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PubMed2005

A family of RS domain proteins with novel subcellular localization and trafficking.

We report the sequence, conservation and cell biology of a novel protein, Psc1, which is expressed and regulated within the embryonic pluripotent cell population of the mouse. The Psc1 sequence includes an RS domain and an RNA recognition motif (RRM), and a sequential arrangement of protein motifs that has not been demonstrated for other RS domain proteins. This arrangement was conserved in a second mouse protein (BAC34721). The identification of Psc1 and BAC34721 homologues in vertebrates and related proteins, more widely throughout evolution, defines a new family of RS domain proteins termed acidic rich RS (ARRS) domain proteins. Psc1 incorporated into the nuclear speckles, but demonstrated novel aspects of subcellular distribution including localization to speckles proximal to the nuclear periphery and localization to punctate structures in the cytoplasm termed cytospeckles. Integration of Psc1 into cytospeckles was dependent on the RRM. Cytospeckles were dynamic within the cytoplasm and appeared to traffic into the nucleus. These observations suggest a novel role in RNA metabolism for ARRS proteins.

Active Transport, Cell Nucleus↗

PubMed2002

Analysis of subcellularly localized mRNAs using in situ hybridization, mRNA amplification, and expression profiling.

Targeting of mRNAs to distinct subcellular regions occurs in all polarized cells. The mechanisms by which RNA transport occurs are poorly understood. With the advent of RNA amplification methodologies and expression profiling it is now possible to catalogue the RNAs that are targeted to particular subcellular regions. In particular, neurons are polarized cells in which dendrites receive signals from presynaptic neurons. Upon stimulation (information receipt) the dendrite processes the information such that an immediate dendritic response is generated as well as a longer-term somatic response. The integrated cellular response results in a signal that can be propagated through the axon to the next post-synaptic neuron. Much previous work has shown that mRNAs can be localized in dendrites and that local translation in dendrites can occur. In this chapter the methods for analysis of RNAs that are localized to dendrites are reviewed and a partial list of dendritically localized RNAs is presented. This information may be useful in identifying RNA regulatory regions that are responsible for specifying rate of RNA transport and the dendritic sites at which targeted RNAs dock so that they can be translated.

Animals↗

PubMed1976

Small RNA species of the HeLa cell: metabolism and subcellular localization.

The small molecular weight RNAs of the HeLa cell have been located in specific subcellular fractions. SnA is located in the nucleolus and is partially bonded to nucleolar 28S RNA. SnD, the most abundant of the small nuclear RNAs, is partially released from the nucleus when the nuclear preparation is briefly warmed. SnF is released from the nuclei when chromatin is digested with the micrococcal nuclease and not when pancreatic DNAase is used. The remainder of the small nuclear species remain in the nucleus following the digestion of chromatin and are concluded to be elements of the "nuclear skeleton." SnK is found predominantly in the cytoplasm, but migrates quantitatively to the nuclear fraction in the presence of high levels of actinomycin D. ScL is totally cytoplasmic and is partially bound to cell membranes. It is the 7S RNA found in oncornavirus virions. All the small nuclear RNAs appear initially in the cytoplasmic fraction before fixation in the nucleus. Two short-lived cytoplasmic species behave kinetically as precursors to the stable nuclear RNAs.

Cell Fractionation↗

PubMed1978

Effect of nutrition on subcellular localization of rat fat-cell lipoprotein lipase.

This study supports the possibility for multiple subcellular forms of lipoprotein lipase. 1. The total activity of lipoprotein lipase per g of intact epididymal adipose tissue from fed rats is much higher than that from starved rats. 2. The isolated fat-cells of fed and of starved rats have lipoprotein lipase of almost the same activity per g of fat-pads. The isolated fat-cells of starved rats have a much higher proportion of total activity per g of the intact tissue than do those of fed rats. 3. Under the conditions of homogenization used, only a small proportion of the total activity per g of intact tissue from fed rats was associated with the fat layer which floated to the top of the homogenate during low-speed centrifugation. The different proportions of the specific enzyme activity found in each subcellular fraction are described. 4. Lipoprotein lipase from plasma membranes and microsomal fractions from starved and fed rats was purified by affinity chromatography. 5. The total activity of microsomal lipoprotein lipase per g of intact adipose tissue is enhanced by a normal diet. 6. In intact epididymal adipose tissue from fed rats, the activity per g of tissue of lipoprotein lipase of plasma membranes is much higher than that in the same fraction from starved rats. By contrast, the activities per g of tissue in plasma membranes obtained from starved or from fed rats by collagenase treatment were similar.

