scholarly journals Corequirement of Specific Phosphoinositides and Small GTP-binding Protein Cdc42 in Inducing Actin Assembly in Xenopus Egg Extracts

1998 ◽  
Vol 140 (5) ◽  
pp. 1125-1136 ◽  
Author(s):  
Le Ma ◽  
Lewis C. Cantley ◽  
Paul A. Janmey ◽  
Marc W. Kirschner
Keyword(s):  
G Proteins ◽  
Membrane Vesicles ◽  
Lipid Vesicles ◽  
Actin Assembly ◽  
Free System ◽  
Gtp Binding ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Egg Extracts ◽  
Rho Family ◽  
Xenopus Egg Extracts

Both phosphoinositides and small GTP-binding proteins of the Rho family have been postulated to regulate actin assembly in cells. We have reconstituted actin assembly in response to these signals in Xenopus extracts and examined the relationship of these pathways. We have found that GTPγS stimulates actin assembly in the presence of endogenous membrane vesicles in low speed extracts. These membrane vesicles are required, but can be replaced by lipid vesicles prepared from purified phospholipids containing phosphoinositides. Vesicles containing phosphatidylinositol (4,5) bisphosphate or phosphatidylinositol (3,4,5) trisphosphate can induce actin assembly even in the absence of GTPγS. RhoGDI, a guanine-nucleotide dissociation inhibitor for the Rho family, inhibits phosphoinositide-induced actin assembly, suggesting the involvement of the Rho family small G proteins. Using various dominant mutants of these G proteins, we demonstrate the requirement of Cdc42 for phosphoinositide-induced actin assembly. Our results suggest that phosphoinositides may act to facilitate GTP exchange on Cdc42, as well as to anchor Cdc42 and actin nucleation activities. Hence, both phosphoinositides and Cdc42 are required to induce actin assembly in this cell-free system.

scholarly journals Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts.

1992 ◽  
Vol 118 (5) ◽  
pp. 1097-1108 ◽  
Author(s):  
F Verde ◽  
M Dogterom ◽  
E Stelzer ◽  
E Karsenti ◽  
S Leibler
Keyword(s):  
Steady State ◽  
Fold Increase ◽  
Cyclin A ◽  
Microtubule Dynamics ◽  
Cyclin B ◽  
Free System ◽  
Egg Extracts ◽  
Dynamical Parameters ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

In eukaryotic cells, the onset of mitosis involves cyclin molecules which interact with proteins of the cdc2 family to produce active kinases. In vertebrate cells, cyclin A dependent kinases become active in S- and pro-phases, whereas a cyclin B-dependent kinase is mostly active in metaphase. It has recently been shown that, when added to Xenopus egg extracts, bacterially produced A- and B-type cyclins associate predominantly with the same kinase catalytic subunit, namely p34cdc2, and induce its histone H1 kinase activity with different kinetics. Here, we show that in the same cell free system, both the addition of cyclin A and cyclin B changes microtubule behavior. However, the cyclin A-dependent kinase does not induce a dramatic shortening of centrosome-nucleated microtubules whereas the cyclin B-dependent kinase does, as previously reported. Analysis of the parameters of microtubule dynamics by fluorescence video microscopy shows that the dramatic shortening induced by the cyclin B-dependent kinase is correlated with a several fold increase in catastrophe frequency, an effect not observed with the cyclin A-dependent kinase. Using a simple mathematical model, we show how the length distributions of centrosome-nucleated microtubules relate to the four parameters that describe microtubule dynamics. These four parameters define a threshold between unlimited microtubule growth and the establishment of steady-state dynamics, which implies that well defined steady-state length distributions can be produced by regulating precisely the respective values of the dynamical parameters. Moreover, the dynamical model predicts that increasing catastrophe frequency is more efficient than decreasing the rescue frequency to reduce the average steady state length of microtubules. These theoretical results are quantitatively confirmed by the experimental data.

scholarly journals Chemotactic Signaling Pathways in Neutrophils: from Receptor to Actin Assembly

