After half a century mitochondrial calcium in- and efflux machineries reveal themselves

Mitochondrial Ca(2+) uptake and release play a fundamental role in the control of different physiological processes, such as cytoplasmic Ca(2+) signalling, ATP production and hormone metabolism, while dysregulation of mitochondrial Ca(2+) handling triggers the cascade of events that lead to cell death. The basic mechanisms of mitochondrial Ca(2+) homeostasis have been firmly established for decades, but the molecular identities of the channels and transporters responsible for Ca(2+) uptake and release have remained mysterious until very recently. Here, we briefly review the main findings that have led to our present understanding of mitochondrial Ca(2+) homeostasis and its integration in cell physiology. We will then discuss the recent work that has unravelled the biochemical identity of three key molecules: NCLX, the mitochondrial Na(+)/Ca(2+) antiporter, MCU, the pore-forming subunit of the mitochondrial Ca(2+) uptake channel, and MICU1, one of its regulatory subunits.     

OCRL controls trafficking through early endosomes via PtdIns4,5P₂-dependent regulation of endosomal actin

Mutations in the phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P(2)) 5-phosphatase OCRL cause Lowe syndrome, which is characterised by congenital cataracts, central hypotonia, and renal proximal tubular dysfunction. Previous studies have shown that OCRL interacts with components of the endosomal machinery; however, its role in endocytosis, and thus the pathogenic mechanisms of Lowe syndrome, have remained elusive. Here, we show that via its 5-phosphatase activity, OCRL controls early endosome (EE) function. OCRL depletion impairs the recycling of multiple classes of receptors, including megalin (which mediates protein reabsorption in the kidney) that are retained in engorged EEs. These trafficking defects are caused by ectopic accumulation of PtdIns4,5P(2) in EEs, which in turn induces an N-WASP-dependent increase in endosomal F-actin. Our data provide a molecular explanation for renal proximal tubular dysfunction in Lowe syndrome and highlight that tight control of PtdIns4,5P(2) and F-actin at the EEs is essential for exporting cargoes that transit this compartment.     

Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure

The pathological form of prion protein (PrP^Sc), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP^Sc extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90–231 (hPrP_90–231) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP^Sc. In the present study we demonstrate that hPrP_90–231, pre-incubated with 10 mM Ca⁺⁺ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP_90–231 bearing pathogenic mutations (D202N and E200K). We also report that Ca⁺⁺ binding to hPrP_90–231 induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP_90–231 cytotoxicity. Finally, by in silico structural analysis, we propose that Ca⁺⁺ binding to hPrP_90–231 modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.     

Ca(2+) dysregulation in neurons from transgenic mice expressing mutant presenilin 2

Mutations in amyloid precursor protein (APP), and presenilin‐1 and presenilin‐2 (PS1 and PS2) have causally been implicated in Familial Alzheimer’s Disease (FAD), but the mechanistic link between the mutations and the early onset of neurodegeneration is still debated. Although no consensus has yet been reached, most data suggest that both FAD‐linked PS mutants and endogenous PSs are involved in cellular Ca²⁺ homeostasis. We here investigated subcellular Ca²⁺ handling in primary neuronal cultures and acute brain slices from wild type and transgenic mice carrying the FAD‐linked PS2‐N141I mutation, either alone or in the presence of the APP Swedish mutation. Compared with wild type, both types of transgenic neurons show a similar reduction in endoplasmic reticulum (ER) Ca²⁺ content and decreased response to metabotropic agonists, albeit increased Ca²⁺ release induced by caffeine. In both transgenic neurons, we also observed a higher ER–mitochondria juxtaposition that favors increased mitochondrial Ca²⁺ uptake upon ER Ca²⁺ release. A model is described that integrates into a unifying hypothesis the contradictory effects on Ca²⁺ homeostasis of different PS mutations and points to the relevance of these findings in neurodegeneration and aging.     

The elusive importance of being a mitochondrial Ca uniporter

The molecular components of the mitochondrial Ca²⁺ uptake machinery have been only recently identified. In the last months, in addition to the pore forming subunit and of one regulatory protein (named MCU and MICU1, respectively) other four components of this complex have been described. In addition, a MCU KO mouse model has been generated and a genetic human disease due to missense mutation of MICU1 has been discovered. In this contribution, we will first summarize the recent findings, discussing the roles of the different subunits of the mitochondrial Ca²⁺ uptake complex, pointing to the current contradictions in the published data, as well as possible explanations. Finally we will speculate on the recent, totally unexpected, results obtained in the MCU knock-out (KO) mice.     

