RESUMO
Understanding the mechanisms of assembly and disassembly of macromolecular structures in cells relies on solving biomolecular interactions. However, those interactions often remain unclear because tools to track molecular dynamics are not sufficiently resolved in time or space. In this study, we present a straightforward method for resolving inter- and intra-molecular interactions in cell adhesive machinery, using quantum dot (QD) based Förster resonance energy transfer (FRET) nanosensors. Using a mechanosensitive protein, talin, one of the major components of focal adhesions, we are investigating the mechanosensing ability of proteins to sense and respond to mechanical stimuli. First, we quantified the distances separating talin and a giant unilamellar vesicle membrane for three talin variants. These variants differ in molecular length. Second, we investigated the mechanosensing capabilities of talin, i.e., its conformational changes due to mechanical stretching initiated by cytoskeleton contraction. Our results suggest that in early focal adhesion, talin undergoes stretching, corresponding to a decrease in the talin-membrane distance of 2.5 nm. We demonstrate that QD-FRET nanosensors can be applied for the sensitive quantification of mechanosensing with a sub-nanometer accuracy.
RESUMO
Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Movimento Celular/genética , Pseudópodes/genética , Pseudópodes/metabolismo , Transdução de Sinais , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Dictyostelium/genética , Dictyostelium/metabolismo , Embrião não Mamífero , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Peixe-Zebra/genéticaRESUMO
The N-terminal part of the inhibitory peptide IF1 interacts with the central γ subunit of mitochondrial isolated extrinsic part of ATP synthase in the inhibited complex (J.R. Gledhill, M.G. Montgomery, G.W. Leslie, J.E. Walker, 2007). To explore its role in the different steps of IF1 binding, kinetics of inhibition of the isolated and membrane-bound enzymes were investigated using Saccharomyces cerevisiae IF1 derivatives modified in N-terminal extremity. First, we studied peptides truncated in Nter up to the amino acid immediately preceding Phe17, a well-conserved residue thought to play a key role. These deletions did not affect or even improve the access of IF1 to its target. They decreased the stability of the inhibited complex but much less than previously proposed. We also mutated IF1-Phe17 and found this amino acid not mandatory for the inhibitory effect. The most striking finding came from experiments in which PsaE, a 8 kDa globular-like protein, was attached in Nter of IF1. Unexpectedly, such a modification did not appreciably affect the rate of IF1 binding. Taken together, these data show that IF1-Nter plays no role in the recognition step but contributes to stabilize the inhibited complex. Moreover, the data obtained using chimeric PsaE-IF1 suggest that before binding IF1 presents to the enzyme with its middle part facing a catalytic interface and its Nter extremity folded in the opposite direction.
Assuntos
Complexos de ATP Sintetase/química , Complexos de ATP Sintetase/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexos de ATP Sintetase/genética , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína Inibidora de ATPaseRESUMO
Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the capping protein for unclear reasons. Here, we show that the WASH complex interacts with dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.
RESUMO
Actin-based comet tails produced by Listeria monocytogenes are considered as representative models for cellular force-producing machineries crucial for cell migration. We here present a proteomic picture of these tails formed in extracts from brain and platelets. This provides a comprehensive view, revealing high molecular complexity and novel host cell proteins as tail components, and suggests the participation of specific multicomponent regulatory complexes. This work forms a new basis to expand current models of cellular protrusion.
