Ubiquitylation by Rab40b/Cul5 regulates Rap2 localization and activity during cell migration.

Cell migration is a complex process that involves coordinated changes in membrane transport and actin cytoskeleton dynamics. Ras-like small monomeric GTPases, such as Rap2, play a key role in regulating actin cytoskeleton dynamics and cell adhesions. However, how Rap2 function, localization, and activation are regulated during cell migration is not fully understood. We previously identified the small GTPase Rab40b as a regulator of breast cancer cell migration. Rab40b contains a suppressor of cytokine signaling (SOCS) box, which facilitates binding to Cullin5, a known E3 ubiquitin ligase component responsible for protein ubiquitylation. In this study, we show that the Rab40b/Cullin5 complex ubiquitylates Rap2. Importantly, we demonstrate that ubiquitylation regulates Rap2 activation as well as recycling of Rap2 from the endolysosomal compartment to the lamellipodia of migrating breast cancer cells. Based on these data, we propose that Rab40b/Cullin5 ubiquitylates and regulates Rap2-dependent actin dynamics at the leading edge, a process that is required for breast cancer cell migration and invasion.

Following the footprints of variability during filopodial growth.

Filopodia are actin-built finger-like dynamic structures that protrude from the cell cortex. These structures can sense the environment and play key roles in migration and cell-cell interactions. The growth-retraction cycle of filopodia is a complex process exquisitely regulated by intra- and extra-cellular cues, whose nature remains elusive. Filopodia present wide variation in length, lifetime and growth rate. Here, we investigate the features of filopodia patterns in fixed prostate tumor cells by confocal microscopy. Analysis of almost a thousand filopodia suggests the presence of two different populations: one characterized by a narrow distribution of lengths and the other with a much more variable pattern with very long filopodia. We explore a stochastic model of filopodial growth which takes into account diffusion and reactions involving actin and the regulatory proteins formin and capping, and retrograde flow. Interestingly, we found an inverse dependence between the filopodial length and the retrograde velocity. This result led us to propose that variations in the retrograde velocity could explain the experimental lengths observed for these tumor cells. In this sense, one population involves a wider range of retrograde velocities than the other population, and also includes low values of this velocity. It has been hypothesized that cells would be able to regulate retrograde flow as a mechanism to control filopodial length. Thus, we propound that the experimental filopodia pattern is the result of differential retrograde velocities originated from heterogeneous signaling due to cell-substrate interactions or prior cell-cell contacts.

Gαs directly drives PDZ-RhoGEF signaling to Cdc42.

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα13 binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα13 Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gαs (GαsQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by Gs-coupled receptors, but not by those coupled to Gi or Gq, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gαs-binding region, PRG-linker. Active Gαs interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with GαsQ227L's ability to guide PRG's interaction with Cdc42. Endogenous Gs-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.

Optimising adjacent membrane segmentation and parameterisation in multicellular aggregates by piecewise active contours.

In fluorescence microscopy imaging, the segmentation of adjacent cell membranes within cell aggregates, multicellular samples, tissue, organs, or whole organisms remains a challenging task. The lipid bilayer is a very thin membrane when compared to the wavelength of photons in the visual spectra. Fluorescent molecules or proteins used for labelling membranes provide a limited signal intensity, and light scattering in combination with sample dynamics during in vivo imaging lead to poor or ambivalent signal patterns that hinder precise localisation of the membrane sheets. In the proximity of cells, membranes approach and distance each other. Here, the presence of membrane protrusions such as blebs; filopodia and lamellipodia; microvilli; or membrane vesicle trafficking, lead to a plurality of signal patterns, and the accurate localisation of two adjacent membranes becomes difficult. Several computational methods for membrane segmentation have been introduced. However, few of them specifically consider the accurate detection of adjacent membranes. In this article we present ALPACA (ALgorithm for Piecewise Adjacent Contour Adjustment), a novel method based on 2D piecewise parametric active contours that allows: (i) a definition of proximity for adjacent contours, (ii) a precise detection of adjacent, nonadjacent, and overlapping contour sections, (iii) the definition of a polyline for an optimised shared contour within adjacent sections and (iv) a solution for connecting adjacent and nonadjacent sections under the constraint of preserving the inherent cell morphology. We show that ALPACA leads to a precise quantification of adjacent and nonadjacent membrane zones in regular hexagons and live image sequences of cells of the parapineal organ during zebrafish embryo development. The algorithm detects and corrects adjacent, nonadjacent, and overlapping contour sections within a selected adjacency distance d, calculates shared contour sections for neighbouring cells with minimum alterations of the contour characteristics, and presents piecewise active contour solutions, preserving the contour shape and the overall cell morphology. ALPACA quantifies adjacent contours and can improve the meshing of 3D surfaces, the determination of forces, or tracking of contours in combination with previously published algorithms. We discuss pitfalls, strengths, and limits of our approach, and present a guideline to take the best decision for varying experimental conditions for in vivo microscopy.

