Force Feedback Controls Motor Activity and Mechanical Properties of Self-Assembling Branched Actin Networks.

Branched actin networks--created by the Arp2/3 complex, capping protein, and a nucleation promoting factor--generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency. Microscopically, increased density reflects increased filament number and altered geometry but no change in average length. Macroscopically, increased density enhances network stiffness and resistance to mechanical failure beyond those of isotropic actin networks. These effects endow branched actin networks with memory of their mechanical history that shapes their material properties and motor activity. This work reveals intrinsic force feedback mechanisms by which mechanical resistance makes self-assembling actin networks stiffer, stronger, and more powerful.

Quantitative Proteomic Analysis Reveals apoE4-Dependent Phosphorylation of the Actin-Regulating Protein VASP.

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease. While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here, we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in vasodilator-stimulated phosphoprotein (VASP) S235 phosphorylation in a protein kinase A (PKA)-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects.

Archaeal actins and the origin of a multi-functional cytoskeleton.

Actin and actin-like proteins form filamentous polymers that carry out important cellular functions in all domains of life. In this review, we sketch a map of the function and regulation of actin-like proteins across bacteria, archaea, and eukarya, marking some of the terra incognita that remain in this landscape. We focus particular attention on archaea because mapping the structure and function of cytoskeletal systems across this domain promises to help us understand the evolutionary relationship between the (mostly) mono-functional actin-like filaments found in bacteria and the multi-functional actin cytoskeletons that characterize eukaryotic cells.

Capping protein increases the rate of actin-based motility by promoting filament nucleation by the Arp2/3 complex.

Capping protein (CP) is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between CP and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex and not by increasing the rate of filament elongation as previously suggested. One consequence of this coupling between capping and nucleation is that, while the rate of motility depends strongly on the concentration of CP and Arp2/3, the net rate of actin assembly is insensitive to changes in either factor. By reorganizing their architecture, dendritic actin networks harness the same assembly kinetics to drive different rates of motility.

WH2 and proline-rich domains of WASP-family proteins collaborate to accelerate actin filament elongation.

WASP-family proteins are known to promote assembly of branched actin networks by stimulating the filament-nucleating activity of the Arp2/3 complex. Here, we show that WASP-family proteins also function as polymerases that accelerate elongation of uncapped actin filaments. When clustered on a surface, WASP-family proteins can drive branched actin networks to grow much faster than they could by direct incorporation of soluble monomers. This polymerase activity arises from the coordinated action of two regulatory sequences: (i) a WASP homology 2 (WH2) domain that binds actin, and (ii) a proline-rich sequence that binds profilin-actin complexes. In the absence of profilin, WH2 domains are sufficient to accelerate filament elongation, but in the presence of profilin, proline-rich sequences are required to support polymerase activity by (i) bringing polymerization-competent actin monomers in proximity to growing filament ends, and (ii) promoting shuttling of actin monomers from profilin-actin complexes onto nearby WH2 domains. Unoccupied WH2 domains transiently associate with free filament ends, preventing their growth and dynamically tethering the branched actin network to the WASP-family proteins that create it. Collaboration between WH2 and proline-rich sequences thus strikes a balance between filament growth and tethering. Our work expands the number of critical roles that WASP-family proteins play in the assembly of branched actin networks to at least three: (i) promoting dendritic nucleation; (ii) linking actin networks to membranes; and (iii) accelerating filament elongation.

Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution.

We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations.

Cryo-EM structure of the bacterial actin AlfA reveals unique assembly and ATP-binding interactions and the absence of a conserved subdomain.

Bacterial actins are an evolutionarily diverse family of ATP-dependent filaments built from protomers with a conserved structural fold. Actin-based segregation systems are encoded on many bacterial plasmids and function to partition plasmids into daughter cells. The bacterial actin AlfA segregates plasmids by a mechanism distinct from other partition systems, dependent on its unique dynamic properties. Here, we report the near-atomic resolution electron cryo-microscopy structure of the AlfA filament, which reveals a strikingly divergent filament architecture resulting from the loss of a subdomain conserved in all other actins and a mode of ATP binding. Its unusual assembly interfaces and nucleotide interactions provide insight into AlfA dynamics, and expand the range of evolutionary variation accessible to actin quaternary structure.

Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer.

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.

Bacterial Tubulins A and B Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis.

Bacteria of the genus Prosthecobacter express homologs of eukaryotic α- and ß-tubulin, called BtubA and BtubB (BtubA/B), that have been observed to assemble into filaments in the presence of GTP. BtubA/B polymers are proposed to be composed in vitro by two to six protofilaments in contrast to that in vivo, where they have been reported to form 5-protofilament tubes named bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may be derived from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is reversible and that BtubA/B folding does not require chaperones. To better understand BtubA/B filament behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro BtubA/B assembly using a combination of polymerization kinetics assays and microscopy. Like eukaryotic microtubules, BtubA/B filaments exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by BtubA/B polymerization drives a stochastic mechanism of filament disassembly that occurs via polymer breakage and/or fast continuous depolymerization. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of BtubA/B filament fragments. Unlike MTs, polymerization of BtubA/B requires KCl, which reduces the critical concentration for BtubA/B assembly and induces it to form stable mixed-orientation bundles in the absence of any additional BtubA/B-binding proteins. The complex dynamics that we observe in stabilized and unstabilized BtubA/B filaments may reflect common properties of an ancestral eukaryotic tubulin polymer.IMPORTANCE Microtubules are polymers within all eukaryotic cells that perform critical functions; they segregate chromosomes, organize intracellular transport, and support the flagella. These functions rely on the remarkable range of tunable dynamic behaviors of microtubules. Bacterial tubulin A and B (BtubA/B) are evolutionarily related proteins that form polymers. They are proposed to be evolved from the ancestral eukaryotic tubulin, a missing link in microtubule evolution. Using microscopy and biochemical approaches to characterize BtubA/B assembly in vitro, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules but differ in how they self-associate into bundles and how this bundling affects their stability. Our results demonstrate the diversity of mechanisms through which tubulin homologs promote filament dynamics and monomer turnover.

Accessory factors promote AlfA-dependent plasmid segregation by regulating filament nucleation, disassembly, and bundling.

In bacteria, some plasmids are partitioned to daughter cells by assembly of actin-like proteins (ALPs). The best understood ALP, ParM, has a core set of biochemical properties that contributes to its function, including dynamic instability, spontaneous nucleation, and bidirectional elongation. AlfA, an ALP that pushes plasmids apart in Bacillus, relies on a different set of underlying properties to segregate DNA. AlfA elongates unidirectionally and is not dynamically unstable; its assembly and disassembly are regulated by a cofactor, AlfB. Free AlfB breaks up AlfA bundles and promotes filament turnover. However, when AlfB is bound to the centromeric DNA sequence, parN, it forms a segrosome complex that nucleates and stabilizes AlfA filaments. When reconstituted in vitro, this system creates polarized, motile comet tails that associate by antiparallel filament bundling to form bipolar, DNA-segregating spindles.

Cell mechanics and the cytoskeleton.

The ability of a eukaryotic cell to resist deformation, to transport intracellular cargo and to change shape during movement depends on the cytoskeleton, an interconnected network of filamentous polymers and regulatory proteins. Recent work has demonstrated that both internal and external physical forces can act through the cytoskeleton to affect local mechanical properties and cellular behaviour. Attention is now focused on how cytoskeletal networks generate, transmit and respond to mechanical signals over both short and long timescales. An important insight emerging from this work is that long-lived cytoskeletal structures may act as epigenetic determinants of cell shape, function and fate.

Design of reversible, cysteine-targeted Michael acceptors guided by kinetic and computational analysis.

