Inmaculada Navarro-Lérida

Biomechanical Remodeling of the Microenvironment by Stromal Caveolin-1 Favors Tumor Invasion and Metastasis

Mechanotransduction is a key determinant of tissue homeostasis and tumor progression. It is driven by intercellular adhesions, cell contractility, and forces generated within the microenvironment and is dependent on extracellular matrix composition, organization, and compliance. We show that caveolin-1 (Cav1) favors cell elongation in three-dimensional cultures and promotes Rho- and force-dependent contraction, matrix alignment, and microenvironment stiffening through regulation of p190RhoGAP. In turn, microenvironment remodeling by Cav1 fibroblasts forces cell elongation. Cav1-deficient mice have disorganized stromal tissue architecture. Stroma associated with human carcinomas and melanoma metastases is enriched in Cav1-expressing carcinoma-associated fibroblasts (CAFs). Cav1 expression in breast CAFs correlates with low survival, and Cav1 depletion in CAFs decreases CAF contractility. Consistently, fibroblast expression of Cav1, through p190RhoGAP regulation, favors directional migration and invasiveness of carcinoma cells in vitro. In vivo, stromal Cav1 remodels peri- and intratumoral microenvironments to facilitate tumor invasion, correlating with increased metastatic potency. Thus, Cav1 modulates tissue responses through force-dependent architectural regulation of the microenvironment.

A palmitoylation switch mechanism regulates Rac1 function and membrane organization

The small GTPase Rac1 plays important roles in many processes, including cytoskeletal reorganization, cell migration, cell‐cycle progression and gene expression. The initiation of Rac1 signalling requires at least two mechanisms: GTP loading via the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle, and targeting to cholesterol‐rich liquid‐ordered plasma membrane microdomains. Little is known about the molecular mechanisms governing this specific compartmentalization. We show that Rac1 can incorporate palmitate at cysteine 178 and that this post‐translational modification targets Rac1 for stabilization at actin cytoskeleton‐linked ordered membrane regions. Palmitoylation of Rac1 requires its prior prenylation and the intact C‐terminal polybasic region and is regulated by the triproline‐rich motif. Non‐palmitoylated Rac1 shows decreased GTP loading and lower association with detergent‐resistant (liquid‐ordered) membranes (DRMs). Cells expressing no Rac1 or a palmitoylation‐deficient mutant have an increased content of disordered membrane domains, and markers of ordered membranes isolated from Rac1‐deficient cells do not correctly partition in DRMs. Importantly, cells lacking Rac1 palmitoylation show spreading and migration defects. These data identify palmitoylation as a mechanism for Rac1 function in actin cytoskeleton remodelling by controlling its membrane partitioning, which in turn regulates membrane organization.

Recognition of novel viral sequences that associate with the dynein light chain LC8 identified through a pepscan technique

Recent data from multiple laboratories indicate that upon infection, many different families of viruses hijack the dynein motor machinery and become transported in a retrograde manner towards the cell nucleus. In certain cases, one of the dynein light chains, LC8, is involved in this interaction. Using a library of overlapping dodecapeptides synthesized on a cellulose membrane (pepscan technique) we have analyzed the interaction of the dynein light chain LC8 with 17 polypeptides of viral origin. We demonstrate the strong binding of two herpesvirus polypeptides, the human adenovirus protease, vaccinia virus polymerase, human papillomavirus E4 protein, yam mosaic virus polyprotein, human respiratory syncytial virus attachment glycoprotein, human coxsackievirus capsid protein and the product of the AMV179 gene of an insect poxvirus to LC8. Our data corroborate the manipulation of the dynein macromolecular complex of the cell during viral infection and point towards the light chain LC8 as one of the most frequently used targets of virus manipulation.

