Disassembly Of Polymers Research Articles

Adaptation of Bacillus subtilis MreB Filaments to Osmotic Stress Depends on Influx of Potassium Ions.

The circumferential motion of MreB filaments plays a key role in cell shape maintenance in many bacteria. It has recently been shown that filament formation of MreB filaments in Bacillus subtilis is influenced by stress conditions. In response to osmotic upshift, MreB molecules were released from filaments, as seen by an increase in freely diffusive molecules, and the peptidoglycan synthesis pattern became less organized, concomitant with slowed-down cell extension. In this study, biotic and abiotic factors were analysed with respect to a possible function in the adaptation of MreB filaments to stress conditions. We show that parallel to MreB, its interactor RodZ becomes more diffusive following osmotic stress, but the remodeling of MreB filaments is not affected by a lack of RodZ. Conversely, mutant strains that prevent efficient potassium influx into cells following osmotic shock show a failure to disassemble MreB filaments, accompanied by less perturbed cell wall extension than is observed in wild type cells. Because potassium ions are known to negatively affect MreB polymerization in vitro, our data indicate that polymer disassembly is directly mediated by the physical consequences of the osmotic stress response. The lack of an early potassium influx response strongly decreases cell survival following stress application, suggesting that the disassembly of MreB filaments may ensure slowed-down cell wall extension to allow for efficient adaptation to new osmotic conditions.

Light-Triggered Disassembly of Molecular Motor-based Supramolecular Polymers Revealed by High-Speed AFM.

Photoresponsive supramolecular polymers have a major potential for applications in responsive materials that are externally triggered by light with spatio-temporal control of their polymerisation state. While changes in macroscopic properties revealed the adaptive nature of these materials, it remains challenging to capture the dynamic depolymerisation process at the molecular level, which requires fast observation techniques combined with in situ irradiation. By implementing in situ UV illumination into a High-Speed Atomic Force Microscope (HS-AFM) setup, we have been able to capture the disassembly of a light-driven molecular motor-based supramolecular polymer. The real-time visualisation of the light-triggered disassembly process not only reveals cooperative depolymerisation, it also shows that this process continues after illumination is halted. Combining the data with cryo-electron microscopy and spectroscopy approaches, we obtain a molecular-level description of the motor-based polymer dynamics reminiscent of actin chain-end depolymerisation. Our detailed understanding of supramolecular depolymerisation will drive the development of future responsive polymer systems.

Studying Microtubule Dynamics in Human Neurons: Two-Dimensional Microtubule Tracing and Kymographs in iPSC- and SH-SY5Y-Derived Neurons for Tau Research.

The study of microtubule (MT) dynamics is essential for the understanding of cellular transport, cell polarity, axon formation, and other neurodevelopmental mechanisms. All these processes rely on the constant transition between assembly and disassembly of tubulin polymers to/from MTs, known as dynamic instability. This process is well-regulated, among others, by phosphorylation of microtubule-associated proteins (MAP), including the Tau protein. Protein kinases, in particular the microtubule affinity regulating kinase (MARK), regulate the MT-Tau interaction, inducingTau dissociation by phosphorylation. Phosphorylated Tau dissociates from microtubules forming insoluble aggregates known as neurofibrillary tangles. These accumulations of hyperphosphorylated Tau in the neurons disrupt the physiological MT-based transport machinery within the cell and can potentially lead to the development of neurodegenerative disorders, such as Alzheimer's disease (AD) and related tauopathies. Further investigations on the MT cytoskeleton dynamics are essential as they may elucidate pathomechanisms of neurodegenerative diseases - particularly tauopathies - as well as fundamental neurodevelopmental processes.The study of thedynamic assembly and disassembly of the MT network requires live-cell imaging rather than conventional immunocytochemistry based on fixed samples. To investigate MT dynamics, we perform live-cell imaging of neurons transfected with a fluorescently tagged version of the microtubule plus-end tracking protein (+TIP) EB3. This protein associates with the growing ends of MTs and thus visualizes MT growth in real time. Our imaging analysis protocol allows the determination of quantity, orientation, and velocity of MT growth in the soma and neurites of transfected neurons,using ImageJ-based tracking software and kymographs. Furthermore, functional effects of Tau and MARK kinases on the MT cytoskeleton can be assessed by overexpression or downregulation experiments of the respective protein prior to the live imaging assay. We use two different human neuronal cell models, naive and differentiated SH-SY5Y neuroblastoma cells, and neurons derived from induced pluripotent stem cells (iPSCs), both of which have shown success as models to study Tau-related pathologies.This protocol describes an optimized method for analysis of microtubule dynamics using fluorescent tagged EB3 protein as microtubule plus end marker. In this chapter, we outline the process of neuronal transfection, live-cell imaging, and necessary time-lapse image analysis based on ImageJ in two human-derived neuronal systems, which are suitable for the analysis of Tau trafficking and sorting studies.

