Dynamic and Regulated Association of Caveolin with Lipid Bodies: Modulation of Lipid Body Motility and Function by a Dominant Negative Mutant

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cavDGV) to study LB formation and to examine its effect on LB function. We now show that the cavDGV mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.

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.

Linking microtubules to the activation of CDC42 in fission yeast

Cell polarity is an essential property of most cell types and relies on a dynamic cytoskeleton of actin filaments and microtubules. In rod-shaped S. pombe cells microtubules are organized along the length of the cell and transport polarity factors to cell tips to regulate cell polarity. An important cell polarity factor is the protein Tea4, which is responsible for correct cell morphogenesis and bipolar growth. During my research I confirmed the known transport mechanism of Tea4 and I also showed alternative localization and anchoring mechanisms at the cell ends. Tea4 contains a conserved SH3 domain, the function of which was unknown and my results show that the SH3 domain of Tea4 is essential for Tea4 function in vivo. First, cells with tea4SH3 mutations show aberrant cell shapes and monopolar growth patterns similar to tea4A and in addition SH3 domain is important for proper localization of multiple cell polarity proteins. Second, I showed that Tea4 associates with Type 1 Phosphatase Dis2 through both its SH3 domain and an RVxF motif. Tea4 also binds the DYRK kinase Pomi through its SH3 domain. In addition Tea4 is proposed to promote the local dephosphorylation of Pomi by Dis2 to induce the formation of a cortical gradient from cell ends essential for cell size homeostasis. Polarized growth is also controlled by cell tip-localized Cdc42. This Rho- family GTPase is activated by the Guanine Exchange Factors Gef1 and Scd1 and inactivated by the Rho GTPase Activating Protein Rga4. In this study, I investigated the mechanisms of how Tea4 promotes Cdc42 activation. My work suggests that Tea4 promotes the local exclusion of Rga4, which in turn allows the accumulation of active Cdc42, which may result in growth. Exclusion of Rga4 by Tea4 is likely to be mediated by Dis2-dependent dephosphorylation. These results suggest a molecular pathway that links the microtubule- associated factor Tea4 with Cdc42 to promote cell polarization and morphogenesis. - La polarité cellulaire est une propriété essentielle de la plupart des types cellulaires et s'appuie sur une dynamique des cytosquelettes d'actine et de microtubules. Dans les cellules en forme de bâtonnet de S. pombe les microtubules sont alignés selon l'axe longitudinal de la cellule et les facteurs de polarité transportés aux extrémité cellulaires afin de réguler la polarité cellulaire. Un facteur important de polarité cellulaire est la protéine Tea4, qui est responsable de la morphogenèse des cellules et leur croissance bipolaire. Au cours de mes recherches, j'ai confirmé les mécanismes connus de transport de Tea4 et j'ai aussi mis en évidence d'autres mechanismes de localisation et d'ancrage de Tea4 aux extrémités cellulaires. Tea4 contient un domaine SH3 conservé, dont la fonction était inconnue et mes résultats montrent que le domaine SH3 est essentiel pour la fonction de Tea4 in vivo. Tout d'abord, les cellules avec des mutations tea4sm ont des formes aberrantes et leur croissance est monopolaire de manière similaire au mutant tea4A. De plus ce domaine SH3 est important pour la localisation correcte de plusieurs protéines de polarité cellulaire. Deuxièmement, j'ai montré que Tea4 s'associe avec la Phosphatase de Type-1 Dis2 par son domaine SH3 et un motif RVxF. Tea4 se lie également la kinase DYRK Pomi par son domaine SH3. De plus, Tea4 pourrait favoriser la déphosphorylation locale de Pomi