2002 ◽  
Vol 13 (3) ◽  
pp. 220-228 ◽  
Author(s):  
Gregor Cicchetti ◽  
Philip G. Allen ◽  
Michael Glogauer
Keyword(s):  
G Proteins ◽  
Neutrophil Function ◽  
Small Gtpases ◽  
Regulatory Proteins ◽  
Heterotrimeric G Proteins ◽  
Actin Assembly ◽  
Receptor Ligand ◽  
Downstream Effectors ◽  
Ligand Interactions ◽  
Rho Family

In this review, we present an overview of the signaling elements between neutrophil chemotactic receptors and the actin cytoskeleton that drives cell motility. From receptor-ligand interactions, activation of heterotrimeric G-proteins, their downstream effectors PLC and PI-3 kinase, the activation of small GTPases of the Rho family, and their regulation of particular cytoskeletal regulatory proteins, we describe pathways specific to the chemotaxing neutrophil and elements documented to be important for neutrophil function.

scholarly journals Clustering of Nck by a 12-residue Tir phosphopeptide is sufficient to trigger localized actin assembly

2004 ◽  
Vol 164 (3) ◽  
pp. 407-416 ◽  
Author(s):  
Kenneth G. Campellone ◽  
Susannah Rankin ◽  
Tony Pawson ◽  
Marc W. Kirschner ◽  
Donald J. Tipper ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Adaptor Protein ◽  
Surface Expression ◽  
Effector Proteins ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Bacterial Surface ◽  
Egg Extracts ◽  
Xenopus Egg Extracts ◽  
Pedestal Formation

Enteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to promote reorganization of the cytoskeleton into filamentous actin pedestals. One effector, Tir, is a transmembrane receptor for the bacterial surface adhesin intimin, and intimin binding by the extracellular domain of Tir is required for actin assembly. The cytoplasmic NH2 terminus of Tir interacts with focal adhesion proteins, and its tyrosine-phosphorylated COOH terminus binds Nck, a host adaptor protein critical for pedestal formation. To define the minimal requirements for EPEC-mediated actin assembly, Tir derivatives were expressed in mammalian cells in the absence of all other EPEC components. Replacement of the NH2 terminus of Tir with a viral membrane-targeting sequence promoted efficient surface expression of a COOH-terminal Tir fragment. Artificial clustering of this fusion protein revealed that the COOH terminus of Tir, by itself, is sufficient to initiate a complete signaling cascade leading to pedestal formation. Consistent with this finding, clustering of Nck by a 12-residue Tir phosphopeptide triggered actin tail formation in Xenopus egg extracts.

scholarly journals Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components.

1992 ◽  
Vol 116 (2) ◽  
pp. 295-306 ◽  
Author(s):  
J Newport ◽  
W Dunphy
Keyword(s):  
Nuclear Envelope ◽  
Molecular Mechanisms ◽  
Membrane Vesicles ◽  
Sperm Chromatin ◽  
Chromosomal Dna ◽  
Free System ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly ◽  
Xenopus Egg

At the end of mitosis membrane vesicles are targeted to the surface of chromatin and fuse to form a continuous nuclear envelope. To investigate the molecular mechanisms underlying these steps in nuclear envelope assembly, we have developed a defined cell-free system in which the binding and fusion steps in nuclear envelope assembly can be examined separately. We have found that extensively boiled Xenopus egg extracts efficiently promote the decondensation of demembranated Xenopus sperm chromatin. When isolated membranes are added to this decondensed chromatin a specific subfraction of membrane vesicles (approximately 70 nM in diameter) bind to the chromatin, but these vesicles do not fuse to each other. Vesicle binding is independent of ATP and insensitive to N-ethylmalamide. Quantitative analysis of these sites by EM suggests that there is at least one vesicle binding site per 100 kb of chromosomal DNA. We show by tryptic digestion that vesicle-chromatin association requires proteins on both the vesicle and on the chromatin. In addition, we show that the vesicles bound under these conditions will fuse into an intact nuclear envelope when incubated with the soluble fraction of a Xenopus egg nuclear assembly extract. With respect to vesicle fusion, we have found that vesicles prebound to chromatin will fuse to each other when ATP and GTP are present in the boiled extract. These results indicate that nuclear envelope assembly is mediated by a subset of approximately 70-nM-diam vesicles which bind to chromatin sites spaced 100 kb apart and that fusion of these vesicles is regulated by membrane-associated GTP-binding proteins.

