Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP.

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and noncoated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgito-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(AWA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-NWASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP¿dependent manner.

Myosin motors and not actin comets are mediators of the actin-based Golgi-to-endoplasmic reticulum protein transport

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2AA). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2AA mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2AA. Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.

Clearance of a hirano body-like F-actin aggresome generated by jasplakinolide

We have reported in a variety of mammalian cells the reversible formation of a filamentous actin (F-actin)-enriched aggresome generated by the actin toxin jasplakinolide (Lázaro-Diéguez et al., J Cell Sci 2008; 121:1415-25). Notably, this F-actin aggresome (FAG) resembles in many aspects the pathological Hirano body, which frequently appears in some diseases such as Alzheimer's and alcoholism. Using selective inhibitors, we examined the molecular and subcellular mechanisms that participate in the clearance of the FAG. Chaperones, microtubules, proteasomes and autophagosomes all actively participate to eliminate the FAG. Here we compile and compare these results and discuss the involvement of each process. Because of its simplicity and high reproducibility, our cellular model could help to test pharmacological agents designed to interfere with the mechanisms involved in the clearance of intracellular bodies and, in particular, of those enriched in F-actin.

Ligand binding to heparan sulfate proteoglycans induces their aggregation and distribution along actin cytoskeleton

Cell surface heparan sulfate proteoglycans (HSPGs) participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study demonstrates that soluble heparin-binding proteins or cross-linking antibodies induce the aggregation of cell surface HSPGs and their distribution along underlying actin filaments. Immunofluorescence and confocal microscopy and immunogold and electron microscopy indicate that, in the absence of ligands, HSPGs are irregularly distributed on the fibroblast cell surface, without any apparent codistribution with the actin cytoskeleton. In the presence of ligand (lipoprotein lipase) or antibodies against heparan sulfate, HSPGs aggregate and colocalize with the actin cytoskeleton. Triton X-100 extraction and immunoelectron microscopy have demonstrated that in this condition HSPGs were clustered and associated with the actin filaments. Crosslinking experiments that use biotinylated lipoprotein lipase have revealed three major proteoglycans as binding sites at the fibroblast cell surface. These cross-linked proteoglycans appeared in the Triton X-100 insoluble fraction. Platinum/carbon replicas of the fibroblast surface incubated either with lipoprotein lipase or antiheparan sulfate showed large aggregates of HSPGs regularly distributed along cytoplasmic fibers. Quantification of the spacing between HSPGs by confocal microscopy confirmed that the nonrandom distribution of HSPG aggregates along the actin cytoskeleton was induced by ligand binding. When cells were incubated either with lipoprotein lipase or antibodies against heparan sulfate, the distance between immunofluorescence spots was uniform. In contrast, the spacing between HSPGs on fixed cells not incubated with ligand was more variable. This highly organized spatial relationship between actin and proteoglycans suggests that cortical actin filaments could organize the molecular machinery involved in signal transduction and molecular movements on the cell surface that are triggered by heparin-binding proteins.

Actin acting at the Golgi

The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in traf- ficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers.

Regulación de la movilidad celular por Profilina 1

Mi proyecto de tesis se basaba en el estudio del papel de profilina 1 en la formación de lamelas, para ello generamos una proteína recombinante y transducible, con el objetivo de poder modificar los niveles endógenos de profilina. Objetivos: i-caracterización bioquímica los tres sitios de union conocidos de la proteína de transducción, el sitio de unión a fosfo-inocitoles (PIP), el de unión a actina (Ac) y el de unión a poli-prolinas (PLP). ii-estudio de la polimerización in-vitro de actina - PTD4-Profilina1 iii-estudio de las proteínas componentes de lamelas inducidas por PTD4-Profilina1. Plan de trabajo: i-Para comprobar la funcionalidad los 3 sitios de unión fueron necesarias las primeras 6 semanas, ya que en primer lugar había que expresar y purificar el peptido Srv2, necesario para el ensayo de PLP. En segundo lugar, se obtuvieron los datos de las concentraciones adecuadas de lípidos para el ensayo de fosfo-inocitoles y por ultimo, se purifico la actina necesaria para el ensayo de unión a actina. Una vez establecida la funcionalidad de la proteína, se procedió a: ii-el estudio de polimerización in-vitro, que llevo 2 semanas. Demostrando que in-vitro era capaz de inhibir la polimerización de una manera similar a la endógena. Una vez terminados estos ensayos, se procedio a: iii-la caracterización inmunohistoquímica de las proteínas componentes de la lamela que fue llevado a cabo en 4 semanas. Para ello se usaron anticuerpos contra: alfa-actinina, talina, vinculina, ENA/Vasp y paxillina. Conclusiones: i-las propiedades bioquímicas de la PTD4-Profilina1 son similares a las de la profilina endógena. ii-los estudios de polimerización indican que la polimerización se produce de manera similar a la endogena. iii-los ensayos de inmunohistoquímica sugieren que, talina esta ausente y que las demás están presentes aunque en menor concentración y con otra distribución comparadas con los controles.

