[Show abstract][Hide abstract] ABSTRACT: Axon initial segments (AISs) and nodes of Ranvier are sites of clustering of voltage-gated sodium channels (VGSCs) in nervous systems of jawed vertebrates that facilitate fast long-distance electrical signaling. We demonstrate that proximal axonal polarity as well as assembly of the AIS and normal morphogenesis of nodes of Ranvier all require a heretofore uncharacterized alternatively spliced giant exon of ankyrin-G (AnkG). This exon has sequence similarity to I-connectin/Titin and was acquired after the first round of whole-genome duplication by the ancestral ANK2/ANK3 gene in early vertebrates before development of myelin. The giant exon resulted in a new nervous system-specific 480-kDa polypeptide combining previously known features of ANK repeats and β-spectrin-binding activity with a fibrous domain nearly 150 nm in length. We elucidate previously undescribed functions for giant AnkG, including recruitment of β4 spectrin to the AIS that likely is regulated by phosphorylation, and demonstrate that 480-kDa AnkG is a major component of the AIS membrane "undercoat' imaged by platinum replica electron microscopy. Surprisingly, giant AnkG-knockout neurons completely lacking known AIS components still retain distal axonal polarity and generate action potentials (APs), although with abnormal frequency. Giant AnkG-deficient mice live to weaning and provide a rationale for survival of humans with severe cognitive dysfunction bearing a truncating mutation in the giant exon. The giant exon of AnkG is required for assembly of the AIS and nodes of Ranvier and was a transformative innovation in evolution of the vertebrate nervous system that now is a potential target in neurodevelopmental disorders.
Proceedings of the National Academy of Sciences 12/2014; 112(4). DOI:10.1073/pnas.1416544112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Septins perform diverse functions through the formation of filaments and higher-order structures. However, the exact architecture of septin structures remains unclear. In the budding yeast Saccharomyces cerevisiae, septins form an 'hourglass' at the mother-bud neck before cytokinesis, which is converted into a 'double ring' during cytokinesis. Here, using platinum-replica electron microscopy, we find that the early hourglass consists of septin double filaments oriented along the mother-bud axis. In the late hourglass, these double filaments are connected by periodic circumferential single filaments on the membrane-proximal side and are associated with centrally located, circumferential, myosin-II thick filaments on the membrane-distal side. The double ring consists of exclusively circumferential septin filaments. Live-cell imaging studies indicate that the hourglass-to-double ring transition is accompanied by loss of septin subunits from the hourglass and reorganization of the remaining subunits into the double ring. This work provides an unparalleled view of septin structures within cells and defines their remodelling dynamics during the cell cycle.
[Show abstract][Hide abstract] ABSTRACT: Background:
Class II myosins generate contractile forces in cells by polymerizing into bipolar filaments and pulling on anchored actin filaments. Nonmuscle myosin II (NMII) plays central roles during cell adhesion, migration, cytokinesis, and tissue morphogenesis. NMII is present in virtually all mammalian cell types as tissue-specific combinations of NMIIA, NMIIB, and NMIIC isoforms. It remains poorly understood how the highly dynamic NMII-actin contractile system begins to assemble at new cellular locations during cell migration and how incorporation of different NMII isoforms into this system is coordinated.
Using platinum replica electron microscopy in combination with immunogold labeling, we demonstrate that individual activated (phosphorylated on the regulatory light chain and unfolded) NMIIA and NMIIB molecules represent a functional form of NMII in motile cells and that NMIIA and NMIIB copolymerize into nascent bipolar filaments during contractile system assembly. Using subdiffraction stimulated emission depletion microscopy together with a pharmacological block-and-release approach, we report that NMIIA and NMIIB simultaneously incorporate into the cytoskeleton during initiation of contractile system assembly, whereas the characteristic rearward shift of NMIIB relative to NMIIA is established later in the course of NMII turnover.
We show existence of activated NMII monomers in cells, copolymerization of endogenous NMIIA and NMIIB molecules, and contribution of both isoforms, rather than only NMIIA, to early stages of the contractile system assembly. These data change the current paradigms about dynamics and functions of NMII and provide new conceptual insights into the organization and dynamics of the ubiquitous cellular machinery for contraction that acts in multiple cellular contexts.