par Dis2 afin d'induire la formation d'un gradient cortical de Pomi essentiel pour l'homéostasie de la longueur des cellules. La croissance polarisée est également contrôlée par la protéine Cdc42 localisée aux extrémités cellulaires. Cette GTPase de la famille de Rho GTPase est activée par les facteurs échange de guanine Gef1 et Scd1 et inactivée par la protéine "Rho GTPase activating" Rga4. Dans cette étude, j'ai étudié les mécanismes d' activation de Cdc42 par Tea4. Mes résultats suggèrent que Tea4 favorise l'exclusion locale de Rga4, ce qui permet l'accumulation de Cdc42 active, nécessaire à la croissance. L' exclusion de Rga4 par Tea4 est vraisemblablement médiée par une déphosphorylation Dis2- dépendente. Ces résultats suggèrent une voie moléculaire qui lie le facteur associé aux microtubules Tea4 à Cdc42 pour promouvoir la polarisation cellulaire et la morphogenèse. - Cell polarity is important for several essential biological functions such as generation of distinct cell fates during development and function of differentiated cells. Defective cell polarity has been related to uncontrolled cell division and subsequently to cancer initiation. Cell polarity depends on a functional cytoskeleton that consists of actin filaments and microtubules, which maintains cell shape, helps cellular motion, enables intracellular protein transport and plays a vital role in cell division. A component of cytoskeleton is microtubules that regulate cell polarization in diverse cell types. During my research, I worked with Schizosaccharomyces pombe, also named fission yeast, a powerful unicellular model organism that allows combination of genetic, biochemical and microscopic analysis for the proper study of cell polarity. Microtubule-associated protein Tea4 is transported to cell tips where it is thought to organize polarized growth. I showed that Tea4 and its evolutionarily conserved SH3 domain play an important role for maintenance of fission yeast cells shape and growth. Furthermore, Tea4 is responsible for the proper localization of multiple polarity proteins and acts as a mediator to control the local activity of an essential polarity regulator called Cdc42. Thus, my results provide a better understanding of the molecular mechanisms that regulate cell polarity. - La polarité cellulaire est importante pour plusieurs fonctions biologiques essentielles telles que la différenciation cellulaires au cours du développement et de la fonction de cellules différenciées. Les défauts de la polarité cellulaire ont été liés à des divisions cellulaires incontrôlées et à l'initiation de tumeur. La polarité cellulaire dépend d'un cytosquelette fonctionnel, qui maintient la forme des cellules, aide à la migration cellulaire, permet le transport intracellulaire des protéines et joue un rôle essentiel dans la division cellulaire. Un composant du cytosquelette est constitué de microtubules qui régissent la polarisation cellulaire dans divers types cellulaires. Au cours de mes recherches, j'ai travaillé avec Schizosaccharomyces pombe, appelé également levure fissipare, un modèle unicellulare puissant qui permet la combinaison de différentes d'approches expérimentales: génétiques, biochimiques et microscopiques pour l'étude de la polarité cellulaire. La protéine Tea4 associée aux microtubules est transportée aux extrémités cellulaires où elle organise la croissance polarisée. J'ai montré que Tea4 et son domaine conservé SH3 jouent un rôle important pour le maintien de la forme des cellules de levure et leur croissance. De plus, Tea4 est responsable de la localisation correcte de multiples facteurs de polarité et agit comme un médiateur pour contrôler l'activité locale d'un régulateur de polarité essentiel appelé Cdc42. Ainsi, mes résultats permettent de mieux comprendre les mécanismes moléculaires qui régulent la polarité cellulaire.