MipZ caps the plus-end of FtsZ polymers to promote their rapid disassembly

The spatiotemporal regulation of cell division is a fundamental issue in cell biology. Bacteria have evolved a variety of different systems to achieve proper division site placement. In many cases, the underlying molecular mechanisms are still incompletely understood. In this study, we investigate the function of the cell division regulator MipZ from Caulobacter crescentus, a P-loop ATPase that inhibits the polymerization of the treadmilling tubulin homolog FtsZ near the cell poles, thereby limiting the assembly of the cytokinetic Z ring to the midcell region. We show that MipZ interacts with FtsZ in both its monomeric and polymeric forms and induces the disassembly of FtsZ polymers in a manner that is not dependent but enhanced by the FtsZ GTPase activity. Using a combination of biochemical and genetic approaches, we then map the MipZ-FtsZ interaction interface. Our results reveal that MipZ employs a patch of surface-exposed hydrophobic residues to interact with the C-terminal region of the FtsZ core domain. In doing so, it sequesters FtsZ monomers and caps the (+)-end of FtsZ polymers, thereby promoting their rapid disassembly. We further show that MipZ influences the conformational dynamics of interacting FtsZ molecules, which could potentially contribute to modulating their assembly kinetics. Together, our findings show that MipZ uses a combination of mechanisms to control FtsZ polymerization, which may be required to robustly regulate the spatiotemporal dynamics of Z ring assembly within the cell.

Raw data for 'Light-induced assembly and disassembly of polymers with PdnL2n-type network junctions'

raw data for 'Light-induced assembly and disassembly of polymers with PdnL2n-type network junctions'

Lipid Surfaces and Glutamate Anions Enhance Formation of Dynamic Biomolecular Condensates Containing Bacterial Cell Division Protein FtsZ and Its DNA-Bound Regulator SlmA.

Dynamic biomolecular condensates formed by liquid-liquid phase separation can regulate the spatial and temporal organization of proteins, thus modulating their functional activity in cells. Previous studies showed that the cell division protein FtsZ from Escherichia coli formed dynamic phase-separated condensates with nucleoprotein complexes containing the FtsZ spatial regulator SlmA under crowding conditions, with potential implications for condensate-mediated spatiotemporal control of FtsZ activity in cell division. In the present study, we assessed formation of these condensates in the presence of lipid surfaces and glutamate ions to better approximate the E. coli intracellular environment. We found that potassium glutamate substantially promoted the formation of FtsZ-containing condensates when compared to potassium chloride in crowded solutions. These condensates accumulated on supported lipid bilayers and eventually fused, resulting in a time-dependent increase in the droplet size. Moreover, the accumulated condensates were dynamic, capturing protein from the external phase. FtsZ partitioned into the condensates at the lipid surface only in its guanosine diphosphate (GDP) form, regardless of whether it came from FtsZ polymer disassembly upon guanosine triphosphate (GTP) exhaustion. These results provide insights into the behavior of these GTP-responsive condensates in minimal membrane systems, which suggest how these membraneless assemblies may tune critical bacterial division events during the cell cycle.

Light-induced assembly and disassembly of polymers with Pd n L2n -type network junctions.

Polymers containing PdnL2n complexes as network junctions were obtained by reaction of poly(ethylene glycol)-linked N-donor ligands with Pd2+. The addition of a metastable state photoacid renders the networks light sensitive, and gel–sol transitions can be achieved by irradiation with light. The inverse process, a light-induced sol–gel transition, was realized by using a molecularly defined Pd complex as an acid-sensitive reservoir for Pd2+. Upon irradiation, Pd2+ ions are released, allowing the formation of an acid-resistant polymer network. Both the gel–sol and the sol–gel transitions are reversed in the dark.

