F-actin binding region of SPIN90 C-terminus is essential for actin polymerization and lamellipodia formation

We recently reported that SPIN90 is able to bind with several proteins involved in regulating actin cytoskeleton networks, including dynamin, WASP, β PIX, and Nck. Based on these findings, we investigated how SPIN90 regulates the actin cytoskeleton and promotes actin assembly. This study demonstrated that aluminium fluoride-induced localization of SPIN90 to lamellipodia requires amino acids 582-722 at the SPIN90 C-terminus, which is also essential for F-actin binding and Arp2/3 complex mediated polymerization of actin into branched actin filaments. Furthermore, after deletion of the F-actin binding region (582-722 SPIN90) failed to localize at the membrane edge and was unable to promote lamellipodia formation, suggesting that the F-actin binding region in the SPIN90 C-terminus is essential for the formation of branched actin networks and regulation of the actin cytoskeleton at the leading edge of cells.     

Assembly and Function of the Actin Cytoskeleton of Yeast: Relationships between Cables and Patches

Actin in eukaryotic cells is found in different pools, with filaments being organized into a variety of supramolecular assemblies. To investigate the assembly and functional relationships between different parts of the actin cytoskeleton in one cell, we studied the morphology and dynamics of cables and patches in yeast. The fine structure of actin cables and the manner in which cables disassemble support a model in which cables are composed of a number of overlapping actin filaments. No evidence for intrinsic polarity of cables was found.To investigate to what extent different parts of the actin cytoskeleton depend on each other, we looked for relationships between cables and patches. Patches and cables were often associated, and their polarized distributions were highly correlated. Therefore, patches and cables do appear to depend on each other for assembly and function.Many cell types show rearrangements of the actin cytoskeleton, which can occur via assembly or movement of actin filaments. In our studies, dramatic changes in actin polarization did not include changes in filamentous actin. In addition, the concentration of actin patches was relatively constant as cells grew. Therefore, cells do not have bursts of activity in which new parts of the actin cytoskeleton are created.     

Reactive oxygen species regulate F-actin dynamics in neuronal growth cones and neurite outgrowth

Reactive oxygen species are well known for their damaging effects due to oxidation of lipids, proteins and DNA that ultimately result in cell death. Accumulating evidence indicates that reactive oxygen species also have important signaling functions in cell proliferation, differentiation, cell motility and apoptosis. Here, we tested the hypothesis whether reactive oxygen species play a physiological role in regulating F-actin structure and dynamics in neuronal growth cones. Lowering cytoplasmic levels of reactive oxygen species with a free radical scavenger, N -tert-butyl-α-phenylnitrone, or by inhibiting specific sources of reactive oxygen species, such as NADPH oxidases or lipoxygenases, reduced the F-actin content in the peripheral domain of growth cones. Fluorescent speckle microscopy revealed that these treatments caused actin assembly inhibition, reduced retrograde actin flow and increased contractility of actin structures in the transition zone referred to as arcs, possibly by activating the Rho pathway. Reduced levels of reactive oxygen species ultimately resulted in disassembly of the actin cytoskeleton. When neurons were cultured overnight in conditions of reduced free radicals, growth cone formation and neurite outgrowth were severely impaired. Therefore, we conclude that physiological levels of reactive oxygen species are critical for maintaining a dynamic F-actin cytoskeleton and controlling neurite outgrowth.     

The Effect of cytoskeleton inhibitors on coccolith morphology in Coccolithus braarudii and Scyphosphaera apsteinii

The calcite platelets of coccolithophores (Haptophyta), the coccoliths, are among the most elaborate biomineral structures. How these unicellular algae accomplish the complex morphogenesis of coccoliths is still largely unknown. It has long been proposed that the cytoskeleton plays a central role in shaping the growing coccoliths. Previous studies have indicated that disruption of the microtubule network led to defects in coccolith morphogenesis in Emiliania huxleyi and Coccolithus braarudii. Disruption of the actin network also led to defects in coccolith morphology in E. huxleyi, but its impact on coccolith morphology in C. braarudii was unclear, as coccolith secretion was largely inhibited under the conditions used. A more detailed examination of the role of actin and microtubule networks is therefore required to address the wider role of the cytoskeleton in coccolith morphogenesis. In this study, we have examined coccolith morphology in C. braarudii and Scyphosphaera apsteinii following treatment with the microtubule inhibitors vinblastine and colchicine (S. apsteinii only) and the actin inhibitor cytochalasin B. We found that all cytoskeleton inhibitors induced coccolith malformations, strongly suggesting that both microtubules and actin filaments are instrumental in morphogenesis. By demonstrating the requirement for the microtubule and actin networks in coccolith morphogenesis in diverse species, our results suggest that both of these cytoskeletal elements are likely to play conserved roles in defining coccolith morphology.     