Sperm Ultrastructure of the Spider Crab Maja brachydactyla (Decapoda: Brachyura)

This study describes the morphology of the sperm cell of Maja brachydactyla, with emphasis on localizing actin and tubulin. The spermatozoon of M. brachydactyla is similar in appearance and organization to other brachyuran spermatozoa. The spermatozoon is a globular cell composed of a central acrosome, which is surrounded by a thin layer of cytoplasm and a cup-shaped nucleus with four radiating lateral arms. The acrosome is a subspheroidal vesicle composed of three concentric zones surrounded by a capsule. The acrosome is apically covered by an operculum. The perforatorium penetrates the center of the acrosome and has granular material partially composed of actin. The cytoplasm contains one centriole in the subacrosomal region. A cytoplasmic ring encircles the acrosome in the subapical region of the cell and contains the structures-organelles complex (SO-complex), which is composed of a membrane system, mitochondria with few cristae, and microtubules. In the nucleus, slightly condensed chromatin extends along the lateral arms, in which no microtubules have been observed. Chromatin fibers aggregate in certain areas and are often associated with the SO-complex. During the acrosomal reaction, the acrosome could provide support for the penetration of the sperm nucleus, the SO-complex could serve as an anchor point for chromatin, and the lateral arms could play an important role triggering the acrosomal reaction, while slightly decondensed chromatin may be necessary for the deformation of the nucleus.

Changes in the enterocyte cytoskeleton in newborn rats exposed to ethanol in utero

BACKGROUND: Cytoskeletal changes after longterm exposure to ethanol have been described in a number of cell types in adult rat and humans. These changes can play a key part in the impairment of nutrient assimilation and postnatal growth retardation after prenatal damage of the intestinal epithelium produced by ethanol intake. AIMS: To determine, in the newborn rat, which cytoskeletal proteins are affected by longterm ethanol exposure in utero and to what extent. ANIMALS: The offspring of two experimental groups of female Wistar rats: ethanol treated group receiving up to 25% (w/v) of ethanol in the drinking fluid and control group receiving water as drinking fluid. METHODS: Single and double electron microscopy immunolocalisation and label density estimation of cytoskeletal proteins on sections of proximal small intestine incubated with monoclonal antibodies against actin, alpha-tubulin, cytokeratin (polypeptides 1, 5, 6, 7, 8, 10, 11, and 18), and with a polyclonal antibody anti-beta 1,4-galactosyl transferase as trans golgi (TG) or trans golgi network (TGN) marker, or both. SDS-PAGE technique was also performed on cytoskeletal enriched fractions from small intestine. Western blotting analysis was carried out by incubation with the same antibodies used for immunolocalisation. RESULTS: Intestinal epithelium of newborn rats from the ethanol treated group showed an overexpression of cytoskeletal polypeptides ranging from 39 to 54 kDa, affecting actin and some cytokeratins, but not tubulin. Furthermore, a cytokeratin related polypeptide of 28-29 kDa was identified together with an increase in free ubiquitin in the same group. It was noteworthy that actin and cytokeratin were abnormally located in the TG or the TGN, or both. CONCLUSIONS: Longterm exposure to ethanol in utero causes severe dysfunction in the cytoskeleton of the developing intestinal epithelium. Actin and cytokeratins, which are involved in cytoskeleton anchoring to plasma membrane and cell adhesion, are particularly affected, showing overexpression, impaired proteolysis, and mislocalisation.

Moesin interacts with the cytoplasmic region of intercellular adhesion molecule-3 and is redistributed to the uropod of T Lymphocytes during cell polarization

During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane-cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the uropods, whereas moesin decorated the most distal part of the uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, ß-actin and ¿-tubulin, clustered at the cell leading edge and uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti-ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin-ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin-ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.

Changes in the enterocyte cytoskeleton in newborn rats exposed to ethanol in utero