Current Biology 08/2014; 24(17). DOI:10.1016/j.cub.2014.07.070 · 9.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microtubule (MT) plus-end tracking proteins (+TIPs) preferentially localize to MT plus ends. End-binding proteins (EBs) are master regulators of the +TIP complex; however, it is unknown whether EBs are regulated by other +TIPs. Here, we show that cytoplasmic linker-associated proteins (CLASPs) modulate EB localization at MTs. In CLASP-depleted cells, EBs localized along the MT lattice in addition to plus ends. The MT-binding region of CLASP was sufficient for restoring normal EB localization, whereas neither EB-CLASP interactions nor EB tail-binding proteins are involved. In vitro assays revealed that CLASP directly functions to remove EB from MTs. Importantly, this effect occurs specifically during MT polymerization, but not at preformed MTs. Increased GTP-tubulin content within MTs in CLASP-depleted cells suggests that CLASPs facilitate GTP hydrolysis to reduce EB lattice binding. Together, these findings suggest that CLASPs influence the MT lattice itself to regulate EB and determine exclusive plus-end localization of EBs in cells.
[Show abstract][Hide abstract] ABSTRACT: The axon initial segment (AIS) of differentiated neurons regulates action potential initiation and axon-dendritic polarity. The latter function depends on actin dynamics, but actin structure and functions at the AIS remain unclear. Using platinum replica electron microscopy (PREM), we have characterized the architecture of the AIS cytoskeleton in mature and developing hippocampal neurons. The AIS cytoskeleton assembly begins with bundling of microtubules and culminates in formation of a dense, fibrillar-globular coat over microtubule bundles. Immunogold PREM revealed that the coat contains a network of known AIS proteins, including ankyrin G, spectrin βIV, neurofascin, neuronal cell adhesion molecule, voltage-gated sodium channels, and actin filaments. Contrary to existing models, we find neither polarized actin arrays, nor dense actin meshworks in the AIS. Instead, the AIS contains two populations of sparse actin filaments: short, stable filaments and slightly longer dynamic filaments. We propose that stable actin filaments play a structural role for formation of the AIS diffusion barrier, whereas dynamic actin may promote AIS coat remodeling.
The Journal of Cell Biology 04/2014; 205(1). DOI:10.1083/jcb.201401045 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Understanding how a particular cell type expresses the lamellipodial or filopodial form of the actin machinery is essential to understanding a cell's functional interactions. To determine how a cell “chooses” among these alternative modes of “molecular hardware,” we tested the role of key proteins that affect actin filament barbed ends. Depletion of capping protein (CP) by short hairpin RNA (shRNA) caused loss of lamellipodia and explosive formation of filopodia. The knockdown phenotype was rescued by a CP mutant refractory to shRNA, but not by another barbed-end capper, gelsolin, demonstrating that the phenotype was specific for CP. In Ena/VASP deficient cells, CP depletion resulted in ruffling instead of filopodia. We propose a model for selection of lamellipodial versus filopodial organization in which CP is a negative regulator of filopodia formation and Ena/VASP has recruiting/activating functions downstream of actin filament elongation in addition to its previously suggested anticapping and antibranching activities.
[Show abstract][Hide abstract] ABSTRACT: Several bacterial pathogens hijack the actin assembly machinery and display intracellular motility in the cytosol of infected cells. At the cell cortex, intracellular motility leads to bacterial dissemination through formation of plasma membrane protrusions that resolve into vacuoles in adjacent cells. Here, we uncover a critical role for actin network disassembly in L. monocytogenes dissemination. We found that defects in the disassembly machinery decreased the rate of actin tail turnover, but did not affect the velocity of the bacteria in the cytosol. By contrast, defects in the disassembly machinery had a dramatic impact on bacterial dissemination. Our results suggest a model of L. monocytogenes dissemination in which the disassembly machinery, through local recycling of the actin network in protrusions, fuels continuous actin assembly at the bacterial pole and concurrently exhausts cytoskeleton components from the network distal to the bacterium, which enables membrane apposition and resolution of protrusions into vacuoles.
