Biomedical subjects

T L Steck

Publications and source records attributed to T L Steck.

At least 19 recordsLinked to original sources

PubMed1999

Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol.

The abundance of cell cholesterol is governed by multiple regulatory proteins in the endoplasmic reticulum (ER) which, in turn, are under the control of the cholesterol in that organelle. But how does ER cholesterol reflect cell (mostly plasma membrane) cholesterol? We have systematically quantitated this relationship for the first time. We found that ER cholesterol in resting human fibroblasts comprised approximately 0.5% of the cell total. The ER pool rose by more than 10-fold in less than 1 h as cell cholesterol was increased by approximately 50% from below to above its physiological value. The curve describing the dependence of ER on plasma membrane cholesterol had a J shape. Its vertex was at the ambient level of cell cholesterol and thus could correspond to a threshold. A variety of class 2 amphiphiles (e.g., U18666A) rapidly reduced ER cholesterol but caused only minor alterations in the J-curve. In contrast, brief exposure of cells to the oxysterol, 25-hydroxycholesterol, elevated and linearized the J-curve, increasing ER cholesterol at all values of cell cholesterol. This finding can explain the rapid action of oxysterols on cholesterol homeostasis. Other functions have also been observed to depend acutely on the level of plasma membrane cholesterol near its physiological level, perhaps reflecting a cholesterol-dependent structural or organizational transition in the bilayer. Such a physical transition could serve as a set-point above which excess plasma membrane cholesterol is transported to the ER where it would signal regulatory proteins to down-regulate its further accumulation.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1998

Circulation of cholesterol between lysosomes and the plasma membrane.

The cholesterol in the lysosomes of cultured human fibroblasts was determined to constitute approximately 6% of the cell total. This pool was enlarged by as much as 10-fold in Niemann-Pick type C cells. Certain amphiphiles (e.g. U18666A, progesterone, and imipramine) caused lysosomal cholesterol to increase to similarly high levels at a rate of approximately 0.8% of cell cholesterol/h. Lysosomal cholesterol accumulated even in the absence of exogenous lipoproteins. Furthermore, nearly all of the lysosomal cholesterol in both of the two perturbed systems was shown to be derived from the plasma membrane. Oxysterols known to alter cholesterol movement and homeostasis blocked lysosomal cholesterol accretion in amphiphile-treated cells, suggesting that this process is regulated physiologically. Treating cells with amphiphiles slightly reduced the efflux of cholesterol from lysosomes and slightly increased the influx from the plasma membrane, causing the lysosomal cholesterol compartment to double in size in approximately 15 h. After more prolonged amphiphile treatments, a population of buoyant lysosomes appeared that exchanged cholesterol with the plasma membrane completely but slowly. Niemann-Pick type C lysosomes were similarly buoyant and sluggish. We conclude that cholesterol circulates bidirectionally between the plasma membrane and lysosomes. The massive accumulation of lysosomal cholesterol in the perturbed cells does not appear to reflect disabled lysosomal transport but rather the formation of lysosomes modified for lipid storage, i.e. lamellar bodies.

Androstenes↗

PubMed1998

Skipper, an LTR retrotransposon of Dictyostelium.

The complete sequence of a retrotransposon from Dictyostelium discoideum , named skipper , was obtained from cDNA and genomic clones. The sequence of a nearly full-length skipper cDNA was similar to that of three other partially sequenced cDNAs. The corresponding retrotransposon is represented in approximately 15-20 copies and is abundantly transcribed. Skipper contains three open reading frames (ORFs) with an unusual sequence organization, aspects of which resemble certain mammalian retroviruses. ORFs 1 and 3 correspond to gag and pol genes; the second ORF, pro, corresponding to protease, was separated from gag by a single stop codon followed shortly thereafter by a potential pseudoknot. ORF3 (pol) was separated from pro by a +1 frameshift. ORFs 2 and 3 overlapped by 32 bp. The computed amino acid sequences of the skipper ORFs contain regions resembling retrotransposon polyprotein domains, including a nucleic acid binding protein, aspartyl protease, reverse transcriptase and integrase. Skipper is the first example of a retrotransposon with a separate pro gene. Skipper is also novel in that it appears to use stop codon suppression rather than frameshifting to modulate pro expression. Finally, skipper and its components may provide useful tools for the genetic characterization of Dictyostelium.

