SEARCH · PubMed Health

Results for “subcellular”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources

PubMed1982

Dolichol metabolism in rat liver. Determination of the subcellular distribution of dolichyl phosphate and its site and rate of de novo biosynthesis.

Two independent experiments indicated that the major subcellular site of de novo dolichyl phosphate biosynthesis in rat liver is in the microsomes. First, when purified subcellular fractions were assayed for long chain prenyltransferase activity, the profile obtained indicated exclusively a microsomal localization. Second, when liver slices were incubated 60 min with [14C]acetate and subcellular fractions were subsequently isolated, the microsomal fraction was found to contain greater than two-thirds of the total radioactivity incorporated into dolichyl phosphate. Chemical analysis of dolichyl phosphate in subcellular fractions showed substantial quantities in both the microsomal and mitochondrial-lysosomal fractions. In contrast, dolichol was found primarily in the mitochondrial-lysosomal fraction. The absolute in vitro rate of hepatic dolichyl phosphate synthesis was determined in liver slices and isolated hepatocytes by [14C]acetate labeling using [3H] water incorporation into cholesterol to correct for the dilution of the labeled acetate by the endogenous acetyl-CoA pool. From this value, an in vivo rate of de novo synthesis was estimated as 1.7-2.6 nmol of dolichyl phosphate/liver/day. This rate is more than 30 times the maximum possible rate of accumulation in liver of dietary dolichol (Keller, R. K., Jehle, E., and Adair, W. L., Jr. (1982) J. Biol. Chem. 257, 8985-8989). From the previously determined concentration of dolichyl phosphate in rate liver (Keller, R. K. Tamkun, J. M., and Adair, W. L. (1981) Biochemistry 20, 5831-5836) and the de novo rate of synthesis, it appears that the half-life of hepatic dolichyl phosphate is on the order of days.

Acetates↗

PubMed1994

Evolution of alanine:glyoxylate aminotransferase 1 peroxisomal and mitochondrial targeting. A survey of its subcellular distribution in the livers of various representatives of the classes Mammalia, Aves and Amphibia.

As part of a wider study on the molecular evolution of alanine:glyoxylate aminotransferase 1 (AGT1) intracellular compartmentalization, we have determined the subcellular distribution of immunoreactive AGT1, using postembedding protein A-gold immunoelectron microscopy, in the livers of various members of the classes Mammalia, Aves, and Amphibia. As far as organellar distribution is concerned, three categories could be distinguished. In members of the first category (type I), all, or nearly all, of the immunoreactive AGT1 was concentrated within the peroxisomes. In the second category (type II), AGT1 was found more evenly distributed in both peroxisomes and mitochondria. In the third category (type III), AGT1 was localized mainly within the mitochondria with much lower, but widely variable, amounts in the peroxisomes. Type I animals include the human, two great apes (gorilla, orangutan), two Old World monkeys (anubis baboon, Japanese macaque), a New World monkey (white-faced Saki monkey), a lago, morph (European rabbit), a bat (Seba's short-tailed fruit bat), two caviomorph rodents (guinea pig, orange-rumped agouti), and two Australian marsupials (koala, Bennett's wallaby). Type II animals include two New World monkeys (common marmoset, cotton-top tamarin), three prosimians (brown lemur, fat-tailed dwarf lemur, pygmy slow loris), five rodents (a hybrid crested porcupine, Colombian ground squirrel, laboratory rat, laboratory mouse, golden hamster), an American marsupial (grey short-tailed opossum), and a bird (raven). Type III animals include the large tree shrew, three insectivores (common Eurasian mole, European hedgehog, house shrew), four carnivores (domestic cat, ocelot, domestic dog, polecat ferret), and an amphibian (common frog). In addition to these categories, some animals (e.g. guinea pig, common frog) possessed significant amounts of cytosolic AGT1. Whereas the subcellular distribution of AGT1 in some orders (e.g. Insectivora and Carnivora) did not appear to vary markedly between the different members, in other orders (e.g. Primates, Rodentia and Marsupialia) it fluctuated widely between the different species. Phylogenetic analysis indicates that the subcellular distribution of AGT1 has changed radically on numerous occasions during the evolution of mammals. The new observations presented in this paper are compatible with our previous demonstration of a relationship between AGT1 subcellular distribution and either present or putative ancestral dietary habit, and our previous suggestion that the molecular evolution of the AGT gene has been markedly influenced by dietary selection pressure.

