Biomedical subjects

Christopher J Staiger

Publications and source records attributed to Christopher J Staiger.

16 recordsLinked to original sources

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

Identification of plant cytoskeleton-interacting proteins by screening for actin stress fiber association in mammalian fibroblasts.

Taking advantage of the high conservation of the cytoskeleton building blocks actin and tubulin between plant and animal kingdoms, we developed a functional genomic screen for the isolation of new plant cytoskeleton-binding proteins that uses a mammalian cell expression system. A yellow fluorescent protein (YFP)-fusion cDNA library from Arabidopsis was inserted into rat fibroblasts and screened for fluorescent chimeras localizing to cytoskeletal structures. The high-throughput screen was performed by an automated microscope. An initial set of candidate genes identified in the screen was isolated, sequenced, the full-length cDNAs were synthesized by RT-PCR and tested by biochemical approaches to verify the ability of the genes to bind actin directly. Alternatively, indirect binding via interaction with other actin-binding proteins was studied. The full-length cDNAs were transferred back to plants as YFP chimeras behind the CAMV-35S promoter. We give here two examples of new plant cytoskeletal proteins identified in the pilot screen. ERD10, a member of the dehydrin family of proteins, was localized to actin stress fibers in rat fibroblasts. Its direct binding to actin filaments was confirmed by several biochemical approaches. Touch-induced calmodulin-like protein, TCH2, was also localized to actin stress fibers in fibroblasts, but was unable to bind actin filaments directly in vitro. Nevertheless, it did bind to the IQ domains of Arabidopsis myosin VIII in a calcium-dependent manner. Further evidence for a cytoskeletal function of ERD10 was obtained in planta; GFP-ERD10 was able to protect the actin cytoskeleton from latrunculin-mediated disruption in Nicotiana benthamiana leaves.

Actins↗

PubMed2006

Actin depolymerization is sufficient to induce programmed cell death in self-incompatible pollen.

Self-incompatibility (SI) prevents inbreeding through specific recognition and rejection of incompatible pollen. In incompatible Papaver rhoeas pollen, SI triggers a Ca2+ signaling cascade, resulting in the inhibition of tip growth, actin depolymerization, and programmed cell death (PCD). We investigated whether actin dynamics were implicated in regulating PCD. Using the actin-stabilizing and depolymerizing drugs jasplakinolide (Jasp) and latrunculin B, we demonstrate that changes in actin filament levels or dynamics play a functional role in initiating PCD in P. rhoeas pollen, triggering a caspase-3-like activity. Significantly, SI-induced PCD in incompatible pollen was alleviated by pretreatment with Jasp. This represents the first account of a specific causal link between actin polymerization status and initiation of PCD in a plant cell and significantly advances our understanding of the mechanisms involved in SI.

Actin Cytoskeleton↗

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↗

PubMed2005

Plant villin, lily P-135-ABP, possesses G-actin binding activity and accelerates the polymerization and depolymerization of actin in a Ca2+-sensitive manner.

From germinating pollen of lily, two types of villins, P-115-ABP and P-135-ABP, have been identified biochemically. Ca(2+)-CaM-dependent actin-filament binding and bundling activities have been demonstrated for both villins previously. Here, we examined the effects of lily villins on the polymerization and depolymerization of actin. P-115-ABP and P-135-ABP present in a crude protein extract prepared from germinating pollen bound to a DNase I affinity column in a Ca(2+)-dependent manner. Purified P-135-ABP reduced the lag period that precedes actin filament polymerization from monomers in the presence of either Ca(2+) or Ca(2+)-CaM. These results indicated that P-135-ABP can form a complex with G-actin in the presence of Ca(2+) and this complex acts as a nucleus for polymerization of actin filaments. However, the nucleation activity of P-135-ABP is probably not relevant in vivo because the assembly of G-actin saturated with profilin, a situation that mimics conditions found in pollen, was not accelerated in the presence of P-135-ABP. P-135-ABP also enhanced the depolymerization of actin filaments during dilution-mediated disassembly. Growth from filament barbed ends in the presence of Ca(2+)-CaM was also prevented, consistent with filament capping activity. These results suggested that lily villin is involved not only in the arrangement of actin filaments into bundles in the basal and shank region of the pollen tube, but also in regulating and modulating actin dynamics through its capping and depolymerization (or fragmentation) activities in the apical region of the pollen tube, where there is a relatively high concentration of Ca(2+).