Multiple actin networks coordinate mechanotransduction at the immunological synapse.

Activation of naive T cells by antigen-presenting cells (APCs) is an essential step in mounting an adaptive immune response. It is known that antigen recognition and T cell receptor (TCR) signaling depend on forces applied by the T cell actin cytoskeleton, but until recently, the underlying mechanisms have been poorly defined. Here, we review recent advances in the field, which show that specific actin-dependent structures contribute to the process in distinct ways. In essence, T cell priming involves a tug-of-war between the cytoskeletons of the T cell and the APC, where the actin cytoskeleton serves as a mechanical intermediate that integrates force-dependent signals. We consider each of the relevant actin-rich T cell structures separately and address how they work together at the topologically and temporally complex cell-cell interface. In addition, we address how this mechanobiology can be incorporated into canonical immunological models to improve how these models explain T cell sensitivity and antigenic specificity.

Aquaporin-2 and Na+ /H+ exchanger isoform 1 modulate the efficiency of renal cell migration.

Aquaporin-2 (AQP2) promotes renal cell migration by the modulation of integrin ß1 trafficking and the turnover of focal adhesions. The aim of this study was to investigate whether AQP2 also works in cooperation with Na+ /H+ exchanger isoform 1 (NHE1), another well-known protein involved in the regulation of cell migration. Our results showed that the lamellipodia of AQP2-expressing cells exhibit significantly smaller volumes and areas of focal adhesions and more alkaline intracellular pH due to increased NHE1 activity than AQP2-null cells. The blockage of AQP2, or its physically-associated calcium channel TRPV4, significantly reduced lamellipodia NHE1 activity. NHE1 blockage significantly reduced the rate of cell migration, the number of lamellipodia, and the assembly of F-actin only in AQP2-expressing cells. Our data suggest that AQP2 modulates the activity of NHE1 through its calcium channel partner TRPV4, thereby determining pH-dependent actin polymerization, providing mechanical stability to delineate lamellipodia structure and defining the efficiency of cell migration.

CTBP1 depletion on prostate tumors deregulates miRNA/mRNA expression and impairs cancer progression in metabolic syndrome mice.

About 20% of prostate cancer (PCa) patients progress to metastatic disease. Metabolic syndrome (MeS) is a pathophysiological disorder that increases PCa risk and aggressiveness. C-terminal binding protein (CTBP1) is a transcriptional corepressor that is activated by high-fat diet (HFD). Previously, our group established a MeS/PCa mice model that identified CTBP1 as a novel link associating both diseases. Here, we integrated in vitro (prostate tumor cell lines) and in vivo (MeS/PCa NSG mice) models with molecular and cell biology techniques to investigate MeS/CTBP1 impact over PCa progression, particularly over cell adhesion, mRNA/miRNA expression and PCa spontaneous metastasis development. We found that CTBP1/MeS regulated expression of genes relevant to cell adhesion and PCa progression, such as cadherins, integrins, connexins, and miRNAs in PC3 xenografts. CTBP1 diminished PCa cell adhesion, membrane attachment to substrate and increased filopodia number by modulating gene expression to favor a mesenchymal phenotype. NSG mice fed with HFD and inoculated with CTBP1-depleted PC3 cells, showed a decreased number and size of lung metastases compared to control. Finally, CTBP1 and HFD reduce hsa-mir-30b-5p plasma levels in mice. This study uncovers for the first time the role of CTBP1/MeS in PCa progression and its molecular targets.