Alanine Transaminase↗

PubMed1994

Contrasting subcellular localization of the Kv1.2 K+ channel subunit in different neurons of rat brain.

In the nervous system, a wide diversity of K+ channels are formed by the oligomeric assembly of subunits encoded by a large number of K+ channel genes. The physiological functions of a specific K+ channel subunit in vivo will be dictated in part by its subcellular location within neurons. We have used a combined in situ hybridization and immunocytochemical approach to determine the subcellular distribution of Kv1.2, a member of the Shaker subfamily of K+ channel genes. In contrast to other characterized K+ channel subunits, Kv1.2 protein shows a complex differential subcellular distribution in neurons of rat brain. In some of these neurons (e.g., hippocampal and cortical pyramidal cells, and Purkinje cells), Kv1.2 is concentrated in dendrites, while in others (e.g., cerebellar basket cells), Kv 1.2 is predominantly, if not exclusively, localized to nerve terminals. Furthermore, Kv1.2 immunoreactivity was also detected in certain axon tracts. We hypothesize that the differential sorting of Kv1.2 could result from association of Kv1.2 with varying heterologous K+ channel subunits in different cell types, with the implication that Kv1.2 may participate in distinct heteromultimeric K+ channels in different subcellular domains. The findings suggest that Kv1.2-containing K+ channels may play diverse functional roles in several neuronal compartments, regulating presynaptic or postsynaptic membrane excitability, depending on the neuronal cell type.

Amino Acid Sequence↗

PubMed1993

Subcellular localization of squalene synthase in rat hepatic cells. Biochemical and immunochemical evidence.

In the present study we investigated the subcellular localization of squalene synthase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21). Squalene synthase catalyzes the formation of squalene from trans-farnesyl diphosphate in two distinct steps and is the first committed enzyme for the biosynthesis of cholesterol. Recently, a truncated form of the enzyme from rat hepatocytes was purified, and monospecific antibodies for squalene synthase were produced. This enabled the subcellular localization of squalene synthase by three different methods: (i) analytical subcellular fractionation and measurements of enzyme activities; (ii) immunodeterminations of squalene synthase in the isolated subcellular fractions with a monospecific antibody; and (iii) immunoelectron microscopy. All three methods gave consistent results. The data clearly illustrate that squalene synthase enzymatic activity and squalene synthase are exclusively localized in the endoplasmic reticulum. In rat hepatic peroxisomes we were not able to detect any squalene synthase. In addition, we also demonstrated that squalene synthase in the microsomal fraction is dramatically regulated by a number of hypolipidemic drugs and dietary treatments.

Animals↗

PubMed2007

Virus-PLoc: a fusion classifier for predicting the subcellular localization of viral proteins within host and virus-infected cells.

Viruses can reproduce their progenies only within a host cell, and their actions depend both on its destructive tendencies toward a specific host cell and on environmental conditions. Therefore, knowledge of the subcellular localization of viral proteins in a host cell or virus-infected cell is very useful for in-depth studying of their functions and mechanisms as well as designing antiviral drugs. An analysis on the Swiss-Prot database (version 50.0, released on May 30, 2006) indicates that only 23.5% of viral protein entries are annotated for their subcellular locations in this regard. As for the gene ontology database, the corresponding percentage is 23.8%. Such a gap calls for the development of high throughput tools for timely annotating the localization of viral proteins within host and virus-infected cells. In this article, a predictor called "Virus-PLoc" has been developed that is featured by fusing many basic classifiers with each engineered according to the K-nearest neighbor rule. The overall jackknife success rate obtained by Virus-PLoc in identifying the subcellular compartments of viral proteins was 80% for a benchmark dataset in which none of proteins has more than 25% sequence identity to any other in a same location site. Virus-PLoc will be freely available as a web-server at http://202.120.37.186/bioinf/virus for the public usage. Furthermore, Virus-PLoc has been used to provide large-scale predictions of all viral protein entries in Swiss-Prot database that do not have subcellular location annotations or are annotated as being uncertain. The results thus obtained have been deposited in a downloadable file prepared with Microsoft Excel and named "Tab_Virus-PLoc.xls." This file is available at the same website and will be updated twice a year to include the new entries of viral proteins and reflect the continuous development of Virus-PLoc.

Cells↗

PubMed1992

Sea urchin small RNA ribonucleoprotein particles: identification, synthesis, and subcellular localization during early embryonic development.

