Regulating the actin cytoskeleton during vesicular transport

Although the actin cytoskeleton is widely believed to play an important role in intracellular protein transport, this role is poorly understood. Recently, progress has been made toward identifying specific actin-binding proteins and signaling molecules involved in regulating actin structures that function in the secretory pathway. Studies on coat protomer I (COPI)-mediated transport at the Golgi apparatus and on clathrin-mediated endocytosis have been particularly informative in identifying such mechanisms. Important similarities between actin regulation at the Golgi and at the plasma membrane have been uncovered. The studies reveal that ADP-ribosylation factor and vesicle coat proteins are able to act through the Rho-family GTP-binding proteins, Cdc42 and Rac, and several specific actin-binding proteins to direct actin assembly through the Arp2/3 complex. Efficient function of the secretory pathway is likely to require precise temporal regulation among transport-vesicle assembly, vesicle scission, and the targeting machinery. It is proposed that numerous actin regulatory mechanisms and the connections between actin signaling and vesicle-coat formation are employed to provide such temporal regulation.     

Interplay between membrane curvature and the actin cytoskeleton

An intimate interplay of the plasma membrane with curvature-sensing and curvature-inducing proteins would allow for defining specific sites or nanodomains of action at the plasma membrane, for example, for protrusion, invagination, and polarization. In addition, such connections are predestined to ensure spatial and temporal order and sequences. The combined forces of membrane shapers and the cortical actin cytoskeleton might hereby in particular be required to overcome the strong resistance against membrane rearrangements in case of high plasma membrane tension or cellular turgor. Interestingly, also the opposite might be necessary, the inhibition of both membrane shapers and cytoskeletal reinforcement structures to relieve membrane tension to protect cells from membrane damage and rupturing during mechanical stress. In this review article, we discuss recent conceptual advances enlightening the interplay of plasma membrane curvature and the cortical actin cytoskeleton during endocytosis, modulations of membrane tensions, and the shaping of entire cells.     

Functional interdependence between septin and actin cytoskeleton

Background  

Septin2 is a member of a highly conserved GTPase family found in fungi and animals. Septins have been implicated in a diversity of cellular processes including cytokinesis, formation of diffusion barriers and vesicle trafficking. Septin2 partially co-localises with actin bundles in mammalian interphase cells and Septin2-filamentmorphology depends upon an intact actin cytoskeleton. How this interaction is regulated is not known. Moreover, evidence that Septin2 is remodelled or redistributed in response to other changes in actin organisation is lacking.     

Functional linkage between the transport characteristics of the MDCK1 cell monolayer and their actin cytoskeleton organization

The dynamics of the actin cytoskeleton spatial organization and transepithelial electric resistance (TEER) in the MDCK1 cell monolayer exposed to arginine–vasopressin (AVP) and forskolin, a protein kinase A (PKA) activator, have been studied. These physiologically active substances are shown to depolymerize filamentous actin in MDCK1 cells (in both the apical and basal cytoplasm) and, concurrently, to considerably decrease the TEER of the cell monolayer. A decrease in TEER suggests an increase in the ion current through the cell monolayer. Correspondingly, the created ion gradient stimulates AVP-sensitive water flow. To clarify the routes of ions and water in MDCK monolayer, the localization of claudin-1 and -2 in tight junctions of ATCC (American Type Culture Collection) MDCK (a low TEER) and MDCK1 (a high TEER) cells was studied by immunofluorescence assay. Claudin-1 and -2 are detectable in the tight junctions of ATCC MDCK cells; however, the tight junctions of MDCK1 cells contain only claudin-1, whereas poreforming claudin-2 is absent. The exposure of MDCK1 cells to forskolin fails to change the distribution of the studied claudins, thereby suggesting that a decrease in TEER caused by forskolin is associated with a change in transcellular, rather than paracellular, permeability of the monolayer     

Mutual regulation between Hippo signaling and actin cytoskeleton

Hippo signaling plays a crucial role in growth control and tumor suppression by regulating cell proliferation, apoptosis, and differentiation. How Hippo signaling is regulated has been under extensive investigation. Over the past three years, an increasing amount of data have supported a model of actin cytoskeleton blocking Hippo signaling activity to allow nuclear accumulation of a downstream effector, Yki/Yap/Taz. On the other hand, Hippo signaling negatively regulates actin cytoskeleton organization. This review provides insight on the mutual regulatory mechanisms between Hippo signaling and actin cytoskeleton for a tight control of cell behaviors during animal development, and points out outstanding questions for further investigations.     

