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 NHE1-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.

To better understand the functional role of the mitochondrial network in shaping the Ca 2؉ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca 2؉ sensors ("Cameleons," "Camgaroos," and "Pericams") and of the classical Ca 2؉ -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 Ca 2؉ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca 2؉ 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 Ca 2؉ release channels (and thus responsible for the fast uptake of Ca 2؉ 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. C ardiac hypertrophy accompanies many forms of heart pathologies, such as genetic or congenital defects, ischemia, and hypertension. The molecular signaling pathways through which the different hypertrophic stimuli modulate cardiac cell size include mitogen-activated protein kinase, Gp130/Stat3, Calmodulin (CaM)-dependent kinases, and the calcineurin-regulated pathway (1). The latter has recently received much attention, in part because it can be the target of therapeutic intervention with well known drugs. For example, in transgenic mice, it has been demonstrated that overexpression of the Ca 2ϩ -dependent phosphatase calcineurin causes a dramatic increase in the size of the heart, inhibited by cyclosporine A (CsA), a calcineurin blocker (2). Along the same line, overexpression of Cain/Cabin (molecules that associate with the calcineurin and inhibit its activity) attenuate cardiac hypertrophy caused not only by calcineurin overexpression but also by pressure overload or ␤-adrenergic receptor stimulation (3). The effect of CsA on cardiac hypertrophy, however, has led to contradictory results in different model systems (4,5).At the cellular level, cardiomyocyte hypertrophy is characterized by an increase in cell size, enhanced protein synthesis, activation of fetal genes, and cytoskeleton reorganization (4, 5). A number of treatments are known to induce cardiac cell hypertrophy in vitro, including angiotensin II (Ang II) (6), catecholamines (7), endothelin (8), and aldosterone (9). Many, but not all, of these hormones are known to be coupled to alterations in Ca 2ϩ homeostasis. In particular, (i) Ang II (6) and endothelin (10) are coupled to IP 3 generation and Ca 2ϩ mobilization from stores; (ii) catecholamines, in particular through ␤1 receptors, are known to induce an increase in the frequency and amplitude of Ca 2ϩ spiking in cardiomyocytes (7); and (iii) the mechanism of aldosterone-induced hypertrophy in vit...

A novel glycolipid of mass 1935 and a phospholipid of mass 1522 are the main residual lipids (along with traces of PGP-Me, S-TGD-1, and PG) specifically associated with "delipidated" bacteriorhodopsin fractions BR I and BR II, prepared by Triton X-100 treatment of purple membrane (PM), from a genetically engineered strain (L33) of Halobacterium salinarum, and chromatography on phenyl-Sepharose CL-4B. The novel glycolipid and phospholipid are components of the PM matrix not previously described. The TLC isolated and purified novel glycolipid and phospholipid were shown, by chemical degradation, mass spectrometry, and NMR analyses, to have the structure, respectively, of a phosphosulfoglycolipid, 3-HSO(3)-Galp-beta1,6Manp-alpha1,2Glcp-alpha1,1-[sn-2, 3-di-O-phytanylglycerol]-6-[phospho-sn-2,3-di-O-phytanylglycero l], and of a glycerol diether analogue of bisphosphatidylglycerol (cardiolipin), sn-2,3-di-O-phytanyl-1-phosphoglycerol-3-phospho-sn-2, 3-di-O-phytanylglycerol.

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