Intracellular levels of calmodulin are increased in transformed cells

By using Hoechst 33342,rabbit anti calmodulin antibody,FITC-labeled goat anti rabbit IgG and SR101(sulfo rhodamine 101)simultaneously to stain individual normal and transformed cells,the microspectrophotometric analysis demonstrated that 3 markers which represented the nucleus,calmodulin and total protein respectively,could be recognized in individualj cells without interference,The phase of the cell cycle was determined by DNA content(Hoechst 33342),We found that in transformed cells(NIH3T3) tsRSV-LA90,cultured at 33℃ and transformed C3H10T1/2 Cells),the ration of calmodulin to total protein (based on the phases of cell cycle)was higher than that in normal cells (NIH3T3 tsRSV-LA90 cells,cultured at 39℃ and C3H10T1/2 cells)in every cell cycle phase,This ration increased obviously only from G1 to S phase in either normal or transformed cells.The results showed that calmodulinreally increased during the transformation,and its increase was specific.In the meantime when cells proceeded from G1 to S.the intraceollular calmodulin content also increased specifically.     

Differential distribution and intracellular targeting of mRNAs corresponding to the three calmodulin genes in rat brain. A quantitative in situ hybridization study.

To investigate the pattern of expression of the three calmodulin (CaM) genes by in situ hybridization, gene-specific [35S]-cRNA probes complementary to the multiple CaM mRNAs were hybridized in rat brain sections and subsequently detected by quantitative film or high-resolution nuclear emulsion autoradiography. A widespread and differential area-specific distribution of the CaM mRNAs was detected. The expression patterns corresponding to the three CaM genes differed most considerably in the olfactory bulb, the cerebral and cerebellar cortices, the diagonal band, the suprachiasmatic and medial habenular nuclei, and the hippocampus. Moreover, the significantly higher CaM I and CaM III mRNA copy numbers than that of CaM II in the molecular layers of certain brain areas revealed a differential dendritic targeting of these mRNAs. The results indicate a differential pattern of distribution of the multiple CaM mRNAs at two levels of cellular organization in the brain: (a) region-specific expression and (b) specific intracellular targeting. A precise and gene-specific regulation of synthesis and distribution of CaM mRNAs therefore exists under physiological conditions in the rat brain.     

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 calcium and calmodulin involvement in protoplast fusion

(45)Ca(2+) uptake was compared between fusogenic and nonfusogenic Daucus carota L. protoplasts. Fusogenic protoplasts took 10 minutes to reach calcium equilibrium compared to 5 minutes in the nonfusogenic protoplasts. Intracellular stores of calcium were manipulated by isolating protoplasts in different calcium regimes. Lowering of intracellular calcium lowered fusion potential, while raising intracellular stores of calcium enhanced fusion potential. Regardless of the amount of calcium sequestered in a store, mobilization with A23187 increased fusion levels within 10 minutes. Calmodulin antagonists were potent inhibitors of protoplast fusion. This inhibition was obtained by treating cells with the calmodulin antagonists during protoplast isolation. A23187, however, only allowed a partial recovery from this inhibition, indicating that calcium flux alone was not sufficient for maximum fusion potential. On the basis of the evidence presented, we propose that calcium fluxes during protoplast isolation activate a calmodulin-mediated biochemical process that is necessary for the formation or maintenance of a fusion permissive state.     

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.     

Intracellular localization of Staphylococcus aureus within primary cultured mouse kidney cells.

