Formation Of Actin Filament Bundles Research Articles

Prostaglandins temporally regulate cytoplasmic actin bundle formation duringDrosophilaoogenesis

Prostaglandins (PGs)--lipid signals produced downstream of cyclooxygenase (COX) enzymes--regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton--temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling.

Circulation Editors’ Picks

<i>Circulation</i> Editors’ Picks

DrosophilaFascin is a novel downstream target of prostaglandin signaling during actin remodeling

Although prostaglandins (PGs)-lipid signals produced downstream of cyclooxygenase (COX) enzymes-regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

P‐15 functionalized porous microspheres as biomimetic habitats for bone tissue engineering applications

AbstractPoly(L‐lactide‐co‐ε‐caprolactone) (PLCL, 75/25) solid and porous microspheres (MS) were prepared using solvent evaporation and gas foaming method, respectively. These MS were characterized for particle size, surface morphology, porosity, and surface area. Porous MS were modified by P‐15 peptide using carbodiimide coupling method. These unmodified solid and P‐15‐modified/‐unmodified porous MS were further evaluated for MG‐63 proliferation, cell alignment, collagen type I expression, and matrix mineralization for 21 days. In vitro cell culture studies of P‐15 functionalized porous MS exhibited enhanced cell viability, increased formation of actin filament bundles and collagen type I expression as compared to solid MS and 2D (polystyrene 24 well plate), and maintained the mineralization potential of MG‐63 cells. The results suggests P‐15‐modified porous PLCL MS were able to grow the cells with proper cell functionality and hold potential to be used as an injectable cell carrier for bone tissue engineering applications. Copyright © 2010 John Wiley &amp; Sons, Ltd.

The C. elegans PH Domain Protein CED-12 Regulates Cytoskeletal Reorganization via a Rho/Rac GTPase Signaling Pathway

The C. elegans gene ced-12 functions in the engulfment of apoptotic cells and in cell migration, acting in a signaling pathway with ced-2 CrkII, ced-5 DOCK180, and ced-10 Rac GTPase and acting upstream of ced-10 Rac. ced-12 encodes a protein with a pleckstrin homology (PH) domain and an SH3 binding motif, both of which are important for ced-12 function. CED-12 acts in engulfing cells for cell corpse engulfment and interacts physically with CED-5, which contains an SH3 domain. CED-12 has Drosophila and human counterparts. Expression of CED-12 and its counterparts in murine Swiss 3T3 fibroblasts induced Rho GTPase-dependent formation of actin filament bundles. We propose that through interactions with membranes and with a CED-2/CED-5 protein complex, CED-12 regulates Rho/Rac GTPase signaling and leads to cytoskeletal reorganization by an evolutionarily conserved mechanism.

Pleckstrin Homology Domains Interact with Filamentous Actin

A fraction of Bruton's tyrosine kinase (Btk) co-localizes with actin fibers upon stimulation of mast cells via the high affinity IgE receptor (FcepsilonRI). In this study, a molecular basis of the Btk co-localization with actin fibers is presented. Btk and other Tec family tyrosine kinases have a pleckstrin homology (PH) domain at their N termini. The PH domain is a short peptide module frequently found in signal-transducing proteins and cytoskeletal proteins. Filamentous actin (F-actin) is shown to be a novel ligand for a subset of PH domains, including that of Btk. The actin-binding site was mapped to a 10-residue region of the N-terminal region of Btk. Basic residues in this short stretch are demonstrated to be involved in actin binding. Isolated PH domains induced actin filament bundle formation. Consistent with these observations, Btk binds F-actin in vitro and in vivo. Wild-type Btk protein is in part translocated to the cytoskeleton upon FcepsilonRI cross-linking, whereas Btk containing a mutated PH domain is not. Phosphatidylinositol 3,4, 5-trisphosphate-mediated membrane translocation of Btk was enhanced in cytochalasin D-pretreated, FcepsilonRI-stimulated mast cells. These data indicate that PH domain-mediated F-actin binding plays a role in Btk co-localization with actin filaments.

Sorting of actin isoforms in chicken auditory hair cells.