Small RNAs in sea urchins were examined in order to characterize developmental changes in their level, subcellular localization, synthesis, and association with proteins and other RNAs. Small RNAs such as the U snRNAs, 5S and 5.8S rRNAs, and 7S RNAs were identified by their mobility on highly cross-linked acrylamide gels. In addition, 7SL and U1 RNAs were identified by northern blot hybridization to cloned human and sea urchin probes, respectively. The level, subcellular localization, and association with proteins or RNA do not change for most small RNAs from fertilization to blastula, even though this is the time when the stored maternal pool of many small RNAs is being supplemented and replaced by embryonically synthesized RNAs. New embryonic synthesis of small RNAs was first detected at the 8-12 hr blastula stage. Although the predicted subsets of the total small RNA pool can be found in the appropriate subcellular compartments, newly synthesized small RNAs have a predominantly cytoplasmic localization: All of the newly synthesized small RNAs were found to be constituents of small RNPs. The RNPs containing newly synthesized small RNAs had sedimentation rates indistinguishable from their maternal counterparts. Thus, on the basis of sedimentation rate, no gross differences could be detected between maternal and embryonic small RNP pools. These small RNPs include a cytoplasmic RNP containing newly synthesized U1 snRNA and the sea urchin signal recognition particle (SRP) containing the 7SL, RNA. We have also identified a small RNP bearing the 5S rRNA which is present in both eggs and embryos. The presence of multiple, abundant, small RNAs and RNPs that are maintained at constant levels in particular subcellular fractions throughout development suggests that small RNAs may be involved in many more cellular activities than have so far been described.

Animals↗

PubMed2003

Subcellular location prediction of apoptosis proteins.

Apoptosis proteins have a central role in the development and homeostasis of an organism. These proteins are very important for understanding the mechanism of programmed cell death. Many efforts in pharmaceutical research have been aimed at understanding their structure and function. Unfortunately, thus far, very few apoptosis protein structures have been determined. In contrast, many apoptosis protein sequences are known, and many more are expected to come in the near future. Because of the extremely unbalanced state, it would be worthwhile to develop a fast sequence-based method to identify their subcellular location so as to gain some insight about their biological function. In view of this, a study was initiated in an attempt to identify the subcellular location of apoptosis proteins according to their sequences by means of the covariant discriminant function, which was established based on the Mahalanobis distance and Chou's invariance theorem (Chou, Proteins 1995;21:319-344). The results were quite promising, indicating that the subcellular location of apoptosis proteins are predictable to a considerably accurate extent if a good training data set can be established. It is expected that, with a continuous improvement of the training data set by incorporating more and more new data, the current method might eventually become a useful tool in this area because the function of an apoptosis protein is closely related to its subcellular location.

Algorithms↗

PubMed1985

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells.

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22 h in the presence of D-[1-3H]galactose or [3H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipid-sialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10-15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [3H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.

Animals↗

PubMed1997

Extracellular Mg2+ regulates intracellular Mg2+ and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe.

Effects of extracellular magnesium ions ([Mg2+]o) on intracellular free Mg2+ ([Mg2+]i) and its subcellular distribution in single fission yeast cells, Schizosaccharomyces pombe, were studied with digital-imaging microscopy and an Mg2+ fluorescent probe (mag-fura-2). Using 0.44 mM [Mg2+]o, [Mg2+]i in yeast cells was 0.91 +/- 0.08 mM. Elevation of [Mg2+]o to 1.97 mM induced rapid (within 5 min) increments in [Mg2+]i (2.18 +/- 0.11 mM). Lowering [Mg2+]o to 0.06 mM, however, exerted no significant effects on [Mg2+]i (0.93 +/- 0.14 mM), at least for periods of up to 30 min. Irrespective of the [Mg2+]o used, the subcellular distribution of [Mg2+]i remained heterogeneous, i.e. where the sub-plasma membrane region > cytoplasm > nucleus. [Mg2+] in all three subcellular compartments increased significantly, two- to threefold, concomitant with [Mg2+] when placed in 1.97 mM [Mg2+]o. We conclude that [Mg2+]i in fission yeast is maintained at a physiologic level when [Mg2+]o is low, but intracellular free Mg2+ rapidly rises when [Mg2+]o is elevated. Like most eukaryotic cells, yeast may have a Mg2+ transport system(s) which functions to maintain gradients of Mg2+ from the outside to inside the cell and among its subcellular compartments.

Animals↗

PubMed2000

Subcellular localization of protein kinase CK2. A key to its function?