Staphylococcus aureus Cowan I was incubated with monolayers of cells derived from several portions of mouse kidney, and found to be ingested by all types of the renal cells. Intracellular localization of S. aureus was determined by resistance of intracellular cocci against lysostaphin digestion and confirmed by electron microscopy. From renal medulla, three morphological variants of the hyperosmolarity-tolerant (HOT) cells were obtained. The rate of cocci-ingesting cells varied from 16.9% to 93.4% among these of the HOT cells at the end of 3-hr incubation. From renal cortex, three morphological variants of epithelial cells grew in medium RK-1. Among them, only the cells on the edge of colony ingested Cowan I, while the epithelial cells on the center of colony ingested few cocci. Transferred from medium RK-1 to MEM supplemented with 10% FBS, part of the cortical cells changed into fibroblast-like appearance and obtained the capacity to ingest Cowan I. This result may indicate the correlation between ingesting capacity and cellular morphology. From a glomerulus, epithelial (GE) cells and fibroblast-like (GF) cells were obtained. The GE cells ingested not only S. aureus Cowan I but Staphylococcus epidermidis and Staphylococcus saprophyticus after 30-min incubation, while the GF cells, like both of the HOT cells and the cortical cells, ingested only S. aureus. These results suggest a possibility that S. aureus is located within nonprofessional phagocytes during its infection and intracellular coccus plays an important role in its pathogenicity.     

Intracellular targeting of the proteasome

Proteasomes are soluble, but can also be found in association with subcellular organelles. Adaptors capable of mediating interactions between proteasomes and intracellular organelles have not yet been identified, although they might exist. Although most proteasomal substrates are soluble, some membrane-bound proteins are also degraded by the proteasome. Processing of such insoluble substrates might cause proteasomes to be organelle-bound by tethering the degradative apparatus to the organelle.     

Intracellular targeting of isoproteins in muscle cytoarchitecture

Part of the muscle creatine kinase (MM-CK) in skeletal muscle of chicken is localized in the M-band of myofibrils, while chicken heart cells containing myofibrils and BB-CK, but not expressing MM-CK, do not show this association. The specificity of the MM-CK interaction was tested using cultured chicken heart cells as "living test tubes" by microinjection of in vitro generated MM-CK and hybrid M-CK/B-CK mRNA with SP6 RNA polymerase. The resulting translation products were detected in injected cells with isoprotein-specific antibodies. M-CK molecules and translation products of chimeric cDNA molecules containing the head half of the B-CK and the tail half of the M-CK coding regions were localized in the M-band of the myofibrils. The tail, but not the head portion of M-CK is essential for the association of M-CK with the M-band of myofibrils. We conclude that gross biochemical properties do not always coincide with a molecule's specific functions like the participation in cell cytoarchitecture which may depend on molecular targeting even within the same cellular compartment.     

The abundance of calmodulin mRNAs is regulated in phosphorylase kinase-deficient skeletal muscle

In the I/Lyn mouse strain a mutation on the X chromosome results in a deficiency of the major calmodulin-regulated enzyme in skeletal muscle, phosphorylase kinase. Calmodulin has been identified as the delta-subunit of phosphorylase kinase, and it is estimated that approximately 40% of the total calmodulin in rabbit skeletal muscle is associated with the phosphorylase kinase hexadecamer (alpha, beta, gamma, delta)4. The absence of phosphorylase kinase in I/Lyn skeletal muscle results in a reduction in the total amount of calmodulin. The mechanisms affecting this reduction were investigated by comparing the abundance and heterogeneities in calmodulin mRNAs between normal and phosphorylase kinase-deficient skeletal muscles. The results demonstrate that in normal tissue there are four species of calmodulin mRNA distinguished by their molecular weight. All four of these species are present in the deficient tissue, and none of them are preferentially reduced. However, there is a 54% reduction in all four mRNAs as well as in calmodulin in the deficient skeletal muscle relative to normal skeletal muscle. These results indicate that the expression of calmodulin mRNAs is coordinated with the expression of its major enzyme target in skeletal muscle.     

