Biomedical subjects

Laurent Blanchoin

Publications and source records attributed to Laurent Blanchoin.

18 recordsLinked to original sources

PubMed2007

Attachment conditions control actin filament buckling and the production of forces.

Actin polymerization is the driving force for a large number of cellular processes. Formation of lamellipodia and filopodia at the leading edge of motile cells requires actin polymerization induced mechanical deformation of the plasma membrane. To generate different types of membrane protrusions, the mechanical properties of actin filaments can be constrained by interacting proteins. A striking example of such constraint is the buckling of actin filaments generated in vitro by the cooperative effect of a processive actin nucleating factor (formin) and a molecular motor (myosin II). We developed a physical model based on equations for an elastic rod that accounts for actin filament buckling. Both ends of the rod were maintained in a fixed position in space and we considered three sets of boundary conditions. The model qualitatively and quantitatively reproduces the shape distribution of actin filaments. We found that actin polymerization counterpoises a force in the range 0.4-1.6 pN for moderate end-to-end distance (approximately 1 microm) and could be as large as 10 pN for shorter distances. If the actin rod attachment includes a spring, we discovered that the stiffness must be in the range 0.1-1.2 pN/nm to account for the observed buckling.

Actin Cytoskeleton↗

PubMed2006

A novel mechanism for the formation of actin-filament bundles by a nonprocessive formin.

Actin-filament bundles (or cables) have a structural role during cell division and morphogenesis, but also serve as important "tracks" for the transport of materials during cytokinesis and polarized cell growth. However, the dynamic formation of these longitudinal actin-filament higher-order structures is not understood. Recently, several lines of evidence suggest that formins provide one avenue for the initiation of actin cables in vivo. A popular model for the mechanism of polymerization of actin filaments by formin involves the processive movement of formin attached at the barbed end of an elongating filament. In the present study, we use an in vitro system to reconstitute the dynamic formation of actin-filament bundles generated by Arabidopsis FORMIN1 (AFH1). To be able to visualize individual events in such a complex system, we used real-time evanescent-wave microscopy. Surprisingly, we find that AFH1 is a nonprocessive formin that moves from the barbed end to the side of an actin filament after the nucleation event. We show why this new mechanism of nucleation by a member of the formin family is important for bundle formation. Finally, we analyze the different parameters controlling the dynamic formation of such longitudinal actin-filament bundles.

Actin Cytoskeleton↗

PubMed2006

Actin dynamics: old friends with new stories.

Actin dynamics, or the rapid turnover of actin filaments, play a central role in numerous cellular processes. A large and diverse cast of characters, accessory proteins known as actin-binding proteins, modulate actin dynamics. They do this by binding to the monomer pool, interacting with the side and ends of filaments, creating breaks along a filament, and generating new filaments de novo. Recent biochemical and single-filament imaging analyses of several conserved classes of plant actin-binding proteins reveal unusual and unexpected properties. Examples that are highlighted in this review include: an abundant monomer-binding protein that catalyzes nucleotide exchange; a barbed-end capping protein that is dissociated from filament ends by the signaling lipid, phosphatidic acid; a villin-like bundling protein that lacks all Ca(2+)-regulated activities; and a formin family member that is non-processive and is sufficient to generate actin filament bundles. These and other stories motivate a careful description of the properties of plant proteins in vitro as a prelude to greater insight into the molecular mechanism(s) underlying the regulation of actin dynamics in vivo.

Actins↗

PubMed2006

Phosphorylation of microtubule-associated protein STOP by calmodulin kinase II.

STOP proteins are microtubule-associated, calmodulin-regulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity.

Actins↗

PubMed2006

Heterodimeric capping protein from Arabidopsis is regulated by phosphatidic acid.

The cytoskeleton is a key regulator of morphogenesis, sexual reproduction, and cellular responses to extracellular stimuli. Changes in the cellular architecture are often assumed to require actin-binding proteins as stimulus-response modulators, because many of these proteins are regulated directly by binding to intracellular second messengers or signaling phospholipids. Phosphatidic acid (PA) is gaining widespread acceptance as a major, abundant phospholipid in plants that is required for pollen tube tip growth and mediates responses to osmotic stress, wounding, and phytohormones; however, the number of identified effectors of PA is rather limited. Here we demonstrate that exogenous PA application leads to significant increases in filamentous actin levels in Arabidopsis suspension cells and poppy pollen grains. To investigate further these lipid-induced changes in polymer levels, we analyzed the properties of a key regulator of actin filament polymerization, the heterodimeric capping protein from Arabidopsis thaliana (AtCP). AtCP binds to PA with a K(d) value of 17 muM and stoichiometry of approximately 1:2. It also binds well to PtdIns(4,5)P(2), but not to several other phosphoinositide or acidic phospholipids. The interaction with PA inhibited the actin-binding activity of CP. In the presence of PA, CP is unable to block the barbed or rapidly growing and shrinking end of actin filaments. Precapped filament barbed ends can also be uncapped by addition of PA, allowing rapid filament assembly from an actin monomer pool that is buffered with profilin. The findings support a model in which the inhibition of CP activity in cells by elevated PA results in the stimulation of actin polymerization from a large pool of profilin-actin. Such regulation may be important for the response of plant cells to extracellular stimuli as well as for the normal process of pollen tube tip growth.

