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Filopodia are actin-filled membrane protrusions that play essential roles in cell motility and cell–cell communication and act as precursors of dendritic spines. IRSp53 is an essential regulator of filopodia formation, which couples Rho-GTPase signaling to actin cytoskeleton and membrane remodeling. IRSp53 has three major domains: an N-terminal inverse-BAR (I-BAR) domain, a Cdc42- and SH3-binding CRIB-PR domain, and an SH3 domain that binds downstream cytoskeletal effectors. Phosphorylation sites in the region between the CRIB-PR and SH3 domains mediate the binding of 14-3-3. Yet the mechanism by which 14-­3-3 regulates filopodia formation and dynamics and its role in cell migration are poorly understood. Here, we show that phosphorylation-dependent inhibition of IRSp53 by 14-3-3 counters activation by Cdc42 and cytoskeletal effectors, resulting in down-regulation of filopodia dynamics and cancer cell migration. In serum-starved cells, increased IRSp53 phosphorylation triggers 14-3-3 binding, which inhibits filopodia formation and dynamics, irrespective of whether IRSp53 is activated by Cdc42 or downstream effectors (Eps8, Ena/VASP). Pharmacological activation or inhibition of AMPK, respectively, increases or decreases the phosphorylation of two of three sites in IRSp53 implicated in 14-3-3 binding. Mutating these phosphorylation sites reverses 14-3-3-dependent inhibition of filopodia dynamics and cancer cell chemotaxis.

Actin cytoskeleton dynamics play vital roles in most forms of intracellular trafficking by promoting the biogenesis and transport of vesicular cargoes. Mounting evidence indicates that actin dynamics and membrane-cytoskeleton scaffolds also have essential roles in macroautophagy, the process by which cellular waste is isolated inside specialized vesicles called autophagosomes for recycling and degradation. Branched actin polymerization is necessary for the biogenesis of autophagosomes from the endoplasmic reticulum (ER) membrane. Actomyosin-based transport is then used to feed the growing phagophore with pre-selected cargoes and debris derived from different membranous organelles inside the cell. Finally, mature autophagosomes detach from the ER membrane by an as yet unknown mechanism, undergo intracellular transport and then fuse with lysosomes, endosomes and multivesicular bodies through mechanisms that involve actin- and microtubule-mediated motility, cytoskeleton-membrane scaffolds and signaling proteins. In this review, we highlight the considerable progress made recently towards understanding the diverse roles of the cytoskeleton in autophagy.

Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) protein slows down actin filament barbed end growth. This inhibition is relieved by CDC42 and counteracted by VASP, which also binds to IRSp53. The VASP:IRSp53 interaction is regulated by activated CDC42 and promotes high-density clustering of VASP, which is required for processive actin filament elongation. The interaction also mediates VASP recruitment to liposomes. In cells, IRSp53 and VASP accumulate at discrete foci at the leading edge, where filopodia are initiated. Genetic removal of IRSp53 impairs the formation of VASP foci, filopodia and chemotactic motility, while IRSp53 null mice display defective wound healing. Thus, IRSp53 dampens barbed end growth. CDC42 activation inhibits this activity and promotes IRSp53-dependent recruitment and clustering of VASP to drive actin assembly. These events result in spatial restriction of VASP filament elongation for initiation of filopodia during cell migration, invasion, and tissue repair.

We have performed molecular dynamics simulations of the phosphorylated (at S-19) and the unphosphorylated 25-residue N-terminal phosphorylation domain of the regulatory light chain (RLC) of smooth muscle myosin to provide insight into the structural basis of regulation. This domain does not appear in any crystal structure, so these simulations were combined with site-directed spin labeling to define its structure and dynamics. Simulations were carried out in explicit water at 310 K, starting with an ideal alpha-helix. In the absence of phosphorylation, large portions of the domain (residues S-2 to K-11 and R-16 through Y-21) were metastable throughout the simulation, undergoing rapid transitions among alpha-helix, pi-helix, and turn, whereas residues K-12 to Q-15 remained highly disordered, displaying a turn motif from 1 to 22.5 ns and a random coil pattern from 22.5 to 50 ns. Phosphorylation increased alpha-helical order dramatically in residues K-11 to A-17 but caused relatively little change in the immediate vicinity of the phosphorylation site (S-19). Phosphorylation also increased the overall dynamic stability, as evidenced by smaller temporal fluctuations in the root mean-square deviation. These results on the isolated phosphorylation domain, predicting a disorder-to-order transition induced by phosphorylation, are remarkably consistent with published experimental data involving site-directed spin labeling of the intact RLC bound to the two-headed heavy meromyosin. The simulations provide new insight into structural details not revealed by experiment, allowing us to propose a refined model for the mechanism by which phosphorylation affects the N-terminal domain of the RLC of smooth muscle myosin.

