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.

Concordance of the location of the innervation zone of the tibialis anterior muscle using voluntary and imposed contractions by electrostimulation.

BACKGROUND: The innervation zone (IZ) corresponds to the location of the neuromuscular junctions. Its location can be determined by using arranged surface linear electrode arrays. Typically, voluntary muscle contractions (VC) are used in this method. However, it also may be necessary to locate the IZ under clinical conditions such as spasticity, in which this type of contraction is difficult to perform. Therefore, contractions imposed by electrostimulation (ES) can be an alternative. There is little background comparing the locations of IZ obtained by two different types of contractions. OBJECTIVE: Evaluate the concordance between using voluntary and imposed contractions from electrostimulation in order to determine the location of the innervation zone of the tibialis anterior muscle in healthy volunteers. METHODS: The tibialis anterior (TA) muscle of sixteen volunteers (men: 8; women: 8; age: 22.1±1.4years, weight: 61.6±7.5kg, height: 167.1±7.5cm) were evaluated using a linear electrode array. The IZ of the TA muscle was located using two types of muscle contractions, voluntary (10% MVC) and imposed contractions by ES. The concordance between both conditions was evaluated using the Bland-Altman method and the concordance correlation coefficient (CCC). The analyses were applied to the absolute and relative positions to the length of an anatomical landmark frame. RESULTS: CCC for absolute position was 0.98 (p<0.0001, 95% CI [0.98-1.00], and CCC for relative positions also was 0.98 (p<0.0001, 95% CI [0.97-1.00]). The Bland-Altman analysis for absolute data showed an average difference of -0.63mm (SD: 4.1). Whereas, for adjusted data, the average difference was -0.20% (SD: 1.2). The power of the results, based on absolute data, was 98%, whereas for relative data, 82%. CONCLUSION: In healthy volunteers, there was a substantially concordance between the location of the IZ of the TA muscle derived from using contractions imposed by ES and the location derived from using VC.

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.

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.

[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.

Detection and sources of nonlinearity in the variability of cardiac R-R intervals and blood pressure in rats.

Beat-to-beat R-R interval (RRV) and systolic blood pressure (SPV) variability signals were obtained from unrestrained rats in baseline and under different pharmacological treatments. The origin and extent of the nonlinearity in both signals, as well as their degree of mutual coupling, was estimated using measurements from the correlation integral (CI) and recurrence quantification analysis (RQA). After the respiratory component of baseline signals was removed, the nonlinearity was lower in the RRV and disappeared in the SPV. This also decreased the RRV-SPV coupling. The nonlinearity of RRV was also reduced after atropine, and the nonlinearity of SPV was strengthened after prazosin and N(omega)-monomethyl-L-arginine (L-NMMA). Atropine and prazosin decreased CI measures of both signals, whereas propranolol, phenylephrine, and L-NMMA decreased only those of SPV. RQA indexes of RRV increased after atropine and decreased after propranolol, whereas the reverse occurred for the RRV-SPV coupling. These results suggest that: 1) the nonlinearity of RRV appears to be very dependent on the parasympathetic activity, whereas that of SPV seems to come from its respiratory component through a nonneural pathway; 2) respiratory component appears to be involved, through the parasympathetic system, in the RRV-SPV coupling; and 3) CI and RQA measures seems to be useful in assessing autonomic mediation of RRV and RRV-SPV coupling.

A novel mutation in the CAG triplet region of exon 1 of androgen receptor gene causes complete androgen insensitivity syndrome in a large kindred.

Complete androgen insensitivity syndrome (CAIS) is an X-linked inherited disease caused by mutations in the androgen receptor (AR) gene. We have previously reported the largest kindred of CAIS, with 17 46,XY psychosexual and phenotypic females who lack secondary sexual hair. Analysis of AR binding indicated a receptor-negative form of complete androgen insensitivity, and DNA linkage analysis indicated that the absent binding was not caused by a large AR gene deletion. Using PCR-single-strand DNA conformational polymorphism, PCR-denaturing gradient gel electrophoresis, and DNA sequencing, we have identified a novel mutation in the polymorphic CAG trinucleotide region of exon 1 of the AR gene, where a single adenine is inserted, or equivalently, a GC-dinucleotide is deleted at this region of the gene. The mutation results in a frameshift at amino acid 60 and a premature termination of the receptor downstream of the mutation. This predicts a mutant AR with only 79 amino acids in the amino-terminal of AR protein, prohibiting binding to the ligand, as well as the cognate DNA. The rest of the encoding regions of the AR gene in the affected subjects are normal. These results are consistent with previous ligand binding and DNA linkage analysis studies. This new mutation in the CAG trinucleotide area of exon 1 of the AR gene represents the first example of a defect in a CAG repeat causing CAIS in this large kindred. All previous reported variants in this region are changes in the number of triplet repeats.

Effects of N omega-monomethyl-L-arginine on short-term RR interval and systolic blood pressure oscillations.

The role of endothelium-derived nitric oxide (EDNO) in short-term regulation of RR interval and arterial blood pressure (BP) in conscious rats was studied with N omega-monomethyl-L-arginine (L-NMMA). RR interval and systolic BP (SBP) variability was investigated by spectral analysis procedures. L-NMMA infused intravenously (i.v.) at 1.2 mg/kg/min elicited a clear increase in blood pressure (BP), RR interval (RRI), and respiratory rate. The main spectral modifications observed during L-NMMA infusion were (a) an increase in power of lower frequency (LF, 0.02-0.20 Hz) systolic BP (SBP) oscillations, (b) a decrease in the power of middle frequency (MF, 0.20-0.60 Hz), SBP oscillations, (c) an increase in the power of respiratory (high-frequency, HF) RR oscillations, and (d) an increase in the SBP-RR correlation in the LF band. These results suggest that L-NMMA infusion induced a rearrangement in the lower frequency oscillations of SBP, in which a decrease in sympathetic activity appears to be involved. The increase in HF oscillations of the RR interval appears to be a consequence of the increase in vagal activity in response to the increase in SBP induced by L-NMMA infusion. The suggested autonomic nervous system alterations could account for the increase in the SBP-RRI correlation in the LF band after L-NMMA administration.