RESUMO
The ability of cells to adhere and to exert contractile forces governs their capacity to move within an organism. The cytoskeletal regulators of the Rho GTPase proteins are involved in control of the contractile forces of cells. To elucidate the basis of cell migration, we analyzed contractile forces and nanoscale adhesion-related particles in single cells expressing constitutively active variants of Rho GTPases by using traction-force microscopy and ultra-high-resolution stimulated emission depletion microscopy, respectively. RhoAV14 induced large increases in the contractile forces of single cells, with Rac1L61 and RhoDV26 having more moderate effects. The RhoAV14- and RhoDV26-induced forces showed similar spatial distributions and were accompanied by reduced or unaltered cell spreading. In contrast, the Rac1L61-induced force had different, scattered, force distributions that were linked to increased cell spreading. All three of these Rho GTPase activities caused a loss of thick stress fibers and focal adhesions and a more homogenous distribution of nanoscale adhesion-related particles over the ventral surface of the cells. Interestingly, only RhoAV14 increased the density of these particles. Our data suggest a Rac1-specific mode for cells to generate contractile forces. Importantly, increased density and a more homogenous distribution of these small adhesion-related particles promote cellular contractile forces.
Assuntos
Adesão Celular/fisiologia , Movimento Celular/fisiologia , Fenômenos Fisiológicos Celulares/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Adesões Focais/metabolismo , Camundongos , Microscopia , Células NIH 3T3 , Suínos , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
Membrane fission is an essential process in membrane trafficking and other cellular functions. While many fissioning and trafficking steps are mediated by the large GTPase dynamin, some fission events are dynamin independent and involve C-terminal-binding protein-1/brefeldinA-ADP ribosylated substrate (CtBP1/BARS). To gain an insight into the molecular mechanisms of CtBP1/BARS in fission, we have studied the role of this protein in macropinocytosis, a dynamin-independent endocytic pathway that can be synchronously activated by growth factors. Here, we show that upon activation of the epidermal growth factor receptor, CtBP1/BARS is (a) translocated to the macropinocytic cup and its surrounding membrane, (b) required for the fission of the macropinocytic cup and (c) phosphorylated on a specific serine that is a substrate for p21-activated kinase, with this phosphorylation being essential for the fission of the macropinocytic cup. Importantly, we also show that CtBP1/BARS is required for macropinocytic internalization and infection of echovirus 1. These results provide an insight into the molecular mechanisms of CtBP1/BARS activation in membrane fissioning, and extend the relevance of CtBP1/BARS-induced fission to human viral infection.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Ligação a DNA/metabolismo , Pinocitose , Quinases Ativadas por p21/metabolismo , Actinas/metabolismo , Oxirredutases do Álcool/ultraestrutura , Linhagem Celular Tumoral , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Enterovirus Humano B/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Humanos , Integrina alfa2beta1/metabolismo , Fosforilação/efeitos dos fármacos , Pinocitose/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Quinases Ativadas por p21/químicaRESUMO
Two problems have hampered the use of light microscopy for structural studies of cellular organelles for a long time: the limited resolution and the difficulty of obtaining true structural boundaries from complex intensity curves. The advent of modern high-resolution light microscopy techniques and their combination with objective image segmentation now provide us with the means to bridge the gap between light and electronmicroscopy in cell biology applications. In this study, we provide the first comparative correlative analysis of three-dimensional structures obtained by 4Pi microscopy and segmented by a zero-crossing procedure with those of transmission electron microscopy (TEM). The distribution within the cisternae of isolated Golgi stacks of the cargo protein procollagen 3 was mapped by both 4Pi microscopy and TEM for a detailed comparative analysis of their imaging capabilities. A high correlation was seen for the structures, indicating the particular accuracy of the 4Pi microscopy. Furthermore, for the first time, transport of a cargo molecule (vesicular stomatitis virus G protein-pEGFP) through individual Golgi stacks (labeled by galactosyl transferase-venus-YFP) was visualized by 4Pi microscopy. Following the procedures validated by the correlative analysis, our transport experiments show that (i) VSVG-pEGFP rapidly enter/exit individual Golgi stacks, (ii) VSVG-pEGFP never fills the GalT-venusYFP compartments completely and (iii) the GalT-venusYFP compartment volume increases upon VSVG-pEGFP arrival. This morphological evidence supports some previous TEM-based observations of intra-Golgi transport of VSVG-pEGFP and provides new insights toward a better understanding of protein progression across Golgi stacks. Our study thus demonstrates the general applicability of super resolution fluorescence microscopy, coupled with the zero-crossing segmentation procedure, for structural studies of suborganelle protein distributions under living cell conditions.