Nucleation promoting factors (NPFs) control the spatio-temporal activity of Arp2/3 complex in cells [1, 2]. Thus, WASP and the WAVE complex direct the formation of branched actin networks at the leading edge during cell motility and endo/exocytosis, whereas the WASH complex is involved in endosomal transport. Less understood are WHAMM and JMY, two NPFs with similar domain architecture. JMY is found in the nucleus and the cytosol, and is involved in transcriptional regulation [3], cell motility [4], and trans-Golgi transport [5]. WHAMM was reported to bind microtubules and to be involved in ER to cis-Golgi transport [6]. Here, we show that WHAMM directs the activity of Arp2/3 complex for autophagosome biogenesis through an actin-comet tail motility mechanism. Macroautophagy – the process by which cytosolic material is engulfed into autophagosomes for degradation and/or recycling – was recently shown to involve actin [7], but the mechanism is unknown. We found that WHAMM forms puncta that colocalize and comigrate with the autophagy markers LC3, DFCP1 and p62 through a WHAMM-dependent actin-comet tail mechanism. Under starvation, WHAMM and actin are observed at the interface between neighboring autophagosomes, whose number and size increases with WHAMM expression. Interfering with actin polymerization, inhibiting Arp2/3 complex, knocking down WHAMM, or blocking its interaction with Arp2/3 complex through mutagenesis, all inhibit comet tail formation and reduce the size and number of autophagosomes. Finally, JMY shows similar localization to WHAMM, and could be involved in similar processes. These results reveal a link between Arp2/3 complex-dependent actin assembly and autophagy.

Actin filament nucleation and branching by Arp2/3 complex is activated by nucleation-promoting factors (NPFs), whose C-terminal WCA region contains binding sites for actin (W) and Arp2/3 complex (CA). It is debated whether one or two NPFs are required for activation. Here, we present conclusive evidence in support of the two-NPF model and show that actin plays a crucial role in the interactions of two mammalian NPFs, N-WASP and WAVE2, with Arp2/3 complex. Competition between actin-WCA and glia maturation factor (GMF) for binding to Arp2/3 complex suggests that during activation the first actin monomer binds at the barbed end of Arp2. Based on distance constrains obtained by time-resolved fluorescence resonance energy transfer, we define the relative position of the two actin-WCAs on Arp2/3 complex and propose an atomic model of the 11-subunit transitional complex.

The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.

Chromatin-remodeling complexes are assembled around a catalytic subunit that contains a central ATPase domain and flanking sequences that recruit auxiliary subunits. The catalytic subunits of SWI/SNF remodelers recruit Arp7/9 through a helicase/SANT-associated (HSA) domain N-terminal to the ATPase domain. Arp7/9-containing remodelers also carry the auxiliary subunit Rtt102, but the role of this subunit is poorly understood. Here, we show that Rtt102 binds with nanomolar affinity to the Arp7/9 heterodimer and modulates its conformation and interactions with the ATPase subunit and nucleotide. When bound to Rtt102, Arp7/9 interacts with a shorter segment of the HSA domain. Structural analysis by small-angle x-ray scattering further shows that when bound to Rtt102, the complex of Arp7/9 with the catalytic subunit assumes a more stable compact conformation. We also found that Arp7, Arp9, and Arp7/9 interact very weakly with ATP, but Rtt102 promotes high-affinity ATP binding to a single site in the heterodimer. Collectively, the results establish a function for subunit Rtt102 as a stabilizing factor for the Arp7/9 heterodimer, enhancing its interaction with nucleotide and controlling the conformation of SWI/SNF remodelers in an Arp7/9-dependent manner.