Actin Capping Proteins↗

PubMed2006

Structural and biochemical characterization of a human adenovirus 2/12 penton base chimera.

The vertex of the adenoviral capsid is formed by the penton, a complex of two proteins, the pentameric penton base and the trimeric fiber protein. The penton contains all necessary components for viral attachment and entry into the host cell. After initial attachment via the head domain of the fiber protein, the penton base interacts with cellular integrins through an Arg-Gly-Asp (RGD) motif located in a hypervariable surface loop, triggering virus internalization. In order to investigate the structural and functional role of this region, we replaced the hypervariable loop of serotype 2 with the corresponding, but much shorter, loop of serotype 12 and compared it to the wild type. Here, we report the 3.6 A crystal structure of a human adenovirus 2/12 penton base chimera crystallized as a dodecamer. The structure is generally similar to human adenovirus 2 penton base, with the main differences localized to the fiber protein-binding site. Fluorescence anisotropy assays using a trimeric fiber protein mimetic called the minifiber and wild-type human adenovirus 2 and chimeric penton base demonstrate that fiber protein binding is independent of the hypervariable loop, with a K(d) for fiber binding estimated in the 1-2 microm range. Interestingly, competition assays using labeled and unlabeled minifiber demonstrated virtually irreversible binding to the penton base, which we ascribe to a conformational change, on the basis of comparisons of all available penton base structures.

Adenoviruses, Human↗

PubMed2005

The formin homology 1 domain modulates the actin nucleation and bundling activity of Arabidopsis FORMIN1.

The organization of actin filaments into large ordered structures is a tightly controlled feature of many cellular processes. However, the mechanisms by which actin filament polymerization is initiated from the available pool of profilin-bound actin monomers remain unknown in plants. Because the spontaneous polymerization of actin monomers bound to profilin is inhibited, the intervention of an actin promoting factor is required for efficient actin polymerization. Two such factors have been characterized from yeasts and metazoans: the Arp2/3 complex, a complex of seven highly conserved subunits including two actin-related proteins (ARP2 and ARP3), and the FORMIN family of proteins. The recent finding that Arabidopsis thaliana plants lacking a functional Arp2/3 complex exhibit rather modest morphological defects leads us to consider whether the large FORMIN family plays a central role in the regulation of actin polymerization. Here, we have characterized the mechanism of action of Arabidopsis FORMIN1 (AFH1). Overexpression of AFH1 in pollen tubes has been shown previously to induce abnormal actin cable formation. We demonstrate that AFH1 has a unique behavior when compared with nonplant formins. The activity of the formin homology domain 2 (FH2), containing the actin binding activity, is modulated by the formin homology domain 1 (FH1). Indeed, the presence of the FH1 domain switches the FH2 domain from a tight capper (Kd approximately 3.7 nM) able to nucleate actin filaments that grow only in the pointed-end direction to a leaky capper that allows barbed-end elongation and efficient nucleation of actin filaments from actin monomers bound to profilin. Another exciting feature of AFH1 is its ability to bind to the side and bundle actin filaments. We have identified an actin nucleator that is able to organize actin filaments directly into unbranched actin filament bundles. We suggest that AFH1 plays a central role in the initiation and organization of actin cables from the pool of actin monomers bound to profilin.

Actins↗

PubMed2005

Plant formin AtFH5 is an evolutionarily conserved actin nucleator involved in cytokinesis.

