Dominant negative effect of cytoplasmic actin isoproteins on cardiomyocyte cytoarchitecture and function

The intracompartmental sorting and functional consequences of ectopic expression of the six vertebrate actin isoforms was investigated in different types of cultured cells. In transfected fibroblasts all isoactin species associated with the endogenous microfilament cytoskeleton, even though cytoplasmic actins also showed partial localization to peripheral submembranous sites. Functional and structural studies were performed in neonatal and adult rat cardiomyocytes. All the muscle isoactin constructs sorted preferentially to sarcomeric sites and, to a lesser extent, also to stress-fiber-like structures. The expression of muscle actins did not interfere with cell contractility, and did not disturb the localization of endogenous sarcomeric proteins. In sharp contrast, ectopic expression of the two cytoplasmic actin isoforms resulted in rapid cessation of cellular contractions and induced severe morphological alterations characterized by an exceptional outgrowth of filopodia and cell flattening. Quantitative analysis in neonatal cardiomyocytes indicated that the levels of accumulation of the different isoactins are very similar and cannot be responsible for the observed isoproteins- specific effects. Structural analysis revealed a remodeling of the cytoarchitecture including a specific alteration of sarcomeric organization; proteins constituting the sarcomeric thin filaments relocated to nonmyofibrillar sites while thick filaments and titin remained unaffected. Experiments with chimeric proteins strongly suggest that isoform specific residues in the carboxy-terminal portion of the cytoplasmic actins are responsible for the dominant negative effects on function and morphology.     

Intracellular targeting of proteins by sumoylation.

A novel host cell posttranslational modification system, termed sumoylation, has recently been characterized. Sumoylation is an enzymatic process that is biochemically analogous to, but functionally distinct from, ubiquitinylation. As in ubiquitinylation, sumoylation involves the covalent attachment of a small protein moiety, SUMO, to substrate proteins. However, conjugation of SUMO does not typically lead to degradation of the substrate and instead has a more diverse array of effects on substrate function. As the list of sumoylation substrates has expanded, a common theme is that many substrates exhibit sumoylation-dependent subcellular distribution. While the molecular mechanisms by which sumoylation targets protein localization are still poorly understood, it is clear that this modification system is an important regulator of intracellular protein localization, particularly involving nuclear uptake and punctate intranuclear accumulation.     

Intracellular fate and nuclear targeting of plasmid DNA

One of the major steps limiting nonviral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of the transfected cells. The nuclear localization signal (NLS) of the SV40 large T antigen is known to efficiently induce nuclear targeting of proteins. We have developed two chemical strategies for covalent coupling of NLS peptides to plasmid DNA. One method involves a site-specific labeling of plasmid DNA by formation of a triple helix with an oligonucleotide–NLS peptide conjugate. After such modification with one NLS peptide per plasmid molecule, plasmid DNA remained fully active in cationic lipid-mediated transfection. In the other method, we randomly coupled 5–115 p-azidotetrafluorobenzyllissamine–NLS peptide molecules per plasmid DNA by photoactivation. Oligonucleotide–NLS and plasmid–lissamine–NLS conjugates interacted specifically with the NLS-receptor importin . Plasmid–lissamine–NLS conjugates were not detected in the nucleus, after cytoplasmic microinjection. Plasmids did not diffuse from the site of injection and plasmid–lissamine–NLS conjugates appeared to be progressively degraded in the cytoplasm. The process of plasmid DNA sequestration/degradation stressed in this study might be as important in limiting the efficiency of nonviral gene transfer as the generally recognized entry step of plasmid DNA from the cytoplasm into the nucleus     

Intracellular targeting of calmodulin mRNAs in primary hippocampal cells.

We investigated the intracellular distribution of the mRNAs corresponding to the three non-allelic CaM genes in cultured hippocampal cells by in situ hybridization with digoxigenin-labeled gene-specific riboprobes. In neurons the perikaryon was heavily stained and strong dendritic mRNA targeting was detected for all three CaM genes. The color labeling exhibited a punctate distribution, suggesting that CaM mRNAs are transported in RNA granules. Immunocytochemistry for S100 demonstrated that glial cells express CaM mRNAs at a very low level. A minority of the cultured cells were negative for either labeling.     

Intracellular pH of frog sartorius muscle

A weak base, morpholine, has been labelled with ³H and tested for its suitability as an indicator for intracellular pH, by distribution in the tissue water of frog sartorius muscle in the species Hyla litoria. Its pK'a at 20°C in a solution of the same of ionic strength as frog Ringer was found to be 8.45 ± 0.02, which is in the range of maximal sensitivity. Morpholine equilibrated with the tissue in 17 h; it was shown that it was not bound to intracellular constituents, that it was not metabolised nor toxic in the concentrations used; it was therefore judged suitable as a pH indicator. Intracellular pH was then measured by distribution of morpholine (6.985 ± 0.08), nicotine (6.915 ± 0.03) and the weak acid 5,5′-dimethyl-2,4-oxazolidinedione (7.10 ± 0.05) and with pH-sensitive microelectrodes (5.9, the equilibrium value). It was shown that the four significantly different values could not be reconciled in terms of experimental error, heterogeneity of intracellular pH, liquid junction potential differences, or binding of indicator molecules inside the fibre. They could, however, be reconciled if the fibre water had different structure and solvent properties from the extracellular water and ions were distributed across the membrane as between two liquid phases containing different solvents. Then the H⁺ would be in equilibrium, as shown by the microelectrode measurement, but intracellular pH would be indeterminable and probably greater than 6.     