Sca2 (surface cell antigen 2) is the only bacterial protein known to promote both actin filament nucleation and profilin-dependent elongation, mimicking eukaryotic formins to assemble actin comet tails for Rickettsia motility. We show that Sca2's functional mimicry of formins is achieved through a unique mechanism. Unlike formins, Sca2 is monomeric, but has N- and C-terminal repeat domains (NRD and CRD) that interact with each other for processive barbed-end elongation. The crystal structure of NRD reveals a previously undescribed fold, consisting of helix-loop-helix repeats arranged into an overall crescent shape. CRD is predicted to share this fold and might form together with NRD, a doughnut-shaped formin-like structure. In between NRD and CRD, proline-rich sequences mediate the incorporation of profilin-actin for elongation, and WASP-homology 2 (WH2) domains recruit actin monomers for nucleation. Sca2's α-helical fold is unusual among Gram-negative autotransporters, which overwhelmingly fold as β-solenoids. Rickettsia has therefore "rediscovered" formin-like actin nucleation and elongation.

Cell migration is a key step in tumor invasion and metastasis, involving dynamic reorganization of the actin cytoskeleton and membranes. BAR domain containing proteins have emerged as important regulators of the cytoskeleton that aid in coupling membrane and cytoskeleton dynamics to signaling pathways. IRSp53 is a BAR domain containing protein that is a potent inducer of lamellipodia- and filopodia-based cell motility under the control of Rho-family GTPases, making it a key player in tumor metastasis. However, little is known about the mechanisms of regulation of BAR domain-containing proteins in general and IRSp53 in particular. Here, we show a structural mechanism for IRSp53 regulation, which involves the release of autoinhibitory interactions by Cdc42. Central to this mechanism is a semi-conserved CRIB-PR motif that is rich in prolines (CRIB-PR) that joins the N-terminal BAR domain to the C-terminal SH3 domain. We have employed several biophysical techniques to confirm that both GTP-Cdc42 and the SH3 domain can bind to a CRIB-PR motif. Furthermore, we have used FRET to show IRSp53 undergoes a conformational change upon Cdc42 binding, consistent with the release of an autoinhibitory interaction. A crystal structure of a complex between activated Cdc42 and the CRIB-PR motif shows that proline-rich region binds to a region on Cdc42, not previously seen by conventional CRIB domains. Mutagenesis studies not only confirm this binding in solution and in cells, but also indicate that the SH3 domain is required for autoinhibition. In conclusion, we show a structural mechanism for IRSp53 regulation that involves a uniquely adapted CRIB motif that serves as a Cdc42-dependent switch for IRSp53 function, which helps to explain it's indispensible role in cell migration.

The use of site-directed spectroscopic probes in elucidating the molecular mechanism of force generation in muscle is reviewed, focusing on recent technical and conceptual advances. High-resolution crystal structures of actin and myosin suggest mechanistic models for force generation, but spectroscopic probe experiments under functional conditions are required to test and refine these models. An array of complementary spectroscopic techniques has been developed that can detect disorder and dynamics directly, resolve multiple conformational states, and can be applied to complex protein assemblies, taking advantage of the beautiful symmetry of the myofibrillar lattice. Recently developed instrumentation has the capacity to resolve simultaneously both biochemical and structural kinetics of myosin and/or actin. These new insights support a model in which structural and biochemical transitions are not tightly coupled, and disorder-to-order transitions play central roles in both force generation and its regulation.

The scaffolding protein insulin receptor tyrosine kinase substrate p53 (IRSp53) is a ubiquitous regulator of the actin cytoskeleton that mediates filopodia and lamellipodia formation under the control of Rho-family GTPases. IRSp53 is comprised of an N-terminal IRSp53/MIM homology domain (IMD) that senses negative membrane curvature and a central semi-CRIB motif followed by an SRC homology 3 (SH3) domain that binds to proline-rich regions of a wide range of actin regulators. Here, we present the crystal structure of a complex between activated CDC42 and the CRIB domain of IRSp53. Isothermal titration calorimetry (ITC) experiments confirm the binding of this domain to CDC42, and exclude the possibility of binding by inactive forms of CDC42 or activated forms of RAC1. Moreover, we demonstrate that the SH3 domain also binds to the CRIB domain, suggesting a mechanism of regulation where binding of CDC42 is required to release autoinhibition of the SH3 domain.

