Transcytosis is a widespread pathway for apical targeting in epithelial cells. MAL2, an essential protein of the machinery for apical transcytosis, functions by shuttling in vesicular carriers between the apical zone and the cell periphery. We have identified INF2, an atypical formin with actin polymerization and depolymerization activities, which is a binding partner of MAL2. MAL2-positive vesicular carriers associate with short actin filaments during transcytosis in a process requiring INF2. INF2 binds Cdc42 in a GTP-loaded-dependent manner. Cdc42 and INF2 regulate MAL2 dynamics and are necessary for apical transcytosis and the formation of lateral lumens in hepatoma HepG2 cells. INF2 and MAL2 are also essential for the formation of the central lumen in organotypic cultures of epithelial MDCK cells. Our results reveal a functional mechanism whereby Cdc42, INF2, and MAL2 are sequentially ordered in a pathway dedicated to the regulation of transcytosis and lumen formation.

The MAL protein is an essential component of the specialized machinery for apical targeting in epithelial cells. The src family kinase Lck plays a pivotal role in T cell signaling. We show that MAL is required in T cells for efficient expression of Lck at the plasma membrane and activation of IL-2 transcription. To investigate the mechanism by which MAL regulates Lck targeting, we analyzed the dynamics of Lck and found that it travels to the plasma membrane in specific transport carriers containing MAL. Coimmunoprecipitation experiments indicated an association of MAL with Lck. Both carrier formation and partitioning of Lck into detergent-insoluble membranes were ablated in the absence of MAL. Polarization of T cell receptor for antigen (TCR) and microtubule-organizing center to immunological synapse (IS) were also defective. Although partial correction of the latter defects was possible by forced expression of Lck at the plasma membrane, their complete correction, formation of transport vesicles, partitioning of Lck, and restoration of signaling pathways, which are required for IL-2 transcription up-regulation, were achieved by exogenous expression of MAL. We concluded that MAL is required for recruitment of Lck to specialized membranes and formation of specific transport carriers for Lck targeting. This novel transport pathway is crucial for TCR-mediated signaling and IS assembly.

The formin INF2 promotes the formation of stabilized, detyrosinated microtubules, which are important for centrosome reorientation to the immunological synapse of T cells.

Expression of the src-family kinase lymphocyte-specific protein tyrosine kinase (Lck) at the plasma membrane is essential for it to fulfill its pivotal role in signal transduction in T lymphocytes. MAL, an integral membrane protein expressed in specific types of lymphoma, has been shown to play an important role in targeting Lck to the plasma membrane. Here we report that MAL interacts with Inverted IntroductionThe activation of the src-family kinase lymphocyte-specific protein tyrosine kinase (Lck) is one of the earliest intracellular events downstream T-cell receptor (TCR) recognition. Activated Lck phosphorylates tyrosine residues of CD3, -chain-associated protein kinase 70, and other substrates, initiating various signaling cascades that result in T-cell activation and proliferation. 1,2 Lck is predominantly associated with the cytosolic side of the plasma membrane, a localization that is consistent with its importance in TCR-mediated early signaling events. 3 Transport of Lck to the plasma membrane relies on the exocytic pathway 4 and requires addition of myristate and palmitate to the glycine and the 2 cysteine residues of its N-terminal Gly-Cys-Val-Cys sequence. 5,6 Palmitoylation/depalmitoylation of Lck can also contribute to exchange Lck between the plasma membrane and the cytosol. 7 Lck acylation is also essential for activation of downstream signaling pathways 6 and partitioning into detergent-resistant membranes 5 that are postulated to contain specialized membrane microdomains. 8 MAL is an integral membrane protein exclusively detected in detergentresistant membranes of epithelial cells and human T lymphocytes. 9,10 Recent work established that MAL, which was originally characterized as a component of the machinery for specialized transport from the Golgi to the apical surface of polarized epithelial cells, 10-12 mediates a specialized route responsible for Lck transport to the plasma membrane of human T cells. 13 The questions remain as to what other machinery collaborates with MAL to transport Lck and how this process is regulated.The important role of MAL in intracellular protein transport implies that alterations in MAL expression or subcellular distribution could be reflected in abnormal functioning of the cells. It has been reported that MAL is overexpressed in cutaneous T-cell lymphoma resistant to ␣-interferon therapy. 14 Although MAL expression is normally absent from normal B lymphocytes, MAL expression has been found in primary mediastinal B-cell lymphoma 15,16 and in a subset of Hodgkin lymphoma with an adverse outcome. 17,18 Therefore, in addition to the interest of the elucidation of the mechanism of MAL-mediated transport of Lck in normal T cells, the understanding of MAL function in normal cells would be of great help to unveil its role in tumor cells.The mechanism and regulation of MAL-mediated Lck transport in T lymphocytes are addressed in the present paper through the identification of human Inverted Formin2 (INF2) as a binding partner of MAL. INF2 domain organization is similar...