SulA is able to block cell division in Escherichia coli by a mechanism different from sequestration

The SOS response is considered to be an extremely important feature of bacterial cells. It helps them to survive bad times, including helping to develop resistance to antibiotics. The SOS response blocks the cell division. For Escherichia coli it is well known that the SulA protein directly interacts with FtsZ – a key division protein. Now it is believed that fission blocking is based on FtsZ sequestration by the SulA protein, which leads to decrease in effective concentration of FtsZ in the cell below a critical value, which in vitro leads to dismantling of FtsZ polymers. In this work, we demonstrate that in order to block the division of E. coli, it is sufficient to have a relatively small amount of SulA in the cell. Moreover, the analysis of structures formed by FtsZ in E. coli cells under the conditions of SulA protein expression or the SOS response showed that there is no complete disassembly of FtsZ polymers, although Z-rings indeed are not formed. The results of the work indicate that the well-known sequestration mechanism is not comprehensive to explain blocking of the division process by SulA in vivo.

Thermal adaptation of mesophilic and thermophilic FtsZ assembly by modulation of the critical concentration.

Cytokinesis is the last stage in the cell cycle. In prokaryotes, the protein FtsZ guides cell constriction by assembling into a contractile ring-shaped structure termed the Z-ring. Constriction of the Z-ring is driven by the GTPase activity of FtsZ that overcomes the energetic barrier between two protein conformations having different propensities to assemble into polymers. FtsZ is found in psychrophilic, mesophilic and thermophilic organisms thereby functioning at temperatures ranging from subzero to >100°C. To gain insight into the functional adaptations enabling assembly of FtsZ in distinct environmental conditions, we analyzed the energetics of FtsZ function from mesophilic Escherichia coli in comparison with FtsZ from thermophilic Methanocaldococcus jannaschii. Presumably, the assembly may be similarly modulated by temperature for both FtsZ orthologs. The temperature dependence of the first-order rates of nucleotide hydrolysis and of polymer disassembly, indicated an entropy-driven destabilization of the FtsZ-GTP intermediate. This destabilization was true for both mesophilic and thermophilic FtsZ, reflecting a conserved mechanism of disassembly. From the temperature dependence of the critical concentrations for polymerization, we detected a change of opposite sign in the heat capacity, that was partially explained by the specific changes in the solvent-accessible surface area between the free and polymerized states of FtsZ. At the physiological temperature, the assembly of both FtsZ orthologs was found to be driven by a small positive entropy. In contrast, the assembly occurred with a negative enthalpy for mesophilic FtsZ and with a positive enthalpy for thermophilic FtsZ. Notably, the assembly of both FtsZ orthologs is characterized by a critical concentration of similar value (1–2 μM) at the environmental temperatures of their host organisms. These findings suggest a simple but robust mechanism of adaptation of FtsZ, previously shown for eukaryotic tubulin, by adjustment of the critical concentration for polymerization.

Cryo-EM structures of the ATP-bound Vps4E233Q hexamer and its complex with Vta1 at near-atomic resolution

The cellular ESCRT-III (endosomal sorting complex required for transport-III) and Vps4 (vacuolar protein sorting 4) comprise a common machinery that mediates a variety of membrane remodelling events. Vps4 is essential for the machinery function by using the energy from ATP hydrolysis to disassemble the ESCRT-III polymer into individual proteins. Here, we report the structures of the ATP-bound Vps4E233Q hexamer and its complex with the cofactor Vta1 (vps twenty associated 1) at resolutions of 3.9 and 4.2 Å, respectively, determined by electron cryo-microscopy. Six Vps4E233Q subunits in both assemblies exhibit a spiral-shaped ring-like arrangement. Locating at the periphery of the hexameric ring, Vta1 dimer bridges two adjacent Vps4 subunits by two different interaction modes to promote the formation of the active Vps4 hexamer during ESCRT-III filament disassembly. The structural findings, together with the structure-guided biochemical and single-molecule analyses, provide important insights into the process of the ESCRT-III polymer disassembly by Vps4.

Structural basis of protein translocation by the Vps4-Vta1 AAA ATPase.

Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 'walks' along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases.