Formins are actin-organizing proteins that are involved in cytokinesis and cell polarity. In the plant Arabidopsis thaliana, there are more than 20 formin homologues, all of which have unknown roles. In this study, we characterize specific cellular and molecular functions of the Arabidopsis formin AtFH5. Despite the low identity of AtFH5 to yeast and mammalian formins, the AtFH5 protein interacts with the barbed end of actin filaments and nucleates actin-filament polymerization in vitro, as is the case in yeast and mammals. In vivo, the AtFH5-GFP fusion protein localizes to the cell plate, a plant-specific membranous component that is assembled at the plane of cell division. Consistent with these data, loss of function of atfh5 compromises cytokinesis in the seed endosperm. Furthermore, endogenous AtFH5 transcripts accumulate in the posterior pole of the endosperm and loss of function of atfh5 perturbs proper morphogenesis of the endosperm posterior pole. Although cytokinesis in animals, yeast and plants occurs through morphologically distinct mechanisms, our study finds that formin recruitment to sites of actin assembly is a common feature of cell division among eukaryotes.

Actins↗

PubMed2005

Arabidopsis VILLIN1 generates actin filament cables that are resistant to depolymerization.

Dynamic cytoplasmic streaming, organelle positioning, and nuclear migration use molecular tracks generated from actin filaments arrayed into higher-order structures like actin cables and bundles. How these arrays are formed and stabilized against cellular depolymerizing forces remains an open question. Villin and fimbrin are the best characterized actin-filament bundling or cross-linking proteins in plants and each is encoded by a multigene family of five members in Arabidopsis thaliana. The related villins and gelsolins are conserved proteins that are constructed from a core of six homologous gelsolin domains. Gelsolin is a calcium-regulated actin filament severing, nucleating and barbed end capping factor. Villin has a seventh domain at its C terminus, the villin headpiece, which can bind to an actin filament, conferring the ability to crosslink or bundle actin filaments. Many, but not all, villins retain the ability to sever, nucleate, and cap filaments. Here we have identified a putative calcium-insensitive villin isoform through comparison of sequence alignments between human gelsolin and plant villins with x-ray crystallography data for vertebrate gelsolin. VILLIN1 (VLN1) has the least well-conserved type 1 and type 2 calcium binding sites among the Arabidopsis VILLIN isoforms. Recombinant VLN1 binds to actin filaments with high affinity (K(d) approximately 1 microM) and generates bundled filament networks; both properties are independent of the free Ca(2+) concentration. Unlike human plasma gelsolin, VLN1 does not nucleate the assembly of filaments from monomer, does not block the polymerization of profilin-actin onto barbed ends, and does not stimulate depolymerization or sever preexisting filaments. In kinetic assays with ADF/cofilin, villin appears to bind first to growing filaments and protects filaments against ADF-mediated depolymerization. We propose that VLN1 is a major regulator of the formation and stability of actin filament bundles in plant cells and that it functions to maintain the cable network even in the presence of stimuli that result in depolymerization of other actin arrays.

Actin Cytoskeleton↗

PubMed2004

Structural basis of actin sequestration by thymosin-beta4: implications for WH2 proteins.

The WH2 (Wiscott-Aldridge syndrome protein homology domain 2) repeat is an actin interacting motif found in monomer sequestering and filament assembly proteins. We have stabilized the prototypical WH2 family member, thymosin-beta4 (Tbeta4), with respect to actin, by creating a hybrid between gelsolin domain 1 and the C-terminal half of Tbeta4 (G1-Tbeta4). This hybrid protein sequesters actin monomers, severs actin filaments and acts as a leaky barbed end cap. Here, we present the structure of the G1-Tbeta4:actin complex at 2 A resolution. The structure reveals that Tbeta4 sequesters by capping both ends of the actin monomer, and that exchange of actin between Tbeta4 and profilin is mediated by a minor overlap in binding sites. The structure implies that multiple WH2 motif-containing proteins will associate longitudinally with actin filaments. Finally, we discuss the role of the WH2 motif in arp2/3 activation.

Actin-Related Protein 2↗

PubMed2004

A gelsolin-like protein from Papaver rhoeas pollen (PrABP80) stimulates calcium-regulated severing and depolymerization of actin filaments.