Intracellular Ca release in skinned smooth muscle

The release of internal Ca from saponin-treated skinned smooth muscle of guinea pig taenia caecum was studied. The amount of Ca released was estimated by the area under the contraction curve during treatment with 25 mM caffeine in the presence of 0.1 mM EGTA. The magnitude of the caffeine response in skinned muscle, after loading with 10(-6) M Ca for 3 min, was similar to that in the depolarized muscle in the presence of EGTA before treatment with saponin. This suggests that Ca in the skinned muscle was in a physiological range after loading. The release of Ca from the storage site could be facilitated by Ca itself when the skinned muscle was exposed to Ca above 3 x 10(-6) M. An increase in environmental MG concentration suppressed the Ca-induced Ca release mechanism. Sudden replacement of propionate with Cl in the bathing solution made it possible to release Ca from the storage site. This "depolarization"-induced Ca release occurred only immediately after the application of Cl; thereafter, the Ca release mechanism seemed to be inactivated by the prolonged presence of Cl. These results suggest that two mechanisms of Ca release operate in smooth muscle: (a) release induced by Ca itself, and (b) release by "depolarization".     

Intracellular distribution of fumarase in rat skeletal muscle

The distribution of fumarase activity between the mitochondrial and cytoplasmic compartments of rat skeletal muscle was studied using the method of Fatania and Dalziel (Biochim. Biophys. Acta 631 (1980) 11–19), fractional extraction technique and a method based on the calculation of mitochondrial protein content in the tissue and on the determination of fumarase activity both in the tissue homogenate and in the isolated mitochondria. We found 10%, 5% and 0% of the total fumarase activity in the cytoplasm using these methods, respectively. The results suggest that no more than 10% of the total fumarase activity is present in the cytosolic fraction of rat skeletal muscle. The metabolic consequences of such distribution of fumarase in skeletal muscle are discussed.     

Intracellular targeting of Gag proteins of the Drosophila telomeric retrotransposons

Drosophila has two non-long-terminal-repeat (non-LTR) retrotransposons that are unique because they have a defined role in chromosome maintenance. These elements, HeT-A and TART, extend chromosome ends by successive transpositions, producing long arrays of head-to-tail repeat sequences. These arrays appear to be analogous to the arrays produced by telomerase on chromosomes of other organisms. While other non-LTR retrotransposons transpose to many chromosomal sites, HeT-A and TART transpose only to chromosome ends. Although HeT-A and TART belong to different subfamilies of non-LTR retrotransposons, they encode very similar Gag proteins, which suggests that Gag proteins are involved in their unique transposition targeting. We have recently shown that both Gags localize efficiently to nuclei where HeT-A Gag forms structures associated with telomeres. TART Gag does not associate with telomeres unless HeT-A Gag is present, suggesting a symbiotic relationship in which HeT-A Gag provides telomeric targeting. We now report studies to identify amino acid regions responsible for different aspects of the intracellular targeting of these proteins. Green fluorescent protein-tagged deletion derivatives were expressed in cultured Drosophila cells. The intracellular localization of these proteins shows the following. (i) Several regions that direct subcellular localizations or cluster formation are found in both Gags and are located in equivalent regions of the two proteins. (ii) Regions important for telomere association are present only in HeT-A Gag. These are present at several places in the protein, are not redundant, and cannot be complemented in trans. (iii) Regions containing zinc knuckle and major homology region motifs, characteristic of retroviral Gags, are involved in protein-protein interactions of the telomeric Gags, as they are in retroviral Gags.     

Intracellular calcium and smooth muscle contraction

Excitation-contraction coupling in smooth muscle involves many processes, some of which are outlined in this article. The total amount of Ca2+ released on excitation is considerably in excess of the free Ca2+ concentration and this implies a high capacity, high affinity Ca2+ buffer system. The two major Ca2+-binding proteins are calmodulin and myosin. Only calmodulin has the appropriate binding affinity to act as a component of the Ca2+-buffer system. The Ca2+-calmodulin complex activates myosin light chain kinase and thus is involved in the regulation of contractile activity. Phosphorylation of myosin stabilizes an active conformation and promotes cross bridge cycling and is essential for the initiation of contraction. During the initial contractile response phosphorylation correlates to tension development and velocity of shortening. However, as contraction continues the extent of myosin phosphorylation and velocity often decreases but tension is maintained. In general, the Ca2+ transient is reflected by the extent of phosphorylation that in turn correlates with shortening velocity. Maintenance of tension at low phosphorylation levels is not accounted for within our understanding of the phosphorylation theory and thus alternative regulatory mechanisms have been implicated. Some of the possibilities are discussed.     