[Show abstract][Hide abstract] ABSTRACT: CARMIL is an approximately 1,370-amino-acid cytoskeletal scaffold that has crucial roles in cell motility and tissue development through interactions with cytoskeletal effectors and regulation of capping protein at the leading edge. However, the mechanism of CARMIL leading edge localization is unknown. Here we show that CARMIL interacts directly with the plasma membrane through its amino-terminal region. The crystal structure of CARMIL1-668 reveals that this region harbours a non-canonical pleckstrin homology (PH) domain connected to a 16-leucine-rich repeat domain. Lipid binding is mediated by the PH domain, but is further enhanced by a central helical domain. Small-angle X-ray scattering reveals that the helical domain mediates antiparallel dimerization, properly positioning the PH domains for simultaneous membrane interaction. In cells, deletion of the PH domain impairs leading edge localization. The results support a direct membrane-binding mechanism for CARMIL localization at the leading edge, where it regulates cytoskeletal effectors and motility.
[Show abstract][Hide abstract] ABSTRACT: Dynamic shape changes of the plasma membrane are fundamental to many processes, ranging from morphogenesis and cell migration to phagocytosis and viral propagation. Here, we demonstrate that Exo70, a component of the exocyst complex, induces tubular membrane invaginations toward the lumen of synthetic vesicles in vitro and generates protrusions on the surface of cells. Biochemical analyses using Exo70 mutants and independent molecular dynamics simulations based on Exo70 structure demonstrate that Exo70 generates negative membrane curvature through an oligomerization-based mechanism. In cells, the membrane-deformation function of Exo70 is required for protrusion formation and directional cell migration. Exo70 thus represents a membrane-bending protein that may couple actin dynamics and plasma membrane remodeling for morphogenesis.
[Show abstract][Hide abstract] ABSTRACT: The actin cytoskeleton is the major force-generating machinery in the cell, which can produce pushing, pulling, and resistance forces. To accomplish these diverse functions, actin filaments, with help of numerous accessory proteins, form higher order ensembles, networks and bundles, adapted to specific tasks. Moreover, dynamic properties of the actin cytoskeleton allow a cell to constantly build, renew, and redesign actin structures according to its changing needs. High resolution architecture of actin filament arrays provides key information for understanding mechanisms of force generation. To generate pushing force, cells use coordinated polymerization of multiple actin filaments organized into branched (dendritic) networks or parallel bundles. This review summarizes our current knowledge of the structural organization of these two actin filament arrays.
Current opinion in cell biology 04/2013; 25(5). DOI:10.1016/j.ceb.2013.04.003 · 8.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system's maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process. We show that inhibition of NMII by a specific inhibitor, blebbistatin, in addition to known effects, such as disassembly of stress fibers and mature focal adhesions, also causes transformation of lamellipodia into unattached ruffles, loss of immature focal complexes, loss of cytoskeleton-associated NMII filaments and peripheral accumulation of activated, but unpolymerized NMII. After blebbistatin washout, assembly of the contractile system begins with quick and coordinated recovery of lamellipodia and focal complexes that occurs before reappearance of NMII bipolar filaments. The initial formation of focal complexes and subsequent assembly of NMII filaments preferentially occurred in association with filopodial bundles and concave actin bundles formed by filopodial roots at the lamellipodial base. Over time, accumulating NMII filaments help to transform the precursor structures, focal complexes and associated thin bundles, into stress fibers and mature focal adhesions. However, semi-sarcomeric organization of stress fibers develops at much slower rate. Together, our data suggest that activation of NMII motor activity by light chain phosphorylation occurs at the cell edge and is uncoupled from NMII assembly into bipolar filaments. We propose that activated, but unpolymerized NMII initiates focal complexes, thus providing traction for lamellipodial protrusion. Subsequently, the mechanical resistance of focal complexes activates a load-dependent mechanism of NMII polymerization in association with attached bundles, leading to assembly of stress fibers and maturation of focal adhesions.