More than 46 years ago, Burnett and Kennedy first described protein kinase CK2 (formerly known as casein kinase 2) in liver extracts. Since then, protein kinase CK2 has been investigated in many organisms from yeast to man. It is now well established that protein kinase CK2 is a pleiotropic and ubiquitous serine or threonine kinase, which is highly conserved during evolution. A great number of studies deal with substrates of CK2, but the fact that over 160 substrates exist is more confusing than elucidatory. The holoenzyme is composed of two regulatory beta-subunits and two catalytic alpha- or alpha'-subunits. There is now increasing evidence for individual functions of the subunits that are different from their functions in the holoenzyme. Furthermore, more and more studies describe interacting partners of the kinase that may be decisive in the regulation of this enzyme. A big step forward has been the determination of the crystal structure of the two subunits of protein kinase CK2. Now the interactions of the catalytic subunit of CK2 with ATP as well as GTP and the interaction between the regulatory subunits can be explained. However, cellular functions of protein kinase CK2 still remain unclear. In the present review we will focus our interest on the subcellular localization of protein kinase CK2. Protein kinase CK2 is found in many organisms and tissues and nearly every subcellular compartment. There is ample evidence that protein kinase CK2 has different functions in these compartments and that the subcellular localization of protein kinase CK2 is tightly regulated. Therefore studying the subcellular localization of protein kinase CK2 may be a key to its function.

Animals↗

PubMed1985

Distribution of gentamicin among subcellular fractions from rat renal cortex.

A substantial amount of data is available to suggest that lysosomal sequestration of aminoglycoside antibiotics plays a role in the pathogenesis of aminoglycoside-induced renal tubule cell injury; however, relatively little information is available on the subcellular distribution of aminoglycosides in the kidney during treatment protocols of the type that ultimately go on to produce extensive lethal renal tubule cell injury and acute renal failure in experimental animals. This study assessed the distribution of gentamicin and subcellular membranes on a discontinuous sucrose density gradient after in vivo exposure of rats to four daily 100 mg/kg doses of gentamicin as compared to in vitro exposure of normal rat renal cortex to gentamicin during tissue homogenization at drug levels comparable to those seen after in vivo treatment. After both in vivo and in vitro exposure, major localization of gentamicin, the lysosomal marker enzyme N-acetyl-beta-D-glucosaminidase (NAG), and the endoplasmic reticulum marker enzyme NADPH-cytochrome c reductase, occurred in a very light membrane fraction. Within this membrane fraction, gentamicin was more closely associated with the NAG than with the NADPH-cytochrome c reductase. The results could not be explained by complete lysosomal disruption during subcellular fractionation after in vivo gentamicin. These data provide additional insights into both the possibilities for subcellular interactions of aminoglycosides in the kidney, and into the methodology required to optimally assess such interactions.

Acetylglucosaminidase↗

PubMed1993

Glut 4 content in the plasma membrane of rat skeletal muscle: comparative studies of the subcellular fractionation method and the exofacial photolabelling technique using ATB-BMPA.

UNLABELLED: Employing subcellular membrane fractionation methods it has been shown that insulin induces a 2-fold increase in Glut 4 protein content in the plasma membrane of skeletal muscle from rats. Data based upon this technique are, however, impeded by poor plasma membrane recovery and cross-contamination with intracellular membrane vesicles. The present study was undertaken to compare the subcellular fractionation technique with the technique using [3H]ATB-BMPA exofacial photolabelling and immunoprecipitation of Glut 4 on soleus muscles from 3-week-old Wistar rats. Maximal insulin stimulation resulted in a 6-fold increase in 3-O-methylglucose uptake, and studies based on the subcellular fractionation method showed a 2-fold increase in Glut 4 content in the plasma membrane, whereas the exofacial photolabelling demonstrated a 6- to 7-fold rise in cell surface associated Glut 4 protein. Glucose transport activity was positively correlated with cell surface Glut 4 content as estimated by exofacial labelling. IN CONCLUSION: (1) the increase in glucose uptake in muscle after insulin exposure is caused by an augmented concentration of Glut 4 protein on the cell surface membrane, (2) at maximal insulin stimulation (20 mU/ml) approximately 40% of the muscle cell content of Glut 4 is at the cell surface, and (3) the exofacial labelling technique is more sensitive than the subcellular fractionation technique in measuring the amount of glucose transporters on muscle cell surface.