Characterization of a novel MYO3A missense mutation associated with a dominant form of late onset hearing loss.

Whole-exome sequencing of samples from affected members of two unrelated families with late-onset non-syndromic hearing loss revealed a novel mutation (c.2090 T > G; NM_017433) in MYO3A. The mutation was confirmed in 36 affected individuals, showing autosomal dominant inheritance. The mutation alters a single residue (L697W or p.Leu697Trp) in the motor domain of the stereocilia protein MYO3A, leading to a reduction in ATPase activity, motility, and an increase in actin affinity. MYO3A-L697W showed reduced filopodial actin protrusion initiation in COS7 cells, and a predominant tipward accumulation at filopodia and stereocilia when coexpressed with wild-type MYO3A and espin-1, an actin-regulatory MYO3A cargo. The combined higher actin affinity and duty ratio of the mutant myosin cause increased retention time at stereocilia tips, resulting in the displacement of the wild-type MYO3A protein, which may impact cargo transport, stereocilia length, and mechanotransduction. The dominant negative effect of the altered myosin function explains the dominant inheritance of deafness.

Association between biomechanical alterations and migratory ability of semaphorin-3A-treated thymocytes.

BACKGROUND: Class 3 semaphorins are soluble proteins involved in cell adhesion and migration. Semaphorin-3A (Sema3A) was initially shown to be involved in neuronal guidance, and it has also been reported to be associated with immune disorders. Both Sema3A and its receptors are expressed by most immune cells, including monocytes, macrophages, and lymphocytes, and these proteins regulate cell function. Here, we studied the correlation between Sema3A-induced changes in biophysical parameters of thymocytes, and the subsequent repercussions on cell function. METHODS: Thymocytes from mice were treated in vitro with Sema3A for 30min. Scanning electron microscopy was performed to assess cell morphology. Atomic force microscopy was performed to further evaluate cell morphology, membrane roughness, and elasticity. Flow cytometry and/or fluorescence microscopy were performed to assess the F-actin cytoskeleton and ROCK2. Cell adhesion to a bovine serum albumin substrate and transwell migration assays were used to assess cell migration. RESULTS: Sema3A induced filopodia formation in thymocytes, increased membrane stiffness and roughness, and caused a cortical distribution of the cytoskeleton without changes in F-actin levels. Sema3A-treated thymocytes showed reduced substrate adhesion and migratory ability, without changes in cell viability. In addition, Sema3A was able to down-regulate ROCK2. CONCLUSIONS: Sema3A promotes cytoskeletal rearrangement, leading to membrane modifications, including increased stiffness and roughness. This effect in turn affects the adhesion and migration of thymocytes, possibly due to a reduction in ROCK2 expression. GENERAL SIGNIFICANCE: Sema3A treatment impairs thymocyte migration due to biomechanical alterations in cell membranes.

Cell adhesion controlled by adhesion G protein-coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo-Dock.

Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gßγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses.

Vimentin as a Marker of Early Differentiating, Highly Motile Corneal Epithelial Cells.

Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial-Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal-like derivatives. Based on previous results showing that epithelial cells co-express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co-expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt+ /Vim+ cell number and ΔNp63α expression when cells reached confluence and formed a 4-5 layered epithelium, while there was a concomitant increase of both Pax-6 expression and Krt+ /Vim- cells. Inhibition of cell proliferation with mitomycin C did not modify cell motility nor the expression of Vim. We studied the distribution and expression of α6 integrin, a protein also involved in cell migration. The results demonstrated that α6 integrin had a distribution which was, in part, co-linear with Vim at the proliferative/migratory rim of cell colonies, suggesting an indirect interaction between these proteins. Immunoprecipitation and immunostaining assays indicated that plectin might be mediating such interaction. These data suggest that Vim expression in corneal epithelium is found in a cell population composed of highly motile cells with a Vim+ /Krt+ /ΔNp63α+ /Pax-6low /α6 integrin+ phenotype. J. Cell. Physiol. 232: 818-830, 2017. © 2016 Wiley Periodicals, Inc.