Integrins and the actin cytoskeleton

The ability to connect to the actin cytoskeleton is a key part of the adhesive function of integrins. This linkage between integrins and the cytoskeleton involves a large complex of integrin-associated proteins that function in both the assembly and disassembly of the link. Genetic evidence has helped to clarify the relative contributions of different components of this link. In different contexts integrins can either stimulate or suppress actin based structures, indicating the variety of pathways leading from integrins to the cytoskeleton. The cytoskeleton also contributes to the extent of the integrin junction, allowing an adhesive contact to attain sufficient strength to resist contractile forces involved in cellular movement and function.     

A functional link between dynamin and the actin cytoskeleton at podosomes

Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2-binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aa(G273D) abolished podosomes while GFP-dynamin(K44A) was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.     

Hydrostatic pressure regulates tight junctions, actin cytoskeleton and transcellular ion transport

In the epithelia and endothelia, tight junctions regulate the movement of several substances through the paracellular pathway, maintaining several gradients between apical and basal compartments including osmolality and hydrostatic pressure. In this study, we show that the change of hydrostatic pressure gradient affected tight junctions as well as actin cytoskeleton, cell height and transcellular ion transport. Hydrostatic pressure gradient from basolateral to apical side increased transepithelial conductance and altered claudin-1 localization within several tens of minutes. These changes were promptly restored by the elimination of hydrostatic pressure gradient. Hydrostatic pressure gradient also induced dynamic changes in the actin structure and cell height. We further found that hydrostatic pressure gradient from basolateral to apical side stimulates transcellular Cl⁻ transport. Our present findings indicate that the epithelial cell structures and functions are regulated by the hydrostatic pressure gradient which is generated and maintained by the epithelia themselves.     

Novel actin cytoskeleton: actin tubules

In spores of Dictyostelium discoideum three actin filaments are bundled to form a novel tubular structure and the tubules are then organized into rods. These tubular structures we will term actin tubules. Actin tubules are reconstructed from the supernatant of spore homogenates, while the usual actin filaments were bundled after incubation of supernatants from growing cells. Alpha-actinin, ABP-120 and EF-1alpha are not essential for rod formation. Cofilin is a component of the cytoplasmic rods but few cofilin molecules are included in the nuclear rods. The viability of spores lacking actin rods is very low, and the spore shape is round instead of capsular. The rods can be fragmented by pressure, indicating that the rods may be effective in absorbing physical pressure. The complex organization of actin filaments, actin tubules and rods may be required for spores to achieve complete dormancy and maintain viability.     

Identification of functional connections between calmodulin and the yeast actin cytoskeleton.

One of four intragenic complementing groups of temperature-sensitive yeast calmodulin mutations, cmd1A, results in a characteristic functional defect in actin organization. We report here that among the complementing mutations, a representative cmd1A mutation (cmd1-226: F92A) is synthetically lethal with a mutation in MYO2 that encodes a class V unconventional myosin with calmodulin-binding domains. Gel overlay assay shows that a mutant calmodulin with the F92A alteration has severely reduced binding affinity to a GST-Myo2p fusion protein. Random replacement and site-directed mutagenesis at position 92 of calmodulin indicate that hydrophobic and aromatic residues are allowed at this position, suggesting an importance of hydrophobic interaction between calmodulin and Myo2p. To analyze other components involved in actin organization through calmodulin, we isolated and characterized mutations that show synthetic lethal interaction with cmd1-226; these "cax" mutants fell into five complementation groups. Interestingly, all the mutations themselves affect actin organization. Unlike cax2, cax3, cax4, and cax5 mutations, cax1 shows allele-specific synthetic lethality with the cmd1A allele. CAX1 is identical to ANP1/GEM3/MCD2, which is involved in protein glycosylation. CAX4 is identical to the ORF YGR036c, and CAX5 is identical to MNN10/SLC2/BED1. We discuss possible roles for Cax proteins in the regulation of the actin cytoskeleton.     

Pathogenicity of bacteria and the actin cytoskeleton

Actin system of eukaryotic cells creates the driving force for alteration of the phagocytic cytoplasmatic membrane shape, which is needed for cell movement in the space and for microorganism capturing. Manipulation by actin cytoskeleton mediated through specialized bacterial products can promote proliferation of bacteria in the host. Published reports indicate that bacterial regulation of the actin system activity can be carried out by two modes: 1) by bacterial interactions with surface receptors regulating the cytoskeleton status and 2) by introduction of bacterial products targeted to the cytoskeleton components into the cells. Intracellular pathogens (Legionella) possess ligands which interact with eukaryotic receptors and type IV secretion system fit for translocation of heretofore unknown effector molecules into the cytoplasm. This can result in stimulation of actin polymerization activity and accelerated phagocytosis of the bacteria with rapid multiplication in tissues. By contrast, representatives of extracellular pathogens (Clostridium) produce substances penetrating inside the eukaryotic cells and destroying the actin network, thus making capturing and intracellular digestion of these microorganisms impossible.     

