Microtubule-severing proteins are involved in flagellar length control and mitosis in Trypanosomatids

Microtubules are key players in the biology of Trypanosomatid parasites, not only as classical components of the mitotic spindle, microtubule-organizing centres and flagellum but also as the essential constituent of the cytoskeleton. Their length dynamics are regulated by, among others, microtubule-severing proteins. Four and six genes encoding microtubule-severing proteins can be found bioinformatically in the Leishmania major and Trypanosoma brucei genome respectively. We investigated all these proteins in these organisms, which include the katanin, katanin-like, spastin and fidgetin, and looked at their subcellular localization as well as their putative function by examining 'loss-of-function' phenotypes. The katanin-like KAT60b was found implicated in flagellar length reduction, but not in its size increase, while the katanin p80 subunit appeared clearly involved in cytokinesis. Fidgetin and spastin homologues were both localized in the nucleus: the first as a discrete and variable number of dots during most of the cell cycle, redistributing to the spindle and midbody during mitosis; the second concentrated as ≤ 5 perinucleolar punctuations, similar to the electron-dense plaques identified in T. brucei , which were assimilated to kinetochores. This first study of microtubule-severing proteins in 'divergent' eukaryotes gives further insight into the multiple functions of these proteins identified in the hitherto studied models.     

Rho and Rac take center stage

Many features of cell behavior are regulated by Rho family GTPases, but the most profound effects of these proteins are on the actin cytoskeleton and it was these that first drew attention to this family of signaling proteins. Focusing on Rho and Rac, we will discuss how their effectors regulate the actin cytoskeleton. We will describe how the activity of Rho proteins is regulated downstream from growth factor receptors and cell adhesion molecules by guanine nucleotide exchange factors and GTPase activating proteins. Additionally, we will discuss how there is signaling crosstalk between family members and how various bacterial pathogens have developed strategies to manipulate Rho protein activity so as to enhance their own survival.     

Regulation of Actin Dynamics in Pollen Tubes: Control of Actin Polymer Level

Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.     

Ena/VASP proteins can regulate distinct modes of actin organization at cadherin-adhesive contacts

Functional interactions between classical cadherins and the actin cytoskeleton involve diverse actin activities, including filament nucleation, cross-linking, and bundling. In this report, we explored the capacity of Ena/VASP proteins to regulate the actin cytoskeleton at cadherin-adhesive contacts. We extended the observation that Ena/vasodilator-stimulated phosphoprotein (VASP) proteins localize at cell-cell contacts to demonstrate that E-cadherin homophilic ligation is sufficient to recruit Mena to adhesion sites. Ena/VASP activity was necessary both for F-actin accumulation and assembly at cell-cell contacts. Moreover, we identified two distinct pools of Mena within individual homophilic adhesions that cells made when they adhered to cadherin-coated substrata. These Mena pools localized with Arp2/3-driven cellular protrusions as well as at the tips of cadherin-based actin bundles. Importantly, Ena/VASP activity was necessary for both modes of actin activity to be expressed. Moreover, selective depletion of Ena/VASP proteins from the tips of cadherin-based bundles perturbed the bundles without affecting the protrusive F-actin pool. We propose that Ena/VASP proteins may serve as higher order regulators of the cytoskeleton at cadherin contacts through their ability to modulate distinct modes of actin organization at those contacts.     

Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin B

To investigate roles of the actin cytoskeleton in growth of the pollen tube of Picea meyeri, we used the actin polymerization inhibitor latrunculin B (LATB) under quantitatively controlled conditions. At low concentrations, LATB inhibited polymerization of the actin cytoskeleton in the growing pollen tube, which rapidly inhibited tip growth. The proteomic approach was used to analyse protein expression-profile changes during pollen germination and subsequent pollen-tube development with disturbed organization of the actin cytoskeleton. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed nearly 600 protein spots. A total of 84 of these were differentially displayed at different hours with varying doses of LATB, and 53 upregulated or downregulated proteins were identified by mass spectrometry. These proteins were grouped into distinct functional categories including signalling, actin cytoskeleton organization, cell expansion and carbohydrate metabolism. Moreover, actin disruption affected the morphology of Golgi stacks, mitochondria and amyloplasts, along with a differential expression of proteins involved in their functions. These findings provide new insights into the multifaceted mechanism of actin cytoskeleton functions and its interaction with signalling, cell-expansion machinery and energy-providing pathways.     