Assuntos
Complexo de Golgi , Microscopia Confocal/métodos , Microscopia Eletrônica/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
The mechanisms of secretory transport through the Golgi apparatus remain an issue of debate. The precise functional importance of calcium ions (Ca(2+)) for intra-Golgi transport has also been poorly studied. Here, using different approaches to measure free Ca(2+) concentrations in the cell cytosol ([Ca(2+)](cyt)) and inside the lumen of the Golgi apparatus ([Ca(2+)](GA)), we have revealed transient increases in [Ca(2+)](cyt) during the late phase of intra-Golgi transport that are concomitant with a decline in the maximal [Ca(2+)](GA) restoration ability. Thus, this redistribution of Ca(2+) from the Golgi apparatus into the cytosol during the movement of cargo through the Golgi apparatus appears to have a role in intra-Golgi transport, and mainly in the late Ca(2+)-dependent phase of SNARE-regulated fusion of Golgi compartments.
Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Complexo de Golgi/metabolismo , Transporte Biológico , Sinalização do Cálcio , Células Cultivadas , Fibroblastos/metabolismo , Células HeLa , Humanos , Pele/citologia , Pele/metabolismo , Frações SubcelularesRESUMO
The association reaction between pairs of proteins proceeds through an encounter complex that develops into the final complex. Here, we combined Brownian dynamics simulations with experimental studies to analyze the structures of the encounter complexes along the association reaction between TEM1-beta-lactamase and its inhibitor, beta-lactamase-inhibitor protein. The encounter complex can be considered as an ensemble of short-lived low free-energy states that are stabilized primarily by electrostatic forces and desolvation. For the wild-type, the simulation showed two main encounter regions located outside the physical binding site. One of these regions was located near the experimentally determined transition state. To validate whether these encounters are fruitful or futile, we examined three groups of mutations that altered the encounter. The first group consisted of mutations that increased the experimental rate of association through electrostatic optimization. This resulted in an increase in the size of the encounter region located near the experimentally determined transition state, as well as a decrease in the energy of this region and an increase in the number of successful trajectories (i.e., encounters that develop into complex). A second group of mutations was specifically designed to either increase or decrease the size and energy of the second encounter complex, but either way it did not affect k(on). A third group of mutations consisted of residues that increased k(on) without significantly affecting the encounter complexes. These results indicate that the size and energy of the encounter regions are only two of several parameters that lead to fruitful association, and that electrostatic optimization is a major driving force in fast association.
Assuntos
Inibidores Enzimáticos/metabolismo , Modelos Moleculares , beta-Lactamases/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Cinética , Mutação , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Termodinâmica , Inibidores de beta-Lactamases , beta-Lactamases/química , beta-Lactamases/genéticaRESUMO
Electron transfer between the water-soluble cytochrome c and the integral membrane protein cytochrome c oxidase (COX) is the terminal reaction in the respiratory chain. The first step in this reaction is the diffusional association of cytochrome c toward COX, and it is still not completely clear whether cytochrome c diffuses in the bulk solution while encountering COX, or whether it prefers to diffuse laterally on the membrane surface. This is a rather crucial question, since in the latter case the association would be strongly dependent on the lipid composition and the presence of additional membrane proteins. We applied Brownian dynamics simulations to investigate the effect of an atomistically modeled dipalmitoyl phosphatidylcholine membrane on the association behavior of cytochrome c toward COX from Paracoccus denitrificans. We studied the negatively charged, physiological electron-transfer partner of COX, cytochrome c(552), and the positively charged horse-heart cytochrome c. As expected, both cytochrome c species prefer diffusion in bulk solution while associating toward COX embedded in a membrane, where the partial charges of the lipids were switched off, and the corresponding optimal association pathways largely overlap with the association toward fully solvated COX. Remarkably, after switching on the lipid partial charges, both cytochrome c species were strongly attracted by the inhomogeneous charge distribution caused by the zwitterionic lipid headgroups. This effect is particularly enhanced for horse-heart cytochrome c and is stronger at lower ionic strength. We therefore conclude that in the presence of a polar or even a charged membrane, cytochrome c diffuses laterally rather than in three dimensions.