Intracellular targeting of ascomycetous catalase-peroxidases (KatG1s)

Bifunctional catalase-peroxidases (KatGs) are heme oxidoreductases widely spread among bacteria, archaea and among lower eukaryotes. In fungi, two KatG groups with different localization have evolved, intracellular (KatG1) and extracellular (KatG2) proteins. Here, the cloning, expression analysis and subcellular localization of two novel katG1 genes from the soil fungi Chaetomium globosum and Chaetomium cochliodes are reported. Whereas, the metalloenzyme from Ch. globosum is expressed constitutively, Ch. cochliodes KatG1 reveals a slight increase in expression after induction of oxidative stress by cadmium ions and hydrogen peroxide. The intronless open reading frames of both Sordariomycetes katG1 genes as well as of almost all fungal katG1s possess two peroxisomal targeting signals (PTS1 and PTS2). Peroxisomal localization of intracellular eukaryotic catalase-peroxidases was verified by organelle separation and immunofluorescence microscopy. Co-localization with the peroxisomal enzyme 3-ketoacyl-CoA-thiolase was demonstrated for KatGs from Magnaporthe grisea, Chaetomium globosum and Chaetomium cochliodes. The physiological role of fungal catalase-peroxidases is discussed.     

Intracellular pH of frog sartorius muscle.

A weak base, morpholine, has been labelled with 3H and tested for its suitability as an indicator for intracellular pH, by distribution in the tissue water of frog sartorius muscle in the species Hyla litoria. Its pK'a at 20 degrees C in a solution of the same ionic strength as frog Ringer was found to be 8.45 +/- 0.02, which is in the range of maximal sensitivity. Morpholine equilibrated with the tissue in 17 h; it was shown that it was not bound to intracellular constituents, that it was not metabolised nor toxic in the concentrations used; it was therefore judged suitable as a pH indcator. Intracellular pH was then measured by distribution of morpholine (6.985 +/- 0.08), nicotine (6.915 +/- 0.03) and the weak acid 5,5'-dimethyl-2,4-oxazolidinedione (7.10 +/- 0.05) and the pH-sensitive microelectrodes (5.9, the equilibrium value). It was shown that the four significantly different values could not be reconciled in terms of experimental error, heterogeneity of intracellular pH, liquid junction potential differences, or binding of indicator molecules inside the fibre. They could, however, be reconciled if the fibre water had different structure and solvent properties from the extracellular water and all ions were distributed across the membrane as between two liquid phases containing different solvents. Then the H+ would be in equilibrium, as shown by the microelectrode measurement, but intracellular pH would be indeterminable and probably greater than 6.     

Intracellular distribution of fish muscle alkaline proteinase

1. Intracellular distribution of a muscle alkaline proteinase was investigated on four kinds of fish. 2. The total activity of the muscle proteinase of carp, Cyprinus carpio, was larger in both myofibrillar (Mf) and microsomal (Mic) fractions than the activity in mitochondrial, lysosomal, and supernatant fractions. The activity found in Mf fraction seemed to due to the Mic enzyme which was not separated from Mf fraction. 3. The relative specific activity was mostly found in Mic fraction in the species tested. 4. The results indicate that the distribution pattern of fish muscle alkaline proteinase is different from those of cathepsin D and acid phosphatase. 5. The Mic fraction hydrolyzed Mf and sarcoplasmic proteins. The rates were 40 and 55%, respectively, of the rate when casein was used as a substrate.     

Intracellular targeting and functional analysis of single-chain Fv fragments in mammalian cells

In the past decade, intracellular antibodies have proven to be a useful tool in obtaining the phenotypic knock-out of selected gene function in different animal and plant systems. This strategy is based on the ectopic expression of recombinant forms of antibodies targeted towards different intracellular compartments, exploiting specific targeting signals to confer the new intracellular location. The functional basis of this technology is closely linked to the ability of intracellular antibodies to interact with their target antigens in vivo. This interaction allows either a direct neutralising effect or the dislodgement of the target protein from its normal intracellular location and, by this mechanism, the inactivation of its function. By using this approach, the function of several antigens has been inhibited in the cytoplasm, the nucleus, and the secretory compartments. In this article, we shall describe all the steps required for expressing single-chain Fv fragments in different subcellular compartments of mammalian cells and their subsequent use in knock-out experiments, starting from a cloned single-chain Fv fragment. This will include the analysis of the solubility properties of the new scFv fragment in transfected mammalian cells, the intracellular distribution of the antigen-antibody complex, and the resulting phenotype.