Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires membrane and released proteases focalized at membrane structures called invadopodia. While extracellular acidification is important in driving tumor invasion, the structure/function mechanisms underlying this regulation are still unknown. Invadopodia are similar in structure and function to osteoclast podosomes responsible for bone degradation, and extracellular acidification is central to podosome action, suggesting that it could also be for invadopodial function. Here, utilizing a novel system for in situ zymography in native matrices, we show that the Na ⁺ /H ⁺ exchanger (NHE1) and NHEl‐generated extracellular acidification are localized at and necessary for invadopodial‐dependent ECM degradation, thereby promoting tumor invasion. Stimulation with EGF increased both NHE1‐dependent proton secretion and ECM degradation. Manipulation of the NHE1 expression by RNA interference or activity via either transport‐deficient mutation or the specific inhibitor cariporide confirmed that NHE1 expression and activity are required for invadopodia‐mediated ECM degradation. Taken together, our data show a concordance among NHE1 localization, the generation of a well‐defined acidic extracellular pH in the nanospace surrounding invadopodia, and matrix‐degrading activity at invadopodia of human malignant breast carcinoma cells, providing a structural basis for the role of NHE1 in invasion and identifying NHE1 as a strategic target for therapeutic intervention.—Busco, G., Cardone, R. A., Greco, M. R., Bellizzi, A., Colella, M., Antelmi, E., Mancini, M. T., Dell'Aquila, M. E., Casavola, V., Paradiso, A., Reshkin, S. J. NHE1 promotes invadopodial ECM proteolysis through acidification of the peri‐invadopodial space. FASEB J. 24, 3903–3915 (2010). www.fasebj.org

To better understand the functional role of the mitochondrial network in shaping the Ca²⁺ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca²⁺ sensors (“Cameleons,” “Camgaroos,” and “Pericams”) and of the classical Ca²⁺-sensitive photoprotein aequorin, all targeted to the mitochondrial matrix. The properties of the green fluorescent protein-based probes in terms of subcellular localization, photosensitivity, and Ca²⁺ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca²⁺ probe for single cell studies, although this probe too is not devoid of problems. The results obtained with ratiometric pericam in single cells, combined with those obtained at the population level with aequorin, provide strong evidence demonstrating that the close vicinity of mitochondria to the Ca²⁺ release channels (and thus responsible for the fast uptake of Ca²⁺ by mitochondria upon receptor activation) are highly stable in time, suggesting the existence of specific interactions between mitochondria and the endoplasmic reticulum.

The role of Ca ²⁺ signaling in triggering hypertrophy was investigated in neonatal rat cardiomyocytes in vitro . We show that an increase in cell size and sarcomere reorganization were elicited by receptor agonists such as Angiotensin II, aldosterone, and norepinephrine and by a small rise in medium KCl concentration, a treatment devoid of direct effects on receptor functions. All these treatments increased the frequency of spontaneous [Ca ²⁺ ] transients, caused nuclear translocation of transfected NFAT(GFP), and increased the expression of a NFAT-sensitive reporter gene. There was no increase in Ca ²⁺ spark frequency in the whole cell or in the perinuclear region under these conditions. Hypertrophy and NFAT translocation but not the increased frequency of [Ca ²⁺ ] transients were inhibited by the calcineurin inhibitor cyclosporine A. Hypertrophy by the different stimuli was insensitive to inhibition of myofilament contraction. We concluded that calcineurin–NFAT can act as integrators of the contractile Ca ²⁺ signal, and that they can decode alterations in the frequency even of rapid Ca ²⁺ oscillations.

FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg(-1) protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min(-1)·mg(-1) protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.