Most nonmuscle cells of higher vertebrates contain two different actin isoforms, beta- and gamma-cytoplasmic actin. The beta-isoform is with few exceptions the predominant isoform in nonmuscle cells and tissues. Perturbation of the beta:gamma ratio has been shown to affect the organization of bundled actin filaments indicating that the beta- and gamma-genes encode functionally distinct cytoarchitectural information. In the present study we localized by immunostaining beta- and gamma-actin in chicken auditory hair cells. These highly specialized cells serve as model system for studying certain developmental and structural aspects of a complex actin filament system with high architectural precision. We show that gamma-actin is the predominant actin isoform in auditory hair cells with an apparent beta:gamma ratio of approximately 1:2. gamma-Actin is not sorted and occurs in all three actin assemblies of the hair border, i.e. the cores of sensory hairs (stereocilia), the subjacent gel-like actin filament meshwork (cuticular plate) and the zonula adherens ring. In contrast to gamma-actin, the beta-isoform is specifically sorted to the actin filament core bundle of stereocilia that is extensively crosslinked by fimbrin. In view of recent studies showing that L-plastin, the leukocyte homolog of fimbrin, has a higher binding affinity for beta-actin than for gamma-actin, a mechanism is proposed for how hair cells might restrict formation of actin filament bundles to a single cellular site (i.e. the stereocilia). The limited level of expression of beta-actin in hair cells may help to prevent ectopic bundle formation in other cellular compartments.

Surface shape changes and cortical actin reorganization associated with the growth of microvilli in the sea urchin egg

This paper investigates the relationship between the assembly of the cortical cytoskeleton and the reorganization of the egg surface following fertilization in the sea urchin. The surface of the unfertilized egg is covered with short microvilli, with dimensions of ∼0.15 × 0.3 μm. Between 30 and 60 sec after insemination, slender finger-like processes, ∼2 μm in length and 0.05 μm in diameter, grow from the tips of these short microvilli. Over the next 30 sec, clusters of these processes transform into broad protrusions of the egg surface, containing networks of actin filaments. By 2 min postinsemination, bundles of filaments start to form within these networks, beginning at the tips of the protrusions and growing inward. As the filament bundles develop, the plasma membrane encases them to form typical microvilli with average dimensions of 0.15 × 2 μm. The formation of the network of cortical actin filaments occurs during the transient increase in free cytoplasmic Ca++ concentration, while the bundling of actin filaments occurs during the period of cytoplasmic alkalization. When cytoplasmic alkalization is inhibited, actin filament networks fail to reorganize into bundles, but microvilli develop by an identical series of surface shape changes. Eggs fertilized in the presence of cytochalasin B develop irregular surface protrusions that gradually transform into microvillar-like structures over a period of ∼30 min without undergoing any definable series of shape changes or assembly of a normal cortical cytoskeleton. These results demonstrate that, while the formation of the large surface protrusions requires an intact network of cortical actin filaments, partitioning of these protrusions into microvilli does not depend on the formation of actin filament bundles. Further, microvillar-like structures are still able to form in eggs in which the normal sequence of surface shape changes has been blocked by inhibiting the assembly of the cortical actin filament network. J. Exp. Zool. 277:230–244, 1997. © 1997 Wiley-Liss, Inc.

Surface shape changes and cortical actin reorganization associated with the growth of microvilli in the sea urchin egg

This paper investigates the relationship between the assembly of the cortical cytoskeleton and the reorganization of the egg surface following fertilization in the sea urchin. The surface of the unfertilized egg is covered with short microvilli, with dimensions of ∼0.15 × 0.3 μm. Between 30 and 60 sec after insemination, slender finger-like processes, ∼2 μm in length and 0.05 μm in diameter, grow from the tips of these short microvilli. Over the next 30 sec, clusters of these processes transform into broad protrusions of the egg surface, containing networks of actin filaments. By 2 min postinsemination, bundles of filaments start to form within these networks, beginning at the tips of the protrusions and growing inward. As the filament bundles develop, the plasma membrane encases them to form typical microvilli with average dimensions of 0.15 × 2 μm. The formation of the network of cortical actin filaments occurs during the transient increase in free cytoplasmic Ca++ concentration, while the bundling of actin filaments occurs during the period of cytoplasmic alkalization. When cytoplasmic alkalization is inhibited, actin filament networks fail to reorganize into bundles, but microvilli develop by an identical series of surface shape changes. Eggs fertilized in the presence of cytochalasin B develop irregular surface protrusions that gradually transform into microvillar-like structures over a period of ∼30 min without undergoing any definable series of shape changes or assembly of a normal cortical cytoskeleton. These results demonstrate that, while the formation of the large surface protrusions requires an intact network of cortical actin filaments, partitioning of these protrusions into microvilli does not depend on the formation of actin filament bundles. Further, microvillar-like structures are still able to form in eggs in which the normal sequence of surface shape changes has been blocked by inhibiting the assembly of the cortical actin filament network. J. Exp. Zool. 277:230–244, 1997. © 1997 Wiley-Liss, Inc.