The Chloroplast Tubulin Homologs FtsZA and FtsZB from the Red Alga Galdieria sulphuraria Co-assemble into Dynamic Filaments

FtsZ is a homolog of eukaryotic tubulin and is present in almost all bacteria and many archaea, where it is the major cytoskeletal protein in the Z ring, required for cell division. Unlike some other cell organelles of prokaryotic origin, chloroplasts have retained FtsZ as an essential component of the division machinery. However, chloroplast FtsZs have been challenging to study because they are difficult to express and purify. To this end, we have used a FATT tag expression system to produce as soluble proteins the two chloroplast FtsZs from Galdieria sulphuraria, a thermophilic red alga. GsFtsZA and GsFtsZB assembled individually in the presence of GTP, forming large bundles of protofilaments. GsFtsZA also assembled in the presence of GDP, the first member of the FtsZ/tubulin superfamily to do so. Mixtures of GsFtsZA and GsFtsZB assembled protofilament bundles and hydrolyzed GTP at a rate approximately equal to the sum of their individual rates, suggesting a random co-assembly. GsFtsZA assembly by itself in limiting GTP gave polymers that remained stable for a prolonged time. However, when GsFtsZB was added, the co-polymers disassembled with enhanced kinetics, suggesting that the GsFtsZB regulates and enhances disassembly dynamics. GsFtsZA-mts (where mts is a membrane-targeting amphipathic helix) formed Z ring-like helices when expressed in Escherichia coli Co-expression of GsFtsZB (without an mts) gave co-assembly of both into similar helices. In summary, we provide biochemical evidence that GsFtsZA assembles as the primary scaffold of the chloroplast Z ring and that GsFtsZB co-assembly enhances polymer disassembly and dynamics.

Combinatorial genetic analysis of a network of actin disassembly-promoting factors.

The patterning of actin cytoskeleton structures in vivo is a product of spatially and temporally regulated polymer assembly balanced by polymer disassembly. While in recent years our understanding of actin assembly mechanisms has grown immensely, our knowledge of actin disassembly machinery and mechanisms has remained comparatively sparse. Saccharomyces cerevisiae is an ideal system to tackle this problem, both because of its amenabilities to genetic manipulation and live‐cell imaging and because only a single gene encodes each of the core disassembly factors: cofilin (COF1), Srv2/CAP (SRV2), Aip1 (AIP1), GMF (GMF1/AIM7), coronin (CRN1), and twinfilin (TWF1). Among these six factors, only the functions of cofilin are essential and have been well defined. Here, we investigated the functions of the nonessential actin disassembly factors by performing genetic and live‐cell imaging analyses on a combinatorial set of isogenic single, double, triple, and quadruple mutants in S. cerevisiae. Our results show that each disassembly factor makes an important contribution to cell viability, actin organization, and endocytosis. Further, our data reveal new relationships among these factors, providing insights into how they work together to orchestrate actin turnover. Finally, we observe specific combinations of mutations that are lethal, e.g., srv2Δ aip1Δ and srv2Δ crn1Δ twf1Δ, demonstrating that while cofilin is essential, it is not sufficient in vivo, and that combinations of the other disassembly factors perform vital functions. © 2015 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.

The Nucleoid Occlusion SlmA Protein Accelerates the Disassembly of the FtsZ Protein Polymers without Affecting Their GTPase Activity.

Division site selection is achieved in bacteria by different mechanisms, one of them being nucleoid occlusion, which prevents Z-ring assembly nearby the chromosome. Nucleoid occlusion in E. coli is mediated by SlmA, a sequence specific DNA binding protein that antagonizes FtsZ assembly. Here we show that, when bound to its specific target DNA sequences (SBS), SlmA reduces the lifetime of the FtsZ protofilaments in solution and of the FtsZ bundles when located inside permeable giant vesicles. This effect appears to be essentially uncoupled from the GTPase activity of the FtsZ protofilaments, which is insensitive to the presence of SlmA·SBS. The interaction of SlmA·SBS with either FtsZ protofilaments containing GTP or FtsZ oligomers containing GDP results in the disassembly of FtsZ polymers. We propose that SlmA·SBS complexes control the polymerization state of FtsZ by accelerating the disassembly of the FtsZ polymers leading to their fragmentation into shorter species that are still able to hydrolyze GTP at the same rate. SlmA defines therefore a new class of inhibitors of the FtsZ ring different from the SOS response regulator SulA and from the moonlighting enzyme OpgH, inhibitors of the GTPase activity. SlmA also shows differences compared with MinC, the inhibitor of the division site selection Min system, which shortens FtsZ protofilaments by interacting with the GDP form of FtsZ.