Differential distribution of MAP1A isoforms in the adult mouse barrel cortex and comparison with the serotonin 5-HT2A receptor.

Microtubule-associated protein 1A (MAP1A) is essential during the late differentiation phase of neuronal development. Here, we demonstrated the presence of two MAP1A isoforms with a differential spatial distribution in the adult mouse barrel cortex. Antibody A stained MAP1A in pyramidal and stellate cells, including dendrites that crossed layer IV in the septa between barrels. The other antibody, BW6 recognized a MAP1A isoform that was mainly confined to the barrel hollow and identified smaller caliber dendrites. Previously, an interaction of MAP1A and the serotonin 5-hydroxytryptamine 2A (5-HT(2A)) receptor was shown in the rat cortex. Here, we identified, by double-immunofluorescent labeling, MAP1A isoform and serotonin 5-HT(2A) receptor distribution. MAP1A co-localized mainly with 5-HT(2A) receptor in larger apical dendrites situated in septa. This differential staining of MAP1A and a serotonin receptor in defined barrel compartments may be due to changes in the expression or processing of MAP1A during dendritic transport as a consequence of functional differences in processing of whisker-related sensory input.

Long-term exercise stabilizes atherosclerotic plaque in apolipoprotein-E deficient mice

PURPOSE: Exercise is known to reduce cardiovascular mortality. However, the precise mechanisms are still unknown. Because atherosclerotic plaque destabilization and rupture leads to dramatic cardiovascular events, stabilization of plaque might be regarded as an important goal of an exercise preventive therapy. The present study examined the plaque-stabilizing effect of long-term exercise in experimental atherosclerosis using apolipoprotein E-deficient mice (ApoE(-/-)). METHODS: ApoE(-/-) mice were subjected to 6 months of swimming exercise. A group of sedentary animals were used as controls. Morphometry and characteristics of atherosclerotic plaque stability were assessed in aortic sinus by immunohistochemistry. Aortic levels of total protein kinase Akt (protein kinase B), phosphorylated Akt at Ser(473) (p-Akt), total endothelial nitric oxide synthase (eNOS), and phosphorylated eNOS at Ser(1177) (p-eNOS) were assessed by Western blotting. RESULTS: Exercised mice developed a more stable plaque phenotype as shown by decreased macrophage and increased smooth muscle cell content. Protein expressions of Akt, p-Akt, eNOS, and p-eNOS were not modulated by exercise. CONCLUSIONS: Long-term exercise promotes plaque stability in ApoE(-/-) mice. The Akt-mediated eNOS phosphorylation pathway seems not to be the primary molecular mechanism.

Neuronal potential of cells in early postnatal rat sciatic nerve.

A population of undifferentiated cells with neuronal potentialities were revealed in rat sciatic nerve. Explant cultures of sciatic nerve were prepared from newborn or early postnatal rat. Cultures were growth in F14 medium supplemented with 10% of fetal calf serum, incubated in a humidified 3% CO2, 97% air atmosphere. Within 2 weeks, refractile cells exhibiting the morphology of neurons were observed in all examined cultures. These cells had ovoid or multipolar refractile cells bodies with extended cytoplasmic processes. The neuronal nature of these cells was confirmed by their immunostaining with specific neuronal markers: neurofilament triplets, neuron-specific enolase, peripherin, microtubule-associated proteins, and brain spectrin. This neuronal population displayed various phenotypes. The CO2 concentration in the incubator plays an important role, since the number of differentiated neurons was lower in cultures incubated in 5% CO2. Since the sciatic nerve is devoid of nerve cell bodies in vivo, we concluded that early postnatal sciatic nerve contains crest cells with neuronal potentialities differentiating into neurons in response to the culture's environmental cues.

Anterior cingulate cortex pathology in schizophrenia and bipolar disorder.

To explore possible morphological abnormalities in the dorsal and subgenual parts of anterior cingulate cortex in mood disorders and schizophrenia, we performed a quantitative postmortem study of 44 schizophrenic patients, 21 patients with sporadic bipolar disorder, 20 patients with sporadic major depression, and 55 age- and sex-matched control cases. All individuals were drug naïve or had received psychotropic medication for less than 6 months, and had no history of substance abuse. Neuron densities and size were estimated on cresyl violet-stained sections using a stereological counting approach. The distribution and density of microtubule-associated (MAP2, MAP1b) and tau proteins were assessed by immunocytochemistry and quantitative immunodot assay. Mean total and laminar cortical thicknesses as well as mean pyramidal neuron size were significantly decreased in the dorsal and subgenual parts of areas 24 (24sg) in schizophrenic cases. Patients with bipolar disorder showed a substantial decrease in laminar thickness and neuron densities in layers III, V, and VI of the subgenual part of area 24, whereas patients with major depression were comparable to controls. Immunodot assay showed a significant decrease of both MAP2 and MAP1b proteins in bipolar patients but not in patients with schizophrenia and major depression. The neuroanatomical and functional significance of these findings are discussed in the light of current hypotheses regarding the role of areas 24 and 24sg in schizophrenia and bipolar disorder.