Intracellular nitric oxide mediates neuroproliferative effect of neuropeptide y on postnatal hippocampal precursor cells

Neuropeptide Y (NPY) is widely expressed in the central and peripheral nervous systems and is proliferative for a range of cells types in vitro. NPY plays a key role in regulating adult hippocampal neurogenesis in vivo under both basal and pathological conditions, although the underlying mechanisms are largely unknown. We have investigated the role of nitric oxide (NO) on the neurogenic effects of NPY. Using postnatal rat hippocampal cultures, we show that the proliferative effect of NPY on nestin(+) precursor cells is NO-dependent. As well as the involvement of neuronal nitric-oxide synthase, the proliferative effect is mediated via an NO/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) and extracellular signal-regulated kinase (ERK) 1/2 signaling pathway. We show that NPY-mediated intracellular NO signaling results in an increase in neuroproliferation. By contrast, extracellular NO had an opposite, inhibitory effect on proliferation. The importance of the NO-cGMP-PKG signaling pathway in ERK1/2 activation was confirmed using Western blotting. This work unites two significant modulators of hippocampal neurogenesis within a common signaling framework and provides a mechanism for the independent extra- and intracellular regulation of postnatal neural precursors by NO.     

Intracellular localization of hyaluronan in proliferating cells.

Hyaluronan is a high molecular weight glycosaminoglycan found in the extracellular matrix of many tissues, where it is believed to promote cell migration and proliferation. It was recently shown that hyaluronan-dependent pericellular matrix formation is a rapid process that occurs as cells detach during mitosis. Growing evidence for intracellular hyaluronan in tissues in vivo, together with evidence of intracellular hyaluronan binding molecules, prompted us to examine hyaluronan distribution and uptake as well as hyaluronan binding sites in cells and their relationship to cell proliferation in vitro, using a biotinylated hyaluronan binding protein and fluorescein-labeled hyaluronan. In permeabilized smooth muscle cells and fibroblasts, hyaluronan staining was seen in the cytoplasm in a diffuse, network-like pattern and in vesicles. Nuclear hyaluronan staining was observed and confirmed by confocal microscopy and was often associated with nucleoli and nuclear clefts. After serum stimulation of 3T3 cells, there was a dramatic increase in cytoplasmic hyaluronan staining, especially during late prophase/early prometaphase of mitosis. In contrast, unstimulated cells were negative. There was a pronounced alteration in the amount and distribution of hyaluronan binding sites, from a mostly nucleolar distribution in unstimulated cells to one throughout the cytoplasm and nucleus after stimulation. Exogenous fluorescein-labeled hyaluronan was taken up avidly into vesicles in growing cells but was localized distinctly compared to endogenous hyaluronan, suggesting that hyaluronan in cells may be derived from an intracellular source. These data indicate that intracellular hyaluronan may be involved in nucleolar function, chromosomal rearrangement, or other events in proliferating cells. (J Histochem Cytochem 47:1331-1341, 1999)     

Differential subcellular localization of particular mRNAs in hippocampal neurons in culture

In situ hybridization was used to assess the subcellular distribution of mRNAs encoding several important neuronal proteins in hippocampal neurons in culture. mRNA encoding GAP-43, a protein that is largely excluded from dendrites, was restricted to nerve cell bodies, as were mRNAs encoding neurofilament-68 and beta-tubulin, which are prominent constituents of dendrites and of axons. In contrast, mRNA encoding MAP-2, a protein that is selectively distributed in dendrites and cell bodies, was present in both dendrites and cell bodies. These results demonstrate that different mRNAs are differentially distributed within individual hippocampal neurons. Taken together with previous findings from other laboratories, our results suggest that only a limited set of mRNAs are available for local translation within dendrites.     

Intracellular trafficking and membrane targeting mechanisms of the human reduced folate carrier in Mammalian epithelial cells

The major pathway for cellular uptake of the water-soluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the reduced folate carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cytoplasmic NH(2)- or COOH-terminal domains of the protein. Further, the integrity of the hRFC backbone is critical for export of the polypeptide from the endoplasmic reticulum to the cell surface. This trafficking is critically dependent on intact microtubules because microtubule disruption inhibits motility of hRFC-containing vesicles as well as final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.