scholarly journals Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts

1999 ◽  
Vol 112 (14) ◽  
pp. 2453-2461 ◽  
Author(s):  
C. Zhang ◽  
M. Hughes ◽  
P.R. Clarke
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Asymmetric Distribution ◽  
Nucleotide Exchange ◽  
Free System ◽  
Nuclear Assembly ◽  
Microtubule Stability ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Ran Gtp ◽  
Xenopus Egg Extracts

Ran is an abundant GTPase of the Ras superfamily that is highly conserved in eukaryotes. In interphase cells, Ran is mainly nuclear and thought to be predominantly GTP-bound, but it is also present in the cytoplasm, probably GDP-bound. This asymmetric distribution plays an important role in directing nucleocytoplasmic transport. Ran has also been implicated in cell cycle control, including the transition from mitosis to interphase when the compartmentalisation of the nucleus is established. Here, we have examined the role of Ran in this transition using a cell-free system of Xenopus egg extracts supplemented with sperm heads that provides a model for microtubule aster formation and post-M phase nuclear assembly. Ran-GTP, added as wild-type protein, a mutant defective in GTPase activity (Q69L), or generated by addition of the specific nucleotide exchange factor RCC1, stabilises large microtubule asters nucleated at the sperm centrosome, prevents the redistribution of NuMA from the aster to the nucleus and blocks chromatin decondensation. In contrast, Ran GDP does not stabilise microtubules or inhibit nuclear assembly. RanT24N and RanBP1, which oppose the generation of Ran-GTP by RCC1, arrest nuclear growth after disappearance of the aster. Ran associates with microtubule asters in egg extracts and with mitotic spindles in somatic Xenopus cells, suggesting that it may affect microtubule stability directly. These results show that Ran has a novel function in the control of microtubule stability that is clearly distinct from nucleocytoplasmic transport. The Ran GDP/GTP switch may play a role in co-ordinating changes in the structure of microtubules and the assembly of the nucleus associated with the transition from mitosis to interphase.

scholarly journals Rho Family Gtpase Cdc42 Is Essential for the Actin-Based Motility of Shigella in Mammalian Cells

1999 ◽  
Vol 191 (11) ◽  
pp. 1905-1920 ◽  
Author(s):  
Toshihiko Suzuki ◽  
Hitomi Mimuro ◽  
Hiroaki Miki ◽  
Tadaomi Takenawa ◽  
Takuya Sasaki ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Actin Dynamics ◽  
Cloud Formation ◽  
Actin Assembly ◽  
Egg Extracts ◽  
Rho Family ◽  
Green Fluorescent

Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. Here, we show that Cdc42 is required for the actin-based motility of Shigella. Microinjection of a dominant active mutant Cdc42, but not Rac1 or RhoA, into Swiss 3T3 cells accelerated Shigella motility. In add-back experiments in Xenopus egg extracts, addition of a guanine nucleotide dissociation inhibitor for the Rho family, RhoGDI, greatly diminished the bacterial motility or actin assembly, which was restored by adding activated Cdc42. In N-WASP–depleted extracts, the bacterial movement almost arrested was restored by adding exogenous N-WASP but not H208D, an N-WASP mutant defective in binding to Cdc42. In pyrene actin assay, Cdc42 enhanced VirG-stimulating actin polymerization by N-WASP–actin-related protein (Arp)2/3 complex. Actually, Cdc42 stimulated actin cloud formation on the surface of bacteria expressing VirG in a solution containing N-WASP, Arp2/3 complex, and G-actin. Immunohistological study of Shigella-infected cells expressing green fluorescent protein–tagged Cdc42 revealed that Cdc42 accumulated by being colocalized with actin cloud at one pole of intracellular bacterium. Furthermore, overexpression of H208D mutant in cells interfered with the actin assembly of infected Shigella and diminished the intra- and intercellular spreading. These results suggest that Cdc42 activity is involved in initiating actin nucleation mediated by VirG–N-WASP–Arp2/3 complex formed on intracellular Shigella.

