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Review TRENDS in Cell Biology Vol.17 No.5 The push and pull of the bacterial cytoskeleton Natalie A. Dye1,2 and Lucy Shapiro2 1 2 Department of Biochemistry, Beckman Center, Stanford University, Stanford, CA 94305, USA Department of Developmental Biology, Beckman Center, Stanford University, Stanford, CA 94305, USA A crucial function for eukaryotic cytoskeletal filaments is to organize the intracellular space: facilitate communication across the cell and enable the active transport of cellular components. It was assumed for many years that the small size of the bacterial cell eliminates the need for a cytoskeleton, because simple diffusion of proteins is rapid over micron-scale distances. However, in the last decade, cytoskeletal proteins have indeed been found to exist in bacteria where they have an important role in organizing the bacterial cell. Here, we review the progress that has been made towards understanding the mechanisms by which bacterial cytoskeletal proteins influence cellular organization. These discoveries have advanced our understanding of bacterial physiology and provided insight into the evolution of the eukaryotic cytoskeleton. Bacterial cytoskeletal proteins Cytoskeletal elements have been identified in prokaryotes based on structural and functional homology to eukaryotic proteins (Table 1 and Ref. [1]). Although there is virtually no primary sequence similarity between prokaryotic and eukaryotic cytoskeletal elements, prokaryotic proteins with actin- and tubulin-like folds have been found. Many of these proteins have been shown to polymerize in vitro in a nucleotide-dependent manner. Furthermore, genetic and cell biological data suggest that, in vivo, these proteins share many of the functions of eukaryotic cytoskeletal proteins: they are involved in cellular organization, division and maintenance of cell shape. In addition to actin, tubulin and intermediate filament-like proteins, a class of proteins, the MinD–ParA superfamily, exists, which, in bacteria, appears to have cytoskeletal functions (Box 1). Although there are eukaryotic ATPases with similar structures and sequences to those of MinD–ParA proteins, the ability to polymerize in vivo and in vitro seems limited to the prokaryotic homologs. Given that passive diffusion alone can enable cellular components to sample a small bacterial cell rapidly, it has been somewhat surprising to find that many of the bacterial cytoskeletal proteins are thought to be involved in positioning proteins and DNA actively (Table 1). Instead of listing what is known about the genetic phenotypes or the polymerization properties of each individual bacterial cytoskeletal protein (reviewed in Ref. [1]), we will here Corresponding author: Shapiro, L. ([email protected]). Available online 16 April 2007. www.sciencedirect.com review the general mechanisms that are emerging for how cytoskeletal proteins in bacteria influence cellular organization. A push in the right direction The most basic way to influence the localization of cellular components is to physically move them through the cytoplasm to a specific location. In eukaryotic cells, there are motor proteins that directly transport vesicles, mRNA and proteins along tracks of actin or tubulin. However, actin and tubulin themselves can also function as motors to propel objects through the cytoplasm (Box 2). In bacteria, the plasmid-encoded actin homolog, ParM, is thought to force actively two clusters of plasmid DNA apart, using its ability to polymerize (Figure 1a) [2–5]. The plasmid R1 exists in low copy number in the Escherichia coli cytoplasm and would be quickly lost from the population without an active mechanism for segregating the DNA into both daughter cells at division. There are three components encoded by the plasmid that are dedicated to its partitioning: the actin-like ParM, the DNAbinding protein ParR, and the centromer-like parC DNA region [6]. These three components form a complex that is necessary and sufficient for the segregation of the DNA in vivo [5–7]. The regulation of par locus expression and the integration of plasmid replication with the host cell cycle are reviewed elsewhere [8]. In the cell, plasmid molecules cluster together so that the DNA appears as tight foci upon labeling [8,9]. Early in the cell cycle, there is only one focus, which colocalizes with foci of ParM and ParR at midcell [5,9]. As the cell cycle progresses, the