Adipose Tissue↗

PubMed1987

Subcellular localization of intracellular forms of human chorionic gonadotropin in first trimester placenta.

To determine the subcellular sites for synthesis and processing of human chorionic gonadotropin subunits in cells, first trimester placental cells were fractionated subcellularly on sucrose density gradients. Analysis of the subcellular fractions by immunobinding techniques revealed that the rough endoplasmic reticulum-rich fraction contained only intermediates having high-mannose oligosaccharides, but the Golgi-rich fraction contained not only intermediates but also mature forms which were resistant to endoglycosidase H but sensitive to neuraminidase. These results show that human chorionic gonadotropin subunits are synthesized in the rough endoplasmic reticulum as forms containing high-mannose oligosaccharides, and their maturation occurs in the Golgi apparatus by trimming with endogenous glycosidases. They are then modified by addition of complex oligosaccharides and terminal sialic acid through glycosyltransferases.

Acetylglucosaminidase↗

PubMed1978

Subcellular Localization of Glycosyl Transferases Involved in Glycoprotein Biosynthesis in the Cotyledons of Pisum sativum L.

Subcellular membrane fractions from 21-day-old pea (Pisum sativum) cotyledons that have associated UDP-N-acetylglucosamine N-acetylglucosaminyl transferase and GDP-mannose mannosyl transferase activities have been isolated and identified. The rough endoplasmic reticulum (RER) is the principal location of glycosyl transferases involved in the assembly of lipid-linked sugar intermediates and glycoproteins. Antimycin A-insensitive NADH-cytochrome c reductase activity was used to identify RER at a density of 1.165 g/cc in sucrose gradients. The high proportion of RER in this fraction was confirmed by electron microscopy.Other mannosyl transferases are found at a density of 1.123 g/cc and 1.201 g/cc but these glycosyl transferases do not appear to be involved with the formation of lipid-linked sugar intermediates utilized in glycoprotein biosynthesis.

Journal Article↗

PubMed1995

Subcellular localization of the GABAA receptor gamma 2 subunit in the rat spinal cord.

The fine subcellular organization of the GABAA receptor complex in the adult rat spinal ventral horn was analysed by immunocytochemistry using a specific polyclonal antiserum raised against the gamma 2 subunit. This subunit confers benzodiazepine sensitivity on the chloride channel of the GABAA receptor. With both fluorescent and peroxidase staining, the immunoreactivity was mainly observed in the grey matter and more specifically in the dorsal and ventral horns on medium and large neurons. A high number of immunostained somata were clustered in regions corresponding to motor nuclei. On the neuronal surface, labelling appeared as fluorescent dots over the more diffuse staining that was present on the soma and proximal part of dendrites. At the ultrastructural level, peroxidase end product was in most cases associated with the internal side of postsynaptic differentiations facing terminal boutons enriched with pleiomorphic small clear vesicles. The positively stained synapses were encountered on proximal dendrites of neurons and throughout the neuropil of the ventral horn (layers VII-IX). An immunoreactivity on the postsynaptic membrane was occasionally found to decorate large pieces of membrane not directly apposed to presynaptic active zones. In addition, presynaptic labelling was observed at axoaxonic contacts and at extrasynaptic sites on membranes within boutons, sometimes themselves apposed to gamma 2 immunoreactivity. Finally, we also observed gamma 2 immunoreactivity at the cytosolic face of the plasma membrane of some glial elements. These results give morphological evidence for the involvement of GABAA receptors in both post- and presynaptic inhibition in the rat spinal ventral horn. The presence of gamma 2 subunit immunoreactivity at these different synaptic contacts suggests that the two types of inhibition can be modulated by benzodiazepine drugs. The findings also provide anatomical evidence for the possible regulation of GABA release through an autoreceptor, and for GABAergic communication between neuronal and glial components.

Animals↗

PubMed1988

Insulin-like growth factor-binding proteins in hypophysectomized rat liver: characterization and subcellular localization.