Regulation of cortical actin cytoskeleton assembly during polarized cell growth in budding yeast

We have established an in vitro assay for assembly of the cortical actin cytoskeleton of budding yeast cells. After permeabilization of yeast by a novel procedure designed to maintain the spatial organization of cellular constituents, exogenously added fluorescently labeled actin monomers assemble into distinct structures in a pattern that is similar to the cortical actin distribution in vivo. Actin assembly in the bud of small-budded cells requires a nucleation activity provided by protein factors that appear to be distinct from the barbed ends of endogenous actin filaments. This nucleation activity is lost in cells that lack either Sla1 or Sla2, proteins previously implicated in cortical actin cytoskeleton function, suggesting a possible role for these proteins in the nucleation reaction. The rate and the extent of actin assembly in the bud are increased in permeabilized delta cap2 cells, providing evidence that capping protein regulates the ability of the barbed ends of actin filaments to grow in yeast cells. Actin incorporation in the bud can be stimulated by treating the permeabilized cells with GTP-gamma S, and, significantly, the stimulatory effect is eliminated by a mutation in CDC42, a gene that encodes a Rho-like GTP-binding protein required for bud formation. Furthermore, the lack of actin nucleation activity in the cdc42 mutant can be complemented in vitro by a constitutively active Cdc42 protein. These results suggest that Cdc42 is closely involved in regulating actin assembly during polarized cell growth.     

Prk1p

The protein kinase Prk1p (standing for p53 regulating kinase 1) of the yeast Saccharomyces cerevisiae is the prototype of a kinase family identified recently as important regulators of the actin cytoskeleton and endocytosis. These kinases all have a highly homologous serine/threonine kinase domain in their N-terminal region but share no significant homology in other regions. Prk1p also contains a proline-rich motif near its C-terminus that is required for the proper subcellular localization of the protein. The kinase activity of Prk1p has been confirmed by both in vitro and in vivo studies and shown to be essential for the protein's function. To date, several proteins that play essential roles in actin cytoskeleton organization and endocytosis have been identified as the regulatory targets of Prk1p. Phosphorylation on the [L/I/V/N]xx[Q/N/T/S]xTG motifs by Prk1p results in a down-regulation of the functions of these target proteins. The observation that many yeast proteins involved in the actin cytoskeleton organization and endocytosis contain the Prk1p phosphorylation motifs has led to the hypothesis that the Prk1p family of kinases are possibly the general regulators of the actin cytoskeleton and endocytosis in yeast.     

Regulation of actin cytoskeleton architecture by Eps8 and Abi1

Background  

The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive.     

The actin-based motility of intracellular Listeria monocytogenes is not controlled by small GTP-binding proteins of the Rho- and Ras-subfamilies

In this study, we analyzed whether the actin-based motility of intracellular Listeria monocytogenes is controlled by the small GTP-binding proteins of the Rho- and Ras-subfamilies. These signalling proteins are key regulatory elements in the control of actin dynamics and their activity is essential for the maintenance of most cellular microfilament structures. We used the Clostridium difficile toxins TcdB-10463 and TcdB-1470 to specifically inactivate these GTP-binding proteins. Treatment of eukaryotic cells with either of these toxins led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the invasion of epithelial cells by L. monocytogenes and had no effect on the actin-based motility of this bacterial parasite. Our data indicate that intracellular Listeria reorganize the actin cytoskeleton in a way that circumvents the control mechanisms mediated by the members of the Rho- and Ras-subfamilies that can be inactivated by the TcdB-10463 and TcdB-1470 toxins.     

Cortactin adopts a globular conformation and bundles actin into sheets

Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching.     