We have performed complementary time-resolved fluorescence resonance energy transfer (TR-FRET) experiments and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of smooth muscle regulatory light chain (RLC) bound to myosin. PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants of RLC, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLC was reconstituted onto myosin subfragment 1 (S1). TR-FRET resolved two simultaneously populated structural states of RLC, closed and open, in both unphosphorylated and phosphorylated biochemical states. All three FRET pairs show that phosphorylation shifts the equilibrium toward the open state, increasing its mol fraction by ∼20%. MD simulations agree with experiments in remarkable detail, confirming the coexistence of two structural states, with phosphorylation shifting the system toward the more dynamic open structural state. This agreement between experiment and simulation validates the additional structural details provided by MD simulations: In the closed state, PD is bent onto the surface of the C-terminal lobe, stabilized by interdomain salt bridges. In the open state, PD is more helical and straight, resides farther from the C-terminal lobe, and is stabilized by an intradomain salt bridge. The result is a vivid atomic-resolution visualization of the first step in the molecular mechanism by which phosphorylation activates smooth muscle.

Spectroscopy of myosin and actin has entered a golden age. High-resolution crystal structures of isolated actin and myosin have been used to construct detailed models for the dynamic actomyosin interactions that move muscle. Improved protein mutagenesis and expression technologies have facilitated site-directed labeling with fluorescent and spin probes. Spectroscopic instrumentation has achieved impressive advances in sensitivity and resolution. Here we highlight the contributions of site-directed spectroscopic probes to understanding the structural dynamics of myosin II and its actin complexes in solution and muscle fibers. We emphasize studies that probe directly the movements of structural elements within the myosin catalytic and light-chain domains, and changes in the dynamics of both actin and myosin due to their alternating strong and weak interactions in the ATPase cycle. A moving picture emerges in which single biochemical states produce multiple structural states, and transitions between states of order and dynamic disorder power the actomyosin engine.

We have engineered site-directed probe pairs in the regulatory light chain (RLC) for exchange into smooth muscle heavy meromyosin (HMM) and subfragment-1 (S1), in order to examine the phosphorylation-induced changes in RLC structural states using time-resolved FRET (TR-FRET). Phosphorylation of the RLC is required for activation of contraction in smooth muscle and modulates force in striated muscle. Force development in smooth muscle is triggered by phosphorylation of the RLC's N-terminal phosphorylation domain (PD) at Ser19, alleviating inhibitory interactions between the two heads. Since crystal structures of the RLC lack the PD, the mechanism by which RLC phosphorylation allosterically triggers disruption of HMM auto-inhibition remains unresolved. Our site-directed FRET probe pairs resolved intramolecular atomic distance measurements between the RLC core and the dynamic PD. Smooth muscle RLC assumes two structural states in both states of activation: phosphorylation shifts the equilibrium of these two structural states from primarily occupying the compact closed state to favoring the extended open state of the PD, relative to the RLC core. Compared to single-headed S1 (Kast et al., PNAS., 2010), the open state RLC conformation of unphosphorylated HMM is more compact, presumably due to head-head or RLC-RLC interactions. All-atom MD simulations corroborate our TR-FRET studies, and reveal specific salt-bridges that stabilize each structural state. The hypotheses generated from our MD simulations are being tested experimentally by TR-FRET via site-directed mutagenesis to manipulate charge. These studies offer the first atomic-resolution insight into the structural dynamics of RLC phosphorylation, and a similar approach should be applicable to striated muscle. Spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota. This work was funded by NIH grants to DDT (R01 AR32961, P30 AR0507220, T32 AR007612).