Electrophilic probes that covalently modify a cysteine thiol often show enhanced pharmacological potency and selectivity. Although reversible Michael acceptors have been reported, the structural requirements for reversibility are poorly understood. Here, we report a novel class of acrylonitrile-based Michael acceptors, activated by aryl or heteroaryl electron-withdrawing groups. We demonstrate that thiol adducts of these acrylonitriles undergo ß-elimination at rates that span more than 3 orders of magnitude. These rates correlate inversely with the computed proton affinity of the corresponding carbanions, enabling the intrinsic reversibility of the thiol-Michael reaction to be tuned in a predictable manner. We apply these principles to the design of new reversible covalent kinase inhibitors with improved properties. A cocrystal structure of one such inhibitor reveals specific noncovalent interactions between the 1,2,4-triazole activating group and the kinase. Our experimental and computational study enables the design of new Michael acceptors, expanding the palette of reversible, cysteine-targeted electrophiles.

The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency.

Enterohaemorrhagic Escherichia coli attaches to the intestine through actin pedestals that are formed when the bacterium injects its protein EspF(U) (also known as TccP) into host cells. EspF(U) potently activates the host WASP (Wiskott-Aldrich syndrome protein) family of actin-nucleating factors, which are normally activated by the GTPase CDC42, among other signalling molecules. Apart from its amino-terminal type III secretion signal, EspF(U) consists of five-and-a-half 47-amino-acid repeats. Here we show that a 17-residue motif within this EspF(U) repeat is sufficient for interaction with N-WASP (also known as WASL). Unlike most pathogen proteins that interface with the cytoskeletal machinery, this motif does not mimic natural upstream activators: instead of mimicking an activated state of CDC42, EspF(U) mimics an autoinhibitory element found within N-WASP. Thus, EspF(U) activates N-WASP by competitively disrupting the autoinhibited state. By mimicking an internal regulatory element and not the natural activator, EspF(U) selectively activates only a precise subset of CDC42-activated processes. Although one repeat is able to stimulate actin polymerization, we show that multiple-repeat fragments have notably increased potency. The activities of these EspF(U) fragments correlate with their ability to coordinate activation of at least two N-WASP proteins. Thus, this pathogen has used a simple autoinhibitory fragment as a component to build a highly effective actin polymerization machine.

On the critical concentration for net assembly of dynamically unstable polymers.

Cytoskeletal and cytomotive filaments are protein polymers that move molecular cargo and organize cellular contents in all domains of life. A key parameter describing the self-assembly of many of these polymers -including actin filaments and microtubules- is the minimum concentration required for polymer formation. This 'critical concentration for net assembly' (ccN) is easy to calculate for eukaryotic actins but more difficult for dynamically unstable filaments such as microtubules and some bacterial polymers. To better understand how cells (especially bacteria) regulate assembly of dynamically unstable polymers I investigate the microscopic parameters that influence their critical concentrations. Assuming simple models for spontaneous nucleation and catastrophe I derive expressions for the monomer-polymer balance. In the absence of concentration-dependent rescue, fixed catastrophe rates do not produce clear critical concentrations. In contrast, simple ATP-/GTP-cap models with concentration-dependent catastrophe rates, generate phenomenological critical concentrations that increase linearly with the rate of nucleotide hydrolysis and decrease logarithmically with the rate of spontaneous nucleation.

Adhesion pilus retraction powers twitching motility in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

Type IV pili are filamentous appendages found in most bacteria and archaea, where they can support functions such as surface adhesion, DNA uptake, aggregation, and motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess PilT homologs, it was thought that archaeal pili cannot retract and that archaea do not exhibit twitching motility. Here, we use live-cell imaging, automated cell tracking, fluorescence imaging, and genetic manipulation to show that the hyperthermophilic archaeon Sulfolobus acidocaldarius exhibits twitching motility, driven by retractable adhesion (Aap) pili, under physiologically relevant conditions (75 °C, pH 2). Aap pili are thus capable of retraction in the absence of a PilT homolog, suggesting that the ancestral type IV pili in the last universal common ancestor (LUCA) were capable of retraction.

Polymer dynamics of Alp7A reveals how two critical concentrations govern assembly of dynamically unstable actin-like proteins.