Mechanotransduction refers to the conversion of mechanical forces into biochemical or electrical signals that initiate structural and functional remodeling in cells and tissues. The heart is a kinetic organ whose form changes considerably during development and disease. This requires cardiomyocytes to be mechanically durable and able to mount coordinated responses to a variety of environmental signals on different time scales, including cardiac pressure loading and electrical and hemodynamic forces. During physiological growth, myocytes, endocardial and epicardial cells have to adaptively remodel to these mechanical forces. Here we review some of the recent advances in the understanding of how mechanical forces influence cardiac development, with a focus on fluid flow forces. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

T cell membrane receptors and signaling molecules assemble at the immunological synapse (IS) in a supramolecular activation cluster (SMAC), organized into two differentiated subdomains: the central SMAC (cSMAC), with the TCR, Lck, and linker for activation of T cells (LAT), and the peripheral SMAC (pSMAC), with adhesion molecules. The mechanism of protein sorting to the SMAC subdomains is still unknown. MAL forms part of the machinery for protein targeting to the plasma membrane by specialized mechanisms involving condensed membranes or rafts. In this article, we report our investigation of the dynamics of MAL during the formation of the IS and its role in SMAC assembly in the Jurkat T cell line and human primary T cells. We observed that under normal conditions, a pool of MAL rapidly accumulates at the cSMAC, where it colocalized with condensed membranes, as visualized with the membrane fluorescent probe Laurdan. Mislocalization of MAL to the pSMAC greatly reduced membrane condensation at the cSMAC and redistributed machinery involved in docking microtubules or transport vesicles from the cSMAC to the pSMAC. As a consequence of these alterations, the raft-associated molecules Lck and LAT, but not the TCR, were missorted to the pSMAC. MAL, therefore, regulates membrane order and the distribution of microtubule and transport vesicle docking machinery at the IS and, by doing so, ensures correct protein sorting of Lck and LAT to the cSMAC.

The formin INF2 is required for stable Glu microtubule formation and inhibition of microtubule dynamics in NIH3T3 cells downstream of mDia1 and LPA. Evidence also shows that the formation of an mDia1/INF2 complex is necessary for microtubule stabilization stimulated by LPA and is regulated by IQGAP1.

The incidence of metabolic syndrome (MetS) is increasing worldwide which makes necessary the finding of new strategies to treat and/or prevent it. The aim of this study was to analyze the possible beneficial effects of a carob fruit extract (CSAT+®) on the cardiometabolic alterations associated with MetS in mice. 16-week-old C57BL/6J male mice were fed for 26 weeks either with a standard diet (chow) or with a diet rich in fats and sugars (HFHS), supplemented or not with 4.8% of CSAT+®. CSAT+® supplementation reduced blood glucose, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) and circulating levels of total cholesterol, low-density lipoprotein (LDL) cholesterol (LDL-c), insulin, and interleukin-6 (IL-6). In adipose tissue and skeletal muscle, CSAT+® prevented MetS-induced insulin resistance, reduced macrophage infiltration and the expression of pro-inflammatory markers, and up-regulated the mRNA levels of antioxidant markers. Supplementation with CSAT+® prevented MetS-induced hypertension and decreased the vascular response of aortic rings to angiotensin II (AngII). Moreover, treatment with CSAT+® attenuated endothelial dysfunction and increased vascular sensitivity to insulin. In the heart, CSAT+® supplementation reduced cardiomyocyte apoptosis and prevented ischemia-reperfusion-induced decrease in cardiac contractility. The beneficial effects at the cardiovascular level were associated with a lower expression of pro-inflammatory and pro-oxidant markers in aortic and cardiac tissues.