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1.
Proc Natl Acad Sci U S A ; 118(47)2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34789575

RESUMO

Here, we present detailed kinetic analyses of a panel of soluble lipid kinases and phosphatases, as well as Ras activating proteins, acting on their respective membrane surface substrates. The results reveal that the mean catalytic rate of such interfacial enzymes can exhibit a strong dependence on the size of the reaction system-in this case membrane area. Experimental measurements and kinetic modeling reveal how stochastic effects stemming from low molecular copy numbers of the enzymes alter reaction kinetics based on mechanistic characteristics of the enzyme, such as positive feedback. For the competitive enzymatic cycles studied here, the final product-consisting of a specific lipid composition or Ras activity state-depends on the size of the reaction system. Furthermore, we demonstrate how these reaction size dependencies can be controlled by engineering feedback mechanisms into the enzymes.

2.
Sci Adv ; 7(35)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34452907

RESUMO

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gßγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gßγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gßγ activation. A nanobody that specifically binds to the p101-Gßγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

3.
STAR Protoc ; 2(2): 100486, 2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34041500

RESUMO

MNase-seq (micrococcal nuclease sequencing) is used to map nucleosome positions in eukaryotic genomes to study the relationship between chromatin structure and DNA-dependent processes. Current protocols require at least two days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for S. cerevisiae and other fungi which takes only three hours. Modified protocols were developed for wild fungi and mammalian cells. This method for rapidly producing sequencing-ready nucleosome footprints from several organisms makes MNase-seq faster and easier, with less chemical waste.

4.
Proc Natl Acad Sci U S A ; 116(30): 15013-15022, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31278151

RESUMO

Phosphorylation reactions, driven by competing kinases and phosphatases, are central elements of cellular signal transduction. We reconstituted a native eukaryotic lipid kinase-phosphatase reaction that drives the interconversion of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol-4,5-phosphate [PI(4,5)P2] on membrane surfaces. This system exhibited bistability and formed spatial composition patterns on supported membranes. In smaller confined regions of membrane, rapid diffusion ensures the system remains spatially homogeneous, but the final outcome-a predominantly PI(4)P or PI(4,5)P2 membrane composition-was governed by the size of the reaction environment. In larger confined regions, interplay between the reactions, diffusion, and confinement created a variety of differentially patterned states, including polarization. Experiments and kinetic modeling reveal how these geometric confinement effects arise from a mechanism based on stochastic fluctuations in the copy number of membrane-bound kinases and phosphatases. The underlying requirements for such behavior are unexpectedly simple and likely to occur in natural biological signaling systems.


Assuntos
Proteínas de Bactérias/química , Fatores de Troca do Nucleotídeo Guanina/química , Fosfatidilinositol 4,5-Difosfato/química , Fosfatos de Fosfatidilinositol/química , Monoéster Fosfórico Hidrolases/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Proteínas de Bactérias/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Cinética , Legionella pneumophila/química , Legionella pneumophila/enzimologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipase C delta/química , Fosfolipase C delta/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Imagem Individual de Molécula , Processos Estocásticos , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo
5.
EMBO J ; 37(1): 102-121, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29141912

RESUMO

WASP-family proteins are known to promote assembly of branched actin networks by stimulating the filament-nucleating activity of the Arp2/3 complex. Here, we show that WASP-family proteins also function as polymerases that accelerate elongation of uncapped actin filaments. When clustered on a surface, WASP-family proteins can drive branched actin networks to grow much faster than they could by direct incorporation of soluble monomers. This polymerase activity arises from the coordinated action of two regulatory sequences: (i) a WASP homology 2 (WH2) domain that binds actin, and (ii) a proline-rich sequence that binds profilin-actin complexes. In the absence of profilin, WH2 domains are sufficient to accelerate filament elongation, but in the presence of profilin, proline-rich sequences are required to support polymerase activity by (i) bringing polymerization-competent actin monomers in proximity to growing filament ends, and (ii) promoting shuttling of actin monomers from profilin-actin complexes onto nearby WH2 domains. Unoccupied WH2 domains transiently associate with free filament ends, preventing their growth and dynamically tethering the branched actin network to the WASP-family proteins that create it. Collaboration between WH2 and proline-rich sequences thus strikes a balance between filament growth and tethering. Our work expands the number of critical roles that WASP-family proteins play in the assembly of branched actin networks to at least three: (i) promoting dendritic nucleation; (ii) linking actin networks to membranes; and (iii) accelerating filament elongation.