Pathogens destabilize the host cytoskeleton for their survival and for motility. Pathogen interaction with host gives an advantage of illustrating the key cytoskeleton functions like phagocytosis, micropinocytosis, autophagy, motility, membrane trafficking and signal transduction. Sca2 is a Rickettsial outer membrane protein is known to nucleate host actin and forms actin comet tails for the pathogen motility. Yet, the mechanism remains a mystery. Sca2 passenger domain is 1482 amino acid length protein and it has three WH2 domains flanked with two proline rich domains. Here, we have used a battery of biophysical approaches such as, Fluorescence, TIRF microscopy, ITC, X-ray crystallography and SAXS to determine the structural basis for nucleation and elongation of actin by Sca2. using biochemical and TIRF assays we have found that Sca2 consists of two domains, where one domain nucleates actin and the other is needed for processivity. We have determined the structure of the processivity domain, which reveals a unique mode of F-actin barbed-end capping, which was confirmed by deletion mutants based on structure. These results suggest that Rickettsia Sca2 has a novel mode of actin nucleation and elongation that involves the coordination of two actin-binding domains.

Actin and actin‐related proteins (Arps) are critical components of chromatin remodelling and modifying complexes, where they are commonly found in pairs. Yet, little is known about their role in these complexes. In this issue of The EMBO Journal , Fenn et al (2011) report the first structural investigation of two nuclear Arps, the INO80 complex subunits Arp4 and Arp8. Importantly, their results also shed light on the critical role that the nucleotide state of actin may have on its interactions with the Arps.

We have developed a high-throughput time-resolved fluorescence spectrometer capable of recording a high-resolution time-domain (sub-nanosecond) fluorescence decay, with high S/N every 100 μs. Coupled with a conventional stopped-flow rapid mixer, this technology has allowed us to measure changes in time-resolved fluorescence decays occurring during the course of a millisecond-resolved biochemical transient experiment. Most instruments used in fluorescence-based kinetic studies are limited to detecting a single fluorescence intensity signal on the millisecond time scale. While this type of fluorescence intensity-based measurement is informative, it provides scant information compared to a full sub-nanosecond resolved fluorescence decay- which is exquisitely sensitive to the structure, dynamics, and interactions of molecules in the sample. Current fluorescence lifetime technology is too slow by a factor of at least 1000 to detect this decay accurately within the millisecond timescale of a typical biochemical transient. Our system, based on a direct acquisition data collection approach, records the entire fluorescence decay with S/N > 100 from a single pulse form a 10 kHz microlaser. Using this approach, we can resolve individual fluorescence lifetime components comprising as little as 10% of a complex multi-exponential fluorescence decay. When used to monitor structural transitions by time resolved FRET, we are capable of isolating individual structural states within complex structural ensembles. Using this approach we were able to simultaneously monitor the binding of myosin to different classes of binding sites on actin filaments. This type of analysis is not possible with conventional fluorescence based stopped flow measurements.

We have performed molecular dynamics simulations of the phosphorylation domain (PD) of the regulatory light chain (RLC) of smooth muscle myosin, to gain insight into the thermodynamic principles governing the phosphorylation-induced disorder-to-order transition. Simulations were performed in explicit water under near-physiological conditions, starting with an ideal alpha-helix. In the absence of phosphorylation, the helical periodicity of the peptide was disrupted at residues T9-K11, while phosphorylation significantly favored the helical periodicity, in agreement with experimental data. Using the MM/PBSA approach, we calculated a relative free energy of -7.1 kcal/mol for the disorder-to-order transition. A large enthalpic decrease was compensated by a large loss of conformational entropy, despite the small helical increase (no more than three residues) upon phosphorylation. Phosphorylation decreased the conformational dynamics of K and R side chains, especially R16, which forms a salt bridge with pS19. Mutation of R16 to A or E prevented this phosphorylation-dependent ordering. We propose that phosphorylation balances the enthalpy-entropy compensation of the disorder-to-order transition of RLC via short and long-range electrostatic interactions with positively charged residues of the phosphorylation domain. We suggest that this balance is necessary to induce a disorder-to-order conformational change through a subtle energy switching.

ATP-dependent chromatin-remodeling complexes (remodelers) modulate chromatin structure and thus play important roles in transcription, DNA replication, and DNA repair. Nuclear actin and actin-related proteins (Arps) are subunits of several such complexes, where they are commonly found in pairs. However, the role of actin/Arps and the mechanism through which they bind the catalytic subunits of their host complexes are unknown. Here, we investigated how budding yeast Arp7 and Arp9 are incorporated into SWI/SNF-family chromatin-remodeling complexes. We present the first biochemical evidence that Arp7 and Arp9 must heterodimerize to bind ATP and interact with the catalytic subunits of the SWI/SNF and RSC complexes. In addition, we present ITC data, which define the remodeler region that interacts with Arp7/9, thus providing a mechanistic framework for understanding how actin/Arps are selectively loaded into their host remodelers.