Heme oxygenase-1 in the forefront of a multi-molecular network that governs cell-cell contacts and filopodia-induced zippering in prostate cancer.

Prostate cancer (PCa) cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown that heme oxygenase 1 (HO-1) is implicated in cell morphology regulation in PCa. Here, through a multi 'omics' approach we define the HO-1 interactome in PCa, identifying HO-1 molecular partners associated with the integrity of the cellular cytoskeleton. The bioinformatics screening for these cytoskeletal-related partners reveal that they are highly misregulated in prostate adenocarcinoma compared with normal prostate tissue. Under HO-1 induction, PCa cells present reduced frequency in migration events, trajectory and cell velocity and, a significant higher proportion of filopodia-like protrusions favoring zippering among neighboring cells. Moreover forced expression of HO-1 was also capable of altering cell protrusions in transwell co-culture systems of PCa cells with MC3T3 cells (pre-osteoblastic cell line). Accordingly, these effects were reversed under siHO. Transcriptomics profiling evidenced significant modulation of key markers related to cell adhesion and cell-cell communication under HO-1 induction. The integration from our omics-based research provides a four molecular pathway foundation (ANXA2/HMGA1/POU3F1; NFRSF13/GSN; TMOD3/RAI14/VWF; and PLAT/PLAU) behind HO-1 regulation of tumor cytoskeletal cell compartments. The complementary proteomics and transcriptomics approaches presented here promise to move us closer to unravel the molecular framework underpinning HO-1 involvement in the modulation of cytoskeleton pathways, pushing toward a less aggressive phenotype in PCa.

Ligand-mediated Galectin-1 endocytosis prevents intraneural H2O2 production promoting F-actin dynamics reactivation and axonal re-growth.

UNLABELLED: Axonal growth cone collapse following spinal cord injury (SCI) is promoted by semaphorin3A (Sema3A) signaling via PlexinA4 surface receptor. This interaction triggers intracellular signaling events leading to increased hydrogen peroxide levels which in turn promote filamentous actin (F-actin) destabilization and subsequent inhibition of axonal re-growth. In the current study, we demonstrated that treatment with galectin-1 (Gal-1), in its dimeric form, promotes a decrease in hydrogen peroxide (H2O2) levels and F-actin repolimerization in the growth cone and in the filopodium of neuron surfaces. This effect was dependent on the carbohydrate recognition activity of Gal-1, as it was prevented using a Gal-1 mutant lacking carbohydrate-binding activity. Furthermore, Gal-1 promoted its own active ligand-mediated endocytosis together with the PlexinA4 receptor, through mechanisms involving complex branched N-glycans. In summary, our results suggest that Gal-1, mainly in its dimeric form, promotes re-activation of actin cytoskeleton dynamics via internalization of the PlexinA4/Gal-1 complex. This mechanism could explain, at least in part, critical events in axonal regeneration including the full axonal re-growth process, de novo formation of synapse clustering, axonal re-myelination and functional recovery of coordinated locomotor activities in an in vivo acute and chronic SCI model. SIGNIFICANCE STATEMENT: Axonal regeneration is a response of injured nerve cells critical for nerve repair in human spinal cord injury. Understanding the molecular mechanisms controlling nerve repair by Galectin-1, may be critical for therapeutic intervention. Our results show that Galectin-1; in its dimeric form, interferes with hydrogen peroxide production triggered by Semaphorin3A. The high levels of this reactive oxygen species (ROS) seem to be the main factor preventing axonal regeneration due to promotion of actin depolymerization at the axonal growth cone. Thus, Galectin-1 administration emerges as a novel therapeutic modality for promoting nerve repair and preventing axonal loss.

Neuronal filopodium formation induced by the membrane glycoprotein M6a (Gpm6a) is facilitated by coronin-1a, Rac1, and p21-activated kinase 1 (Pak1).