Assuntos
Citoesqueleto de Actina/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Domínios Proteicos Ricos em Prolina , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Humanos , Ligação Proteica
6.
Biomacromolecules ; 18(11): 3706-3713, 2017 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-28934548

RESUMO

The ability of styrene maleic acid copolymers to dissolve lipid membranes into nanosized lipid particles is a facile method of obtaining membrane proteins in solubilized lipid discs while conserving part of their native lipid environment. While the currently used copolymers can readily extract membrane proteins in native nanodiscs, their highly disperse composition is likely to influence the dispersity of the discs as well as the extraction efficiency. In this study, reversible addition-fragmentation chain transfer was used to control the polymer architecture and dispersity of molecular weights with a high-precision. Based on Monte Carlo simulations of the polymerizations, the monomer composition was predicted and allowed a structure-function analysis of the polymer architecture, in relation to their ability to assemble into lipid nanoparticles. We show that a higher degree of control of the polymer architecture generates more homogeneous samples. We hypothesize that low dispersity copolymers, with control of polymer architecture are an ideal framework for the rational design of polymers for customized isolation and characterization of integral membrane proteins in native lipid bilayer systems.


Assuntos
Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Proteínas de Membrana/química , Polímeros/química , Maleatos/química , Peso Molecular , Nanopartículas/química , Polimerização , Estireno/química
7.
J Bacteriol ; 199(19)2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28716960

RESUMO

Bacteria of the genus Prosthecobacter express homologs of eukaryotic α- and ß-tubulin, called BtubA and BtubB (BtubA/B), that have been observed to assemble into filaments in the presence of GTP. BtubA/B polymers are proposed to be composed in vitro by two to six protofilaments in contrast to that in vivo, where they have been reported to form 5-protofilament tubes named bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may be derived from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is reversible and that BtubA/B folding does not require chaperones. To better understand BtubA/B filament behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro BtubA/B assembly using a combination of polymerization kinetics assays and microscopy. Like eukaryotic microtubules, BtubA/B filaments exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by BtubA/B polymerization drives a stochastic mechanism of filament disassembly that occurs via polymer breakage and/or fast continuous depolymerization. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of BtubA/B filament fragments. Unlike MTs, polymerization of BtubA/B requires KCl, which reduces the critical concentration for BtubA/B assembly and induces it to form stable mixed-orientation bundles in the absence of any additional BtubA/B-binding proteins. The complex dynamics that we observe in stabilized and unstabilized BtubA/B filaments may reflect common properties of an ancestral eukaryotic tubulin polymer.IMPORTANCE Microtubules are polymers within all eukaryotic cells that perform critical functions; they segregate chromosomes, organize intracellular transport, and support the flagella. These functions rely on the remarkable range of tunable dynamic behaviors of microtubules. Bacterial tubulin A and B (BtubA/B) are evolutionarily related proteins that form polymers. They are proposed to be evolved from the ancestral eukaryotic tubulin, a missing link in microtubule evolution. Using microscopy and biochemical approaches to characterize BtubA/B assembly in vitro, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules but differ in how they self-associate into bundles and how this bundling affects their stability. Our results demonstrate the diversity of mechanisms through which tubulin homologs promote filament dynamics and monomer turnover.


Assuntos
Bactérias/metabolismo , Proteínas do Citoesqueleto/fisiologia , Guanosina Trifosfato/metabolismo , Tubulina (Proteína)/fisiologia , Proteínas de Bactérias/fisiologia , Citoesqueleto/fisiologia , Transferência Genética Horizontal , Hidrólise , Cinética , Microscopia , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Moleculares , Polimerização , Tubulina (Proteína)/química
8.
Nat Commun ; 8: 15061, 2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28452363

RESUMO

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.


Assuntos
Modelos Moleculares , Domínios Proteicos , Proteína SOS1/química , Proteína SOS1/metabolismo , Sítio Alostérico , Sítios de Ligação , Membrana Celular/metabolismo , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência , Ligação Proteica , Proteínas ras/química , Proteínas ras/metabolismo
9.
Proc Natl Acad Sci U S A ; 113(29): 8218-23, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27370798

RESUMO

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Fosfotirosina/metabolismo , Proteínas Son Of Sevenless/metabolismo , Proteína Adaptadora GRB2/metabolismo , Cinética , Membranas Artificiais , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteínas ras
10.
Elife ; 42015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26295568

RESUMO

Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Multimerização Proteica , Humanos , Ligação Proteica
11.
Science ; 345(6192): 50-4, 2014 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-24994643

RESUMO

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average.