plasmid is replicated by host machinery and split into two foci that move to opposite poles before division. As the plasmid foci move, they appear to be connected by a line of ParM [5,9]. The formation of these ParM filaments in vivo requires the ParR–parC complex and is essential for plasmid segregation [10]. Additionally, these filaments must be dynamic. A mutant ParM with altered ATPase activity still forms filaments, but they often inhibit cell division and abolish plasmid segregation [7,9,10]. In vitro, ParM forms filaments with strikingly similar ultrastructure to eukaryotic actin filaments [10,11]. In vitro-filament formation can be stimulated by the complex of ParR bound to parC [10]. This result was first interpreted to mean that ParR accelerates the nucleation of ParM polymerization on DNA, because the nucleation step is a kinetic hurdle in the polymerization of eukaryotic 0962-8924/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2007.03.005 240 Review TRENDS in Cell Biology Vol.17 No.5 Table 1. Functions and mechanisms of selected bacterial cytoskeletal proteinsa a The proteins listed above have been categorized according to their homology to eukaryotic cytoskeletal proteins. In white are actin homologs, in yellow, the tubulin homolog; in green, intermediate filament homolog; in orange, the MinD–ParA superfamily, which does not have any counterpart in the eukaryotic cytoskeleton. b The references listed provide recent insights into the molecular mechanism of these proteins in contributing to cellular organization. More information on the bacterial cytoskeletal elements that are not discussed here can be found in a separate recent review [1]. actin. However, when the rate of ParM nucleation was directly measured in vitro, it was found to be 300 times faster than that of eukaryotic actin [2]. The critical concentration for polymerization of ParM in vitro was also found to be much lower than its in vivo cellular concentration Box 1. The MinD–ParA family of bacterial-specific cytoskeletal proteins In bacteria, members of a superfamily of Walker ATPases have cytoskeletal properties and regulate the localization of both DNA and protein. Members of this family all share the deviant Walker A motif, GXGGXHK[TS], within the P-loop ATP-binding pocket [66]. MinD and ParA are two members of this family that were characterized early and represent two groups distinct in sequence and function. MinD regulates the position of the cytokinetic FtsZring by controlling the localization of a FtsZ inhibitor, MinC (reviewed in Refs [1,27,37]). The ParA subgroup functions in the segregation of DNA [64]. Three subclasses of the ParA group can be identified, based on protein sequence: chromosomal and plasmid Type Ia and Ib [65]. Although it shares its ‘Par’ name with ParM, ParA does not resemble actin in structure. To date, five members of the MinD–ParA superfamily have been shown in vitro to polymerize into filaments in a nucleotidedependent manner: MinD (E. coli), Soj (B. subtilis), ParA (plasmid pB171), ParF (plasmid TP228), and SopA (F plasmid) [23,24,63,67– 69]. All but Soj were shown to form long, thin protofilaments assembled into large, bundled structures with a frayed appearance at one end. Soj polymers do not resemble these bundled structures, and it is not yet known whether this difference is due to an inherent difference between the homologs or variation in the experimental conditions. In vivo, MinD–ParA homologs tend to form large-scale filamentous structures that follow a helical path in the cell, often with oscillatory dynamics [22–24,55,70–72]. E. coli MinD, for example, forms a helix at one cell pole that dissociates and reforms at the opposite pole approximately every 20 s, a process dependent on a regulatory protein, MinE [55,72]. Mutations that affect nucleotide hydrolysis do not prevent the formation of such helical structures but do affect their dynamic behavior. www.sciencedirect.com [2–4,10]. Finally, ParM filaments in vitro were found to be unstable, constantly alternating between phases of elongation and rapid disassembly, a process called dynamic instability [2–4]. In light of these in vitro findings, it now seems likely that ParR–parC regulates ParM filament stability, rather than nucleation. These data suggest an in vivo model (Figure 1a) whereby ParM filaments are bound at both ends to two different plasmid clusters through ParR, which protects them from their inherent instability and enables them