Protein interactions at the C‐terminus of essential cell division protein FtsZ modulate Z‐ring dynamics in Escherichia coli

Highly coordinated spatiotemporal regulation of the cell division pathway ensures the production of two identical daughter cells in Escherichia coli. Constriction and septation at midcell require the assembly of a ring-like dynamic protein structure, called the Z-ring, containing FtsZ, a structural homolog of tubulin that exhibits GTP-dependent polymerization. FtsZ monomers contain an N-terminal polymerization domain, an unstructured linker region, and a conserved C-terminal core domain important for protein interactions. Residues near the FtsZ C-terminus have been implicated in direct interactions with proteins that tether the Z-ring to the membrane, including FtsA and ZipA, and proteins that promote disassembly of FtsZ polymers, including MinC and chaperone-protease ClpXP. To test if protein interactions at the FtsZ C-terminus are important for regulating the dynamic exchange of FtsZ subunits into and out of the Z-ring, we monitored the fluorescence recovery of Z-rings containing Gfp-FtsZ in dividing cells after photobleaching. We demonstrate that multiple mutations near the FtsZ C-terminus lead to the formation of Z-rings that have significantly impaired dynamics in vivo. We also deleted several genes encoding proteins that promote the disassembly of FtsZ polymers in vitro, including MinC and ClpXP, and have observed that cells deleted for these genes contain Z-rings that exchange subunits more slowly than wild type cells. These results show that proteins promoting disassembly of FtsZ polymers have a positive effect on enhancing the exchange of subunits within the FtsZ-ring. Moreover, these results also suggest that proteins modulate Z-ring dynamics through direct interactions with the FtsZ C-terminus.

The bacterial cell division regulators MinD and MinC form polymers in the presence of nucleotide

The Min system of proteins, consisting of MinC, MinD and MinE, is essential for normal cell division in Escherichia coli. MinC forms a polar gradient to restrict placement of the division septum to midcell. MinC localization occurs through a direct interaction with MinD, a membrane-associating Par-like ATPase. MinE stimulates ATP hydrolysis by MinD, thereby releasing MinD from the membrane. Here, we show that MinD forms polymers with MinC and ATP without the addition of phospholipids. The topological regulator MinE induces disassembly of MinCD polymers. Two MinD mutant proteins, MinD(K11A) and MinD(ΔMTS15), are unable to form polymers with MinC.

Deconstructing formin-dependent actin cable assembly

Eukaryotic cells are organized in three dimensions to allow for both cell division and for them to perform their physiological functions. This organization is dependent upon the arrangement of the cytoskeleton, which can either generate force by subunit polymerization or serve as tracks for molecular motors to deliver protein complexes, RNAs, or membrane-bound compartments. In general, long-range transport is mediated by microtubules, whereas microfilaments often contribute more local structural and motile roles, being especially prominent in association with the plasma membrane. Microtubules and microfilaments can be quite dynamic, and this requires the appropriate spatial regulation of polymer assembly, then association with factors to generate functional structures, and also control of polymer disassembly. For microfilaments, two major nucleators of actin assembly are the actin-related protein 2/3 (Arp2/3) complex, which generates branched filaments and formins that generate unbranched filaments (1). Formins are present in all eukaryotes—humans have 15 different ones—so understanding their biology is of significant importance. Miao et al. (2) have now established a formin-dependent actin cable assembly reaction using yeast cell extracts that will permit a dissection of how formin activity is regulated in vivo.