Das Bakterien/L-Formen-Verhältnis in den aus Mäusen isolierten Proteus-und Salmonella-Stämmen

Schlußfolgerungen und zusammenfassung Aus dem bei einigen Bakterienstämmen durchgeführten Studium des Bakterien/L-Formen-Verhältnisses (B/L-Verhältnis) ging hervor, daß bei einer Bakterienart immer eine gleiche Zahl von Zellen vorhanden ist, die L-Umwandlungen aufweisen. Diese Zahl ist je nach der Art verschieden. Bei zwei Proteus-Stämmen und vier Salmonella-Stämmen und den gleichen aus der Maus isolierten Stämmen war das Bakterienkolonien/L-Kolonien-Verhältnis für beide Proteusstämme sehr niedrig, und zwar für P ₁₈: 16, für Ahmed: 250; dagegen zeigen Salmonellen ein hohes Verhältnis (S. 240: 850000; S. 168: 20 Millionen).Außerdem wurde beobachtet, daß die gleichen Bakterienstämme nach Aufenthalt in der Maus eine höhere Zahl von L-Kolonien erzeugen. Es wird angenommen, daß im tierischen Organismus die widerstandsfähigsten Zellen der Bakterienstämme ausgewählt werden und daß diese Zellen auch die Fähigkeit haben, das Überleben der Art durch den L-Cyclus zu sichern.Vorstand: G. F. De Gaetani     

Ocean acidification boosts larval fish development but reduces the window of opportunity for successful settlement

Locating appropriate settlement habitat is a crucial step in the life cycle of most benthic marine animals. In marine fish, this step involves the use of multiple senses, including audition, olfaction and vision. To date, most investigations of larval fish audition focus on the hearing thresholds to various frequencies of sounds without testing an ecological response to such sounds. Identifying responses to biologically relevant sounds at the development stage in which orientation is most relevant is fundamental. We tested for the existence of ontogenetic windows of reception to sounds that could act as orientation cues with a focus on vulnerability to alteration by human impacts. Here we show that larvae of a catadromous fish species (barramundi, Lates calcarifer) were attracted towards sounds from settlement habitat during a surprisingly short ontogenetic window of approximately 3 days. Yet, this auditory preference was reversed in larvae reared under end-of-century levels of elevated CO₂, such that larvae are repelled from cues of settlement habitat. These future conditions also reduced the swimming speeds and heightened the anxiety levels of barramundi. Unexpectedly, an acceleration of development and onset of metamorphosis caused by elevated CO₂ were not accompanied by the earlier onset of attraction towards habitat sounds. This mismatch between ontogenetic development and the timing of orientation behaviour may reduce the ability of larvae to locate habitat or lead to settlement in unsuitable habitats. The misinterpretation of key orientation cues can have implications for population replenishment, which are only exacerbated when ontogenetic development decouples from the specific behaviours required for location of settlement habitats.     

Effect of cocultivation of the levels of spontaneous and induced sister chromatid exchanges in chinese hamster cells

The level of sister chromatid exchanges, both spontaneous and induced by UV light or ethylmethanesulphonate (EMS), in Chinese hamster V79/AP4 cell line, is partially reduced upon cocultivation with human cells. Normal and xeroderma pigmentosum (XP) cells are equally effective. On the basis of these observations, cocultivation may represent a good tool for investigating the SCE's formation processes.     

Mechanism of active shrinkage in mitochondria II. Coupling between strong electrolyte fluxes

1. Addition of succinate to valinomycin-treated mitochondria incubated in KCl causes a large electrolyte penetration. The process depends on a steady supply of energy and involves a continuous net extrusion of protons. Rates of respiration and of electrolyte penetration proceed in a parallel manner.2. A passive penetration of K⁺ salt of permeant anions occurs in respiratory-inhibited mitochondria after addition of valinomycin. Addition of succinate at the end of the passive swelling starts an active extrusion of anions and cations with restoration of the initial volume. The shrinkage is accompanied by a slow reuptake of protons. The initiation of the active shrinkage correlates with the degree of stretching of the inner membrane. The extrusion of electrolytes is inhibited by nigericin, while it is only slightly sensitive to variations of the valinomycin concentration larger than two orders of magnitude.3. Passive swelling and active shrinkage occurs also when K⁺ is replaced by a large variety of organic cations. The rate of organic cation penetration is enhanced by tetraphenylboron, while the rate of electrolyte extrusion is insensitive to variation of the tetraphenylboron concentration.4. Active shrinkage, either with K⁺ or organic cation salts, is inhibited by weak acids. The phosphate inhibition is removed by SH inhibitors. The active shrinkage is also inhibited by mersalyl to an extent of about 60%.5. Three models of active shrinkage are discussed: (a) mechanoprotein, (b) electrogenic proton pump, and (c) proton-driven cation anion pump.