to elongate to sufficient lengths to separate the plasmids to opposite ends of the cell [2–4]. The polymerization of ParM in-between ParR would provide the force required to separate the plasmid clusters (Box 2) [2–4]. Recently, this model was confirmed with an in vitro reconstitution of plasmid segregation with purified ParM, ParR and parC [3]. In the presence of ATP and ParR, ParM forms dynamic astral filaments on the surface of the parCcoated microspheres. In addition, it is possible to see ParM filaments connecting two or more beads, forming a spindlelike structure similar to that observed in vivo, which elongates to push the beads apart. It is even possible to witness ‘search and capture’ events, where filaments extending off of the surface of one bead reach out and contact a neighboring bead. ParM filaments in between beads then elongate as the beads separate from one another (Box 2). This in vitro reconstitution experiment demonstrates that ParM polymerization, coupled to ParR–parC, can directly exert force on an object, mirroring actin polymerization driving the intracellular movement of Listeria monocytogenes and whole cell motility in eukaryotic cells (Box 2) [12]. Furthermore, the reconstitution experiments provide additional support for a model in which ParRbinding to ParM stabilizes the filaments in-between two segregating pieces of DNA. Long filaments of ParM were Review TRENDS in Cell Biology Box 2. Force generation by polymerization The polymerization of a cytoskeletal protein can be used to move a load through a mechanism referred to as thermal or Brownian ratcheting (reviewed in Ref. [12]). Spontaneous elongation of a polymer occurs when the free energy of the subunit in its polymer state is lower than the free energy of the subunit in its monomer state, which is true above a certain critical concentration (Ccrit). If the polymer is attached to an object, for example another protein or piece of DNA, the energy that is released upon polymerization can be used to displace the object in the direction of polymerization. The maximum amount of force a polymer can exert is related to the thermodynamics of its polymerization: F max ¼ kBT d ½C ln C crit [I] In the above equation (Equation I), kB is the Boltzmann’s constant; T is the absolute temperature; d is the distance the object is moved by the insertion of a single subunit; [C] is the monomer concentration; and Ccrit is the critical concentration for polymerization, which is normally equal to the ratio of koff to kon, the rate constants for the depolymerization and polymerization, respectively. We can now estimate the maximum force that can be generated by ParM polymerization, given the measured parameters of d = 2.45 nm (1/2 the diameter of a monomer, because ParM is a double stranded polymer) [11], T = 25 8C, Ccrit of ATP-bound ParM = 0.6 mM (ATP is in excess in the cell) [2,4], and [C] = 2.3 mM (steady-state concentration of monomer) [2,3]. These values predict that single filaments of ParM can exert up to 2.3 pN of force. Interestingly, the nucleation rate of ParM is rapid and the critical concentration of ATP-bound ParM (ATP-ParM) is much lower than its cellular concentration of 12–14 mM [10]. Therefore, much of the ParM in the cell should in theory be in filament form at steady state and unable to provide any force. In fact, in eukaryotic cells, actin is also present at concentrations well above its critical concentration; however, the presence of multiple depolymerizing proteins and monomer-sequestering proteins in the cell maintains a high steady state concentration of monomers. In the case of ParM, however, there is no need for other proteins to regulate polymerization because the filaments themselves exhibit dynamic instability. Once hydrolysis of ATP occurs, the ParM filament is unstable. This is demonstrated by the fact that the critical concentration of ADP-bound ParM (ADP-ParM) is at least two orders of magnitude larger than the ATP-ParM, meaning that the offrate of monomers from the filament is much faster than the on-rate [2]. The balance between rapid nucleation of ATP-ParM and rapid depolymerization of ADP-ParM upon hydrolysis results in dynamic instability, which maintains a steady-state monomer concentration of 2.3 mM [2,3]. The in vitro reconstitution of the ParM-mediated movement of DNA enabled the direct demonstration of the requirement of dynamic instability to power force generation [3]. With a steady-state concentration of