Direct observation of cortactin protecting Arp2/3-actin filament branch junctions from GMF-mediated destabilization.

How cells tightly control the formation and turnover of branched actin filament arrays to drive cell motility, endocytosis, and other cellular processes is still not well understood. Here, we investigated the mechanistic relationship between two binding partners of the Arp2/3 complex, glia maturation factor (GMF) and cortactin. Individually, GMF and cortactin have opposite effects on the stability of actin filament branches, but it is unknown how they work in concert with each other to govern branch turnover. Using TIRF microscopy, we observe that GMF's branch destabilizing activities are potently blocked by cortactin (IC50 = 1.3 nM) and that this inhibition requires direct interactions of cortactin with Arp2/3 complex. The simplest model that would explain these results is competition for binding Arp2/3 complex. However, we find that cortactin and GMF do not compete for free Arp2/3 complex in solution. Further, we use single molecule analysis to show that cortactin's on-rate (3 ×107 s-1 M-1) and off-rate (0.03 s-1) at branch junctions are minimally affected by excess GMF. Together, these results show that cortactin binds with high affinity to branch junctions, where it blocks the destabilizing effects of GMF, possibly by a mechanism that is allosteric in nature. In addition, the affinities we measure for cortactin at actin filament branch junctions (Kd = 0.9 nM) and filament sides (Kd = 206 nM) are approximately 20-fold stronger than previously reported. These observations contribute to an emerging view of molecular complexity in how Arp2/3 complex is regulated through the integration of multiple inputs.

Cyclase-associated protein interacts with actin filament barbed ends to promote depolymerization and formin displacement.

Cyclase-associated protein (CAP) has emerged as a central player in cellular actin turnover, but its molecular mechanisms of action are not yet fully understood. Recent studies revealed that the N terminus of CAP interacts with the pointed ends of actin filaments to accelerate depolymerization in conjunction with cofilin. Here, we use in vitro microfluidics-assisted TIRF microscopy to show that the C terminus of CAP promotes depolymerization at the opposite (barbed) ends of actin filaments. In the absence of actin monomers, full-length mouse CAP1 and C-terminal halves of CAP1 (C-CAP1) and CAP2 (C-CAP2) accelerate barbed end depolymerization. Using mutagenesis and structural modeling, we show that these activities are mediated by the WH2 and CARP domains of CAP. In addition, we observe that CAP collaborates with profilin to accelerate barbed end depolymerization and that these effects depend on their direct interaction, providing the first known example of CAP-profilin collaborative effects in regulating actin. In the presence of actin monomers, CAP1 attenuates barbed end growth and promotes formin dissociation. Overall, these findings demonstrate that CAP uses distinct domains and mechanisms to interact with opposite ends of actin filaments and drive turnover. Further, they contribute to the emerging view of actin barbed ends as sites of dynamic molecular regulation, where numerous proteins compete and cooperate with each other to tune polymer dynamics, similar to the rich complexity seen at microtubule ends.

BORG family proteins in physiology and human disease.

The binder of rho GTPases (BORG)/Cdc42 effector proteins (Cdc42EP) family is composed of five Rho GTPase binding proteins whose functions and mechanism of actions are of emerging interest. Here, we review recent findings pertaining to the family as a whole and consider how these change our understanding of cellular organization. Recent studies have implicated BORGs in both fundamental physiology and in human diseases, mainly cancers. An emerging pattern suggests that BORG family members cancer-promoting properties are related to their ability to regulate the cytoskeleton, with many impacting the organization of acto-myosin stress fibers. This is consistent with the broader literature indicating that BORG family members are regulators of both the septin and actin cytoskeleton networks. The exact mechanism through which BORGs modify the cytoskeleton is not clear, but we consider here a few data-supported and speculative possibilities. Finally, we delve into how the Rho GTPase Cdc42 modifies BORG function in cells. This remains open-ended as Cdc42's effects on BORGs appear cell type- and cell state-dependent. Collectively, these data point to the importance of the BORG family and suggest broader themes in their function and regulation.

Structure of VanS from vancomycin-resistant enterococci: A sensor kinase with weak ATP binding.

The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci. VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from vancomycin-resistant enterococci types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins' binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with KD values in the low millimolar range. Since these KD values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.

Septins mediate a microtubule-actin crosstalk that enables actin growth on microtubules.

Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons. Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin. Septins are filamentous guanosine-5'-triphosphate (GTP) binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin, and intermediate filaments. Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Superresolution and platinum replica electron microscopy (PREM) show that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We demonstrate that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings show that septins directly mediate microtubule interactions with actin filaments, and reveal a mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis.

Production and analysis of a mammalian septin hetero-octamer complex.

