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Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water‐accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein‐rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox‐to‐condensed transition or a mitochondrial unfolded protein response. CAP‐treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants. Synopsis: It is still unclear whether presence of macromolecules in the highly protein‐rich mitochondrial matrix fluid affects solute diffusion and biochemical reactions remains unclear. This study identifies macromolecular crowding as a stress‐dependent determinant of matrix solute mobility and solvent viscosity, suggesting additional regulatory layers of mitochondrial biology.Mitochondrial matrix‐targeted fusion peptides of increasing molecular weight show a decreasing solvent‐dependent diffusion constant indicative of macromolecular crowding.Mitochondrial impairment by chloramphenicol decreases the number of cristae and increases matrix solvent crowding and viscosity.The mitochondrial matrix solvent exhibits macromolecular crowding, suggesting that the kinetics of matrix biochemical reactions depend on the molecular weight of the involved crowders and reactants. [ABSTRACT FROM AUTHOR]

Translation of extracellular hormonal input into cellular responses is often mediated by repetitive increases in cytosolic free Ca2+ concentration ([Ca2+]c). Amplitude, duration and frequency of these so-called [Ca2+]c oscillations then carry information about the nature and concentration of the extracellular signalling molecule. At present, there are different hypotheses concerning the induction and control of these oscillations. Here, we investigated the role of agonist-induced receptor phosphorylation in this process using Chinese hamster ovary cells stably expressing a variant of the cholecystokinin 1 receptor ( CCK1R) lacking the four consensus sites for protein kinase C ( PKC) phosphorylation and deficient in CCK-induced receptor phosphorylation ( CCK1R-mt cells). In the presence of cholecystokinin-(26-33)-peptide amide ( CCK-8), these cells displayed Ca2+ oscillations with a much more pronounced bursting dynamics rather than the dominant spiking dynamics observed in Chinese hamster ovary cells stably expressing the wild-type CCK1R. The bursting behaviour returned to predominantly spiking behaviour following removal of extracellular Ca2+, suggesting that CCK-8-induced, PKC-mediated CCK1R phosphorylation inhibits Ca2+ influx across the plasma membrane. To gain mechanistic insight into the underlying mechanism we developed a mathematical model able to reproduce the experimental observations. From the model we conclude that binding of CCK-8 to the CCK1R leads to activation of PKC which subsequently phosphorylates the receptor to inhibit the receptor-mediated influx of Ca2+ across the plasma membrane. Receptor-specific differences in this feedback mechanism may, at least in part, explain the observation that different agonists evoke [Ca2+]c oscillations with different kinetics in the same cell type. [ABSTRACT FROM AUTHOR]

Mitochondrial oxidative phosphorylation (OXPHOS) represents the final step in the conversion of nutrients into cellular energy. Genetic defects in the OXPHOS system have an incidence between 1:5,000 and 1:10,000 live births. Inherited isolated deficiency of the first complex (CI) of this system, a multisubunit assembly of 45 different proteins, occurs most frequently and originates from mutations in either the nuclear DNA, encoding 38 structural subunits and several assembly factors, or the mitochondrial DNA, encoding 7 structural subunits. The deficiency is associated with devastating multisystemic disorders, often affecting the brain, with onset in early childhood. There are currently no rational treatment strategies. Here, we present an overview of the genetic origins and cellular consequences of this deficiency and discuss how these insights might aid future development of treatment strategies. [ABSTRACT FROM AUTHOR]

Objective There are marked mitochondrial abnormalities in parkin-knock-out Drosophila and other model systems. The aim of our study was to determine mitochondrial function and morphology in parkin-mutant patients. We also investigated whether pharmacological rescue of impaired mitochondrial function may be possible in parkin-mutant human tissue. Methods We used three sets of techniques, namely, biochemical measurements of mitochondrial function, quantitative morphology, and live cell imaging of functional connectivity to assess the mitochondrial respiratory chain, the outer shape and connectivity of the mitochondria, and their functional inner connectivity in fibroblasts from patients with homozygous or compound heterozygous parkin mutations. Results Parkin-mutant cells had lower mitochondrial complex I activity and complex I-linked adenosine triphosphate production, which correlated with a greater degree of mitochondrial branching, suggesting that the functional and morphological effects of parkin are related. Knockdown of parkin in control fibroblasts confirmed that parkin deficiency is sufficient to explain these mitochondrial effects. In contrast, 50% knockdown of parkin, mimicking haploinsufficiency in human patient tissue, did not result in impaired mitochondrial function or morphology. Fluorescence recovery after photobleaching assays demonstrated a lower level of functional connectivity of the mitochondrial matrix, which further worsened after rotenone exposure. Treatment with experimental neuroprotective compounds resulted in a rescue of the mitochondrial membrane potential. Interpretation Our study demonstrates marked abnormalities of mitochondrial function and morphology in parkin-mutant patients and provides proof-of-principle data for the potential usefulness of this new model system as a tool to screen for disease-modifying compounds in genetically homogenous parkinsonian disorders. Ann Neurol 2008;64:555-565 [ABSTRACT FROM AUTHOR]