monomers above the critical concentration for ATP-ParM, polymerization can be coupled to the generation of force. The only protein required for the coupling is ParR, which selectively stabilizes the ParM filaments on DNA. Therefore, the kinetic parameters of ParM polymerization are aptly tuned so that the ParM–ParR–parC complex can function in the absence of other cellular factors, a property that is undoubtedly favorable for a plasmid-encoded system. only observed in-between two beads bound by ParR. If the spindle is severed with laser ablation, the newly generated free end of the filament, which is not bound to ParR, rapidly depolymerizes [3]. It is not yet known whether other plasmids use a partitioning mechanism similar to the ParM-pushing mechanism. However, a newly discovered actin homolog from Bacillus subtilis, AlfA, which is required for the segregation of plasmid pLS32, resembles ParM in its www.sciencedirect.com Vol.17 No.5 241 intracellular localization pattern and dynamics [13]. This result suggests that AlfA might also be able to push plasmids through the cytoplasm like ParM, although this hypothesis has yet to be tested. A mitotic-like pull Just as polymerization can generate a pushing force, depolymerization can generate a pulling force. In eukaryotic cells, for example, the energy released upon depolymerization of microtubules can be harnessed by a complex of proteins attached to chromosomes, driving their segregation [14]. Recent evidence from the bacterium Vibrio cholerae suggests an analogous pulling mechanism might exist in prokaryotes as well [15]. V. cholerae has two chromosomes, each encoding its own partitioning genes and exhibiting distinct dynamic behaviors upon segregation [16,17]. The mechanism of segregation of chromosome II is not yet known, but recent work indicates that chromosome I might be pulled to the pole by a ParA homolog (Figure 1b) [15]. Although they are similarly named (‘Par’ referring to a role in partitioning), ParA and ParM are different at both the sequence and structural levels (Box 1). At the beginning of the cell cycle, ParA of chromosome I (ParAI) localizes to the oldest pole of the cell with a DNAbinding protein ParB and the origin of replication [15]. ParAI is also found in the opposite half of the cell in a haze that can be resolved into filamentous structures with deconvolution microscopy. Once the origin duplicates, one copy stays at the old pole, along with foci of ParB and ParAI. The other ParB–origin complex moves across the cell at the edge of the filamentous ParAI structure. As the migration of the chromosome progresses, the ParAI zone shrinks towards the new pole, finally condensing into a polar focus. In the absence of ParAI, the origin does not exhibit the same dynamic behavior [15]. The two ParB–origin complexes can be seen ‘floating’ in the cytoplasm, no longer restricted to the pole. This data suggests that not only does ParAI have a role in directing one of the origins to the new pole, but also in keeping the other origin at the old pole. Interestingly, in the absence of ParAI, the two origins are still able to move apart from one another, but they do so inefficiently and bidirectionally, rather than asymmetrically as in the wild-type cells. It is possible that a partitioning mechanism that directs chromosome II movement is able to compensate partially for the absence of ParAI. Unlike ParM, ParAI does not localize in-between the two separating DNA loci; instead it localizes in-between one of the duplicated loci and the new pole of the cell (Figure 1). It seems clear, therefore, that ParAI and ParM act through distinct mechanisms. In V. cholerae, the data are consistent with a model in which ParAI is pulling the duplicated origin in, toward the new pole, similarly to the way in which microtubules direct the motion of chromosomes in eukaryotic cells. It has not yet been shown that this particular ParA homolog can self-associate into filaments in vitro (Box 1), but it has been shown to make filamentous structures in vivo. Additionally, mutations in the ATPase domain of ParAI abolish its dynamic behavior and its ability to position the origin, which is consistent 242 Review TRENDS in Cell Biology Vol.17 No.5 Figure 1. Cytoskeletal-mediated movement of DNA in bacteria. Schematics of the in vivo segregation dynamics, on the left, and a molecular model, on the right, are presented for two distinct cytoskeletal partitioning mechanisms. (a) The actin-like