The septins are filament-forming proteins found in diverse eukaryotes from fungi to vertebrates, with roles in cytokinesis, shaping of membranes and modifying cytoskeletal organization. These GTPases assemble into rod-shaped soluble hetero-hexamers and hetero-octamers in mammals, which polymerize into filaments and higher order structures. While the cell biology and pathobiology of septins are advancing rapidly, mechanistic study of the mammalian septins is limited by a lack of recombinant hetero-octamer materials. We describe here the production and characterization of a recombinant mammalian septin hetero-octamer of defined stoichiometry, the SEPT2/SEPT6/SEPT7/SEPT3 complex. Using a fluorescent protein fusion to the complex, we observed filaments assembled from this complex. In addition, we used this novel tool to resolve recent questions regarding the organization of the soluble septin complex. Biochemical characterization of a SEPT3 truncation that disrupts SEPT3-SEPT3 interactions is consistent with SEPT3 occupying a central position in the complex while the SEPT2 subunits are at the ends of the rod-shaped octameric complexes. Consistent with SEPT2 being on the complex ends, we find that our purified SEPT2/SEPT6/SEPT7/SEPT3 hetero-octamer copolymerizes into mixed filaments with separately purified SEPT2/SEPT6/SEPT7 hetero-hexamer. We expect this new recombinant production approach to lay essential groundwork for future studies into mammalian septin mechanism and function.

Sequence Determinants of Intracellular Phase Separation by Complex Coacervation of a Disordered Protein.

Liquid-liquid phase separation, driven by collective interactions among multivalent and intrinsically disordered proteins, is thought to mediate the formation of membrane-less organelles in cells. Using parallel cellular and in vitro assays, we show that the Nephrin intracellular domain (NICD), a disordered protein, drives intracellular phase separation via complex coacervation, whereby the negatively charged NICD co-assembles with positively charged partners to form protein-rich dense liquid droplets. Mutagenesis reveals that the driving force for phase separation depends on the overall amino acid composition and not the precise sequence of NICD. Instead, phase separation is promoted by one or more regions of high negative charge density and aromatic/hydrophobic residues that are distributed across the protein. Many disordered proteins share similar sequence characteristics with NICD, suggesting that complex coacervation may be a widely used mechanism to promote intracellular phase separation.

The antitumor toxin CD437 is a direct inhibitor of DNA polymerase α.

CD437 is a retinoid-like small molecule that selectively induces apoptosis in cancer cells, but not in normal cells, through an unknown mechanism. We used a forward-genetic strategy to discover mutations in POLA1 that coincide with CD437 resistance (POLA1(R)). Introduction of one of these mutations into cancer cells by CRISPR-Cas9 genome editing conferred CD437 resistance, demonstrating causality. POLA1 encodes DNA polymerase α, the enzyme responsible for initiating DNA synthesis during the S phase of the cell cycle. CD437 inhibits DNA replication in cells and recombinant POLA1 activity in vitro. Both effects are abrogated by the identified POLA1 mutations, supporting POLA1 as the direct antitumor target of CD437. In addition, we detected an increase in the total fluorescence intensity and anisotropy of CD437 in the presence of increasing concentrations of POLA1 that is consistent with a direct binding interaction. The discovery of POLA1 as the direct anticancer target for CD437 has the potential to catalyze the development of CD437 into an anticancer therapeutic.

On the acquisition and analysis of microscale thermophoresis data.

A comprehensive understanding of the molecular mechanisms underpinning cellular functions is dependent on a detailed characterization of the energetics of macromolecular binding, often quantified by the equilibrium dissociation constant, KD. While many biophysical methods may be used to obtain KD, the focus of this report is a relatively new method called microscale thermophoresis (MST). In an MST experiment, a capillary tube filled with a solution containing a dye-labeled solute is illuminated with an infrared laser, rapidly creating a temperature gradient. Molecules will migrate along this gradient, causing changes in the observed fluorescence. Because the net migration of the labeled molecules will depend on their liganded state, a binding curve as a function of ligand concentration can be constructed from MST data and analyzed to determine KD. Herein, simulations demonstrate the limits of KD that can be measured in current instrumentation. They also show that binding kinetics is a major concern in planning and executing MST experiments. Additionally, studies of two protein-protein interactions illustrate challenges encountered in acquiring and analyzing MST data. Combined, these approaches indicate a set of best practices for performing and analyzing MST experiments. Software for rigorous data analysis is also introduced.

A novel role for WAVE1 in controlling actin network growth rate and architecture.

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 ("V") domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.

Biochemical reconstitution of the WAVE regulatory complex.