Assuntos
Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/agonistas , Proteína Son Of Sevenless de Drosófila/química , Regulação Alostérica , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Humanos , Cinética , Nucleotídeos/química , Proteína Son Of Sevenless de Drosófila/genética
12.
J Biol Chem ; 289(19): 13589-601, 2014 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-24692547

RESUMO

The classical cadherin·ß-catenin·α-catenin complex mediates homophilic cell-cell adhesion and mechanically couples the actin cytoskeletons of adjacent cells. Although α-catenin binds to ß-catenin and to F-actin, ß-catenin significantly weakens the affinity of α-catenin for F-actin. Moreover, α-catenin self-associates into homodimers that block ß-catenin binding. We investigated quantitatively and structurally αE- and αN-catenin dimer formation, their interaction with ß-catenin and the cadherin·ß-catenin complex, and the effect of the α-catenin actin-binding domain on ß-catenin association. The two α-catenin variants differ in their self-association properties: at physiological temperatures, αE-catenin homodimerizes 10× more weakly than does αN-catenin but is kinetically trapped in its oligomeric state. Both αE- and αN-catenin bind to ß-catenin with a Kd of 20 nM, and this affinity is increased by an order of magnitude when cadherin is bound to ß-catenin. We describe the crystal structure of a complex representing the full ß-catenin·αN-catenin interface. A three-dimensional model of the cadherin·ß-catenin·α-catenin complex based on these new structural data suggests mechanisms for the enhanced stability of the ternary complex. The C-terminal actin-binding domain of α-catenin has no influence on the interactions with ß-catenin, arguing against models in which ß-catenin weakens actin binding by stabilizing inhibitory intramolecular interactions between the actin-binding domain and the rest of α-catenin.


Assuntos
Caderinas/química , Complexos Multiproteicos/química , alfa Catenina/química , beta Catenina/química , Animais , Cristalografia por Raios X , Camundongos , Estrutura Quaternária de Proteína , Termodinâmica
13.
Proc Natl Acad Sci U S A ; 111(8): 2996-3001, 2014 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-24516166

RESUMO

The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein-protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization Kd is measured to be ∼1 × 10(3) molecules/µm(2), and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.


Assuntos
Membrana Celular/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas ras/química , Sequência de Aminoácidos , Dimerização , Polarização de Fluorescência , Humanos , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas ras/metabolismo
14.
Mol Biol Cell ; 24(23): 3710-20, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24068324

RESUMO

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell-cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell-cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , alfa Catenina/química , alfa Catenina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Fatores de Despolimerização de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Microscopia Crioeletrônica , Cães , Processamento de Imagem Assistida por Computador , Células Madin Darby de Rim Canino , Camundongos , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
15.
Methods Mol Biol ; 1046: 145-70, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23868587

RESUMO

The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility, cytokinesis, and endocytosis. Actin polymerization is tightly regulated by its numerous cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. Here we describe how to use the pyrene actin assembly assay to determine the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleation or capping factors. Secondly, we show how fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how proteins regulate actin filament structure. Finally, we describe a method for visualizing dynamic assembly and disassembly of single actin filaments and fluorescently labeled actin binding proteins using total internal reflection fluorescence (TIRF) microscopy.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Citoplasma/metabolismo , Biologia Molecular/métodos , Pirenos/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Movimento Celular , Citocinese/genética , Endocitose/genética , Humanos , Cinética , Microscopia de Fluorescência , Faloidina/química , Polimerização
16.
Curr Opin Cell Biol ; 25(1): 6-13, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23267766

RESUMO

Now that many genomes have been sequenced, a central concern of cell biology is to understand how the proteins they encode work together to create living matter. In vitro studies form an essential part of this program because understanding cellular functions of biological molecules often requires isolating them and reconstituting their activities. In particular, many elements of the actin cytoskeleton were first discovered by biochemical methods and their cellular functions deduced from in vitro experiments. We highlight recent advances that have come from in vitro studies, beginning with studies of actin filaments, and ending with multi-component reconstitutions of complex actin-based processes, including force-generation and cell spreading. We describe both scientific results and the technical innovations that made them possible.


Assuntos
Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/metabolismo , Materiais Biomiméticos , Forma Celular , Movimento
17.
Cancer Cell ; 22(5): 615-30, 2012 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-23153535

RESUMO

Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.