Assuntos
Membrana Celular/metabolismo , Grupo dos Citocromos c/metabolismo , Citocromos c/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Algoritmos , Animais , Simulação por Computador , Difusão , Entropia , Cavalos , Membranas Artificiais , Modelos Químicos , Oxirredução , Paracoccus denitrificans/enzimologia , Conformação Proteica , Eletricidade EstáticaRESUMO
The conformation and correlations of amphiphilic and antimicrobial peptides and the associated changes of lipid bilayers can be studied in oriented lipid membranes deposited on solid substrates. Here we review recent work on these systems, as studied by modern interface-sensitive X-ray and neutron scattering methods. Density profile, short range order of acyl chains and molecular conformations of peptides and lipids are probed in the fluid state of the bilayer. With an emphasis on technical aspects, we review recent work illustrating the potential of the methods and discuss its potential in the field.
Assuntos
Lipídeos/química , Peptídeos/química , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Espalhamento de Radiação , Raios XRESUMO
We present a structural study of biomimetic lipid bilayers interacting with the antimicrobial peptide magainin 2 amide, using grazing incidence X-ray diffraction and reciprocal space mapping (RSM) techniques. The short-range order of lipid chains in lecithin is found to be strongly reduced by the peptides. From the scattering intensity of the chain correlation peak, we can quantify the lateral length scale R over which the bilayer structure is affected by peptide binding. The non-local perturbation of the bilayer is discussed in the framework of bilayer elasticity theory.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Proteínas de Xenopus , Sequência de Aminoácidos , Magaininas , Dados de Sequência Molecular , Conformação Proteica , Difração de Raios X/métodosRESUMO
The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression-maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes.
Assuntos
Albuminas/metabolismo , Complexo de Golgi/metabolismo , alfa 1-Antitripsina/metabolismo , Transporte Biológico , Simulação por Computador , Difusão , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Células Hep G2 , Humanos , Luz , Microscopia Confocal , Microscopia Imunoeletrônica , Microscopia de Vídeo , Transporte ProteicoRESUMO
Newly synthesized proteins exit the endoplasmic reticulum (ER) via coat protein complex II (COPII) vesicles. Procollagen (PC), however, forms prefibrils that are too large to fit into typical COPII vesicles; PC thus needs large transport carriers, which we term megacarriers. TANGO1 assists PC packing, but its role in promoting the growth of megacarriers is not known. We found that TANGO1 recruited Sedlin, a TRAPP component that is defective in spondyloepiphyseal dysplasia tarda (SEDT), and that Sedlin was required for the ER export of PC. Sedlin bound and promoted efficient cycling of Sar1, a guanosine triphosphatase that can constrict membranes, and thus allowed nascent carriers to grow and incorporate PC prefibrils. This joint action of TANGO1 and Sedlin sustained the ER export of PC, and its derangement may explain the defective chondrogenesis underlying SEDT.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Pró-Colágeno/metabolismo , Fatores de Transcrição/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Linhagem Celular , Condrogênese/genética , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Mutação , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Transporte Proteico , Fatores de Transcrição/genéticaRESUMO
We present an analysis of trajectories from Brownian dynamics simulations of diffusional protein-protein encounter for the well-studied system of barnase and barstar. This analysis reveals details about the optimal association pathways, the regions of the encounter complex, possible differences of the pathways for dissociation and association, the coupling of translational and rotation motion, and the effect of mutations on the trajectories. We found that a small free-energy barrier divides the energetically most favorable region into a region of the encounter complex above the barnase binding interface and a region around a second energy minimum near the RNA binding loop. When entering the region of the encounter complex from the region near the RNA binding loop, barstar has to change its orientation to increase the electrostatic attraction between the proteins. By concentrating the analysis on the successful binding trajectories, we found that the region of the second minimum is not essential for the binding of barstar to barnase. Nevertheless, this region may be helpful to steer barstar into the region of the encounter complex. When applying the same analysis to several barnase mutants, we found that single mutations may drastically change the free-energy landscape and may significantly alter the population of the two minima. Therefore, certain protein-protein pairs may require careful adaptation of the positions of encounter and transition states when interpreting mutation effects on kinetic rates of association and/or dissociation.