The WAVE regulatory complex (WRC) is a 400-kDa heteropentameric protein assembly that plays a central role in controlling actin cytoskeletal dynamics in many cellular processes. The WRC acts by integrating diverse cellular cues and stimulating the actin nucleating activity of the Arp2/3 complex at membranes. Biochemical and biophysical studies of the underlying mechanisms of these processes require large amounts of purified WRC. Recent success in recombinant expression, reconstitution, purification, and crystallization of the WRC has greatly advanced our understanding of the inhibition, activation, and membrane recruitment mechanisms of this complex. But many important questions remain to be answered. Here, we summarize and update the methods developed in our laboratory, which allow reliable and flexible production of tens of milligrams of recombinant WRC of crystallographic quality, sufficient for many biochemical and structural studies.

The bacterial effector VopL organizes actin into filament-like structures.

VopL is an effector protein from Vibrio parahaemolyticus that nucleates actin filaments. VopL consists of a VopL C-terminal domain (VCD) and an array of three WASP homology 2 (WH2) motifs. Here, we report the crystal structure of the VCD dimer bound to actin. The VCD organizes three actin monomers in a spatial arrangement close to that found in the canonical actin filament. In this arrangement, WH2 motifs can be modeled into the binding site of each actin without steric clashes. The data suggest a mechanism of nucleation wherein VopL creates filament-like structures, organized by the VCD with monomers delivered by the WH2 array, that can template addition of new subunits. Similarities with Arp2/3 complex and formin proteins suggest that organization of monomers into filament-like structures is a general and central feature of actin nucleation.

Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation.

During cell locomotion and endocytosis, membrane-tethered WASP proteins stimulate actin filament nucleation by the Arp2/3 complex. This process generates highly branched arrays of filaments that grow toward the membrane to which they are tethered, a conflict that seemingly would restrict filament growth. Using three-color single-molecule imaging in vitro we revealed how the dynamic associations of Arp2/3 complex with mother filament and WASP are temporally coordinated with initiation of daughter filament growth. We found that WASP proteins dissociated from filament-bound Arp2/3 complex prior to new filament growth. Further, mutations that accelerated release of WASP from filament-bound Arp2/3 complex proportionally accelerated branch formation. These data suggest that while WASP promotes formation of pre-nucleation complexes, filament growth cannot occur until it is triggered by WASP release. This provides a mechanism by which membrane-bound WASP proteins can stimulate network growth without restraining it. DOI:http://dx.doi.org/10.7554/eLife.01008.001.

Purification of Arp2/3 complex from Saccharomyces cerevisiae.

Much of the cellular control over actin dynamics comes through regulation of actin filament initiation. At the molecular level, this is accomplished through a collection of cellular protein machines, called actin nucleation factors, which position actin monomers to initiate a new actin filament. The Arp2/3 complex is a principal actin nucleation factor used throughout the eukaryotic family tree. The budding yeast Saccharomyces cerevisiae has proven to be not only an excellent genetic platform for the study of the Arp2/3 complex, but also an excellent source for the purification of endogenous Arp2/3 complex. Here we describe a protocol for the preparation of endogenous Arp2/3 complex from wild type Saccharomyces cerevisiae. This protocol produces material suitable for biochemical study and yields milligram quantities of purified Arp2/3 complex.

Measurement and analysis of in vitro actin polymerization.

The polymerization of actin underlies force generation in numerous cellular processes. While actin polymerization can occur spontaneously, cells maintain control over this important process by preventing actin filament nucleation and then allowing stimulated polymerization and elongation by several regulated factors. Actin polymerization, regulated nucleation, and controlled elongation activities can be reconstituted in vitro, and used to probe the signaling cascades cells use to control when and where actin polymerization occurs. Introducing a pyrene fluorophore allows detection of filament formation by an increase in pyrene fluorescence. This method has been used for many years and continues to be broadly used, owing to its simplicity and flexibility. Here we describe how to perform and analyze these in vitro actin polymerization assays, with an emphasis on extracting useful descriptive parameters from kinetic data.

Purification of native Arp2/3 complex from bovine thymus.

The Arp2/3 complex is an actin filament nucleator involved in cell motility and vesicle trafficking. Owing to the role the complex plays in important and fundamental cell biological processes, the purified complex is used in biochemical assays, reconstituted motility assays, and structural biology. As this is a eukaryotic complex assembled from seven polypeptides, the complex is purified from eukaryotic sources. Described here is a detailed method for purification of the complex from a mammalian tissue, bovine thymus.

GMF severs actin-Arp2/3 complex branch junctions by a cofilin-like mechanism.