ParM forms a bundle of filaments in-between duplicated plasmids that elongates as plasmids segregate to the poles. These filaments are bound through the DNA-binding protein ParR (green) to a specific site on the plasmid, parC. Insertion of ATPmonomers (red) at the growing tip of filaments is proposed to exert force on the plasmids, separating the two copies (in the direction of the arrows). ADP-monomers (blue) accumulate in the filament, owing to its hydrolysis activity, making the filament unstable and subject to rapid depolymerization. When bound to the DNA through ParR, however, the filaments are presumably protected from depolymerization. (b) V. cholerae ParAI (yellow) forms filaments that extend from the cell pole to attach to an origin proximal region of chromosome I as it is segregating. Again, a DNA-binding protein, ParB (green), appears to mediate the interaction between ParA and the DNA. As the migration of the chromosome continues, the ParAI filaments shrink toward the new pole (in the direction of the arrow). This pattern is consistent with a model that ParAI exerts force on the DNA, pulling it to the pole, although this mechanism has not been confirmed. with a role for depolymerization in mediating segregation [15]. Visualization of ParAI filamentous structures in vivo using high resolution electron microscopy would confirm the existence of a cytoskeletal structure and help clarify the mechanism of chromosome movement. Interestingly, in Caulobacter crescentus, clusters of filaments of unknown protein composition have been detected, tethered to the pole of the cell [18]. C. crescentus and V. cholerae both localize a chromosomal origin to the pole [16,17,19]. Additionally, C. crescentus ParA is essential for chromosome segregation and shares considerable homology with V. cholerae ParAI [20]. Therefore, it is possible that these two ParA homologs use similar mechanisms and that the electron micrographs have captured the C. crescentus ParA in the act of separating the chromosome. To confirm this hypothesis, it will be necessary to see if the ‘polar filaments’ are missing in a ParA-depleted C. crescentus strain. Recently, a ParA homolog in Rhodobacter sphaeroides, PpfA, was found to be required for the segregation of clusters of a chemotaxis protein, TlpT [21]. This discovery is the first example of a ParA homolog mediating the segregation of a protein, rather than DNA. The mechanism www.sciencedirect.com of this segregation is unknown. It is likely to be distinct, however, from that of V. cholerae ParAI, given their different localization patterns. TlpT clusters segregate to the quarter-cell positions, rather than at the poles [21]. The pattern of segregation actually resembles that of V. cholerae chromosome II [16,17], in addition to that of certain plasmids in E. coli, such as F and pB171, which are also regulated by related by filament-forming ParA homologs [22–24]. Dynamic scaffolding Cytoskeletal proteins do not have to physically move proteins or DNA through the cytoplasm to influence cellular organization. A higher order cellular structure formed by cytoskeletal proteins can act as a scaffold to direct the localization of other cellular components. Just one of many possible examples from eukaryotic cells is the microtubule network in plants, which forms a scaffold for cell wall synthesis, directing the placement of the synthetic enzymes and the orientation of the cellulose microfibrils [25]. Theoretically, cells can form scaffolds with anything capable of forming a higher order structure. The polymerization of a cytoskeletal protein, however, is coupled to Review TRENDS in Cell Biology nucleotide hydrolysis. Therefore, the assembly and disassembly of cytoskeletal scaffolds is rapid and can be easily regulated with modulators of polymerization. This dynamic property enables the structure to be intimately integrated with the cell cycle and to be responsive to its intracellular environment. In bacteria, there are at least three examples of such dynamic cytoskeletal scaffolds. FtsZ – organizing the division apparatus The tubulin homolog, FtsZ, is a scaffold for several proteins involved in cytokinesis. Its filaments form a ring-like structure that defines the division plane [26]. In the absence of FtsZ, all of the other proteins involved in division fail to localize correctly and the cell cannot initiate constriction (reviewed in Refs [1,27]). This Z-ring structure is stabilized and bound to the membrane