Monomeric G‐actin is uniformly distributed in pollen tubes and is rapidly redistributed via cytoplasmic streaming during pollen tube growth

Dynamic assembly and disassembly of the actin cytoskeleton has been implicated in the regulation of pollen germination and subsequent tube growth. It is widely accepted that actin filaments are arrayed into distinct structures within different regions of the pollen tube. Maintenance of the equilibrium between monomeric globular actin (G‐actin) and filamentous actin (F‐actin) is crucial for actin assembly and array construction, and the local concentration of G‐actin thus directly impacts actin assembly. The localization and dynamics of G‐actin in the pollen tube, however, remain to be determined conclusively. To address this question, we created a series of fusion proteins between green fluorescent protein (GFP) and the Arabidopsis reproductive actin ACT11. Expression of a fusion protein with GFP inserted after methionine at position 49 within the DNase I‐binding loop of ACT11 (GFP_Met49–ACT11) rescued the phenotypes in act11 mutants. Consistent with the notion that the majority of actin is in its monomeric form, GFP_Met49–ACT11 and GFP fusion proteins of four other reproductive actins generated with the same strategy do not obviously label filamentous structures. In further support of the functionality of these fusion proteins, we found that they can be incorporated into filamentous structures in jasplakinolide (Jasp)‐treated pollen tubes. Careful observations showed that G‐actin is distributed uniformly in the pollen tube and is rapidly redistributed via cytoplasmic streaming during pollen tube growth. Our study suggests that G‐actin is readily available in the cytoplasm to support continuous actin polymerization during rapid pollen tube growth.     

植物肌动蛋白异型体研究进展

肌动蛋白在真核生物中广泛存在,由肌动蛋白参与形成的动态微丝骨架系统是细胞生命活动的基础。在植物细胞中,肌动蛋白由多基因家族编码,从而产生了多种肌动蛋白异型体。本文综述了拟南芥肌动蛋白异型体的分类、体内分布与功能,详细介绍了豌豆卷须肌动蛋白异型体的研究现状,并讨论了该领域的研究方向。     

Regulated actin cytoskeleton assembly at filopodium tips controls their extension and retraction.

The extension and retraction of filopodia in response to extracellular cues is thought to be an important initial step that determines the direction of growth cone advance. We sought to understand how the dynamic behavior of the actin cytoskeleton is regulated to produce extension or retraction. By observing the movement of fiduciary marks on actin filaments in growth cones of a neuroblastoma cell line, we found that filopodium extension and retraction are governed by a balance between the rate of actin cytoskeleton assembly at the tip and retrograde flow. Both assembly and flow rate can vary with time in a single filopodium and between filopodia in a single growth cone. Regulation of assembly rate is the dominant factor in controlling filopodia behavior in our system.     

Glycine-rich region regulates cysteine-rich protein 1 binding to actin cytoskeleton

Cysteine-rich protein 1 (CRP1) has a unique structure with two well separated LIM domains, each followed by a glycine-rich region. Although CRP1 has been shown to interact with actin-binding proteins and actin filaments, the mechanism regulating localization to the actin cytoskeleton in cells is not clear. Experiments using truncated forms showed that the first LIM domain and glycine-rich region are necessary for CRP1 bundling of actin filaments and localization to the actin cytoskeleton. Furthermore, domain swapping experiments replacing the first glycine-rich region with the second resulted in the loss of CRP1 bundling activity and localization to the actin cytoskeleton, identifying seven critical amino acid residues. These results highlight the importance of the first glycine-rich region for CRP1 bundling activity and localization to the actin cytoskeleton. In addition, this work identifies the first LIM domain and glycine-rich region as a distinct actin filament bundling module.     

Growth cone collapse through coincident loss of actin bundles and leading edge actin without actin depolymerization

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse.     

Gelsolin deficiency blocks podosome assembly and produces increased bone mass and strength

Osteoclasts are unique cells that utilize podosomes instead of focal adhesions for matrix attachment and cytoskeletal remodeling during motility. We have shown that osteopontin (OP) binding to the alpha(v)beta(3) integrin of osteoclast podosomes stimulated cytoskeletal reorganization and bone resorption by activating a heteromultimeric signaling complex that includes gelsolin, pp(60c-src), and phosphatidylinositol 3'-kinase. Here we demonstrate that gelsolin deficiency blocks podosome assembly and alpha(v)beta(3)-stimulated signaling related to motility in gelsolin-null mice. Gelsolin-deficient osteoclasts were hypomotile due to retarded remodeling of the actin cytoskeleton. They failed to respond to the autocrine factor, OP, with stimulation of motility and bone resorption. Gelsolin deficiency was associated with normal skeletal development and endochondral bone growth. However, gelsolin-null mice had mildly abnormal epiphyseal structure, retained cartilage proteoglycans in metaphyseal trabeculae, and increased trabecular thickness. With age, the gelsolin-deficient mice expressed increased trabecular and cortical bone thickness producing mechanically stronger bones. These observations demonstrate the critical role of gelsolin in podosome assembly, rapid cell movements, and signal transduction through the alpha(v)beta(3) integrin.     

Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast

Formins are actin filament nucleators regulated by Rho-GTPases. In budding yeast, the formins Bni1p and Bnr1p direct the assembly of actin cables, which guide polarized secretion and growth. From the six yeast Rho proteins (Cdc42p and Rho1-5p), we have determined that four participate in the regulation of formin activity. We show that the essential function of Rho3p and Rho4p is to activate the formins Bni1p and Bnr1p, and that activated alleles of either formin are able to bypass the requirement for these Rho proteins. Through a separate signaling pathway, Rho1p is necessary for formin activation at elevated temperatures, acting through protein kinase C (Pkc1p), the major effector for Rho1p signaling to the actin cytoskeleton. Although Pkc1p also activates a MAPK pathway, this pathway does not function in formin activation. Formin-dependent cable assembly does not require Cdc42p, but in the absence of Cdc42p function, cable assembly is not properly organized during initiation of bud growth. These results show that formin function is under the control of three distinct, essential Rho signaling pathways.     

The carboxy-terminus of the formin FMNL1ɣ bundles actin to potentiate adenocarcinoma migration

The formin family of proteins contributes to spatiotemporal control of actin cytoskeletal rearrangements during motile cell activities. The FMNL subfamily exhibits multiple mechanisms of linear actin filament formation and organization. Here we report novel actin-modifying functions of FMNL1 in breast adenocarcinoma migration models. FMNL1 is required for efficient cell migration and its three isoforms exhibit distinct localization. Suppression of FMNL1 protein expression results in a significant impairment of cell adhesion, migration, and invasion. Overexpression of FMNL1ɣ, but not FMNL1β or FMNL1α, enhances cell adhesion independent of the FH2 domain and FMNL1ɣ rescues migration in cells depleted of all three endogenous isoforms. While FMNL1ɣ inhibits actin assembly in vitro, it facilitates bundling of filamentous actin independent of the FH2 domain. The unique interactions of FMNL1ɣ with filamentous actin provide a new understanding of formin domain functions and its effect on motility of diverse cell types suggest a broader role than previously realized.     

The role of vasodilator‐stimulated phosphoprotein in podocyte functioning

Vasodilator‐stimulated phosphoprotein (VASP) is a 39‐kDa protein belonging to the Ena/VASP protein family, which is involved in adhesion, migration, cell–cell interaction, and regulation of pathways connected with actin cytoskeleton remodeling. VASP is phosphorylated at Tyr39, Ser157, Ser239, Thr278, and Ser322 mainly by tyrosine kinase Abl, cAMP‐dependent protein kinase, protein kinase G, AMP‐activated protein kinase, and protein kinase D1, respectively. VASP phosphorylation, as a regulator of actin dynamics, may lead to impaired reorganization of the podocyte actin cytoskeleton not only by indirect interaction of VASP with actin but also by regulation of other signaling pathways. A few studies have shown that VASP participates in the development of renal diseases and mediates podocyte movement through its interaction with proteins of the slit diaphragm. VASP phosphorylation may cause reduced actin filament assembly in podocytes and mediate disturbances in regulation of filtration barrier permeability as a consequence of podocyte foot process effacement. In this paper, we describe the role of VASP in podocyte function, mainly in the context of actin dynamics and glomerular filtration barrier permeability. In addition, we discuss the involvement of VASP and its phosphorylated forms in the development of kidney diseases.     

Two proteins from Saccharomyces cerevisiae: Pfy1 and Pkc1, play a dual role in activating actin polymerization and in increasing cell viability in the adaptive response to oxidative stress

In this work, we show that the proteins Pkc1 and Pfy1 play a role in the repolarization of the actin cytoskeleton and in cell survival in response to oxidative stress. We have also developed an assay to determine the actin polymerization capacity of total protein extracts using fluorescence recovery after photobleaching techniques and actin purified from rabbit muscle. This assay allowed us to demonstrate that Pfy1 promotes actin polymerization under conditions of oxidative stress, while Pkc1 induces actin polymerization and cell survival under all the conditions tested. Our assay also points to a relationship between Pkc1 and Pfy1 in the actin cytoskeleton polymerization that is required to adapt to oxidative stress.     

Fascin,may the Forked be with you

The FGFR pathway triggers a wide range of key biological responses. Among others, the Breathless (Btl, Drosophila FGFR1) receptor cascade promotes cell migration during embryonic tracheal system development. However, how the actin cytoskeleton responds to Btl pathway activation to induce cell migration has remained largely unclear. Our recent results shed light into this issue by unveiling a link between the actin-bundling protein Singed (Sn) and the Btl pathway. We showed that the Btl pathway regulates sn, which leads to the stabilization of the actin bundles required for filopodia formation and actin cytoskeleton rearrangement. This regulation contributes to tracheal migration, tracheal branch fusion and tracheal cell elongation. Parallel actin bundles (PABs) are usually cross-linked by more than one actin-bundling protein. Accordingly, we have also shown that sn synergistically interacts with forked (f), another actin crosslinker. In this Extra View we extend f analysis and hypothesize how both actin-bundling proteins may act together to regulate the PABs during tracheal embryonic development. Although both proteins are required for similar tracheal events, we suggest that Sn is essential for actin bundle initiation and stiffening, while F is required for the lengthening and further stabilization of the PABs.