BACKGROUND: Branched actin filament networks driving cell motility, endocytosis, and intracellular transport are assembled in seconds by the Arp2/3 complex and must be equally rapidly debranched and turned over. One of the only factors known to promote debranching of actin networks is the yeast homolog of glia maturation factor (GMF), which is structurally related to the actin filament-severing protein cofilin. However, the identity of the molecular mechanism underlying debranching and whether this activity extends to mammalian GMF have remained open questions. RESULTS: Using scanning mutagenesis and total internal reflection fluorescence microscopy, we show that GMF depends on two separate surfaces for debranching. One is analogous to the G-actin and F-actin binding site on cofilin, but we show using fluorescence anisotropy and chemical crosslinking that it instead interacts with actin-related proteins in the Arp2/3 complex. The other is analogous to a second F-actin binding site on cofilin, which in GMF appears to contact the first actin subunit in the daughter filament. We further show that GMF binds to the Arp2/3 complex with low nanomolar affinity and promotes the open conformation. Finally, we show that this debranching activity and mechanism are conserved for mammalian GMF. CONCLUSIONS: GMF debranches filaments by a mechanism related to cofilin-mediated severing, but in which GMF has evolved to target molecular junctions between actin-related proteins in the Arp2/3 complex and actin subunits in the daughter filament of the branch. This activity and mechanism are conserved in GMF homologs from evolutionarily distant species.

Multi-signal sedimentation velocity analysis with mass conservation for determining the stoichiometry of protein complexes.

Multi-signal sedimentation velocity analytical ultracentrifugation (MSSV) is a powerful tool for the determination of the number, stoichiometry, and hydrodynamic shape of reversible protein complexes in two- and three-component systems. In this method, the evolution of sedimentation profiles of macromolecular mixtures is recorded simultaneously using multiple absorbance and refractive index signals and globally transformed into both spectrally and diffusion-deconvoluted component sedimentation coefficient distributions. For reactions with complex lifetimes comparable to the time-scale of sedimentation, MSSV reveals the number and stoichiometry of co-existing complexes. For systems with short complex lifetimes, MSSV reveals the composition of the reaction boundary of the coupled reaction/migration process, which we show here may be used to directly determine an association constant. A prerequisite for MSSV is that the interacting components are spectrally distinguishable, which may be a result, for example, of extrinsic chromophores or of different abundances of aromatic amino acids contributing to the UV absorbance. For interacting components that are spectrally poorly resolved, here we introduce a method for additional regularization of the spectral deconvolution by exploiting approximate knowledge of the total loading concentrations. While this novel mass conservation principle does not discriminate contributions to different species, it can be effectively combined with constraints in the sedimentation coefficient range of uncomplexed species. We show in theory, computer simulations, and experiment, how mass conservation MSSV as implemented in SEDPHAT can enhance or even substitute for the spectral discrimination of components. This should broaden the applicability of MSSV to the analysis of the composition of reversible macromolecular complexes.

Arp2/3 complex is bound and activated by two WASP proteins.

Actin related protein 2/actin related protein 3 (Arp2/3) complex nucleates new actin filaments in eukaryotic cells in response to signals from proteins in the Wiskott-Aldrich syndrome protein (WASP) family. The conserved VCA domain of WASP proteins activates Arp2/3 complex by inducing conformational changes and delivering the first actin monomer of the daughter filament. Previous models of activation have invoked a single VCA acting at a single site on Arp2/3 complex. Here we show that activation most likely involves engagement of two distinct sites on Arp2/3 complex by two VCA molecules, each delivering an actin monomer. One site is on Arp3 and the second is on ARPC1 and Arp2. The VCAs at these sites have distinct roles in activation. Our findings reconcile apparently conflicting literature on VCA activation of Arp2/3 complex and lead to a new model for this process.

Evaluating the stoichiometry of macromolecular complexes using multisignal sedimentation velocity.

Gleaning information regarding the molecular physiology of macromolecular complexes requires knowledge of their component stoichiometries. In this work, a relatively new means of analyzing sedimentation velocity (SV) data from the analytical ultracentrifuge is examined in detail. The method depends on collecting concentration profile data simultaneously using multiple signals, like Rayleigh interferometry and UV spectrophotometry. If the cosedimenting components of a complex are spectrally distinguishable, continuous sedimentation-coefficient distributions specific for each component can be calculated to reveal the molar ratio of the complex's components. When combined with the hydrodynamic information available from the SV data, a stoichiometry can be derived. Herein, the spectral properties of sedimenting species are systematically explored to arrive at a predictive test for whether a set of macromolecules can be spectrally resolved in a multisignal SV (MSSV) experiment. Also, a graphical means of experimental design and criteria to judge the success of the spectral discrimination in MSSV are introduced. A detailed example of the analysis of MSSV experiments is offered, and the possibility of deriving equilibrium association constants from MSSV analyses is explored. Finally, successful implementations of MSSV are reviewed.