We describe a high-performance time-resolved fluorescence (HPTRF) spectrometer that dramatically increases the rate at which precise and accurate subnanosecond-resolved fluorescence emission waveforms can be acquired in response to pulsed excitation. The key features of this instrument are an intense (1 μJ/pulse), high-repetition rate (10 kHz), and short (1 ns full width at half maximum) laser excitation source and a transient digitizer (0.125 ns per time point) that records a complete and accurate fluorescence decay curve for every laser pulse. For a typical fluorescent sample containing a few nanomoles of dye, a waveform with a signal/noise of about 100 can be acquired in response to a single laser pulse every 0.1 ms, at least 10(5) times faster than the conventional method of time-correlated single photon counting, with equal accuracy and precision in lifetime determination for lifetimes as short as 100 ps. Using standard single-lifetime samples, the detected signals are extremely reproducible, with waveform precision and linearity to within 1% error for single-pulse experiments. Waveforms acquired in 0.1 s (1000 pulses) with the HPTRF instrument were of sufficient precision to analyze two samples having different lifetimes, resolving minor components with high accuracy with respect to both lifetime and mole fraction. The instrument makes possible a new class of high-throughput time-resolved fluorescence experiments that should be especially powerful for biological applications, including transient kinetics, multidimensional fluorescence, and microplate formats.

We are using time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate nucleotide binding of the Ca-ATPase (SERCA). Based on biochemistry and crystallography, it has been proposed that SERCA has two distinct modes of nucleotide binding. To extend this observation from the crystal to the functional sarcoplasmic reticulum membrane, we have performed TR-FRET to measure the distance between C674 (labeled with the dye IAEDANS) and the fluorescent nucleotide TNP-ADP, in the presence and absence of inhibitors. In TR-FRET, a pulsed laser excites the AEDANS donor, and the emission is detected with sub-nanosecond time resolution, yielding a detailed description of the distance distribution between donor and acceptor. To determine whether these modes of nucleotide binding occur in solution during the Ca-ATPase reaction mechanism, we performed transient TR-FRET ([TR]2FRET) experiments, in which a complete subnanosecond TR-FRET decay (from 0 to 100 ns after the pulse) was recorded every 0.1 ms after rapid mixing of AEDANS-SERCA and TNP-ADP in a stopped-flow instrument. We observed clearly a biphasic reaction with a fast component (rate constant proportional to nucleotide concentration, presumably representing the nucleotide binding reaction) and a slower component (14s−1) representing a conformational change within SERCA, apparently corresponding to the predicted nucleotide rearrangement. TR-FRET is a powerful technique for connecting structural dynamics of SERCA with its static crystal structures.

We have used site-directed time-resolved FRET to determine light-chain domain transitions in expressed Dictyostelium full-length myosin S1. Myosin S1 is proposed to undergo large rotations of the lever-arm in response to the nucleotide state. Previous fluorescence experiments (Shih et al., 2000) have shown that S1 adopts two populations of pre-stroke states and the equilibrium between these states is driven by nucleotide. To observe these population changes in detail, and to observe the effect of actin, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to measure the distribution of distances between the labeled catalytic domain (A250C) and a labeled RLC (M129C). In the present work, we show that the lever arm adopts multiple orientations in solution. In the absence of nucleotide, the light-chain domain probe is at a mean distance of 8.4 nm from the catalytic domain probe. This distance extends to >9.0 nm when flS1 is bound to actin. The addition of ATP gives three roughly equal populations of distances at 3.6, 4.3, and >9.0 nm. ADP.Vi stabilizes the shorter of these populations, indicating that ADP.Pi induces a conformational change of the light-chain domain. This work was supported by grants from NIH (AR32961, AR07612). We thank Igor Negrashov for excellent technical assistance.