Coronin 1A promotes a cytoskeletal‐based feedback loop that facilitates Rac1 translocation and activation

The activation of the Rac1 GTPase during cell signalling entails its translocation from the cytosol to membranes, release from sequestering Rho GDP dissociation inhibitors (RhoGDI), and GDP/GTP exchange. In addition to those steps, we show here that optimal Rac1 activation during cell signalling requires the engagement of a downstream, cytoskeletal‐based feedback loop nucleated around the cytoskeletal protein coronin 1A and the Rac1 exchange factor ArhGEF7. These two proteins form a cytosolic complex that, upon Rac1‐driven F‐actin polymerization, translocates to juxtamembrane areas where it expands the pool of activated, membrane‐bound Rac1. Such activity requires the formation of an F‐actin/ArhGEF7‐dependent physical complex of coronin 1A with Pak1 and RhoGDIα that, once assembled, promotes the Pak1‐dependent dissociation of Rac1 from the Rac1/RhoGDIα complex and subsequent Rac1 activation. Genetic evidence demonstrates that this relay circuit is essential for generating sustained Rac1 activation levels during cell signalling.

Palmitoylation of inducible nitric-oxide synthase at Cys-3 is required for proper intracellular traffic and nitric oxide synthesis.

A number of cell types express inducible nitric-oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide or proinflammatory cytokines. Although it has been known for some time that the N-terminal end of NOS2 suffers a post-translational modification, its exact identification has remained elusive. Using radioactive fatty acids, we show herein that NOS2 becomes thioacylated at Cys-3 with palmitic acid. Site-directed mutagenesis of this single residue results in the absence of the radiolabel incorporation. Acylation of NOS2 is completely indispensable for intracellular sorting and ·NO synthesis. In fact, a C3S mutant of NOS2 is completely inactive and accumulates to intracellular membranes that almost totally co-localize with the Golgi marker β-cop. Likewise, low concentrations of the palmitoylation blocking agents 2-Br-palmitate or 8-Br-palmitate severely affected the ·NO synthesis of both NOS2 induced in muscular myotubes and transfected NOS2. However, unlike endothelial NOS, palmitoylation of inducible NOS is not involved in its targeting to caveolae. We have created 16 NOS2-GFP chimeras to inspect the effect of the neighboring residues of Cys-3 on the degree of palmitoylation. In this regard, the hydrophobic residue Pro-4 and the basic residue Lys-6 seem to be indispensable for palmitoylation. In addition, agents that block the endoplasmic reticulum to Golgi transit such as brefeldin A and monensin drastically reduced NOS2 activity leading to its accumulation in perinuclear areas. In summary, palmitoylation of NOS2 at Cys-3 is required for both its activity and proper intracellular localization.

Rac1 Nucleocytoplasmic Shuttling Drives Nuclear Shape Changes and Tumor Invasion

Nuclear membrane microdomains are proposed to act as platforms for regulation of nuclear function, but little is known about the mechanisms controlling their formation. Organization of the plasma membrane is regulated by actin polymerization, and the existence of an actin pool in the nucleus suggests that a similar mechanism might operate here. We show that nuclear membrane organization and morphology are regulated by the nuclear level of active Rac1 through actin polymerization-dependent mechanisms. Rac1 nuclear export is mediated by two internal nuclear export signals and through its interaction with nucleophosmin-1 (B23), which acts as a Rac1 chaperone inside the nucleus. Rac1 nuclear accumulation alters the balance between cytosolic Rac1 and Rho, increasing RhoA signaling in the cytoplasm and promoting a highly invasive phenotype. Nuclear Rac1 shuttling is a finely tuned mechanism for controlling nuclear shape and organization and cell invasiveness.

Interplay between hepatic mitochondria-Associated membranes, lipid metabolism and caveolin-1 in mice

The mitochondria-associated membrane (MAM) is a specialized subdomain of the endoplasmic reticulum (ER) which acts as an intracellular signaling hub. MAM dysfunction has been related to liver disease. We report a high-throughput mass spectrometry-based proteomics characterization of MAMs from mouse liver, which portrays them as an extremely complex compartment involved in different metabolic processes, including steroid metabolism. Interestingly, we identified caveolin-1 (CAV1) as an integral component of hepatic MAMs, which determine the relative cholesterol content of these ER subdomains. Finally, a detailed comparative proteomics analysis between MAMs from wild type and CAV1-deficient mice suggests that functional CAV1 contributes to the recruitment and regulation of intracellular steroid and lipoprotein metabolism-related processes accrued at MAMs. The potential impact of these novel aspects of CAV1 biology on global cell homeostasis and disease is discussed.