The potent inhibitor of Ca24. Mg24-ATPase activity, thapsigargin, has been used to investigate the effect of ambient free Ca24 on basal Mg24-dependent ATPase activity in rat liver microsomes. Thapsigargin non-competitively inhibited both Ca24-stimulated ATP-dependent Ca24 uptake and Ca24-stimulated Mg24-dependent ATPase activity. At a concentration of I μM, thapsigargin completely inhibited the Ca24 L-pump activity, without affecting Mg24-dependent ATPase activity measured in the absence of Ca24. Increasing the ambient free Ca24 concentration did not alter the basal Mg24-dependent ATPase activity: The data presented indicate that ATPase activity measured in the absence of Ca24 is a reliable measure for the basal Mg24-dependent ATPase activity and that, consequently, the Ca24-stimulated Mg24-dependent ATPase activity can indeed be defined as the difference between the ATPase activity measured in the presence and the absence of Ca24. [ABSTRACT FROM AUTHOR]

Treatment of rabbit pancreatic acini with the phorbol ester, 12-O-etradecanoylphorbol 13-acetate (TPA), resulted in a time- and dose-dependent decrease of soluble protein kinase C activity coinciding with an increase of protein kinase C activity in the particulate fraction. After 5 min, soluble protein kinase C activity had decreased to almost 10% of the corresponding control. Total extractable protein kinase C activity, however, remained unchanged, indicating that the decrease of soluble protein kinase C activity was not due to TPA-induced inactivation of the enzyme. The biologically inactive phorbol ester, 4α-phorbol 12,1 3-didecanoate, did not induce such a translocation of protein kinase C. The half-maximal concentration for TPA-induced translocation of protein kinase C was 40 nM, and was equal to that for TPA-induced amylase secretion from isolated acini. This suggests that translocation of protein kinase C to the particulate fraction is an important step in TPA-induced activation of protein kinase C and enzyme secretion. On the other hand, cholecystokinin, a secretagogue of the calcium-mobilizing type, whose secretory action is thought to be mediated, at least in part. by protein kinase C, did not change the subcellular distribution of protein kinase C. In the presence of R59022 6-(2-¦(4- fluorophenyl)phenylmethylene]-1 -piperidinyl) ethyl-7-methyl-5H-thiazolo[3 ,2-a]pyrimidin-5-one, an inhibitor of diacylglycerol kinase activity, cholecystokinin produced a small but significant translocation of protein kinase C, suggesting that the inability of the hormone to induce translocation is not due to a rapid conversion of the diacylglycerol formed into phosphatidic acid. [ABSTRACT FROM AUTHOR]

Digital-imaging microscopy was performed to study the effect of Coxsackie B3 virus infection on the cytosolic free Ca2+ concentration and the Ca2+ content of the endoplasmic reticulum (ER). During the course of infection a gradual increase in the cytosolic free Ca2+ concentration was observed, due to the influx of extracellular Ca2+. The Ca2+ content of the ER decreased in time with kinetics inversely proportional to those of viral protein synthesis. Individual expression of protein 2B was sufficient to induce the influx of extracellular Ca2+ and to release Ca2+ from ER stores. Analysis of mutant 2B proteins showed that both a cationic amphipathic α-helix and a second hydrophobic domain in 2B were required for these activities. Consistent with a presumed ability of protein 2B to increase membrane permeability, viruses carrying a mutant 2B protein exhibited a defect in virus release. We propose that 2B gradually enhances membrane permeability, thereby disrupting the intracellular Ca2+ homeostasis and ultimately causing the membrane lesions that allow release of virus progeny. [ABSTRACT FROM AUTHOR]