Association of brain spectrin isoforms with microtubules.

The relationship of rat brain spectrin isoforms to microtubules of newborn and adult animals was studied. Spectrins were minor components in microtubule preparations. The microtubule-associated spectrin is a major calmodulin-binding protein. Radiolabelled brain spectrin(240/235) revealed specific microtubule binding activity in vitro, possibly via a tubulin.

The human estrogen receptor can regulate exogenous but not endogenous vitellogenin gene promoters in a Xenopus cell line.

Transfection of a human estrogen receptor cDNA expression vector (HEO) into cultured Xenopus kidney cells confers estrogen responsiveness to the recipient cells as demonstrated by the hormone dependent expression of co-transfected Xenopus vitellogenin-CAT chimeric genes. The estrogen stimulation of these vit-CAT genes is dependent upon the presence of the vitellogenin estrogen responsive element (ERE) in their 5' flanking region. Thus, functional human estrogen receptor (hER) can be synthesized in heterologous lower vertebrate cells and can act as a trans-acting regulatory factor that is necessary, together with estradiol, for the induction of the vit-CAT constructs in these cells. In addition, vitellogenin minigenes co-transfected with the HEO expression vector also respond to hormonal stimulation. Their induction is not higher than that of the vit-CAT chimeric genes. It suggests that in the Xenopus kidney cell line B 3.2, the structural parts of the vitellogenin minigenes do not play a role in the induction process. Furthermore, no stabilizing effect of estrogen on vitellogenin mRNA is observed in these cells. In contrast to the transfected genes, the endogenous chromosomal vitellogenin genes remain silent, demonstrating that in spite of the presence of the hER and the hormone, the conditions necessary for their activation are not fulfilled.

Amine-reactive OVA multimers for auto-vaccination against cytokines and other mediators: perspectives illustrated for GCP-2 in L. major infection.

Anticytokine auto-vaccination is a powerful tool for the study of cytokine functions in vivo but has remained rather esoteric as a result of numerous technical difficulties. We here describe a two-step procedure based on the use of OVA multimers purified by size exclusion chromatography after incubation with glutaraldehyde at pH 6. When such polymers are incubated with a target protein at pH 8.5 to deprotonate reactive amines, complexes are formed that confer immunogenicity to self-antigens. The chemokine GCP-2/CXCL6, the cytokines GM-CSF, IL-17F, IL-17E/IL-25, IL-27, and TGF-β1, and the MMP-9/gelatinase B are discussed as examples. mAb, derived from such immunized mice, have obvious advantages for in vivo studies of the target proteins. Using a mAb against GCP-2, obtained by the method described here, we provide the first demonstration of the major role played by this chemokine in rapid neutrophil mobilization after Leishmania major infection. Pre-activated OVA multimers reactive with amine residues thus provide an efficient carrier for auto-vaccination against 9-90 kDa autologous proteins.

Phosphorylation of neurofilament subunit NF-M is regulated by activation of NMDA receptors and modulates cytoskeleton stability and neuronal shape.