PLoS ONE 07/2012; 7(7):e40814. DOI:10.1371/journal.pone.0040814 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Directional cell migration requires the coordination of actin assembly and membrane remodeling. The exocyst is an octameric protein complex essential for exocytosis and plasma membrane remodeling. A component of the exocyst, Exo70, directly interacts with the Arp2/3 complex, a core nucleating factor for the generation of branched actin networks for cell morphogenesis and migration. Using in vitro actin polymerization assay and time-lapse total internal reflection fluorescence microscopy, we found that Exo70 functions as a kinetic activator of the Arp2/3 complex that promotes actin filament nucleation and branching. We further found that the effect of Exo70 on actin is mediated by promoting the interaction of the Arp2/3 complex with WAVE2, a member of the N-WASP/WAVE family of nucleation promoting factors. At the cellular level, the stimulatory effect of Exo70 on the Arp2/3 complex is required for lamellipodia formation and maintaining directional persistence of cell migration. Our findings provide a novel mechanism for regulating actin polymerization and branching for effective membrane protrusion during cell morphogenesis and migration.
Current biology: CB 06/2012; 22(16):1510-5. DOI:10.1016/j.cub.2012.05.055 · 9.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Axon branching is fundamental to the development of the peripheral and central nervous system. Branches that sprout from the axon shaft are termed collateral or interstitial branches. Collateral branching of axons requires the formation of filopodia from actin microfilaments (F-actin) and their engorgement with microtubules (MTs) that splay from the axon shaft. The mechanisms that drive and coordinate the remodeling of actin and MTs during branch morphogenesis are poorly understood. Septins comprise a family of GTP-binding proteins that oligomerize into higher-order structures, which associate with membranes and the actin and microtubule cytoskeleton. Here, we show that collateral branching of axons requires SEPT6 and SEPT7, two interacting septins. In the axons of sensory neurons, both SEPT6 and SEPT7 accumulate at incipient sites of filopodia formation. We show that SEPT6 localizes to axonal patches of F-actin and increases the recruitment of cortactin, a regulator of Arp2/3-mediated actin polymerization, triggering the emergence of filopodia. Conversely, SEPT7 promotes the entry of axonal MTs into filopodia, enabling the formation of collateral branches. Surprisingly, septins provide a novel mechanism for the collateral branching of axons by coordinating the remodeling of the actin and microtubule cytoskeleton.
Current biology: CB 05/2012; 22(12):1109-15. DOI:10.1016/j.cub.2012.04.019 · 9.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polyphosphoinositides (PPIs) and in particular phosphatidylinositol-(4,5)-bisphosphate (PI4,5P2), control many cellular events and bind with variable levels of specificity to hundreds of intracellular proteins in vitro. The much more restricted targeting of proteins to PPIs in cell membranes is thought to result in part from the formation of spatially distinct PIP2 pools, but the mechanisms that cause formation and maintenance of PIP2 clusters are still under debate. The hypothesis that PIP2 forms submicrometer-sized clusters in the membrane by electrostatic interactions with intracellular divalent cations is tested here using lipid monolayer and bilayer model membranes. Competitive binding between Ca(2+) and Mg(2+) to PIP2 is quantified by surface pressure measurements and analyzed by a Langmuir competitive adsorption model. The physical chemical differences among three PIP2 isomers are also investigated. Addition of Ca(2+) but not Mg(2+), Zn(2+), or polyamines to PIP2-containing monolayers induces surface pressure drops coincident with the formation of PIP2 clusters visualized by fluorescence, atomic force, and electron microscopy. Studies of bilayer membranes using steady-state probe-partitioning fluorescence resonance energy transfer (SP-FRET) and fluorescence correlation spectroscopy (FCS) also reveal divalent metal ion (Me(2+))-induced cluster formation or diffusion retardation, which follows the trend: Ca(2+) ≫ Mg(2+) > Zn(2+), and polyamines have minimal effects. These results suggest that divalent metal ions have substantial effects on PIP2 lateral organization at physiological concentrations, and local fluxes in their cytoplasmic levels can contribute to regulating protein-PIP2 interactions.
Journal of the American Chemical Society 02/2012; 134(7):3387-95. DOI:10.1021/ja208640t · 12.11 Impact Factor