BACKGROUND: Cytoskeletal changes after longterm exposure to ethanol have been described in a number of cell types in adult rat and humans. These changes can play a key part in the impairment of nutrient assimilation and postnatal growth retardation after prenatal damage of the intestinal epithelium produced by ethanol intake. AIMS: To determine, in the newborn rat, which cytoskeletal proteins are affected by longterm ethanol exposure in utero and to what extent. ANIMALS: The offspring of two experimental groups of female Wistar rats: ethanol treated group receiving up to 25% (w/v) of ethanol in the drinking fluid and control group receiving water as drinking fluid. METHODS: Single and double electron microscopy immunolocalisation and label density estimation of cytoskeletal proteins on sections of proximal small intestine incubated with monoclonal antibodies against actin, alpha-tubulin, cytokeratin (polypeptides 1, 5, 6, 7, 8, 10, 11, and 18), and with a polyclonal antibody anti-beta 1,4-galactosyl transferase as trans golgi (TG) or trans golgi network (TGN) marker, or both. SDS-PAGE technique was also performed on cytoskeletal enriched fractions from small intestine. Western blotting analysis was carried out by incubation with the same antibodies used for immunolocalisation. RESULTS: Intestinal epithelium of newborn rats from the ethanol treated group showed an overexpression of cytoskeletal polypeptides ranging from 39 to 54 kDa, affecting actin and some cytokeratins, but not tubulin. Furthermore, a cytokeratin related polypeptide of 28-29 kDa was identified together with an increase in free ubiquitin in the same group. It was noteworthy that actin and cytokeratin were abnormally located in the TG or the TGN, or both. CONCLUSIONS: Longterm exposure to ethanol in utero causes severe dysfunction in the cytoskeleton of the developing intestinal epithelium. Actin and cytokeratins, which are involved in cytoskeleton anchoring to plasma membrane and cell adhesion, are particularly affected, showing overexpression, impaired proteolysis, and mislocalisation.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions

Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB¿/¿ mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA¿/¿ MEFs, but not FlnB¿/¿ MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

Micropatterning of single endothelial cell shape reveals a tight coupling between nuclear volume in G1 and proliferation

Shape-dependent local differentials in cell proliferation are considered to be a major driving mechanism of structuring processes in vivo, such as embryogenesis, wound healing, and angiogenesis. However, the specific biophysical signaling by which changes in cell shape contribute to cell cycle regulation remains poorly understood. Here, we describe our study of the roles of nuclear volume and cytoskeletal mechanics in mediating shape control of proliferation in single endothelial cells. Micropatterned adhesive islands were used to independently control cell spreading and elongation. We show that, irrespective of elongation, nuclear volume and apparent chromatin decondensation of cells in G1 systematically increased with cell spreading and highly correlated with DNA synthesis (percent of cells in the S phase). In contrast, cell elongation dramatically affected the organization of the actin cytoskeleton, markedly reduced both cytoskeletal stiffness (measured dorsally with atomic force microscopy) and contractility (measured ventrally with traction microscopy), and increased mechanical anisotropy, without affecting either DNA synthesis or nuclear volume. Our results reveal that the nuclear volume in G1 is predictive of the proliferative status of single endothelial cells within a population, whereas cell stiffness and contractility are not. These findings show that the effects of cell mechanics in shape control of proliferation are far more complex than a linear or straightforward relationship. Our data are consistent with a mechanism by which spreading of cells in G1 partially enhances proliferation by inducing nuclear swelling and decreasing chromatin condensation, thereby rendering DNA more accessible to the replication machinery.

Mapping cell-matrix stresses during stretch reveals inelastic reorganization of the cytoskeleton.

The mechanical properties of the living cell are intimately related to cell signaling biology through cytoskeletal tension. The tension borne by the cytoskeleton (CSK) is in part generated internally by the actomyosin machinery and externally by stretch. Here we studied how cytoskeletal tension is modified during stretch and the tensional changes undergone by the sites of cell-matrix interaction. To this end we developed a novel technique to map cell-matrix stresses during application of stretch. We found that cell-matrix stresses increased with imposition of stretch but dropped below baseline levels on stretch release. Inhibition of the actomyosin machinery resulted in a larger relative increase in CSK tension with stretch and in a smaller drop in tension after stretch release. Cell-matrix stress maps showed that the loci of cell adhesion initially bearing greater stress also exhibited larger drops in traction forces after stretch removal. Our results suggest that stretch partially disrupts the actin-myosin apparatus and the cytoskeletal structures that support the largest CSK tension. These findings indicate that cells use the mechanical energy injected by stretch to rapidly reorganize their structure and redistribute tension.

Mechanical properties of cultured human airway smooth muscle cells from 0.05 to 0.4 Hz.

We investigated the rheological properties of living human airway smooth muscle cells in culture and monitored the changes in rheological properties induced by exogenous stimuli. We oscillated small magnetic microbeads bound specifically to integrin receptors and computed the storage modulus (G') and loss modulus (G") from the applied torque and the resulting rotational motion of the beads as determined from their remanent magnetic field. Under baseline conditions, G' increased weakly with frequency, whereas G" was independent of the frequency. The cell was predominantly elastic, with the ratio of G" to G' (defined as eta) being ~0.35 at all frequencies. G' and G" increased together after contractile activation and decreased together after deactivation, whereas eta remained unaltered in each case. Thus elastic and dissipative stresses were coupled during changes in contractile activation. G' and G" decreased with disruption of the actin fibers by cytochalasin D, but eta increased. These results imply that the mechanisms for frictional energy loss and elastic energy storage in the living cell are coupled and reside within the cytoskeleton.