We have characterized binding proteins for insulin-like growth factors (IGFs) in hepatic subcellular fractions and in the washed supernatants of these fractions in normal and hypophysectomized (hypox) rats. In the course of assessing IGF-II-binding sites on rat liver microsomes, we observed that [125I] IGF-II binding to the microsomal membranes of hypox rats was much lower than that in normal rats. Paradoxically, binding increased in hypox animals at low concentrations (0.5-5 ng/ml) of unlabeled IGF-II. After resuspension and centrifugation (washing) of the microsomes, no difference was found in [125I]IGF-II binding to hypox vs. normal microsomes. However, the binding of [125I]IGF-II to the washing supernatant (SN) from hypox rat microsomes was greater than binding to that from normal animals. Binding to SN was inhibited by unlabeled IGF-II in a dose-dependent manner. Scatchard analyses indicated that the affinity constant for binding by hypox SN was higher than that of normal SN and the microsomal fractions of both hypox and normal rats. After further subfractionation of the liver, no binding activity was found in SN from plasmalemma, whereas it was about 20% of input counts per min of [125I]IGF-II in SN from combined Golgi-endosome fractions of hypox rat liver. We next compared IGF-binding moieties in microsomal SN with those in plasma using cross-linking of [125I]IGF-II followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal rat plasma, we observed the presence of 42K, 39K, 31K, and 27K binding complexes. In hypox rat plasma only a 42-39K doublet was found. In the SN of normal rat microsomes, the predominant complex migrated at 39K and was distinguishable only after acidification. In the SN of hypox rat microsomes, the 42K complex was predominant, with a minor 34K complex. These studies have identified IGF-binding moieties in hepatic tissues, particularly in hepatic vesicular elements, which interfere in the binding of IGF-II to membrane receptors. Their presence in these receptor-rich elements may influence IGF binding to intracellular receptors and, hence, the biological activity of the peptide.

Animals↗

PubMed1969

The subcellular localization of calcium ion in mammalian myocardium.

This study was designed to investigate the proposition that subcellular calcium is sequestered in specific sites in mammalian myocardium. 29 functioning dog papillary muscles were fixed through the intact vascular supply by means of osmium tetroxide containing a 2% concentration of potassium pyroantimonate (K(2)H(2)Sb(2)O(7).(4)H(2)O). Tissue examined in the electron microscope showed a consistent and reproducible localization of the electron-opaque pyroantimonate salts of sodium and calcium to distinct sites in the tissue. Sodium pyroantimonate was found exclusively in the extracellular space and clustered at the sarcolemmal membrane. Calcium pyroantimonate, on the other hand, identified primarily by its susceptibility to removal by chelation with EGTA and EDTA, was consistently found densely concentrated in the lateral sacs of the sarcoplasmic reticulum and over the sarcomeric I bands. M zones were virtually free of precipitate. The implications of these findings with respect to various parameters of muscle function are discussed.

Animals↗

PubMed1996

Cellular and subcellular localization of SALMFamide (S1)-like immunoreactivity within the central nervous system of the nematode Ascaris suum (Nematoda, Ascaroidea).

The localization and distribution of SALMFamide (S1)-like immunoreactivity (IR), was determined at both the cellular and subcellular level in the central nervous system (CNS) of the nematode roundworm Ascaris suum. The techniques of indirect immunofluorescence in conjunction with confocal scanning laser microscopy and post-embedding, IgG-conjugated colloidal gold immunostaining were used, respectively. Immunostaining was widespread in the CNS of adult A. suum, with immunoreactivity (IR) being localized in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures. At the subcellular level, gold labeling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the neuropile of the anterior nerve ring, main ganglia and nerve cords in the CNS. Double-labeling demonstrated an apparent co-localization of S1- and FMRFamide-IR-together with S1- and pancreatic polypeptide (PP)-IR in the same dense-cored vesicles. Antigen preabsorption experiments indicated little cross-reactivity, if any, between the three antisera; indeed, neither FMRFamide nor PP antigens abolished S1 immunostaining.

Animals↗

PubMed1991

Aldehyde dehydrogenase (EC 1.2.1.3): comparison of subcellular localization of the third isozyme that dehydrogenates gamma-aminobutyraldehyde in rat, guinea pig and human liver.

