Monitoring spatiotemporal changes in chaperone-mediated autophagy in vivo.

Autophagy malfunctioning occurs in multiple human disorders, making attractive the idea of chemically modulating it with therapeutic purposes. However, for many types of autophagy, a clear understanding of tissue-specific differences in their activity and regulation is missing because of lack of methods to monitor these processes in vivo. Chaperone-mediated autophagy (CMA) is a selective type of autophagy that until now has only been studied in vitro and not in the tissue context at single cell resolution. Here, we develop a transgenic reporter mouse that allows dynamic measurement of CMA activity in vivo using image-based procedures. We identify previously unknown spatial and temporal differences in CMA activity in multiple organs and in response to stress. We illustrate the versatility of this model for monitoring CMA in live animals, organotypic cultures and cell cultures from these mice, and provide practical examples of multiorgan response to drugs that modulate CMA.

Profilin1 regulates invadopodium maturation in human breast cancer cells.

Invadopodia are actin-driven membrane protrusions that show oscillatory assembly and disassembly causing matrix degradation to support invasion and dissemination of cancer cells in vitro and in vivo. Profilin1, an actin and phosphoinositide binding protein, is downregulated in several adenocarcinomas and it is been shown that its depletion enhances invasiveness and motility of breast cancer cells by increasing PI(3,4)P2 levels at the leading edge. In this study, we show for the first time that depletion of profilin1 leads to an increase in the number of mature invadopodia and these assemble and disassemble more rapidly than in control cells. Previous work by Sharma et al. (2013a), has shown that the binding of the protein Tks5 with PI(3,4)P2 confers stability to the invadopodium precursor causing it to mature into a degradation-competent structure. We found that loss of profilin1 expression increases the levels of PI(3,4)P2 at the invadopodium and as a result, enhances recruitment of the interacting adaptor Tks5. The increased PI(3,4)P2-Tks5 interaction accelerates the rate of invadopodium anchorage, maturation, and turnover. Our results indicate that profilin1 acts as a molecular regulator of the levels of PI(3,4)P2 and Tks5 recruitment in invadopodia to control the invasion efficiency of invadopodia.

[Neurotransmitters, calcium signalling and neuronal communication]. / Neurotransmisores, señales de calcio y comunicación neuronal.

In this article we show some recent findings that constitute a great progress in the molecular knowledge of synaptic dynamics. To communicate, neurons use a code that includes electrical (action potentials) and chemical signals (neurotransmitters, neuromodulators). At the moment a great variety of molecules are known, whose neurotransmitter function in brain and the peripheral nervous system are out of question. Monoamines like acetylcholine, dopamine, noradrenaline, adrenaline, histamine, serotonin, glutamate, aspartate, glycine, ATP and GABA are good examples. Opioid neuropeptides, vasoactive intestinal peptide (VIP), neurokinines (substance P), somatostatin, neurotensin, neuropeptide Y, cholecystokinine, vasopressin or oxitocin have been related to the control of the stress response, sexual behaviour, food intake, pain, learning and memory, qualities that are also related to nitric oxide (NO). A great part of the molecular structure of the secretory machinery is known to be responsible for fast neurotransmitter release at the synapse, in response to action potentials. Proteins like sinaptobrevin (located in the membrane of the synaptic vesicle), sintaxin and SNAP-25 (both located at the presynaptic plasma membrane) constitute a trimeric complex which is responsible of the vesicular docking at the active sites for exocytosis. From this strategic location, vesicles release their neurotransmitter within few milliseconds, when the action potential invades the nerve terminal and activates the opening of the different subtypes of voltage-dependent Ca2+ channels. The asymmetric geographical distribution of each type of channel, in different neurons, rose the hypothesis that Ca2+ that enters through each subtype of channel is compartmentalised, thus favouring the generation of Ca2+ microdomains, in the cytosol and the nucleus, involved in different cellular functions. This great biochemical synaptic heterogeneity is facilitating the selection of many biological targets to develop drugs with potential therapeutic applications in neuropsychiatric diseases i.e. Alzheimer's, Parkinson, epilepsies, stroke, vascular dementia, depression, schizophrenia, anxiety and so on.

Macrophage contact induces RhoA GTPase signaling to trigger tumor cell intravasation.

Most cancer patients die as a result of metastasis, thus it is important to understand the molecular mechanisms of dissemination, including intra- and extravasation. Although the mechanisms of extravasation have been vastly studied in vitro and in vivo, the process of intravasation is still unclear. Furthermore, how cells in the tumor microenvironment facilitate tumor cell intravasation is still unknown. Using high-resolution imaging, we found that macrophages enhance tumor cell intravasation upon physical contact. Macrophage and tumor cell contact induce RhoA activity in tumor cells, triggering the formation of actin-rich degradative protrusions called invadopodia, enabling tumor cells to degrade and break through matrix barriers during tumor cell transendothelial migration. Interestingly, we show that macrophage-induced invadopodium formation and tumor cell intravasation also occur in patient-derived tumor cells and in vivo models, revealing a conserved mechanism of tumor cell intravasation. Our results illustrate a novel heterotypic cell contact-mediated signaling role for RhoA, as well as yield mechanistic insight into the ability of cells within the tumor microenvironment to facilitate steps of the metastatic cascade.

Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation.

Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells. The mechanisms regulating invadopodium assembly and maturation are not understood. We have dissected the stages of invadopodium assembly and maturation and show that invadopodia use cortactin phosphorylation as a master switch during these processes. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex-dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent actin polymerization. After barbed end formation, cortactin is dephosphorylated, which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation.