motil. Cytoskeleton 66: 909-914, 2009. '

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Dynamic Instability in a DNA-Segregating Prokaryotic Actin Homolog

Cited by 253 publications

References 29 publications

Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the ParasiteToxoplasma gondii

Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the ParasiteToxoplasma gondii

The structure of bacterial ParM filaments

Dynamics of bacterial cytoskeletal elements

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