Stress-responsive neuronal membrane glycoprotein M6a (Gpm6a) functions in neurite extension, filopodium and spine formation and synaptogenesis. The mechanisms of Gpm6a action in these processes are incompletely understood. Previously, we identified the actin regulator coronin-1a (Coro1a) as a putative Gpm6a interacting partner. Here, we used co-immunoprecipitation assays with the anti-Coro1a antibody to show that Coro1a associates with Gpm6a in rat hippocampal neurons. By immunofluorescence microscopy, we demonstrated that in hippocampal neurons Coro1a localizes in F-actin-enriched regions and some of Coro1a spots co-localize with Gpm6a labeling. Notably, the over-expression of a dominant-negative form of Coro1a as well as its down-regulation by siRNA interfered with Gpm6a-induced filopodium formation. Coro1a is known to regulate the plasma membrane translocation and activation of small GTPase Rac1. We show that Coro1a co-immunoprecipitates with Rac1 together with Gpm6a. Pharmacological inhibition of Rac1 resulted in a significant decrease in filopodium formation by Gpm6a. The same was observed upon the co-expression of Gpm6a with the inactive GDP-bound form of Rac1. In this case, the elevated membrane recruitment of GDP-bound Rac1 was detected as well. Moreover, the kinase activity of the p21-activated kinase 1 (Pak1), a main downstream effector of Rac1 that acts downstream of Coro1a, was required for Gpm6a-induced filopodium formation. Taken together, our results provide evidence that a signaling pathway including Coro1a, Rac1, and Pak1 facilitates Gpm6a-induced filopodium formation. Formation of filopodia by membrane glycoprotein M6a (Gpm6a) requires actin regulator coronin-1a (Coro1a), known to regulate plasma membrane localization and activation of Rac1 and its downstream effector Pak1. Coro1a associates with Gpm6a. Blockage of Coro1a, Rac1, or Pak1 interferes with Gpm6a-induced filopodium formation. Moreover, Gpm6a facilitates Rac1 membrane recruitment. Altogether, a mechanistic insight into the process of Gpm6a-induced neuronal filopodium formation is provided.

Auditory Brainstem Response in Term and Preterm Infants with Neonatal Complications: The Importance of the Sequential Evaluation

Introduction Literature data are not conclusive as to the influence of neonatal complications in the maturational process of the auditory system observed by auditory brainstem response (ABR) in infants at term and preterm. Objectives Check the real influence of the neonatal complications in infants by the sequential auditory evaluation. Methods Historical cohort study in a tertiary referral center. A total of 114 neonates met inclusion criteria: treatment at the Universal Neonatal Hearing Screening Program of the local hospital; at least one risk indicator for hearing loss; presence in both evaluations (the first one after hospital discharge from the neonatal unit and the second one at 6 months old); all latencies in ABR and transient otoacoustic emissions present in both ears. Results The complications that most influenced the ABR findings were Apgar scores less than 6 at 5 minutes, gestational age, intensive care unit stay, peri-intraventricular hemorrhage, and mechanical ventilation. Conclusion Sequential auditory evaluation is necessary in premature and term newborns with risk indicators for hearing loss to correctly identify injuries in the auditory pathway. .

Fatores psicossociais e socioeconômicos relacionados à insônia e menopausa: Estudo Pró-Saúde / Psychosocial and socioeconomic factors related to insomnia and menopause: Pró-Saúde Study / Factores psicosociales y socioeconómicos relacionados con el insomnio y la menopausia: Estudio Pro-Salud

