RESUMO
Force arising from actin polymerization and myosin activity drives a number of different actin-based cell movements. Several new reports support previous data suggesting that actin polymerization drives lamellipodial protrusion and bacterial propulsion, and one report describes a more indirect role for actin assembly in axonal elongation. The major new findings of the past year concerning possible motility roles for myosin describe myosin-driven protrusion of cell margins.
Assuntos
Actinas/fisiologia , Movimento Celular/fisiologia , Miosinas/fisiologia , Actinas/metabolismo , Animais , Humanos , Substâncias Macromoleculares , Modelos Biológicos , Miosinas/metabolismoRESUMO
Polymerizing networks of actin filaments are capable of exerting significant mechanical forces, used by eukaryotic cells and their prokaryotic pathogens to change shape or to move. Here we show that small beads coated uniformly with a protein that catalyses actin polymerization are initially surrounded by symmetrical clouds of actin filaments. This symmetry is broken spontaneously, after which the beads undergo directional motion. We have developed a stochastic theory, in which each actin filament is modelled as an elastic brownian ratchet, that quantitatively accounts for the observed emergent symmetry-breaking behaviour. Symmetry-breaking can only occur for polymers that have a significant subunit off-rate, such as the biopolymers actin and tubulin.
Assuntos
Actinas/metabolismo , Movimento Celular , Simulação por Computador , Modelos Biológicos , Animais , Biopolímeros/metabolismo , Extratos Celulares , Difusão , Elasticidade , Cinética , Listeria monocytogenes , Microesferas , Movimento (Física) , Poliestirenos , Ligação Proteica , Processos Estocásticos , Tubulina (Proteína)/metabolismo , Xenopus laevisRESUMO
Most of the fascinating phenomena studied in cell biology emerge from interactions among highly organized multimolecular structures embedded into complex and frequently dynamic cellular morphologies. For the exploration of such systems, computer simulation has proved to be an invaluable tool, and many researchers in this field have developed sophisticated computational models for application to specific cell biological questions. However, it is often difficult to reconcile conflicting computational results that use different approaches to describe the same phenomenon. To address this issue systematically, we have defined a series of computational test cases ranging from very simple to moderately complex, varying key features of dimensionality, reaction type, reaction speed, crowding, and cell size. We then quantified how explicit spatial and/or stochastic implementations alter outcomes, even when all methods use the same reaction network, rates, and concentrations. For simple cases, we generally find minor differences in solutions of the same problem. However, we observe increasing discordance as the effects of localization, dimensionality reduction, and irreversible enzymatic reactions are combined. We discuss the strengths and limitations of commonly used computational approaches for exploring cell biological questions and provide a framework for decision making by researchers developing new models. As computational power and speed continue to increase at a remarkable rate, the dream of a fully comprehensive computational model of a living cell may be drawing closer to reality, but our analysis demonstrates that it will be crucial to evaluate the accuracy of such models critically and systematically.
Assuntos
Células/metabolismo , Simulação por Computador , Divisão Celular , Relógios Circadianos/genética , Difusão , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Fosforilação , Ligação Proteica , Processos Estocásticos , Fatores de TempoRESUMO
The actin cytoskeleton is intimately involved in the motile behaviour of animal cells. The structure and dynamic behaviour of actin and its binding proteins have been intensively studied in vitro over the past several decades, culminating in achievements such as an atomic model of the actin filament. Despite this progress, it is not yet clear how the behaviour of these purified proteins in vitro relates to the dynamic behaviour of actin inside living, moving cells. Here we discuss a new model that relates the known dynamic parameters for pure actin to the observed behaviour of actin filaments inside motile cells.
RESUMO
We have investigated the dynamic behavior of actin in fibroblast lamellipodia using photoactivation of fluorescence. Activated regions of caged resorufin (CR)-labeled actin in lamellipodia of IMR 90 and MC7 3T3 fibroblasts were observed to move centripetally over time. Thus in these cells, actin filaments move centripetally relative to the substrate. Rates were characteristic for each cell type; 0.66 +/- 0.27 microns/min in IMR 90 and 0.36 +/- 0.16 microns/min in MC7 3T3 cells. In neither case was there any correlation between the rate of actin movement and the rate of lamellipodial protrusion. The half-life of the activated CR-actin filaments was approximately 1 min in IMR 90 lamellipodia, and approximately 3 min in MC7 3T3 lamellipodia. Thus continuous filament turnover accompanies centripetal movement. In both cell types, the length of time required for a section of the actin meshwork to traverse the lamellipodium was several times longer than the filament half-life. The dynamic behavior of the dorsal surface of the cell was also observed by tracking lectin-coated beads on the surface and phase-dense features within lamellipodia of MC7 3T3 cells. The movement of these dorsal features occurred at rates approximately three times faster than the rate of movement of the underlying bulk actin cytoskeleton, even when measured in the same individual cells. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton.
Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Células 3T3 , Actinas/ultraestrutura , Animais , Transporte Biológico , Fibroblastos/ultraestrutura , Fluorescência , Substâncias Macromoleculares , Camundongos , Oxazinas , Pseudópodes/metabolismoRESUMO
The ActA protein is responsible for the actin-based movement of Listeria monocytogenes in the cytosol of eukaryotic cells. Analysis of mutants in which we varied the number of proline-rich repeats (PRR; consensus sequence DFPPPPTDEEL) revealed a linear relationship between the number of PRRs and the rate of movement, with each repeat contributing approximately 2-3 microns/min. Mutants lacking all functional PRRs (generated by deletion or point mutation) moved at rates 30% of wild-type. Indirect immunofluorescence indicated that the PRRs were directly responsible for binding of vasodilator-stimulated phosphoprotein (VASP) and for the localization of profilin at the bacterial surface. The long repeats, which are interdigitated between the PRRs, increased the frequency with which actin-based motility occurred by a mechanism independent of the PRRs, VASP, and profilin. Lastly, a mutant which expressed low levels of ActA exhibited a phenotype indicative of a threshold; there was a very low percentage of moving bacteria, but when movement did occur, it was at wild-type rates. These results indicate that the ActA protein directs at least three separable events: (1) initiation of actin polymerization that is independent of the repeat region; (2) initiation of movement dependent on the long repeats and the amount of ActA; and (3) movement rate dependent on the PRRs.
Assuntos
Proteínas de Bactérias/genética , Moléculas de Adesão Celular/análise , Proteínas Contráteis/análise , Listeria monocytogenes/fisiologia , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/análise , Fosfoproteínas/análise , Sequências Repetitivas de Ácido Nucleico/genética , Actinas/análise , Actinas/biossíntese , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/fisiologia , Linhagem Celular , DNA Bacteriano/genética , Humanos , Dose Letal Mediana , Listeria monocytogenes/química , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Polímeros , Profilinas , ProlinaRESUMO
The bacterial pathogen, Listeria monocytogenes, grows in the cytoplasm of host cells and spreads intercellularly using a form of actin-based motility mediated by the bacterial protein ActA. Tightly adherent monolayers of MDCK cells that constitutively express GFP-actin were infected with L. monocytogenes, and intercellular spread of bacteria was observed by video microscopy. The probability of formation of membrane-bound protrusions containing bacteria decreased with host cell monolayer age and the establishment of extensive cell-cell contacts. After their extension into a recipient cell, intercellular membrane-bound protrusions underwent a period of bacterium-dependent fitful movement, followed by their collapse into a vacuole and rapid vacuolar lysis. Actin filaments in protrusions exhibited decreased turnover rates compared with bacterially associated cytoplasmic actin comet tails. Recovery of motility in the recipient cell required 1-2 bacterial generations. This delay may be explained by acid-dependent cleavage of ActA by the bacterial metalloprotease, Mpl. Importantly, we have observed that low levels of endocytosis of neighboring MDCK cell surface fragments occurs in the absence of bacteria, implying that intercellular spread of bacteria may exploit an endogenous process of paracytophagy.
Assuntos
Membrana Celular/metabolismo , Citoplasma/microbiologia , Endocitose , Listeria monocytogenes/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caderinas/metabolismo , Linhagem Celular , Membrana Celular/microbiologia , Tamanho Celular , Citoplasma/metabolismo , Cães , Concentração de Íons de Hidrogênio , Junções Intercelulares/metabolismo , Junções Intercelulares/microbiologia , Cinética , Listeria monocytogenes/enzimologia , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Microscopia de Vídeo , Modelos Biológicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologiaRESUMO
Polymerization of actin filaments is necessary for both protrusion of the leading edge of crawling cells and propulsion of certain intracellular pathogens, and it is sufficient for generating force for bacterial motility in vitro. Motile intracellular pathogens are associated with actin-rich comet tails containing many of the same molecular components present in lamellipodia, and this suggests that these two systems use a similar mechanism for motility. However, available structural evidence suggests that the organization of comet tails differs from that of lamellipodia. Actin filaments in lamellipodia form branched arrays, which are thought to arise by dendritic nucleation mediated by the Arp2/3 complex. In contrast, comet tails have been variously described as consisting of short, randomly oriented filaments, with a higher degree of alignment at the periphery, or as containing long, straight axial filaments with a small number of oblique filaments. Because the assembly of pathogen-associated comet tails has been used as a model system for lamellipodial protrusion, it is important to resolve this apparent discrepancy. Here, using a platinum replica approach, we show that actin filament arrays in comet tails in fact have a dendritic organization with the Arp2/3 complex localizing to Y-junctions as in lamellipodia. Thus, comet tails and lamellipodia appear to share a common dendritic nucleation mechanism for protrusive motility. However, comet tails differ from lamellipodia in that their actin filaments are usually twisted and appear to be under significant torsional stress.