Actin in action: the interplay between the actin cytoskeleton and synaptic efficacy

Synapse regulation exploits the capacity of actin to function as a stable structural component or as a dynamic filament. Beyond its well-appreciated role in eliciting visible morphological changes at the synapse, the emerging picture points to an active contribution of actin to the modulation of the efficacy of pre- and postsynaptic terminals. Moreover, by engaging distinct pools of actin and divergent signalling pathways, actin-dependent morphological plasticity could be uncoupled from modulation of synaptic strength. The aim of this Review is to highlight some of the recent progress in elucidating the role of the actin cytoskeleton in synaptic function.     

Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton in Saccharomyces cerevisiae

The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1 gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, we screened for mutations that were synthetically lethal in combination with a bni1 mutation and isolated two genes. They were the previously identified PAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150(Glued), a component of the dynein-activated dynactin complex. Disruption of BNI1 in either the pac1 or nip100 mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1 mutant showed a synthetic lethal phenotype while the cin8 bni1 mutant did not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibited a random distribution of cortical actin patches. Cells of the pac1 act1-4 mutant showed temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region did not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.     

Mythical origins of the actin cytoskeleton

The origin of the eukaryotic cell is one of the greatest mysteries in modern biology. Eukaryotic-wide specific biological processes arose in the lost ancestors of eukaryotes. These distinctive features, such as the actin cytoskeleton, define what it is to be a eukaryote. Recent sequencing, characterization, and isolation of Asgard archaea have opened an intriguing window into the pre-eukaryotic cell. Firstly, sequencing of anaerobic sediments identified a group of uncultured organisms, Asgard archaea, which contain genes with homology to eukaryotic signature genes. Secondly, characterization of the products of these genes at the protein level demonstrated that Asgard archaea have related biological processes to eukaryotes. Finally, the isolation of an Asgard archaeon has produced a model organism in which the morphological consequences of the eukaryotic-like processes can be studied. Here, we consider the consequences for the Asgard actin cytoskeleton and for the evolution of a regulated actin system in the archaea-to-eukaryotic transition.     

Interactions between auxin transport and the actin cytoskeleton in developmental polarity of Fucus distichus embryos in response to light and gravity

Land plants orient their growth relative to light and gravity through complex mechanisms that require auxin redistribution. Embryos of brown algae use similar environmental stimuli to orient their developmental polarity. These studies of the brown algae Fucus distichus examined whether auxin and auxin transport are also required during polarization in early embryos and to orient growth in already developed tissues. These embryos polarize with the gravity vector in the absence of a light cue. The auxin, indole-3-acetic acid (IAA), and auxin efflux inhibitors, such as naphthylphthalamic acid (NPA), reduced environmental polarization in response to gravity and light vectors. Young rhizoids are negatively phototropic, and NPA also inhibits rhizoid phototropism. The effect of IAA and NPA on gravity and photopolarization is maximal within 2.5 to 4.5 h after fertilization (AF). Over the first 6 h AF, auxin transport is relatively constant, suggesting that developmentally controlled sensitivity to auxin determines the narrow window during which NPA and IAA reduce environmental polarization. Actin patches were formed during the first hour AF and began to photolocalize within 3 h, coinciding with the time of NPA and IAA action. Treatment with NPA reduced the polar localization of actin patches but not patch formation. Latrunculin B prevented environmental polarization in a time frame that overlaps the formation of actin patches and IAA and NPA action. Latrunculin B also altered auxin transport. Together, these results indicate a role for auxin in the orientation of developmental polarity and suggest interactions between the actin cytoskeleton and auxin transport in F. distichus embryos.     

Integrin signaling to the actin cytoskeleton

Integrin engagement stimulates the activity of numerous signaling molecules, including the Rho family of GTPases, tyrosine phosphatases, cAMP-dependent protein kinase and protein kinase C, and stimulates production of PtdIns(4,5)P2. Integrins promote actin assembly via the recruitment of molecules that directly activate the actin polymerization machinery or physically link it to sites of cell adhesion.