Actins↗

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

DISTORTED3/SCAR2 is a putative arabidopsis WAVE complex subunit that activates the Arp2/3 complex and is required for epidermal morphogenesis.

In a plant cell, a subset of actin filaments function as a scaffold that positions the endomembrane system and acts as a substrate on which organelle motility occurs. Other actin filament arrays appear to be more dynamic and reorganize in response to growth signals and external cues. The distorted group of trichome morphology mutants provides powerful genetic tools to study the control of actin filament nucleation in the context of morphogenesis. In this article, we report that DISTORTED3 (DIS3) encodes a plant-specific SCAR/WAVE homolog. Null alleles of DIS3, like those of other Arabidopsis thaliana WAVE and Actin-Related Protein (ARP) 2/3 subunit genes, cause trichome distortion, defects in cell-cell adhesion, and reduced hypocotyl growth in etiolated seedlings. DIS3 efficiently activates the actin filament nucleation and branching activity of vertebrate Arp2/3 and functions within a WAVE-ARP2/3 pathway in vivo. DIS3 may assemble into a WAVE complex via a physical interaction with a highly diverged Arabidopsis Abi-1-like bridging protein. These results demonstrate the utility of the Arabidopsis trichome system to understand how the WAVE and ARP2/3 complexes translate signaling inputs into a coordinated morphogenetic response.

Actin-Related Protein 2↗

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

Identification and characterization of a Ca2+-dependent actin filament-severing protein from lily pollen.

It is well known that a tip-focused intracellular Ca2+ gradient and the meshwork of short actin filaments at the tip region are necessary for pollen tube growth. However, little is known about the connections between the two factors. Here, a novel Ca2+-dependent actin-binding protein with molecular mass of 41 kD from lily (Lilium davidii) pollen (LdABP41) was isolated and purified with DNase I chromatography. Our purification procedure yielded about 0.6 mg of LdABP41 with >98% purity from 10 g of lily pollen. At least two isoforms with isoelectric points of 5.8 and 6.0 were detected on two-dimensional gels. The results of N-terminal sequencing and mass-spectrometry analysis of LdABP41 showed that both isoforms shared substantial similarity with trumpet lily (Lilium longiflorum) villin and other members of the gelsolin superfamily. Negative-stained electron microscope images showed that LdABP41 severed in vitro-polymerized lily pollen F-actin into short actin filaments in a Ca2+-sensitive manner. Microinjection of the anti-LdABP41 antibody into germinated lily pollen demonstrated that the protein was required for pollen tube growth. The results of immunolocalization of the protein showed that it existed in the cytoplasm of the pollen tube, especially focused in the tip region. Our results suggest that LdABP41 belongs to the gelsolin superfamily and may play an important role in controlling actin organization in the pollen tube tip by responding to the oscillatory, tip-focused Ca2+ gradient.

Actin Cytoskeleton↗

PubMed2004

A plant-specific kinesin binds to actin microfilaments and interacts with cortical microtubules in cotton fibers.

A novel kinesin, GhKCH1, has been identified from cotton (Gossypium hirsutum) fibers. GhKCH1 has a centrally located kinesin catalytic core, a signature neck peptide of minus end-directed kinesins, and a unique calponin homology (CH) domain at its N terminus. GhKCH1 and other CH domain-containing kinesins (KCHs) belong to a distinct branch of the minus end-directed kinesin subfamily. To date the KCH kinesins have been found only in higher plants. Because the CH domain is often found in actin-binding proteins, we proposed that GhKCH1 might play a role in mediating dynamic interaction between microtubules and actin microfilaments in cotton fibers. In an in vitro actin-binding assay, GhKCH1's N-terminal region including the CH domain interacted directly with actin microfilaments. In cotton fibers, GhKCH1 decorated cortical microtubules in a punctate manner. Occasionally GhKCH1 was found to be associated with transverse-cortical actin microfilaments, but never with axial actin cables in cotton fibers. Localization of GhKCH1 on cortical microtubules was independent of the integrity of actin microfilaments. Thus, GhKCH1 may play a role in organizing the actin network in coordination with the cortical microtubule array. These data also suggest that flowering plants may employ unique KCHs to coordinate actin microfilaments and microtubules during cell growth.