Assuntos
Actinas/ultraestrutura , Dendritos/ultraestrutura , Microscopia Eletrônica , Microscopia de FluorescênciaAssuntos
Citoesqueleto/fisiologia , Animais , Citoesqueleto/química , Citoesqueleto/metabolismo , HumanosRESUMO
Recent advances in optical imaging have dramatically expanded the capabilities of the light microscope and its usefulness in microbiology research. Some of these advances include improved fluorescent probes, better cameras, new techniques such as confocal and deconvolution microscopy, and the use of computers in imaging and image analysis. These new technologies have now been applied to microbiological problems with resounding success.
Assuntos
Microscopia/métodos , Compartimento Celular , Corantes Fluorescentes , Proteínas de Fluorescência Verde , Processamento de Imagem Assistida por Computador , Proteínas Luminescentes , Microscopia/instrumentação , Microscopia Confocal , Microscopia de Fluorescência/métodos , Microscopia de Vídeo/métodosAssuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fatores de Despolimerização de Actina , Animais , Arabidopsis/genética , Citoesqueleto/ultraestrutura , Destrina , Proteínas Fúngicas/metabolismo , Cinética , Listeria monocytogenes/efeitos dos fármacos , Substâncias Macromoleculares , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Oócitos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Xenopus laevisAssuntos
Actinas/fisiologia , Fenômenos Fisiológicos Virais , Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Infecções Bacterianas/fisiopatologia , Fenômenos Fisiológicos Bacterianos , Evolução Biológica , Citoesqueleto/fisiologia , Citoesqueleto/virologia , Humanos , Movimento/fisiologia , Vaccinia virus/patogenicidade , Vaccinia virus/fisiologia , Viroses/fisiopatologia , Viroses/virologia , Vírus/patogenicidadeAssuntos
Actinas/metabolismo , Listeria monocytogenes/fisiologia , Proteínas dos Microfilamentos/isolamento & purificação , Animais , Extratos Celulares , Citoplasma , Listeria monocytogenes/química , Movimento , Polímeros/metabolismo , Poliestirenos/metabolismo , Ligação Proteica , Rodaminas/metabolismo , XenopusRESUMO
Morphology is an important large-scale manifestation of the global organizational and physiological state of cells, and is commonly used as a qualitative or quantitative measure of the outcome of various assays. Here we evaluate several different basic representations of cell shape - binary masks, distance maps and polygonal outlines - and different subsequent encodings of those representations - Fourier and Zernike decompositions, and the principal and independent components analyses - to determine which are best at capturing biologically important shape variation. We find that principal components analysis of two-dimensional shapes represented as outlines provide measures of morphology which are quantitative, biologically meaningful, human interpretable and work well across a range of cell types and parameter settings.
Assuntos
Caulobacter/citologia , Biologia Celular , Células Cultivadas/citologia , Animais , Tamanho CelularRESUMO
The bacterial pathogen Listeria monocytogenes propels itself in the cytoplasm of the infected cells by forming a filamentous comet tail assembled by the polymerization of the cytoskeletal protein actin. Although a great deal is known about the molecular processes that lead to actin-based movement, most macroscale aspects of motion, including the nature of the trajectories traced out by the motile bacteria, are not well understood. Here, we present 2D trajectories of Listeria moving between a glass-slide and coverslip in a Xenopus frog egg extract motility assay. We observe that the bacteria move in a number of fascinating geometrical trajectories, including winding S curves, translating figure eights, small- and large-amplitude sine curves, serpentine shapes, circles, and a variety of spirals. We then develop a dynamic model that provides a unified description of these seemingly unrelated trajectories. A key ingredient of the model is a torque (not included in any microscopic models of which we are aware) that arises from the rotation of the propulsive force about the body axis of the bacterium. We show that a large variety of trajectories with a rich mathematical structure are obtained by varying the rate at which the propulsive force moves about the long axis. The trajectories of bacteria executing both steady and saltatory motion are found to be in excellent agreement with the predictions of our dynamic model. When the constraints that lead to planar motion are removed, our model predicts motion along regular helical trajectories, observed in recent experiments.
Assuntos
Actinas/metabolismo , Listeria monocytogenes/citologia , Animais , Fenômenos Biomecânicos , Bactérias Gram-Negativas , Modelos Biológicos , XenopusRESUMO
The dynamic behavior of pure actin in vitro is more complex than that of most simple polymers, due to the energy input from the irreversible nucleotide hydrolysis associated with polymerization. However, the dynamic behavior of actin is vastly more complicated inside cells, where dozens of different types of actin-binding proteins alter every rate constant for actin polymerization and the chemical environment is inhomogeneous both temporally and spatially. Actin dynamics in cells are tightly regulated, so that rapid filament polymerization can occur in response to external signals or at the front of an active lamellipodium, while rapid depolymerization occurs simultaneously elsewhere in the cell. Although more direct observations of actin dynamics in vivo are accumulating, it is not yet clear how to reconcile the behavior of actin in cells with its well-documented in vitro properties.