largely by the protein FtsA, another actin homolog [28–30]. Numerous different molecules are then recruited to this site. In E. coli, FtsA is thought to recruit downstream effectors directly to the site of the FtsZ ring [27,31,32], whereas in B. subtilis, the newly discovered proteins SepF and YlmF are also involved in the recruitment process [28,33,34]. FtsZ might also be directly exerting force on the membrane during constriction, although there is currently little direct evidence to support this idea. The FtsZ ring, perhaps more than any other cytoskeletal structure, demonstrates the importance of dynamic behavior in a scaffold. Photobleaching experiments have shown that the Z-ring of E. coli and B. subtilis is continually remodeling with a half-time of 8–9 s [35]. Additionally, low levels of filaments can be seen rapidly traversing the cell along a helical path [36]. Mutations that affect the GTPase activity of FtsZ alter its dynamic behavior and do not fully support cytokinesis [35]. Its dynamic behavior enables FtsZ to sample the cell quickly and to position itself optimally between the replicated chromosomes. Specific mechanisms for integrating Z-ring assembly with chromosome segregation in E. coli, B. subtilis and C. crescentus have been characterized [27,37,38]. Although each organism accomplishes this task differently, they all link a sensor for the intracellular position of DNA to a regulator of FtsZ polymerization. For example, in C. crescentus, the origin of replication recruits a direct inhibitor of FtsZ polymerization, MipZ [38]. When the origins duplicate and segregate to the poles, MipZ travels with them and FtsZ is forced to polymerize at midcell, the area of lowest inhibitor concentration. This mechanism ensures a dynamic coupling between chromosome segregation and cytokinesis. The FtsZ ring is also responsive to variations in intracellular conditions. In B. subtilis, the initiation of sporulation causes the Z-ring to be moved from a midcell to a polar site [39]. In E. coli, DNA damage induces the expression of a specific FtsZ inhibitor, SulA, that arrests cell division [40]. Again, the rapid turnover of FtsZ filaments enables these responses to occur within seconds. MreB and MreC – directing helical cell wall growth The actin homolog MreB (and MreB-like proteins in B. subtilis), in rod-shaped bacteria, forms a dynamic helical scaffold that controls the localization of cell wall growth [41–43]. Enzymes involved in cell wall synthesis and www.sciencedirect.com Vol.17 No.5 243 peptidoglycan precursor molecules localize in the cell following a helical pattern that is similar to that of MreB [41–46]. In the absence of MreB, these enzymes and precursors are incorrectly localized, resulting in misshapen cells [41–46]. Therefore, it has been suggested that MreB creates helical tracks onto which the cell wall machinery localizes, perhaps in a similar fashion to the way plants use the microtubule cytoskeleton as tracks for cellulose synthesis [25]. Though the mechanism by which MreB controls cell wall growth is not fully understood, it is known to involve another helical scaffold composed of the MreC protein, whose gene lies in the mreB operon [45–48]. Unlike MreB, MreC is not an actin homolog, nor does it resemble any other known protein, yet, in the cell, it is able to localize in a helical pattern that resembles that of MreB [45–48]. In C. crescentus, this MreC spiral resides in the periplasmic space and is non-overlapping with the MreB pattern [45,46]. MreC is required for the localization of an enzyme for cell wall synthesis, Pbp2, in addition to several outer membrane proteins, suggesting that it might have a general role in the organization of the periplasmic and outer membrane compartments [45,46]. Like FtsZ, the MreB structure inside cells has been shown to exhibit a rapid dynamic behavior that is probably due to its ability to hydrolyze nucleotide [49–53]. C. crescentus MreB undergoes regulated transitions in its cellular localization, making a helical structure early in the cell cycle and then reorganizing into a ring structure positioned at the division plane later in the cell cycle. [43,54]. The mechanism and function of this large-scale reorganization is not known. It could conceivably be involved in a switch between elongation and division-specific phases of cell wall growth. In E. coli, MreB is also able to form a variety of helical and ring structures, but a similar coordination with