Foi avaliada a associação entre menopausa e insônia e a influência de variáveis socioeconômicas e psicossociais, em estudo transversal com 2.190 funcionárias de uma universidade (Estudo Pró-Saúde), a partir de um questionário autopreenchível com variáveis sobre menopausa, insônia, transtorno mental comum, eventos de vida estressantes, apoio social e variáveis socioeconômicas. Odds ratios foram calculados por meio de regressão logística multivariada, com desfecho politômico. Após ajuste para potenciais confundidoras sociodemográficas, mulheres na menopausa há mais de 60 meses apresentaram maior chance de reportar queixas de sono frequentes (OR entre 1,53 e 1,86) do que as que estavam na menopausa há menos de 60 meses. Após os ajustes, no primeiro grupo, para as variáveis psicossociais, a magnitude dos ORs reduziu para 1,53 (IC95%: 0,92-2,52) para dificuldade em iniciar o sono, 1,81 (IC95%: 1,09-2,98) para dificuldade em manter o sono e 1,71 (IC95%: 1,08-2,73) para queixa geral de insônia. Fatores psicossociais podem mediar a manifestação da insônia em mulheres na menopausa.

This study evaluated the association between insomnia and menopausal status and the influence of socioeconomic and psychosocial variables on this association in a cross-sectional analysis of 2,190 university employees (the Pró-Saúde Study). A self-administered questionnaire was used, covering menopausal status, complaints of insomnia, common mental disorders, stressful life events, social support, and socioeconomic variables. Odds ratios were calculated using logistic regression with a polytomous outcome. After adjusting for potential socio-demographic confounders, women who had entered menopause more than 60 months previously were more likely to report complaints with sleep (OR 1.53-1.86) as compared to women in menopause for less than 60 months. After adjusting for psychosocial variables, in the first group the ORs decreased to 1.53 (95%CI: 0.92-2.52) for difficulty initiating sleep, 1.81 (95%CI: 1.09-2.98) for difficulty maintaining sleep, and 1.71 (95%CI: 1.08-2.73) for general complaints of insomnia. Psychosocial factors can mediate the manifestation of insomnia among menopausal women.

En este estudio se evaluó la asociación entre insomnio y menopausia y la influencia de las variables socioeconómicas y psicosociales, en un estudio transversal con 2.190 mujeres de una universidad (Estudio Pro-Salud), a partir de un cuestionario autoadministrado con variables de la menopausia, insomnio, trastornos mentales, situaciones de estrés vital, apoyo social y variables socioeconómicas. Se calcularon los odds ratio mediante regresión logística multivariante con desenlace politómico. Después de ajustar por factores de confusión sociodemográficos potenciales, las mujeres menopáusicas desde hace más de 60 meses fueron más propensas a reportar quejas frecuentes de sueño (OR entre 1,53 y 1,86) que las menopáusicas hace menos de 60 meses. Después de los ajustes, en el primer grupo, para las variables psicosociales la magnitud de los OR se redujo a 1,53 (IC95%: 0,92-2,52) para la dificultad para iniciar el sueño, un 1,81 (IC95%: 1,09-2,98) para mantener el sueño y un 1,71 (IC95%: 1,08-2,73) para las quejas de insomnio en general. Los factores psicosociales pueden mediar en la manifestación del insomnio en las mujeres menopáusicas.

Sapocribrum chincoteaguense n. gen. n. sp.: A Small, Scale-bearing Amoebozoan with Flabellinid Affinities.

The isolate American Type Culture Collection (ATCC)® 50979™ is a small amoebozoan whose actin gene was previously characterized, but did not allow a stable phylogenetic placement. This isolate was originally mis-identified upon deposition, and subsequently mis-illustrated in a recent publication. Here, we provide both a detailed morphological description as well as additional molecular analyses in order to clarify the isolate's phylogenetic relationships. The amoeba is minute (less than 5 µm), and presents the behavior of staying in a fixed location, while emitting one or two thin pseudopods. Transmission electron microscopy reveals that the cell is covered in a layer with embedded scales, giving the cell an armored appearance. Molecular phylogenetic analyses of data (actin, alpha- and beta-tubulin, elongation factor 2, and 14-3-3) from transcriptomes of this and four other isolates reveals that ATCC® 50979(™) is closely related to the recently described Squamamoeba japonica and in a novel, stable clade. Due to the unique nature of the scale covering, as well as other gross morphological characters and the molecular phylogenetic analyses, we formally describe the isolate as Sapocribrum chincoteaguense n. gen. n. sp.

Tyrosine 251 at the C-terminus of neuronal glycoprotein M6a is critical for neurite outgrowth.