1. Subcellular fractionation of rat, guinea pig and human livers showed that aldehyde dehydrogenase metabolizing gamma-aminobutyraldehyde was exclusively localized in the cytoplasmic fraction in all three mammalian species. 2. Total gamma-aminobutyraldehyde activity of aldehyde dehydrogenase was found to be ca 0.41, 0.3 and 0.24 mumol NADH min-1 g-1 tissue, respectively in rat, guinea pig and human liver, with more than 95% of activity in the cytoplasm. 3. Partially purified cytoplasmic isozyme from rat liver showed similar chromatographic behavior and kinetic properties to the E3 isozyme isolated from human liver. 4. The rat isozyme was insensitive to disulfiram (40 microM) and to magnesium (160 microM) and had Km values of 5 microM (pH 7.4) for gamma-aminobutyraldehyde, 7.5 microM (pH 9.0) for propionaldehyde and 4 microM (pH 7.4) for NAD.

Aldehyde Dehydrogenase↗

PubMed1979

Studies on the subcellular localization of human neutrophil alkaline phosphatase.

The intracellular localization of alkaline phosphatase has been determined in human neutrophils with analytical subcellular fractionation by density gradient centrifugation and EM cytochemistry. Centrifugation on sucrose gradients containing 1 mM DETA and 5 units/ml of heparin showed that alkaline phosphatase was associated with a membranous component distinct from plasma membrane, mitochondria, specific granules and azurophil granules. There was no resolution from the endoplasmic reticulum. Density gradient centrifugation on a sucrose-imidazole-heparin gradient showed a clear resolution of the alkaline phosphatase-containing membranes from the Golgi and endoplasmic reticulum. Density gradient centrifugation of neutrophils that had been disrupted in the presenceof 0.12 mmol/l. digitonin clearly separated alkaline phosphatase-containing membranes from the endoplasmic reticulum. Part of the gamma-glutamyl transferase has a similar localization to that of alkaline phosphatase. EM cytochemistry of neutrophils, neutrophil homogenates and of the density gradient fractions identified alkaline phosphatase-containing granules as irregular-shaped, often tubular, structures. It is suggested that alkaline phosphatase and part of the gamma-glutamyl transferase activity are localized to a unique organelle in the human neutrophil.

Alkaline Phosphatase↗

PubMed1994

Subcellular localization of p-boronophenylalanine-delivered boron-10 in the rat 9L gliosarcoma: cryogenic preparation in vitro and in vivo.

A well-characterized in vitro cryogenic preparation for ion microscopic isotope imaging, which minimizes redistribution of diffusible species, was used to determine the distribution of boron in GS-9L gliosarcoma cells incubated with the boron neutron capture therapy agent, p-boronophenylalanine (BPA). At the subcellular level, boron from BPA distributes relatively homogeneously within the glioma cell. Boron from BPA was eliminated rapidly, indicating that most is unbound. Thus a large pool of boron is susceptible to diffusion artifact. Removal of this artifact increases the degree of confidence in microdosimetric results inferred from the homogeneous subcellular distribution. The ion microscopic imaging of boron in subcutaneous tumors cryofixed in situ was achieved in rats treated with BPA. Boron signals from BPA were adequate to image microdistributions at the 1-micron resolution level. As in the in vitro case, boron did not localize discretely at the subcellular level. However, boron heterogeneity was seen at the tissue level. Physiologically valid cellular potassium and sodium levels were seen, which demonstrates minimized redistribution artifact. Future tissue studies designed to correlate ion microscopic boron images to microscopic structure are feasible using cryogenic sample preparation and ion microscopy.

Animals↗

PubMed1993

Subcellular localization of a low Mr GTP-binding protein, c25KG, in resting and stimulated human platelets.

Subcellular fractions were prepared from human platelets by sucrose density gradient centrifugation and the presence of a GTP-binding protein, c25KG, was analyzed by immunoblotting. c25KG was found to be located in plasma membrane and alpha-granule fractions in resting platelets by immunoblot analysis. Disappearance of the protein by the pronase treatment of intact alpha-granules indicated that c25KG is exposed on the cytoplasmic face of the granules. Activation of platelets by thrombin induced the redistribution of c25KG from alpha-granules to plasma membranes. On the other hand, Gi2, which is the main pertussis toxin-substrate in human platelets, was predominantly present in plasma membranes and thrombin-stimulation did not alter its distribution.

Blood Platelets↗

PubMed2000

Subcellular localization of presenilins: association with a unique membrane pool in cultured cells.