Assuntos
Proteínas de Bactérias/química , Modelos Químicos , Modelos Moleculares , Mapeamento de Interação de Proteínas/métodos , Ribonucleases/química , Sítios de Ligação , Simulação por Computador , Difusão , Ativação Enzimática , Inibidores Enzimáticos/química , Modelos Estatísticos , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Over the past years Brownian dynamics (BD) simulations have been proven to be a suitable tool for the analysis of protein-protein association. The computed rates and relative trends for protein mutants and different ionic strength are generally in good agreement with experimental results, e.g. see ref 1. By design, BD simulations correspond to an intensive sampling over energetically favorable states, rather than to a systematic sampling over all possible states which is feasible only at rather low resolution. On the example of barnase and barstar, a well characterized model system of electrostatically steered diffusional encounter, we report here the computation of the 6-dimensional free energy landscape for the encounter process of two proteins by a novel, careful analysis of the trajectories from BD simulations. The aim of these studies was the clarification of the encounter state. Along the trajectories, the individual positions and orientations of one protein (relative to the other) are recorded and stored in so-called occupancy maps. Since the number of simulated trajectories is sufficiently high, these occupancy maps can be interpreted as a probability distribution which allows the calculation of the entropy landscape by the use of a locally defined entropy function. Additionally, the configuration dependent electrostatic and desolvation energies are recorded in separate maps. The free energy landscape of protein-protein encounter is finally obtained by summing the energy and entropy contributions. In the free energy profile along the reaction path, which is defined as the path along the minima in the free energy landscape, a minimum shows up suggesting this to be used as the definition of the encounter state. This minimum describes a state of reduced diffusion velocity where the electrostatic attraction is compensated by the repulsion due to the unfavorable desolvation of the charged residues and the entropy loss due to the increasing restriction of the motional freedom. In the simulations the orientational degrees of freedom at the encounter state are found to be less restricted than the translational degrees of freedom. Therefore, the orientational alignment of the two binding partners seems to take place beyond this free energy minimum. The free energy profiles along the reaction pathway are compared for different ionic strength and temperature. This novel analysis technique facilitates mechanistic interpretation of protein-protein encounter pathways which should be useful for interpretation of experimental results as well.
RESUMO
We present a study of the short range ordering of hydrocarbon chains in phospholipid bilayers. The x-ray peak associated with the hydrocarbon chains has been probed by means of reciprocal space mappings. Using 20 keV undulator radiation and samples of negligible mosaicity (orientational disorder), the intensity distribution is probed as a function of two coordinates, the momentum transfer parallel and perpendicular to the bilayer, over a wide range and at high resolution. Structural results are obtained concerning the distribution of tilted segments, the correlation length and the radial distribution function of the quasi two-dimensional liquid structure. A comparison is made with published molecular dynamics data (H. Heller, M. Schaefer, and K. Schulten. 1993. J. Phys. Chem. 97:8343-8360) by direct Fourier transformation of the atomic coordinates. The exact prefactor in the relationship between interchain distance and peak position is derived.
Assuntos
Hidrocarbonetos/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Difração de Raios X/métodos , Fenômenos Biofísicos , Biofísica , Dimiristoilfosfatidilcolina/química , Membranas Artificiais , Modelos Teóricos , Fosfatidilcolinas/química , Éteres Fosfolipídicos/química , Espalhamento de Radiação , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Although the antimicrobial, fungal peptide alamethicin has been extensively studied, the conformation of the peptide and the interaction with lipid bilayers as well as the mechanism of channel gating are still not completely clear. As opposed to studies of the crystalline state, the polypeptide structures in the environment of fluid bilayers are difficult to probe. We have investigated the conformation of alamethicin in highly aligned stacks of model lipid membranes by synchrotron-based x-ray scattering. The (wide-angle) scattering distribution has been measured by reciprocal space mappings. A pronounced scattering signal is observed in samples of high molar peptide/lipid ratio which is distinctly different from the scattering distribution of an ideal helix in the transmembrane state. Beyond simple models of ideal helices, the data is analyzed in terms of models based on atomic coordinates from the Brookhaven Protein Data Bank, as well as from published molecular dynamics simulations. The results can be explained by assuming a wide distribution of helix tilt angles with respect to the membrane normal and a partial insertion of the N-terminus into the membrane.