Neuronal glycoprotein M6a is involved in neuronal plasticity, promoting neurite and filopodia outgrowth and, likely, synaptogenesis. Polymorphisms in the human M6a gene GPM6A have recently been associated with mental illnesses such as schizophrenia, bipolar disorders, and claustrophobia. Nevertheless, the molecular bases underlying these observations remain unknown. We have previously documented that, to induce filopodia formation, M6a depends on the association of membrane lipid microdomains and the activation of Src and mitogen-activated protein kinase kinases. Here, in silico analysis of the phosphorylation of tyrosine 251 (Y251) at the C-terminus of M6a showed that it could be a target of Src kinases. We examined whether phosphorylation of M6a at Y251 affects neurite and filopodia outgrowth and the targets involved in its signal propagation. This work provides evidence that the Src kinase family and the phosphatidylinositide 3-kinase (PI3K), but not Ras, participate in M6a signal cascade leading to neurite/filopodia outgrowth in hippocampal neurons and murine neuroblastoma N2a cells. Phosphorylation of M6a at Y251 is essential only for neurite outgrowth by the PI3K/AKT-mediated pathway and, moreover, rescues the inhibition caused by selective Src inhibitor and external M6a monoclonal antibody treatment. Thus, we suggest that phosphorylation of M6a at Y251 is critical for a specific stage of neuronal development and triggers redundant signaling pathways leading to neurite extension.

Molecular cloning, sequence analysis, prokaryotic expression, and function prediction of foot-specific peroxidase in Hydra magnipapillata Chinese strain.

The cDNA sequence of foot-specific peroxidase PPOD1 from the Chinese strain of Hydra magnipapillata was cloned by reverse transcription-polymerase chain reaction. The cDNA sequence contained a coding region with an 873-bp open reading frame, a 31-bp 5'-untranslated region, and a 36-bp 3'-untranslated region. The structure prediction results showed that PPOD1 contains 10.34% of α-helix, 38.62% of extended strand, 12.41% of ß-turn, and 38.62% of random coil. The structural core was α-helix at the N terminus. The GenBank protein blast server showed that PPOD1 contains 2 fascin-like domains. In addition, high-level PPOD1 activity was only present in the ectodermal epithelial cells located on the edge of the adhesive face of the basal disc, and that these cells extended lamellipodia and filopodia when the basal disc was tightly attached to a glass slide. The fascin-like domains of Hydra PPOD1 might contribute to the bundling of the actin filament of these cells, and hence, the formation of filopodia. In conclusion, these cells might play an important role in strengthening the adsorbability of the basal disc to substrates.

New roles of filopodia and podosomes in the differentiation and fusion process of osteoclasts.

The cytoskeleton mediates various cellular processes such as differentiation and fusion, including in the filopodia and podosomes. However, apart from cell migration and formation of the sealing zone, little is known regarding the changes and related regulatory mechanisms of the cytoskeleton and additional roles of the filopodia and podosomes during the differentiation and fusion of osteoclasts. The cytomorphology and cytoskeleton of osteoclasts in the differentiation process were evaluated using tartrate-resistant acid phosphatase staining and immunofluorescence staining. Moreover, the expression levels of Rho GTPases and enzymes related to osteoclast differentiation and bone resorption were detected by quantitative reverse transcription-polymerase chain reaction. We detected 3 types of filopodia in osteoclast precursors and only 1 type of filopodia in undifferentiated cells. Mature osteoclasts were completely devoid of filopodia. Interestingly, cell fusion was highly specific, and the fusion initially occurred to the filopodia. Confocal images revealed that F-actin and microtubules significantly differed among fused cells. These results suggest that filopodia and podosomes not only play important roles in cell migration and the formation of sealing zones but also in the pre-fusion selectivity of 2 cells and the movement direction of the cell nucleus and cytoplasm during the fusion process. In addition, cdc42v1, RhoU, and RhoF regulate the formation of 3 types of filopodia during various stages of differentiation, while Rac1, Rac2, and filament A may be associated with cell selectivity during the fusion process.