Formation of actin filament bundles in the ring canals of developing Drosophila follicles.

Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay the groundwork for our understanding of how a noncontractile ring increases in thickness, diameter, and length during development.

Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles.

The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion.

Interaction of calponin with actin and its functional implications

Titration of F-actin with calponin causes the formation of two types of complexes. One, at saturation, contains a lower ratio of calponin to actin (0.5:1) and is insoluble at physiological ionic strength. The another is soluble, with a higher ratio of calponin to actin (1:1). Electron microscopy revealed that the former complex consists of paracrystalline bundles of actin filaments, whereas the latter consists of separate filaments. Ca(2+)-calmodulin causes dissociation of bundles with simultaneous increase in the number of separate calponin-containing filaments. Further increase in the calmodulin concentration results in full release of calponin from actin filaments. In motility assays, calponin, when added together with ATP to actin filaments complexed with immobilized myosin, evoked a decrease in both the number and velocity of moving actin filaments. Addition of calponin to actin filaments before their binding to myosin resulted in a formation of actin filament bundles which were dissociated by ATP.

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension.

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.

Immunohistochemical demonstration of villin in the normal human pancreas and in chronic pancreatitis

Human pancreatic tissue was investigated by immunohistochemistry using a polyclonal antibody against the actin binding protein villin, which participates in the formation of actin filament bundles in the microvilli. In cells of the different parts of the pancreatic duct system as well as in the acinar cells villin immunoreactivity was located mainly at the apical cell surface. This was confirmed by the ultrastructural demonstration of microvilli on the surface of duct and acinar cells, which exhibited the typical actin bundles. In chronic pancreatitis the staining for villin in duct-like structures of degenerative pancreatic tissue was irregular or even absent. This correlated with the electron microscopic observation of duct-like structures known as tubular complexes composed of cells devoid of microvilli at the apical cell surface. At the light microscopical level degenerative structures without lumen and of unknown origin showed a strong staining for villin at their basal cell surface.

A substrate of the cell-attachment sequence of fibronectin (Arg-Gly-Asp-Ser) is sufficient to promote transition of arterial smooth muscle cells from a contractile to a synthetic phenotype

Extracellular matrix components strongly influence the differentiated properties of isolated rat arterial smooth muscle cells during in vitro cultivation. The attachment and spreading of the cells on a substrate of fibronectin or a 105-kDa cell-binding fragment of fibronectin are accompanied by a structural and functional transformation, referred to as a transition or modulation from a contractile to a synthetic phenotype. Here, the ability of the cell-attachment sequence of fibronectin, Arg-Gly-Asp-Ser (RGDS), to promote this process was studied. The results demonstrate that freshly isolated smooth muscle cells attached to a substrate of the synthetic peptide Gly-Arg-Gly-Asp-Ser-Cys (GRGDSC) in a specific manner and as well as to substrates of fibronectin and the 105-kDa fragment. Subsequent spreading of the cells on the peptide substrate followed the same kinetics and was as extensive as on fibronectin, even if protein synthesis was blocked by treatment of the cultures with cycloheximide. Like fibronectin, the peptide substrate induced formation of actin filament bundles, again without ongoing protein synthesis. Moreover, it was as efficient as fibronectin in supporting the transition of the cells from a contractile to a synthetic phenotype as analyzed by electron microscopy. Antibodies against the β subunit of the fibronectin receptor interfered with the attachment, spreading, and fine structural reorganization of the cells in a similar manner on substrates of fibronectin, the 105-kDa fragment, and GRGDSC. Taken together, the findings indicate that the cell-attachment sequence (RGDS) mimics intact fibronectin in promoting a change in the differentiated properties of arterial smooth muscle cells and does so by interacting with a cell surface receptor for fibronectin.

Calmodulin inhibits interaction of actin with MAP2 and Tau, two major microtubule-associated proteins.