3-O-Methylglucose↗

PubMed1996

Acetaminophen-arylated proteins are detected in hepatic subcellular fractions and numerous extra-hepatic tissues in CD-1 and C57B1/6J mice.

To identify acetaminophen (APAP)-bound proteins in addition to the major 44 and 58 kDa APAP-binding proteins (Bartolone et al., 1992, Toxicol. Appl. Pharmacol. 113. 19-9; Pumford et al., 1992, Biochem. Biophys. Res. Commun. 182, 1348-1355; Bulera et al., 1995, Toxicol, Appl. Pharmacol. 134, 313-320), we investigated subcellular localization of liver proteins and tissue distribution of proteins arylated by a hepatotoxic dose of APAP in CD-1 and C57B1/6J mice. Western blot analysis with affinity-purified, anti-APAP antibodies allowed the detection of covalently bound proteins in liver mitochondria, nuclei, membrane, cytosol, and microsomes. Enzyme market assays revealed that subcellular fractions were 90-98% pure. The lack of contamination from other isolated subcellular fractions indicates that covalently bound proteins were specific to the particular subcellular fraction. APAP-arylated proteins with molecular weights similar to those detected in the liver were found in cytosolic fractions from kidney, lung, pancreas, heart, skeletal muscle, and stomach. The presence of arylated proteins in extra-hepatic organs suggests that other organs may be susceptible to APAP toxicity and may contain critical protein targets that are important in APAP toxicity. In contrast, covalently bound proteins were not detected in cytosols isolated from spleen, small intestine, brain, and testis. The characterization of the APAP-arylated proteins identified in this study will aid in elucidating the mechanism of APAP-induced toxicity.

Acetaminophen↗

PubMed1980

Subcellular localization of adenylate cyclase in thymocytes.

In almost all cell types, adenylate cyclase is located in the plasma membrane. In lymphocytes, however, this enzyme has been claimed to be largely present in intracellular compartments. In this study, the distribution of adenylate cyclase activity in subcellular fractions of calf thymocytes was reinvestigated by a balance sheet approach. When subcellular fractionation was performed in the absence of ATP and dithiothreitol, less than a half of the homogenate basal activity could be recovered in the fractions, and this amount was distributed almost equally in three main compartments: the plasma membrane fraction, the microsomal and mitochondrial fractions and the nuclear fraction. However, if enzyme activity in the above fractions was measured in the presence of the stimulatory agents NaF, guanylylimidophosphate or guanosine 5'-O-(3-thio)triphosphate, or if the subcellular fractionation was performed in media containing ATP and dithiothreitol, the overall recovered activity greatly increased (up to 90%) and the distribution was shifted in favour of the plasma membrane fraction (up to 65% of the recovered activity). The adenylate cyclase properties were similar in all fractions. The ionophore alamethicin did not alter the subcellular distribution of the enzyme. The localization of adenylate cyclase in thymocytes thus appears to be primarily, if not uniquely, in the plasma membrane, as generally found in other cell types.

Adenosine Triphosphate↗

PubMed2005

Lipopolysaccharide changes the subcellular distribution of aquaporin 5 and increases plasma membrane water permeability in mouse lung epithelial cells.

Aquaporin-5 (AQP5), a major water channel in lung epithelial cells, plays an important role in maintaining water homeostasis in the lungs. Cell surface expression of AQP5 is regulated by not only mRNA and protein synthesis but also changes in subcellular distribution. We investigated the effect of lipopolysaccharide (LPS) on the subcellular distribution of AQP5 in a mouse lung epithelial cell line (MLE-12). LPS caused significant increases in AQP5 in the plasma membrane at 0.5-2 h. Immunofluorescence and Western blotting strongly suggested that LPS altered AQP5 subcellular distribution from an intracellular vesicular compartment to the plasma membrane. The specific p38 MAP kinase inhibitor SB 203580 apparently prevented LPS-induced changes in AQP5 distribution. Furthermore, LPS increased the osmotic water permeability of MLE-12 cells. These findings demonstrate that LPS increases cell surface AQP5 expression by changing its subcellular distribution and increases membrane osmotic water permeability through activation of p38 MAP kinase.

Animals↗

PubMed2005

Subcellular localization and dynamics of MysPDZ (Myo18A) in live mammalian cells.