Association and interactions of GTP-binding proteins with rat medullary H(+)-ATPase

1994 ◽  
Vol 267 (6) ◽  
pp. F944-F951
Author(s):  
N. J. Brunskill ◽  
J. J. Morrissey ◽  
S. Klahr
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Binding Proteins ◽  
Rat Kidney ◽  
Membrane Vesicles ◽  
Intercalated Cells ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Nephron Segment ◽  
G Protein Alpha Subunit

Guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are expressed in a heterogeneous manner in the mammalian kidney. In particular, cells of the medullary collecting tubule demonstrate a complex pattern of G protein expression both between cell types and between the polarized surfaces of individual cells. Intercalated cells expressing the H(+)-ATPase are also prevalent in this nephron segment. To examine interactions between G proteins and the H(+)-ATPase, we performed immunocytochemical studies on perfusion-fixed sections of rat kidney using polyclonal anti-G protein antibodies and E11, a mouse monoclonal antibody to the 31-kDa subunit of the vacuolar H(+)-ATPase. G alpha s subunits were consistently not associated with cells containing the H(+)-ATPase in this nephron segment, whereas G alpha i-2, G alpha i-3, and G alpha q/11 were. Some intercalated cells that stained prominently for the proton pump in the apical membrane did not, however, stain for any G protein alpha-subunit. We prepared medullary membrane vesicles highly enriched for the H(+)-ATPase to examine possible functional interactions of G proteins with the H(+)-ATPase by the acridine orange method. These vesicles were also highly enriched for G protein subunits. Proton transport was significantly increased in the presence of guanosine 5'-O-(3-thiotriphosphate), and this held true in the absence of chloride. This excludes an effect on chloride conductance indirectly stimulating the H(+)-ATPase. Guanine nucleotides did not affect the proton leak of the vesicles. Thus some G proteins are associated with the H(+)-ATPase and can regulate its function; however, the particular G proteins involved remain to be identified.

scholarly journals Mitotic spindle assembly by two different pathways in vitro.

1991 ◽  
Vol 112 (5) ◽  
pp. 925-940 ◽  
Author(s):  
K E Sawin ◽  
T J Mitchison
Keyword(s):  
Spindle Assembly ◽  
Sperm Nucleus ◽  
Free System ◽  
Microtubule Stabilization ◽  
Egg Extracts ◽  
A Cell ◽  
Xenopus Egg ◽  
Sperm Nuclei ◽  
Xenopus Egg Extracts

We have used Xenopus egg extracts to study spindle morphogenesis in a cell-free system and have identified two pathways of spindle assembly in vitro using methods of fluorescent analogue cytochemistry. When demembranated sperm nuclei are added to egg extracts arrested in a mitotic state, individual nuclei direct the assembly of polarized microtubule arrays, which we term half-spindles; half-spindles then fuse pairwise to form bipolar spindles. In contrast, when sperm nuclei are added to extracts that are induced to enter interphase and arrested in the following mitosis, a single sperm nucleus can direct the assembly of a complete spindle. We find that microtubule arrays in vitro are strongly biased towards chromatin, but this does not depend on specific kinetochore-microtubule interactions. Indeed, although we have identified morphological and probably functional kinetochores in spindles assembled in vitro, kinetochores appear not to play an obligate role in the establishment of stable, bipolar microtubule arrays in either assembly pathway. Features of the two pathways suggest that spindle assembly involves a hierarchy of selective microtubule stabilization, involving both chromatin-microtubule interactions and antiparallel microtubule-microtubule interactions, and that fundamental molecular interactions are probably the same in both pathways. This in vitro reconstitution system should be useful for identifying the molecules regulating the generation of asymmetric microtubule arrays and for understanding spindle morphogenesis in general.

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