The cytoskeleton is essential for the structural organization of neurons and is influenced during development by excitatory stimuli such as activation of glutamate receptors. In particular, NMDA receptors are known to modulate the function of several cytoskeletal proteins and to influence cell morphology, but the underlying molecular and cellular mechanisms remain unclear. Here, we characterized the neurofilament subunit NF-M in cultures of developing mouse cortical neurons chronically exposed to NMDA receptor antagonists. Western blots analysis showed that treatment of cortical neurons with MK801 or AP5 shifted the size of NF-M towards higher molecular weights. Dephosphorylation assay revealed that this increased size of NF-M observed after chronic exposure to NMDA receptor antagonists was due to phosphorylation. Neurons treated with cyclosporin, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, also showed increased levels of phosphorylated NF-M. Moreover, analysis of neurofilament stability revealed that the phosphorylation of NF-M, resulting from NMDA receptor inhibition, enhanced the solubility of NF-M. Finally, cortical neurons cultured in the presence of the NMDA receptor antagonists MK801 and AP5 grew longer neurites. Together, these data indicate that a blockade of NMDA receptors during development of cortical neurons increases the phosphorylation state and the solubility of NF-M, thereby favoring neurite outgrowth. This also underlines that dynamics of the neurofilament and microtubule cytoskeleton is fundamental for growth processes.

Interfacial growth in driven diffusive systems

We have studied the growth of interfaces in driven diffusive systems well below the critical temperature by means of Monte Carlo simulations. We consider the region beyond the linear regime and of large values of the external field which has not been explored before. The simulations support the existence of interfacial traveling waves when asymmetry is introduced in the model, a result previously predicted by a linear-stability analysis. Furthermore, the generalization of the Gibbs-Thomson relation is discussed. The results provide evidence that the external field is a stabilizing effect which can be considered as effectively increasing the surface tension.

Ocular biocompatibility of novel Cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers.

Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.

Prescription footwear for severe injuries of foot and ankle: effect on regularity and symmetry of the gait assessed by trunk accelerometry.

After foot and/or ankle fracture, the restoration of optimal gait symmetry is one of the criteria of recovery. Orthotic insoles and orthopaedic shoes improve gait symmetry and regularity by controlling joint motion and improving alignment. The aim of the present study was to assess the effect of prescription footwear on gait quality by using accelerometers attached to the lower back. Sixteen adult patients with persistent disability after ankle and/or foot fractures performed two 30-s walking trials with and without prescription footwear (insoles and stabilizing shoes). Sixteen control subjects were also tested for comparison. The autocorrelation function was computed from the acceleration signal and the first two dominant periods were assessed (d1 and d2). Two parameters were used: (1) Stride Regularity (SR) which expresses the similarity between strides over time (d2), and (2) Stride Symmetry (SS) a ratio (d1/d2) which expresses the left/right similarity of gait independently of repeatability in the successive movements of each limb. In control subjects, SR and SS were 0.86+/-0.05 (correlation coefficient) and 81+/-10%, respectively. In the patient group, the effect of footwear was significant (SR: 0.88+/-0.06 vs. 0.90+/-0.05, SS: 38+/-23% vs. 46+/-27%). Pain was also significantly reduced (-34%). By using a rapid and low-cost method, we objectively quantified gait quality improvement after footwear intervention, concomitant to pain reduction. Substantial inter-patient variability in the footwear outcome was observed. In conclusion, we believe that trunk accelerometry can be a useful tool in the field of gait rehabilitation.

Shaping fission yeast cells by rerouting actin-based transport on microtubules.

Kinesins and myosins transport cargos to specific locations along microtubules and actin filaments, respectively. The relative contribution of the two transport systems for cell polarization varies extensively in different cell types, with some cells relying exclusively on actin-based transport while others mainly use microtubules. Using fission yeast, we asked whether one transport system can substitute for the other. In this organism, microtubules and actin cables both contribute to polarized growth by transporting cargos to cell poles, but with distinct roles: microtubules transport landmarks to label cell poles for growth and actin assembly but do not directly contribute to the growth process [1]. Actin cables serve as tracks for myosin V delivery of growth vesicles to cell poles [2-4]. We engineered a chimera between the motor domain of the kinesin 7 Tea2 and the globular tail of the myosin V Myo52, which we show transports Ypt3, a myosin cargo receptor, to cell poles along microtubules. Remarkably, this chimera restores polarized growth and viability to cells lacking actin cables. It also bypasses the normal microtubule-dependent marking of cell poles for polarized growth, but not for other functions. Thus, a synthetic motor protein successfully redirects cargos along a distinct cytoskeletal route.