We have investigated the subcellular distribution of presenilin-1 (PS1) and presenilin-2 (PS2) in a variety of mammalian cell lines. In Iodixanol-based density gradients, PS1 derivatives show a biphasic distribution, cofractionating with membranes containing ER-resident proteins and an additional population of membranes with low buoyant density that do not contain markers of the Golgi complex, ERGIC, COP II vesicles, ER exit compartment, COP II receptor, Golgi SNARE, trans-Golgi network, caveolar membranes, or endocytic vesicles. Confocal immunofluorescence and immunoelectron microscopy studies fully supported the fractionation studies. These data suggest that PS1 fragments accumulate in a unique subcompartment(s) of the ER or ER to Golgi trafficking intermediates. Interestingly, the FAD-linked PS1 variants show a marked redistribution toward the heavier region of the gradient. Finally, and in contrast to PS1, PS2 fragments are detected preponderantly in more densely sedimenting membranes, suggesting that the subcellular compartments in which these molecules accumulate are distinct.

Alzheimer Disease↗

PubMed1995

Cellular and subcellular localization of AMPA-selective glutamate receptors in the mammalian peripheral vestibular system.

The cellular and subcellular distribution of AMPA-selective glutamate receptors in the mammalian peripheral vestibular system was examined using antibodies against peptides corresponding to the C-terminal portions of AMPA receptor subunits: GluR1, GluR2/R3 and GluR4. The light and electron microscopic immunocytochemical studies were carried out on Vibratome sections of rat and guinea pig vestibular sensory epithelial and ganglia. In the epithelium, GluR1 subunit immunoreactivity appeared as accumulations of patches outlining the baso-lateral periphery of the type I sensory cells. The GluR1-immunoreactive microareas were postsynaptically distributed on the membranes of calyceal afferent fibers. GluR2/R3 immunoreactivity was present in the sensory cells. GluR4 was not detected. In the vestibular ganglion, the neurons were densely stained with antibodies to GluR2/R3 and GluR4. The fibroblasts and the Schwann cells were also intensely stained with antibodies to GluR2/R3 and GluR4. In the sensory cells, the AMPA receptors, GluR2/R3, may function as (1) autoreceptors controlling afferent neurotransmitter release or (2) 'postsynaptic' receptors activated by the neurotransmitter release of the afferent calyx. The detection of GluR1 at postsynaptic sites in the afferent fibers provides anatomical evidence for the role of glutamate as a neurotransmitter of sensory cells. In the ganglion neurons, GluR2/R3 and GluR4 may represent reserve intracytoplasmic pools of receptor subunits in transit to the postsynaptic sites. In the Schwann cells, GluR2/R3 and GluR4 may be involved in neuronal-glial signalling at the nodes of Ranvier.

Animals↗

PubMed1985

Glycogen synthase (casein) kinase-1: tissue distribution and subcellular localization.

The distribution of glycogen synthase (casein) kinase-1 (CK-1) among different rat tissues and subcellular fractions was investigated. Using casein, glycogen synthase and phosphorylase kinase as substrates, CK-1 activity was detected in kidney, spleen, liver, testis, lung, brain, heart, skeletal muscle and adipose tissue. The distribution of CK-1 among different subcellular fractions of rat liver was; cytosol (72.1%), microsome (17.6%), mitochondria (9.6%) and nuclei (0.7%). CK-1 from rat tissues was shown to have a similarly wide substrate specificity as highly purified CK-1 from rabbit skeletal muscle. Such wide substrate specificity and distribution among different mammalian tissues and subcellular organelles indicate that CK-1 may be involved in the regulation of diverse cellular functions.

Adipose Tissue↗

PubMed1994

Subcellular localization of Pfs16, a Plasmodium falciparum gametocyte antigen.

We have used immunoelectron microscopy to investigate the subcellular location of Pfs16 in Plasmodium falciparum. It was detected in the outer membrane region of gametocytes and more specifically on the parasitophorous vacuole membrane (pvm), since, during gametogenesis when the pvm disintegrates, the majority of the antigen was detected on the remains of this membrane in multilaminated whorls and not on the gamete plasma membrane. The antigen was also present on other gametocyte cellular structures, including those which we believe to be Garnham bodies, present in the host cell cytoplasm of some gametocytes. The antigen was present too on the membrane surrounding cytosomes and the resulting food vacuoles in the parasite cytoplasm.

Animals↗