MysPDZ is an unconventional myosin belonging to the class XVIII myosin containing a KE (lysine and glutamine)-rich domain and a PDZ domain, which codistributes with actin fibers partially without any canonical actin binding sequence in its myosin head domain. Recently, we reported the identification of a novel isoform of MysPDZ lacking these domains and exhibiting subcellular localization and expression profile different from the original form of MysPDZ. In order to delineate domains directing the subcellular localization of MysPDZ, we performed co-immunoprecipitation experiments and image analyses using mutants of MysPDZ fused with enhanced yellow fluorescent protein. Co-immunoprecipitation analyses showed that MysPDZ can self-associate through its C-terminus coiled-coil domain and the KE-rich domain mediates the interaction with actin. We observed by image analyses that the codistribution with actin fibers and the localization in inner surface of cell membrane of MysPDZ are controlled by the KE-rich domain and the PDZ domain, respectively. Time lapse video microscopy showed that MysPDZ in the cytoplasm moves randomly and rapidly within short range and is allocated to a subcellular compartment without ATP hydrolysis by MysPDZ. This suggests that MysPDZ is a protein which is unlike most unconventional myosins. Our study uncovers a novel role of the KE-rich and PDZ domains in directing subcellular localization and also contributes to a better understanding of functional differences in MysPDZ isoforms.

Actins↗

PubMed2006

The subcellular fractionation properties and function of insulin receptor substrate-1 (IRS-1) are independent of cytoskeletal integrity.

Efficient insulin action requires spatial and temporal coordination of signaling cascades. The prototypical insulin receptor substrate, IRS-1 plays a central role in insulin signaling. By subcellular fractionation IRS-1 is enriched in a particulate fraction, termed the high speed pellet (HSP), and its redistribution from this fraction is associated with signal attenuation and insulin resistance. Anecdotal evidence suggests the cytoskeleton may underpin the localization of IRS-1 to the HSP. In the present study we have taken a systematic approach to examine whether the cytoskeleton contributes to the subcellular fractionation properties and function of IRS-1. By standard microscopy or immunoprecipitation we were unable to detect evidence to support a specific interaction between IRS-1 and the major cytoskeletal components actin (microfilaments), vimentin (intermediate filaments), and tubulin (microtubules) in 3T3-L1 adipocytes or in CHO.IR.IRS-1 cells. Pharmacological disruption of microfilaments and microtubules, individually or in combination, was without effect on the subcellular distribution of IRS-1 or insulin-stimulated tyrosine phosphorylation in either cell type. Phosphorylation of Akt was modestly reduced (20-35%) in 3T3-L1 adipocytes but not in CHO.IR.IRS-1 cells. In cells lacking intermediate filaments (Vim(-/-)) IRS-1 expression, distribution and insulin-stimulated phosphorylation appeared normal. Even after depolymerisation of microfilaments and microtubules, insulin-stimulated phosphorylation of IRS-1 and Akt were maintained in Vim(-/-) cells. Taken together these data indicate that the characteristic subcellular fractionation properties and function of IRS-1 are unlikely to be mediated by cytoskeletal networks and that proximal insulin signaling does not require an intact cytoskeleton.

Actin Cytoskeleton↗

PubMed2005

Subcellular localization and signaling properties of dishevelled in developing vertebrate embryos.

The Dishevelled protein mediates several diverse biological processes. Intriguingly, within the same tissues where Xenopus Dishevelled (Xdsh) controls cell fate via canonical Wnt signaling, it also controls cell polarity via the vertebrate planar cell polarity (PCP) cascade [1, 2, 3, 4, 5, 6, 7, 8 and 9]. The relationship between subcellular localization of Dishevelled and its signaling activities remains unclear; conflicting results have been reported depending upon the organism and cell types examined [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20]. We have approached this issue by developing new reagents to sequester wild-type Dishevelled protein either at the cell membrane or away from the cell membrane. Removal of Dishevelled from the cell membrane disrupts convergent extension by preventing Rho/Rac activation and mediolateral cell polarization. By manipulating the subcellular localization of K-->M (dsh1), we show that this mutation inhibits Dishevelled activation of Rac, regardless of its subcellular localization. These data demonstrate that membrane localization of Dishevelled is a prerequisite for vertebrate PCP signaling. However, both membrane-targeted and cytoplasm-targeted Dishevelled can potently activate canonical Wnt signaling, suggesting that local concentration of Dishevelled protein, but not its spatial localization, is central to canonical Wnt signaling. These results suggest that in vertebrate embryos, subcellular localization is insufficient to account for the pathway specificity of Dishevelled in the canonical Wnt versus PCP signaling cascades.

Adaptor Proteins, Signal Transducing↗