Actin Cytoskeleton↗

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

Structure of the actin crosslinking core of fimbrin.

Filamentous actin is organized into bundles and orthogonal networks by the fimbrin/alpha-actinin superfamily of F-actin crosslinking proteins. The crystal structure of the Arabidopsis thaliana and Schizosaccharomyces pombe fimbrin cores provides the first description of a functional F-actin crosslinking protein and highlights the compact and distinctly asymmetric organization of the fimbrin molecule, in which the two actin binding domains present distinct surfaces to solvent. The mapping of functionally important residues onto the structure affords new insights into the binding process and provides additional constraints which must be accommodated by models for F-actin binding and crosslinking. Most strikingly, this work provides unique insight into the mechanistic features of conditional-lethal mutants and their extragenic suppressors, which highlight conformational and dynamic properties required for fimbrin function. These results underscore the power of jointly considering structural and genetic suppressor data for obtaining unexpected and biologically relevant mechanistic information.

Actins↗

PubMed2004

Independent genetic mechanisms mediate turgor generation and penetration peg formation during plant infection in the rice blast fungus.

The first barrier to infection encountered by foliar pathogens is the host cuticle. To traverse this obstacle, many fungi produce specialized infection cells called appressoria. MST12 is essential for appressorium-mediated penetration and infectious growth by the rice pathogen Magnaporthe grisea. In this study, we have characterized in detail the penetration defects of an mst12 deletion mutant. Appressoria formed by the mst12 mutant developed normal turgor pressure and ultrastructure but failed to form penetration pegs either on cellophane membranes or on plant epidermal cells. Deletion and site-directed mutagenesis analyses indicated that both the homeodomain and zinc finger domains, but not the middle region, of MST12 are essential for appressorial penetration and plant infection. The mst12 mutant appeared to be defective in microtubule reorganization associated with penetration peg formation. In mature appressoria, the mutant lacked vertical microtubules observed in the wild type. The mst12 mutant also failed to elicit localized host defence responses, including papilla formation and autofluorescence. Our data indicate that generation of appressorium turgor pressure and formation of the penetration peg are two independent processes. MST12 may play important roles in regulating penetration peg formation and directing the physical forces exerted by the appressorium turgor in mature appressoria.

Actins↗

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↗

PubMed2002

Signal-mediated depolymerization of actin in pollen during the self-incompatibility response.

Signal perception and the integration of signals into networks that effect cellular changes is essential for all cells. The self-incompatibility (SI) response in field poppy pollen triggers a Ca(2+)-dependent signaling cascade that results in the inhibition of incompatible pollen. SI also stimulates dramatic alterations in the actin cytoskeleton. By measuring the amount of filamentous (F-) actin in pollen before and during the SI response, we demonstrate that SI stimulates a rapid and large reduction in F-actin level that is sustained for at least 1 h. This represents quantitative evidence for stimulus-mediated depolymerization of F-actin in plant cells by a defined biological stimulus. Surprisingly, there are remarkably few examples of sustained reductions in F-actin levels stimulated by a biologically relevant ligand. Actin depolymerization also was achieved in pollen by treatments that increase cytosolic free Ca(2+) artificially, providing evidence that actin is a target for the Ca(2+) signals triggered by the SI response. By determining the cellular concentrations and binding constants for native profilin from poppy pollen, we show that profilin has Ca(2+)-dependent monomeric actin-sequestering activity. Although profilin is likely to contribute to stimulus-mediated actin depolymerization, our data suggest a role for additional actin binding proteins. We propose that Ca(2+)-mediated depolymerization of F-actin may be a mechanism whereby SI-induced tip growth inhibition is achieved.

Actins↗