Role of actin in regulated exocytosis and compensatory membrane retrieval: insights from an old acquaintance

This review summarizes new insights into the role of the actin cytoskeleton in exocytosis and compensatory membrane retrieval from mammalian regulated secretory cells. Data from our lab and others now indicate that the actin cytoskeleton is involved in exocytosis both as a negative regulator of membrane fusion under resting conditions and as a facilitator of movement of secretory granules to their site of fusion with the apical plasmalemma. Coating of docked secretory granules with actin filaments correlates with the dissociation of secretory-granule-associated rab3D, pointing out a novel role for rab proteins in modulating the actin cytoskeleton during regulated exocytosis. Compensatory membrane retrieval following regulated exocytosis is also critically dependent on the actin cytoskeleton both in initiating the formation of clathrin-coated retrieval vesicles and subsequent trafficking back into the cell. We propose that insertion of secretory granule membrane into the plasmalemma initiates a trigger for membrane retrieval, possibly by exposing sites where proteins involved in compensatory membrane retrieval are assembled. The results summarized in this review were derived primarily from investigations on the pancreatic acinar cell, an old friend who is providing modern wisdom not attainable in other simpler systems.     

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.     

Bending over backwards: BAR proteins and the actin cytoskeleton in mammalian receptor-mediated endocytosis

The role of the actin cytoskeleton during receptor-mediated endocytosis (RME) has been well characterized in yeast for many years. Only more recently has the interplay between the actin cytoskeleton and RME been extensively explored in mammalian cells. These studies have revealed the central roles of BAR proteins in RME, and have demonstrated significant roles of BAR proteins in linking the actin cytoskeleton to this cellular process. The actin cytoskeleton generates and transmits mechanical force to promote the extension of receptor-bound endocytic vesicles into the cell. Many adaptor proteins link and regulate the actin cytoskeleton at the sites of endocytosis. This review will cover key effectors, adaptors and signalling molecules that help to facilitate the invagination of the cell membrane during receptor-mediated endocytosis, including recent insights gained on the roles of BAR proteins. The final part of this review will explore associations of alterations to genes encoding BAR proteins with cancer.     

Actin-binding Verprolin Is a Polarity Development Protein Required for the Morphogenesis and Function of the Yeast Actin Cytoskeleton

Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70– amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.     

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.     

Mammalian homolog of the yeast cyclase associated protein, CAP/Srv2p, regulates actin filament assembly

Control of cell shape and motility requires rearrangements of the actin cytoskeleton. One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56). CAP was first isolated from yeast as an adenylyl cyclase associated protein required for RAS regulation of cAMP signaling. In addition, CAP also regulates the actin cytoskeleton primarily through an actin monomer binding activity. CAP homologs are found in many eukaryotes, including mammals where they also bind actin, but little is known about their biological function. We, therefore, designed experiments to address CAP1 regulation of the actin cytoskeleton. CAP1 localized to membrane ruffles and actin stress fibers in fixed cells of various types. To address localization in living cells, we constructed GFP-CAP1 fusion proteins and found that fusion proteins lacking the actin-binding region localized like the wild type protein. We also performed microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts and found that the antibodies attenuated serum stimulation of stress fibers. Finally, CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulated the formation of stress fiber-like filaments when it was microinjected into serum-starved Swiss 3T3 cells. Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton.     

Dynamic actin remodeling in response to lysophosphatidic acid

Abstract

Lysophosphatidic acid (LPA) is a multifunctional regulator of actin cytoskeleton that exerts a dramatic impact on the actin cytoskeleton to build a platform for diverse cellular processes including growth cone guidance, neurite retraction and cell motility. It has been implicated in the formation and dissociation of complexes between actin and actin binding proteins, supporting its role in actin remodeling. Several studies point towards its ability to facilitate formation of special cellular structures including focal adhesions and actin stress fibres by phosphoregulation of several actin associated proteins and their multiple regulatory kinases and phosphatases. In addition, multiple levels of crosstalk among the signaling cascades activated by LPA, affect actin cytoskeleton-mediated cell migration and chemotaxis which in turn play a crucial role in cancer metastasis. In the current review, we have attempted to highlight the role of LPA as an actin modulator which functions by controlling activities of specific cellular proteins that underlie mechanisms employed in cytoskeletal and pathophysiological events within the cell. Further studies on the actin affecting/remodeling activity of LPA in different cell types will no doubt throw up many surprises essential to gain a full understanding of its contribution in physiological processes as well as in diseases.