The cytoskeleton is a key regulator of plant morphogenesis, sexual reproduction, and cellular responses to extracellular stimuli. During the self-incompatibility response of Papaver rhoeas L. (field poppy) pollen, the actin filament network is rapidly depolymerized by a flood of cytosolic free Ca2+ that results in cessation of tip growth and prevention of fertilization. Attempts to model this dramatic cytoskeletal response with known pollen actin-binding proteins (ABPs) revealed that the major G-actin-binding protein profilin can account for only a small percentage of the measured depolymerization. We have identified an 80-kDa, Ca(2+)-regulated ABP from poppy pollen (PrABP80) and characterized its biochemical properties in vitro. Sequence determination by mass spectrometry revealed that PrABP80 is related to gelsolin and villin. The molecular weight, lack of filament cross-linking activity, and a potent severing activity are all consistent with PrABP80 being a plant gelsolin. Kinetic analysis of actin assembly/disassembly reactions revealed that substoichiometric amounts of PrABP80 can nucleate actin polymerization from monomers, block the assembly of profilin-actin complex onto actin filament ends, and enhance profilin-mediated actin depolymerization. Fluorescence microscopy of individual actin filaments provided compelling, direct evidence for filament severing and confirmed the actin nucleation and barbed end capping properties. This is the first direct evidence for a plant gelsolin and the first example of efficient severing by a plant ABP. We propose that PrABP80 functions at the center of the self-incompatibility response by creating new filament pointed ends for disassembly and by blocking barbed ends from profilin-actin assembly.

Actins↗

PubMed2004

Interactions of tobacco microtubule-associated protein MAP65-1b with microtubules.

Tobacco microtubule associated protein (MAP65) (NtMAP65s) constitute a family of microtubule-associated proteins with apparent molecular weight around 65 kDa that collectively induce microtubule bundling and promote microtubule assembly in vitro. They are associated with most of the tobacco microtubule arrays in situ. Recently, three NtMAP65s belonging to the NtMAP65-1 subfamily have been cloned. Here we investigated in vitro the biochemical properties of one member of this family, the tobacco NtMAP65-1b. We demonstrated that recombinant NtMAP65-1b is a microtubule-binding and a microtubule-bundling protein. NtMAP65-1b has no effect on microtubule polymerization rate and binds microtubules with an estimated equilibrium constant of dissociation (K(d)) of 0.57 micro m. Binding of NtMAP65-1b to microtubules occurs through the carboxy-terminus of tubulin, as NtMAP65-1b was no longer able to bind subtilisin-digested tubulin. In vitro, NtMAP65-1b stabilizes microtubules against depolymerization induced by cold, but not against katanin-induced destabilization. The biological implications of these results are discussed.

Adenosine Triphosphatases↗

PubMed2003

Arabidopsis capping protein (AtCP) is a heterodimer that regulates assembly at the barbed ends of actin filaments.

The precise regulation of actin filament polymerization and depolymerization is essential for many cellular processes and is choreographed by a multitude of actin-binding proteins (ABPs). In higher plants the number of well characterized ABPs is quite limited, and some evidence points to significant differences in the biochemical properties of apparently conserved proteins. Here we provide the first evidence for the existence and biochemical properties of a heterodimeric capping protein from Arabidopsis thaliana (AtCP). The purified recombinant protein binds to actin filament barbed ends with Kd values of 12-24 nM, as assayed both kinetically and at steady state. AtCP prevents the addition of profilin actin to barbed ends during a seeded elongation reaction and suppresses dilution-mediated depolymerization. It does not, however, sever actin filaments and does not have a preference for the source of actin. During assembly from Mg-ATP-actin monomers, AtCP eliminates the initial lag period for actin polymerization and increases the maximum rate of polymerization. Indeed, the efficiency of actin nucleation of 0.042 pointed ends created per AtCP polypeptide compares favorably with mouse CapZ, which has a maximal nucleation of 0.17 pointed ends per CapZ polypeptide. AtCP activity is not affected by calcium but is sensitive to phosphatidylinositol 4,5-bisphosphate. We propose that AtCP is a major regulator of actin dynamics in plant cells that, together with abundant profilin, is responsible for maintaining a large pool of actin subunits and a surprisingly small population of F-actin.

Actin Depolymerizing Factors↗

PubMed2003

Phosphorylation of the WASP-VCA domain increases its affinity for the Arp2/3 complex and enhances actin polymerization by WASP.

Wiskott-Aldrich syndrome protein (WASP) and neural (N)-WASP regulate dynamic actin structures through the ability of their VCA domains to bind to and stimulate the actin nucleating activity of the Arp2/3 complex. Here we identify two phosphorylation sites in the VCA domain of WASP at serines 483 and 484. S483 and S484 are substrates for casein kinase 2 in vitro and in vivo. Phosphorylation of these residues increases the affinity of the VCA domain for the Arp2/3 complex 7-fold and is required for efficient in vitro actin polymerization by the full-length WASP molecule. We propose that constitutive VCA domain phosphorylation is required for optimal stimulation of the Arp2/3 complex by WASP.

3T3 Cells↗

PubMed2003

The putative Arabidopsis arp2/3 complex controls leaf cell morphogenesis.