the cell cycle has not been observed [55]. MamK – lining up magnetosensing organelles A different actin-like protein, MamK, forms a linear scaffold in magnetotactic bacteria [56]. These bacteria form crystals of either Fe3O4 or Fe3S4 within membrane-bound clusters that line up along the long axis of the cell (reviewed in Ref. [57]). Using both cryoelectron tomography and fluorescence microscopy, MamK can be seen forming filaments right under the magnetosensing organelles in Magnetospirillum magneticum [56]. In the absence of MamK, no filaments are observed, the organelles disperse throughout the cytoplasm or aggregate, and the organism is no longer magnetotactic [56]. The polymerization of MamK has not yet been studied in vitro. In addition, its dynamic behavior in vivo is only beginning to be investigated [58]. Presumably the structure must be able to disassemble at division to enable separation of the two daughter cells, but this process has not yet been observed in live dividing cells. It is also not known whether MamK is involved in segregating the magnetosensing organelles at division. Concluding remarks Both prokaryotes and eukaryotes seem capable of using cytoskeletal polymers to actively and dynamically organize their intracellular space. Moreover, archeal homologs of 244 Review TRENDS in Cell Biology Vol.17 No.5 FtsZ and MreB have been identified, although little is known about their function in vivo. Nonetheless, the presence of this activity in eukaryotic and prokaryotic organisms argues that there is a fundamental requirement for dynamic polymers in cellular life and ancient mechanisms of polymer motors and scaffolds. Dynamic cytoskeletal polymers were thought to be vitally important to the evolution of eukaryotic cells, enabling them to grow in size by orders of magnitude and accomplish more complex tasks than the morphologically simpler bacteria and archaea [59]. Now that it has become clear that prokaryotic organisms have cytoskeletal proteins that are fundamentally similar in structure and activity to eukaryotic actin and tubulin, how have eukaryotes evolved a cellular biology that is so much more complex? Currently, there is not enough known about the bacterial cytoskeleton to answer definitively this question. As it stands, there does seem to be a difference in the functional diversity between eukaryotic and prokaryotic cytoskeletons. Eukaryotic actin and tubulin are responsible for the specific localization of thousands of molecules and are involved in many different mechanical processes. To accomplish this feat, eukaryotes have evolved a huge repertoire of binding partners and regulatory proteins for actin and tubulin. By contrast, prokaryotic cytoskeletal elements have so far been shown to be specialized for one particular function, for example the segregation of a particular DNA locus or the localization of one particular protein. MamK, for example, is only involved in lining up the magnetosensing clusters and cannot substitute for E. coli MreB in cell wall synthesis [58,60]. The one key exception to this observation is C. crescentus MreB, which has been implicated in chromosome segregation, protein localization and cell wall synthesis [43,45,46,53,54,61]. There is not yet sufficient evidence to show that MreB has a direct role in all these processes. The effect MreB has on polar positioning of proteins might be secondary to its role in directing cell wall growth [60]. It will be important in the future to determine whether MreB is able to participate directly in more than one cellular process and, if so, to use different types of mechanisms. The ability to use the same cytoskeletal proteins in many different processes might have been crucially important in the evolution of the complex eukaryotic organisms that we see today [59]. Motor proteins that move along cytoskeletal filaments, such as myosin and kinesin, might have also been key in that development. Such proteins have not yet been found in bacteria and might not exist. They might have been a recent development that evolved to improve the efficiency of intracellular motility and organization and thereby enable more complex functions [59]. Acknowledgements We thank Erin Barnhart, Matt Footer, Ethan Garner, Erin Goley, Antonio Iniesta, Kinneret Keren, Julie Theriot and Esteban Toro for critical reading of the manuscript and inspiring discussions. References 1 Shih, Y.L. and Rothfield, L. 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