Communicated by Ramaswamy H. Sarma     

Controlling cytoskeleton structure by phosphoinositide-protein interactions: phosphoinositide binding protein domains and effects of lipid packing

Cell movement and resistance to mechanical forces are largely governed by the cytoskeleton, a three-dimensional network of protein filaments that form viscoelastic networks within the cytoplasm. The cytoskeleton underlying the plasma membrane of most cells is rich in actin filaments whose assembly and disassembly are regulated by actin binding proteins that are stimulated or inhibited by signals received and transmitted at the membrane/cytoplasm interface. Inositol phospholipids, or phosphoinositides, are potent regulators of many actin binding proteins, and changes in the phosphorylation of specific phosphoinositide species or in their spatial localization are associated with cytoskeletal remodeling in vitro. This review will focus on recent studies directed at defining the structural features of phosphoinositide binding sites in actin binding proteins and on the influence of the physical state of phosphoinositides on their ability to interact with their target proteins.     

crossveinless-c is a RhoGAP required for actin reorganisation during morphogenesis

Members of the Rho family of small GTPases are required for many of the morphogenetic processes required to shape the animal body. The activity of this family is regulated in part by a class of proteins known as RhoGTPase Activating Proteins (RhoGAPs) that catalyse the conversion of RhoGTPases to their inactive state. In our search for genes that regulate Drosophila morphogenesis, we have isolated several lethal alleles of crossveinless-c (cv-c). Molecular characterisation reveals that cv-c encodes the RhoGAP protein RhoGAP88C. During embryonic development, cv-c is expressed in tissues undergoing morphogenetic movements; phenotypic analysis of the mutants reveals defects in the morphogenesis of these tissues. Genetic interactions between cv-c and RhoGTPase mutants indicate that Rho1, Rac1 and Rac2 are substrates for Cv-c, and suggest that the substrate specificity might be regulated in a tissue-dependent manner. In the absence of cv-c activity, tubulogenesis in the renal or Malpighian tubules fails and they collapse into a cyst-like sack. Further analysis of the role of cv-c in the Malpighian tubules demonstrates that its activity is required to regulate the reorganisation of the actin cytoskeleton during the process of convergent extension. In addition, overexpression of cv-c in the developing tubules gives rise to actin-associated membrane extensions. Thus, Cv-c function is required in tissues actively undergoing morphogenesis, and we propose that its role is to regulate RhoGTPase activity to promote the coordinated organisation of the actin cytoskeleton, possibly by stabilising plasma membrane/actin cytoskeleton interactions.     

Crucial role for the LSP1–myosin1e bimolecular complex in the regulation of Fcγ receptor–driven phagocytosis

Actin cytoskeleton remodeling is fundamental for Fcγ receptor–driven phagocytosis. In this study, we find that the leukocyte-specific protein 1 (LSP1) localizes to nascent phagocytic cups during Fcγ receptor–mediated phagocytosis, where it displays the same spatial and temporal distribution as the actin cytoskeleton. Down-regulation of LSP1 severely reduces the phagocytic activity of macrophages, clearly demonstrating a crucial role for this protein in Fcγ receptor–mediated phagocytosis. We also find that LSP1 binds to the class I molecular motor myosin1e. LSP1 interacts with the SH3 domain of myosin1e, and the localization and dynamics of both proteins in nascent phagocytic cups mirror those of actin. Furthermore, inhibition of LSP1–myosin1e and LSP1–actin interactions profoundly impairs pseudopodial formation around opsonized targets and their subsequent internalization. Thus the LSP1–myosin1e bimolecular complex plays a pivotal role in the regulation of actin cytoskeleton remodeling during Fcγ receptor–driven phagocytosis.