The evolutionarily conserved Arp2/3 complex has been shown to activate actin nucleation and branching in several eukaryotes, but its biological functions are not well understood in multicellular organisms. The model plant Arabidopsis provides many advantages for genetic dissection of the function of this conserved actin-nucleating machinery, yet the existence of this complex in plants has not been determined. We have identified Arabidopsis genes encoding homologs of all of the seven Arp2/3 subunits. The function of the putative Arabidopsis Arp2/3 complex has been studied using four homozygous T-DNA insertion mutants for ARP2, ARP3, and ARPC5/p16. All four mutants display identical defects in the development of jigsaw-shaped epidermal pavement cells and branched trichomes in the leaf. These loss-of-function mutations cause mislocalization of diffuse cortical F-actin to the neck region and inhibit lobe extension in pavement cells. The mutant trichomes resemble those treated with the actin-depolymerizing drug cytochalasin D, exhibiting stunted branches but dramatically enlarged stalks due to depolarized growth suggesting defects in the formation of a fine actin network. Our data demonstrate that the putative Arabidopsis Arp2/3 complex controls cell morphogenesis through its roles in cell polarity establishment and polar cell expansion. Furthermore, our data suggest a novel function for the putative Arp2/3 complex in the modulation of the spatial distribution of cortical F-actin and provide evidence that the putative Arp2/3 complex may activate the polymerization of some types of actin filaments in specific cell types.

Actin-Related Protein 2↗

PubMed2002

Xenopus actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends.

Xenopus actin-interacting protein 1 (XAip1) is thought to promote fragmentation of actin filaments by cofilin. To examine the mechanism of XAip1, we measured polymer lengths by fluorescence microscopy and the concentration of filament ends with an elongation assay. Cofilin creates ends by severing actin filaments. XAip1 alone does not sever actin filaments or prevent annealing/redistribution of mechanically severed filaments and has no effect on the concentration of ends available for subunit addition. In the presence of XAip1, the apparent filament fragmentation by cofilin is enhanced, but XAip1 reduces rather than increases the concentration of ends capable of adding subunits. Electron microscopy with gold-labeled antibodies showed that a low concentration of XAip1 bound preferentially to one end of the filament. A high concentration of XAip1 bound along the length of the filament. In the presence of gelsolin-actin to cap filament barbed ends, XAip1 does not enhance cofilin activity. We conclude that XAip1 caps the barbed end of filaments severed by cofilin. This capping blocks annealing and depolymerization and allows more extensive severing by cofilin.

Actin Depolymerizing Factors↗

PubMed2002

Hydrolysis of ATP by polymerized actin depends on the bound divalent cation but not profilin.

Growing evidence suggests that the nucleotide bound to actin filaments serves as a timer to control actin filament turnover during cell motility (Pollard, T. D., Blanchoin, L., and Mullins, R. D. (2000) Annu. Rev. Biophys. Biomol. Struct. 29, 545-576). We re-examined the hydrolysis of ATP by polymerized actin using mechanical quenched-flow methods to improve temporal resolution. The rate constant for ATP hydrolysis by polymerized Mg actin is 0.3 s(-1), 3-fold faster than that measured manually. The ATP hydrolysis rate is similar when Mg ATP actin elongates either the pointed end or the barbed end of filaments. Polymerized Ca actin hydrolyzes ATP at 0.05 s(-1). Mg ATP actin saturated with profilin can elongate barbed ends at >60 s(-1), 2 orders of magnitude faster than ATP hydrolysis (0.3 s(-1)). Given that profilin binds to a surface on actin that is buried in the Holmes model of the actin filament, we expect that profilin will block subunit addition at the barbed end of a filament. Profilin must move from this site at rates much faster than it dissociates from monomers (4 s(-1)). ATP hydrolysis is not required for this movement.

Actins↗

PubMed2002

Actin polymerization processes in plant cells.

Growing evidence shows that the actin cytoskeleton is a key effector of signal transduction, which controls and maintains the shape of plant cells, as well as playing roles in plant morphogenesis. Recently, several signaling pathways, including those triggered by hormones, Ca(2+), and cAMP, have been reported to be connected to the reorganization of the actin cytoskeleton. The molecular mechanisms involved in such signaling cascades are, however, largely unknown. The Arabidopsis genome sequence is a valuable tool for identifying some of the highly conserved molecules that are involved in such signaling cascades. Recent work has begun to unravel these complex pathways using a panoply of techniques, including genetic analysis, live-cell imaging of intracellular actin dynamics, in vivo localization of factors that are involved in the control of actin dynamics, and the biochemical characterization of how these factors function.

Abscisic Acid↗