We have previously shown that microtubule-associated protein 2 (MAP2) and Tau, two major microtubule-associated proteins, interact with actin differently as measured by low-shear viscosity and that their activities are modified by phosphorylation (Nishida, E., Kotani, S., Kuwaki, T., and Sakai, H. (1982 in Biological Functions of Microtubules and Related Structures (Sakai, H., Mohri, H., and Borisy, G. G., eds) pp. 297-309, Academic Press, Japan). In the present study we further examined their interaction using turbidimetry, electron microscopy, low- and high-shear viscometry. MAP2 increased the low-shear viscosity of actin filament but had weaker effect on high-shear viscosity and turbidity of actin filaments. In contrast, Tau reduced high-shear viscosity of actin filaments and enhanced the turbidity which were due to formation of actin filament bundles as shown by electron microscopy. We conclude that MAP2 is a gelation factor, while Tau is a bundling factor. A well-known Ca2+-dependent regulatory protein, calmodulin, inhibited both MAP2-actin and Tau-actin interaction in a Ca2+-dependent manner. The calmodulin-dependent inhibition was canceled by higher concentrations of MAP2 or Tau, and calmodulin had no effect on the viscosity of actin filament alone, indicating that this inhibition is based on the stoichiometric interaction of calmodulin with MAP2 or Tau.

Skeletal muscle myosin subfragment-1 induces bundle formation by actin filaments.

As is well known, the light scattering intensity of F-actin solutions increases immediately upon formation of the rigor complex with subfragment-1 (S-1). We have found that after the initial rise in scattering, there is a further gradual increase in scattering (we call it "super-opalescence"). Fluorescence and electron microscopic observations of acto-S-1 solutions showed that super-opalescence results from formation of actin filament bundles once S-1 binds to F-actin. The actin bundles possessed transverse stripes with a periodicity of about 350 A, which suggested that in the bundles actin filaments are arranged in parallel register. The rate of the initial process of bundle formation (i.e. side-by-side dimerization) could be approximately estimated by measuring the initial rate of super-opalescence (V0). V0 had a maximum (V0m) at a molar ratio of S-1 to actin of 1;6-1;7, regardless of the actin concentration, pH (6-8.5), Mg2+ concentration (up to 5 mM), or ionic strength (up to 0.3 M KC1). Lower pH, higher Mg2+ concentration, and higher ionic strength increased V0m; V0 was proportional to the square of the actin concentration, regardless of the solution conditions.

Isolation of actin-binding protein and villin from toad oocytes

Two actin-modulating proteins have been purified from toad oocytes. A high-molecular weight protein, similar in structure and function to macrophage actin-binding protein, accounts for the isotropic actin-crosslinking activity in oocyte homogenates. A calcium-dependent activity in toad oocyte homogenates which shortens actin filaments is accounted for by a 95,000-dalton protein which resembles villin, an actin-severing and -bundling protein of avian epithelial brush borders. In the presence of high (⩾ μM) calcium, this protein shortens actin filaments in a concentration-dependent fashion and stimulates filament assembly when added to monomeric actin. In the absence of calcium the protein promotes the formation of actin filament bundles. Therefore, in the toad oocyte actin can be crosslinked into a network by actin-binding protein. Calcium regulation of the actin network may be mediated by villin. These results are different from those reported in echinoderm eggs.

Structural organization of actin in the sea urchin egg cortex: microvillar elongation in the absence of actin filament bundle formation.

We have investigated the relationship between the formation of actin filament bundles and the elongation of microvilli (MV) after fertilization in sea urchin eggs. In a previous study (1979, J Cell Biol. 83:241-248) we demonstrated that increased pH induced the formation of actin filaments in isolated sea urchin egg cortices with the concomitant elongation of MV. On the basis of these results we suggested that increased cytoplasmic pH after fertilization causes a reorganization of cortical actin, which in turn provides the force for MV elongation. To test this hypothesis, we compared the morphology of microvilli in eggs activated with and without the release of fertilization acid. Activation of eggs in normal sea water with the calcium ionophore A23187 causes the release of fertilization acid and the elongation of MV containing core bundles of actin filaments. Eggs activated with A23187 in NA(+)-free water do not undergo normal fertilization acid release but develop elongated, flaccid MV. These MV contain an irregular network of actin filaments rather than the parallel bundles of filaments found in normal MV. The addition of 40 mM NaCl to these eggs results in the release of H(+) and the concomitant conversion of flaccid MV to erect MV containing typical core bundles of actin filaments. Identical results are obtained when 10 mM NH(4)Cl is substituted for NaCl. The induction of cytoplasmic alkalinization in unactivated eggs with NH(4)Cl does not cause either MV elongation or the formation of actin filament bundles . These results suggest that: (a) the elongation of MV is stimulated by a rise in intracellular free Ca(++) concentration; (b) actin filament bundle formation is triggered by an increase in cytoplasmic pH; and (c) the formation of actin filament bundles is not necessary for MV elongation but is required to provide rigid support for MV.