Free Energy Changes and Components Implicit in the MWC Allosteric Model for the Cooperative Oxygen Binding of Hemoglobin

Hill's plots of oxygen binding isotherms reveal the presence of a transition between two different oxygen affinities at the beginning and end of the isotherm. They correspond to the two conformations anticipated by the MWC model, namely, the T and R conformations at the beginning and end of oxygen binding, when the lower affinity of the T form develops into the higher affinity of the R form. The difference between the binding Gibbs free energy changes of the two affinities (Δ G(L)) is the free energy of binding cooperativity. Notably, Δ G(L) is positive in favor of the T form, which moves to a higher energy level upon oxygen release. Osmotic stress reveals a higher volume/surface ratio of deoxyhemoglobin, with a positive Δ G(W) also in favor of the T form. An increasing protein concentration shifts the isotherms to the right, indicating the formation of intermediate polymeric forms. The enthalpy of the intermediates shows a strong absorption of heat at the third oxygenation step because of polymer formation with quinary, and higher-order, structures. The disassembly of intermediate polymers releases energy with a negative Δ G that compensates and allows the positive values of Δ G(L). High-energy polymers are the barrier preventing the relaxation of the T and R conformations into one another. The MWC allosteric model is the best justification of oxygen binding cooperativity.

Divalent Recognition of the E. Coli Cytoskeletal Protein FtsZ by the ATP-dependent Protease ClpXP

Cell division in bacteria requires the coordinated interactions of cytoskeletal motor proteins, their regulators and proteins that remodel the outer cell layers. In Escherichia coli, the tubulin homolog FtsZ is essential for cell division. FtsZ forms a large ring-like structure, called the Z-ring, inside the cell at the site of constriction. In vitro FtsZ hydrolyzes GTP and assembles into dynamic polymers when GTP is present. One modulator of FtsZ assembly is the ATP-dependent protease ClpXP. ClpX, a AAA+ ATPase, forms a hexameric ring that unfolds proteins with specific recognition signals. ClpX associates with ClpP, a barrel-shaped compartmentalized protease, to degrade proteins in a regulated manner. ClpXP degrades FtsZ in vivo and in vitro and promotes the disassembly of FtsZ polymers. Site-directed mutagenesis of FtsZ indicated that two discrete regions of FtsZ near the C-terminus are important for degradation by ClpXP. One region is within the C-terminal four amino acids of FtsZ and overlaps with the recognition site for several other FtsZ-interacting proteins. A second region is located ∼30 residues proximal to the C-terminus, in a linker region connecting the polymerization domain to the C-terminal conserved core region. We determined that amino acid residues from both regions are important for FtsZ degradation in the absence and presence of GTP, the condition that promotes polymerization. We also showed that the amount of FtsZ degraded by ClpXP in vitro is reduced in the presence of excess MinC, an inhibitor of FtsZ polymerization. Our results suggest that MinC competes with ClpXP for the FtsZ C-terminus and prevents unfolding and degradation by ClpXP. These studies indicate that two negative regulators of FtsZ assembly, ClpXP and MinC, both utilize the FtsZ C-terminal region for destabilization and/or degradation of FtsZ polymers.

The instability of stabilization

Actin filaments and microtubules control the shape of cells and organize their contents. The proteins that form these cytoskeletal polymers were identified and isolated decades ago—actin in 1942 (1) and tubulin in 1968 (2)—but we still argue about some of their basic properties. New work from Niedermayer et al. (3), however, puts us on the right track, or more properly back on the right track, to understanding how actin filaments fall apart. After years of study, how is it still possible for otherwise reasonable people to disagree about how actin filaments and microtubules assemble and disassemble? The shortest answer I can think of has two parts: (i) the details matter, and (ii) the experiments are hard. The details matter because small differences in the rate, mechanism, or geometry of polymer assembly and disassembly produce cytoskeletal networks with different architectures. Ultimately, we want to understand how cells manipulate such basic biochemical properties to produce different cytoskeletal structures with different biological functions. How, for example, does one cell construct a 3D pseudopod that pushes forward the plasma membrane; a linear stress fiber that pulls on cell adhesions; and a contractile ring that splits the cell in two—all from actin filaments? The experiments are hard, in part, because cytoskeletal polymers are constructed from large numbers of weak, noncovalent interactions, and they are dynamic, sometimes even ephemeral structures. To study assembly and disassembly of cytoskeletal polymers, we require techniques that maintain their native structure: techniques that often push technical boundaries in both biochemistry and physics. It is not surprising then, that the work of Niedermayer et al. represents the joint effort of two groups, one led by biochemist Marie France Carlier and the other by physicist Rheinhard Lipowsky.