Dynamically unstable polymers capture and move cellular cargos in bacteria and eukaryotes, but regulation of their assembly remains poorly understood. Here we describe polymerization of Alp7A, a bacterial actin-like protein (ALP) that distributes copies of plasmid pLS20 among daughter cells in Bacillus subtilis. Purified ATP-Alp7A forms dynamically unstable polymers with a high critical concentration for net assembly (ccN = 10.3 µM), but a much lower critical concentration for filament elongation (ccE = 0.6 µM). Rapid nucleation and stabilization of Alp7A polymers by the accessory factor, Alp7R, decrease ccN into the physiological range. Stable populations of Alp7A filaments appear under two conditions: (i) when Alp7R slows catastrophe rates or (ii) when Alp7A concentrations are high enough to promote filament bundling. These results reveal how dynamic instability maintains high steady-state concentrations of monomeric Alp7A, and how accessory factors regulate Alp7A assembly by modulating ccN independently of ccE.

Sulfolobus acidocaldarius adhesion pili power twitching motility in the absence of a dedicated retraction ATPase.

Type IV pili are ancient and widespread filamentous organelles found in most bacterial and archaeal phyla where they support a wide range of functions, including substrate adhesion, DNA uptake, self aggregation, and cell motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess homologs of PilT, it was thought that archaeal pili cannot retract. Here, we employ live-cell imaging under native conditions (75°C and pH 2), together with automated single-cell tracking, high-temperature fluorescence imaging, and genetic manipulation to demonstrate that S. acidocaldarius exhibits bona fide twitching motility, and that this behavior depends specifically on retractable adhesion pili. Our results demonstrate that archaeal adhesion pili are capable of retraction in the absence of a PilT retraction ATPase and suggests that the ancestral type IV pilus machinery in the last universal common ancestor (LUCA) relied on such a bifunctional ATPase for both extension and retraction.

Architecture and assembly of a divergent member of the ParM family of bacterial actin-like proteins.

Eubacteria and archaea contain a variety of actin-like proteins (ALPs) that form filaments with surprisingly diverse architectures, assembly dynamics, and cellular functions. Although there is much data supporting differences between ALP families, there is little data regarding conservation of structure and function within these families. We asked whether the filament architecture and biochemical properties of the best-understood prokaryotic actin, ParM from plasmid R1, are conserved in a divergent member of the ParM family from plasmid pB171. Previous work demonstrated that R1 ParM assembles into filaments that are structurally distinct from actin and the other characterized ALPs. They also display three biophysical properties thought to be essential for DNA segregation: 1) rapid spontaneous nucleation, 2) symmetrical elongation, and 3) dynamic instability. We used microscopic and biophysical techniques to compare and contrast the architecture and assembly of these related proteins. Despite being only 41% identical, R1 and pB171 ParMs polymerize into nearly identical filaments with similar assembly dynamics. Conservation of the core assembly properties argues for their importance in ParM-mediated DNA segregation and suggests that divergent DNA-segregating ALPs with different assembly properties operate via different mechanisms.

Regulatory interactions between two actin nucleators, Spire and Cappuccino.

Spire and Cappuccino are actin nucleation factors that are required to establish the polarity of Drosophila melanogaster oocytes. Their mutant phenotypes are nearly identical, and the proteins interact biochemically. We find that the interaction between Spire and Cappuccino family proteins is conserved across metazoan phyla and is mediated by binding of the formin homology 2 (FH2) domain from Cappuccino (or its mammalian homologue formin-2) to the kinase noncatalytic C-lobe domain (KIND) from Spire. In vitro, the KIND domain is a monomeric folded domain. Two KIND monomers bind each FH2 dimer with nanomolar affinity and strongly inhibit actin nucleation by the FH2 domain. In contrast, formation of the Spire-Cappuccino complex enhances actin nucleation by Spire. In Drosophila oocytes, Spire localizes to the cortex early in oogenesis and disappears around stage 10b, coincident with the onset of cytoplasmic streaming.