N-terminal palmitoylation within the appropriate amino acid environment conveys on NOS2 the ability to progress along the intracellular sorting pathways

We have analysed the mechanism by which palmitoylation permits the progression of nitric oxide synthase 2 (NOS2) along the ER-Golgi-TGN pathway. Introduction of an additional myristoylation site at the N-terminus of NOS2 resulted in a chimera that displayed an enhanced association with the particulate fraction and with the plasma membrane but did not display increased enzymatic activity. In the absence of palmitoylation, introduction of a surrogate myristoylation site resulted in a mutant NOS2 with only 25% activity compared with the wild-type enzyme. Hence, the novel surrogate myristoyl moiety not only failed to increase NOS2 activity when introduced in a wild-type sequence environment, but was also unable to rescue the inactive phenotype of the Cys3Ser mutant. Introduction of an additional palmitoylatable Cys at position 2 of the wild-type sequence resulted in a chimera that associated to a larger degree with membranes and displayed decreased activity. Our data indicate that palmitoylation of inducible NOS at position 3 exquisitely determines its transit along the secretory pathway following a route that cannot be mimicked by a surrogate myristoylation or by a palmitate at position 2. In addition, the exit of NOS2 from the TGN and the accumulation in the cellular plasma membrane per se did not correlate with increased ·NO synthesis.

Induction of nitric oxide synthase-2 proceeds with the concomitant downregulation of the endogenous caveolin levels.

Several cell types express inducible nitric oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide (LPS) or proinflammatory cytokines. For instance, muscular cells treated with LPS and interferon γ (IFN-γ) respond by increasing the mRNA and protein levels of NOS2, and synthesize large amounts of nitric oxide. We show here that transcriptional induction of NOS2 in muscular cells proceeds with a concomitant decrease in the levels of caveolin-1, -2 and -3. Addition of ·NO-releasing compounds to C2C12 muscle cells reveals that this downregulation of the caveolin (cav) levels is due to the presence of ·NO itself in the case of caveolin-3 and to the action of the LPS/IFN-γ in the case of cav-1 and cav-2. Likewise, muscle cells obtained from NOS2-/- knockout mice challenged with LPS/IFN-γ could downregulate their levels of cav-1 but not of cav-3, unlike wild-type animals, in which both cav-1 and cav-3 levels diminished in the presence of the proinflammatory insult. Laser confocal immunofluorescence analysis proves that ·NO exerts autocrine and paracrine actions, hence diminishing the cav-3 levels. When the induced NOS2 was purified using an affinity resin or immunoprecipitated from muscular tissues, it appears strongly bound not only to calmodulin but also to cav-1, and marginally to cav-2 and cav-3. When the cav levels where reduced using antisense oligonucleotides, an increase in the NOS2-derived ·NO levels could be measured, demonstrating the inhibitory role of the three cav isoforms. Our results show that cells expressing NOS2 diminish their cav levels when the synthesis of ·NO is required.

Caveolin-1-dependent nanoscale organization of the BCR regulates B cell tolerance

Caveolin-1 (Cav1) regulates the nanoscale organization and compartmentalization of the plasma membrane. Here we found that Cav1 controlled the distribution of nanoclusters of isotype-specific B cell antigen receptors (BCRs) on the surface of B cells. In mature B cells stimulated with antigen, the immunoglobulin M BCR (IgM-BCR) gained access to lipid domains enriched for GM1 glycolipids, by a process that was dependent on the phosphorylation of Cav1 by the Src family of kinases. Antigen-induced reorganization of nanoclusters of IgM-BCRs and IgD-BCRs regulated BCR signaling in vivo. In immature Cav1-deficient B cells, altered nanoscale organization of IgM-BCRs resulted in a failure of receptor editing and a skewed repertoire of B cells expressing immunoglobulin-μ heavy chains with hallmarks of poly- and auto-reactivity, which ultimately led to autoimmunity in mice. Thus, Cav1 emerges as a cell-intrinsic regulator that prevents B cell–induced autoimmunity by means of its role in plasma-membrane organization.