Assuntos
Actinas/metabolismo , Movimento Celular , Listeria monocytogenes/fisiologia , Listeriose/metabolismo , Proteoma , Plaquetas/microbiologia , Encéfalo/microbiologia , Proteínas de Ligação a Calmodulina/metabolismo , Células HeLa , Humanos , Neuropeptídeos/metabolismoRESUMO
Toxofilin is a 27 kDa protein isolated from the human protozoan parasite Toxoplasma gondii, which causes toxoplasmosis. Toxofilin binds to G-actin, and in vitro studies have shown that it controls elongation of actin filaments by sequestering actin monomers. Toxofilin affinity for G-actin is controlled by the phosphorylation status of its Ser53, which depends on the activities of a casein kinase II and a type 2C serine/threonine phosphatase (PP2C). To get insights into the functional properties of toxofilin, we undertook a structure-function analysis of the protein using a combination of biochemical techniques. We identified a domain that was sufficient to sequester G-actin and that contains three peptide sequences selectively binding to G-actin. Two of these sequences are similar to sequences present in several G- and F-actin-binding proteins, while the third appears to be specific to toxofilin. Additionally, we identified two toxofilin domains that interact with PP2C, one of which contains the Ser53 substrate. In addition to characterizing the interacting domains of toxofilin with its partners, the present study also provides information on an in vivo-based approach to selectively and competitively disrupt the protein-protein interactions that are important to parasite motility.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Actinas/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Proteínas de Capeamento de Actina/química , Actinas/química , Sequência de Aminoácidos , Animais , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Fosfoproteínas Fosfatases/química , Ligação Proteica , Proteína Fosfatase 2C , Estrutura Terciária de Proteína , Proteínas de Protozoários/químicaRESUMO
The evolutionarily conserved Arp2/3 complex plays a central role in nucleating the branched actin filament arrays that drive cell migration, endocytosis, and other processes. To better understand Arp2/3 complex regulation, we used single-particle electron microscopy to compare the structures of Arp2/3 complex bound to three different inhibitory ligands: glia maturation factor (GMF), Coronin, and Arpin. Although the three inhibitors have distinct binding sites on Arp2/3 complex, they each induced an "open" nucleation-inactive conformation. Coronin promoted a standard (previously described) open conformation of Arp2/3 complex, with the N-terminal ß-propeller domain of Coronin positioned near the p35/ARPC2 subunit of Arp2/3 complex. GMF induced two distinct open conformations of Arp2/3 complex, which correlated with the two suggested binding sites for GMF. Furthermore, GMF synergized with Coronin in inhibiting actin nucleation by Arp2/3 complex. Arpin, which uses VCA-related acidic (A) motifs to interact with the Arp2/3 complex, induced the standard open conformation, and two new masses appeared at positions near Arp2 and Arp3. Furthermore, Arpin showed additive inhibitory effects on Arp2/3 complex with Coronin and GMF. Together, these data suggest that Arp2/3 complex conformation is highly polymorphic and that its activities can be controlled combinatorially by different inhibitory ligands.
Assuntos
4-Butirolactona/análogos & derivados , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Proteínas de Transporte/farmacologia , Fator de Maturação da Glia/farmacologia , 4-Butirolactona/farmacologia , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Sítios de Ligação , Movimento Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Humanos , Imageamento Tridimensional , Conformação ProteicaRESUMO
The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins.
Assuntos
Actinas/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Glucosiltransferases/metabolismo , Pseudomonas aeruginosa/metabolismo , Actinas/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Glucosiltransferases/genética , Mutação , Ligação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Virulência/genéticaRESUMO
Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein-Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endocitose , Receptores ErbB/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/genética , Cães , Técnicas de Silenciamento de Genes , Humanos , Proteínas com Domínio LIM/genética , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Proteínas dos Microfilamentos , Oxigenases de Função Mista , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Apicomplexa are obligate intracellular parasites that actively invade host cells using their membrane-associated, actin-myosin motor. The current view is that host cell invasion by Apicomplexa requires the formation of a parasite-host cell junction, which has been termed the moving junction, but does not require the active participation of host actin. Using Toxoplasma gondii tachyzoites and Plasmodium berghei sporozoites, we show that host actin participates in parasite entry. Parasites induce the formation of a ring-shaped F-actin structure in the host cell at the parasite-cell junction, which remains stable during parasite entry. The Arp2/3 complex, an actin-nucleating factor, is recruited at the ring structure and is important for parasite entry. We propose that Apicomplexa invasion of host cells requires not only the parasite motor but also de novo polymerization of host actin at the entry site for anchoring the junction on which the parasite pulls to penetrate the host cell.