Assuntos
Citoesqueleto de Actina/metabolismo , Movimento Celular , Neoplasias/patologia , Profilinas/fisiologia , Citoesqueleto de Actina/ultraestrutura , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/ultraestrutura , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Miosinas/metabolismo , Miosinas/fisiologia , Gradação de Tumores , Invasividade Neoplásica/genética , Neoplasias/genética , Neoplasias/metabolismo , Profilinas/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Interferência de RNA
18.
J Cell Biol ; 193(2): 285-94, 2011 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-21482719

RESUMO

During mitosis, chromosome passenger complexes (CPCs) exhibit a well-conserved association with the anaphase spindle and have been implicated in spindle stability. However, their precise effect on the spindle is not clear. In this paper, we show, in budding yeast, that a CPC consisting of CBF3, Bir1, and Sli15, but not Ipl1, is required for normal spindle elongation. CPC mutants slow spindle elongation through the action of the bipolar kinesins Cin8 and Kip1. The same CPC mutants that slow spindle elongation also result in the enrichment of Cin8 and Kip1 at the spindle midzone. Together, these findings argue that CPCs function to organize the spindle midzone and potentially switch motors between force generators and molecular brakes. We also find that slowing spindle elongation delays the mitotic exit network (MEN)-dependent release of Cdc14, thus delaying spindle breakdown until a minimal spindle size is reached. We propose that these CPC- and MEN-dependent mechanisms are important for coordinating chromosome segregation with spindle breakdown and mitotic exit.


Assuntos
Anáfase , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fuso Acromático , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Motores Moleculares/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
J Cell Biol ; 191(3): 571-84, 2010 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21041447

RESUMO

Ena/VASP proteins regulate the actin cytoskeleton during cell migration and morphogenesis and promote assembly of both filopodial and lamellipodial actin networks. To understand the molecular mechanisms underlying their cellular functions we used total internal reflection fluorescence microscopy to visualize VASP tetramers interacting with static and growing actin filaments in vitro. We observed multiple filament binding modes: (1) static side binding, (2) side binding with one-dimensional diffusion, and (3) processive barbed end tracking. Actin monomers antagonize side binding but promote high affinity (K(d) = 9 nM) barbed end attachment. In low ionic strength buffers, VASP tetramers are weakly processive (K(off) = 0.69 s(-1)) polymerases that deliver multiple actin monomers per barbed end-binding event and effectively antagonize filament capping. In higher ionic strength buffers, VASP requires profilin for effective polymerase and anti-capping activity. Based on our observations, we propose a mechanism that accounts for all three binding modes and provides a model for how VASP promotes actin filament assembly.


Assuntos
Actinas/química , Actinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Sítios de Ligação , Biopolímeros/biossíntese , Biopolímeros/química , Biopolímeros/metabolismo , Humanos , Profilinas/metabolismo
20.
J Cell Biol ; 171(5): 773-84, 2005 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-16330709

RESUMO

In budding yeast, the kinetochore scaffold complex centromere binding factor 3 (CBF3) is required to form kinetochores on centromere DNA and to allow proper chromosome segregation. We have previously shown that SKP1 and SGT1 balance the assembly and turnover of CBF3 complexes, a cycle that we suggest is independent of its role in chromosome segregation (Rodrigo-Brenni, M.C., S. Thomas, D.C. Bouck, and K.B. Kaplan. 2004. Mol. Biol. Cell. 15:3366-3378). We provide evidence that this cycle contributes to a second, kinetochore-independent function of CBF3. In this study, we show that inhibiting the assembly of CBF3 causes disorganized septins and defects in cell polarity that give rise to cytokinesis failures. Specifically, we show that septin ring separation and disassembly is delayed in anaphase, suggesting that CBF3 regulates septin dynamics. Only mutations that affect the CBF3 cycle, and not mutants in outer kinetochore subunits, cause defects in septins. These results demonstrate a novel role for CBF3 in regulating cytokinesis, a role that is reminiscent of passenger proteins. Consistent with this possibility, we find that CBF3 interacts with Bir1p, the homologue of the passenger protein Survivin. Mutants in Bir1p similarly affect septin organization, leading us to propose that CBF3 and Bir1p act as passenger proteins to coordinate chromosome segregation with cytokinesis.


Assuntos
Polaridade Celular/fisiologia , Citocinese/fisiologia , Proteínas do Citoesqueleto/fisiologia , Cinetocoros/fisiologia , Proteínas Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Anáfase/genética , Anáfase/fisiologia , Polaridade Celular/genética , Segregação de Cromossomos/genética , Segregação de Cromossomos/fisiologia , Citocinese/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mutação , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
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