Using site-directed fluorescence labeling, transient time resolved FRET ([TR]2FRET) and time resolved fluorescence anisotropy, we have measured actin- and nucleotide-induced structural changes within the actin binding cleft of Dictyostelium myosin II. In a recent report using time-resolved pulsed EPR spectroscopy, distances were measured between paramagnetic probes attached to the upper and lower 50 kDa subdomains of Dictyostelium myosin II (Klein et al., PNAS, 105:12867-72). These results support the hypothesis that the actin-binding cleft closes partially upon actin binding, but also suggested that both open and closed conformations are simultaneously present, with nucleotides and actin controlling the open-closed equilibrium. Due to technical constraints, those EPR distance measurements were limited to frozen samples. In the present study, time-resolved fluorescence is used to probe the cleft in solution under more physiologically relevant conditions, including the transient phase of the ATPase reaction. Fluorescent probes were attached to engineered Cys residues in the upper and lower 50 kDa subdomains and used to measure the distance across the cleft. Single probes attached to either subdomain were used in combination with fluorescent nucleotides to monitor the coupling between the actin-binding cleft and the active site. Here we use a combination of transient time resolved FRET and transient time resolved fluorescence anisotropy to probe the equilibrium between open and closed cleft conformations. In the key experiments, a complete nanosecond time-resolved fluorescence decay was measured, defining the detailed distance distribution between probes, every 0.1 ms following rapid mixing (stopped flow), thus yielding high-resolution structural information on the sub-millisecond time scale. The results provide new insights into the coupling between the actin-binding cleft, the active site, and actin binding.

SummaryNucleation-promoting factors (NPFs) control the spatio-temporal activity of Arp2/3 complex in cells [1, 2]. Thus, WASP and the WAVE complex direct the formation of branched actin networks at the leading edge during cell motility and endo/exocytosis, whereas the WASH complex is involved in endosomal transport. Less understood are WHAMM and JMY, two NPFs with similar domain architecture. JMY is found in the nucleus and the cytosol and is involved in transcriptional regulation [3], cell motility [4], and trans-Golgi transport [5]. WHAMM was reported to bind microtubules and to be involved in ER to cis-Golgi transport [6]. Here, we show that WHAMM directs the activity of Arp2/3 complex for autophagosome biogenesis through an actin-comet tail motility mechanism. Macroautophagy—the process by which cytosolic material is engulfed into autophagosomes for degradation and/or recycling—was recently shown to involve actin [7], but the mechanism is unknown. We found that WHAMM forms puncta that colocalize and comigrate with the autophagy markers LC3, DFCP1, and p62 through a WHAMM-dependent actin-comet tail mechanism. Under starvation, WHAMM and actin are observed at the interface between neighboring autophagosomes, whose number and size increase with WHAMM expression. Interfering with actin polymerization, inhibiting Arp2/3 complex, knocking down WHAMM, or blocking its interaction with Arp2/3 complex through mutagenesis all inhibit comet tail formation and reduce the size and number of autophagosomes. Finally, JMY shows similar localization to WHAMM and could be involved in similar processes. These results reveal a link between Arp2/3-complex-dependent actin assembly and autophagy.

We have combined time-resolved fluorescence resonance energy transfer (TR-FRET) and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of chicken gizzard smooth muscle regulatory light chain (RLC) bound to smooth muscle myosin. The PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLCs, with and without phosphorylation at S19, were reconstituted into myosin S1. Time-resolved FRET identified two structural states of the RLC, closed and open, which are present in both unphosphorylated and phosphorylated biochemical states. Phosphorylation shifts the equilibrium toward the open state by 20-30%. Molecular dynamics simulations, guided by these and previous spectroscopic studies, confirm the existence of the open and closed structural states and produce atomic resolution models of these states. In the closed state, the PD interacts with the surface of the C-terminal lobe. In the open state, the PD is more helical and straight, resides farther from the C-terminal lobe, and is more mobile. Phosphorylation stabilizes the open state by forming a specific salt bridge between R16 and phosphorylated S19. This conformational shift is consistent with a mechanism of regulation that catalyzes large structural changes within myosin at a low energetic cost. This work was supported by grants from NIH (AR32961, AR07612) and the Minnesota Supercomputing Institute. We thank Igor Negrashov for excellent technical assistance.