Amino Acid Sequence↗

PubMed1998

Four cholesterol-sensing proteins.

What is the connection among the following three medical conditions: Niemann-Pick type C disease (a cause of mental retardation and early death), systemic lipidosis (in which an obscure side effect of numerous drugs transforms lysosomes into lamellar bodies), and holoprosencephaly (a catastrophe in embryonic development)? Recent evidence suggests that the pathogenesis in each use involves impaired sensing of cellular cholesterol.

Carrier Proteins↗

PubMed1997

An NADH-dependent disulfide reductase activity in the endoplasmic reticulum of Dictyostelium discoideum.

A novel disulfide reductase activity was observed in the amoeba, Dictyostelium discoideum. Both 5,5'-dithiobis(2-nitro-benzoate) (DTNB) and insulin were substrates for reduction. NADH was utilized in preference to NADPH. The activity comigrated with NADPH-dependent cytochrome c reductase on sucrose gradients, suggesting localization in the endoplasmic reticulum (ER). The buoyant density of the disulfide reductase was not shifted when ribosomes were released from the ER nor was the activity solubilized when membranes were shocked with low osmotic buffer. It therefore appears that the reductase was membrane-bound in the lumen of the smooth ER.

Animals↗

PubMed1997

Quantitation of the pool of cholesterol associated with acyl-CoA:cholesterol acyltransferase in human fibroblasts.

The esterification of cholesterol in homogenates of human fibroblasts was explored as a means of estimating the size of the pool of cholesterol associated with the endoplasmic reticulum (ER) in vivo. The rationale was that the acyl-coenzyme A:cholesterol acyltransferase (ACAT) in homogenates should have access only to cholesterol associated with the (rough) ER membrane fragments in which it resides. Reacting whole homogenates to completion with an excess of [14C]oleoyl-CoA converted approximately 0.1-2% of total cell-free cholesterol to [14C]cholesteryl esters. Control studies indicated that membranes not associated with ACAT did not contribute cholesterol to this reaction. The extent of in vitro cholesterol esterification varied with pretreatment of the cells. Exposing intact cells to serum lipoproteins, oxysterols, or sphingomyelinase increased cholesterol esterification in homogenates severalfold; exposing the cells to mevinolin or cholesterol oxidase had the opposite effect. The variation in cholesterol esterification did not correlate with either the total cellular cholesterol or the intrinsic activity of ACAT, neither of which was changed significantly by the pretreatments. Rather, the total amount of cholesterol esterified in homogenates paralleled the rate of cholesterol esterification in the corresponding intact cells. The pool of cholesterol esterified in vitro therefore appears to reflect that associated with the ER in vivo. Since several of the mechanisms keeping cell cholesterol under tight feedback control are themselves located in the ER, this pool might not only be regulated physiologically, but could, in turn, help to regulate homeostatic effector pathways.

Cells, Cultured↗

PubMed1997

Late events in the intracellular sorting of major histocompatibility complex class II molecules are regulated by the 80-82 segment of the class II beta chain.

The molecular mechanisms that regulate sorting of major histocompatibility complex (MHC) class II molecules into the endocytic pathway are poorly understood. For many proteins, access to endosomal compartments is regulated by cytosolically expressed sequences. We present evidence that a sequence in the lumenal domain of the MHC class II molecule regulates a very late event in class II biogenesis. Class II molecules containing single amino acid changes in the highly conserved 80-82 region of the beta chain were introduced into invariant chain (Ii)-negative fibroblasts with wild-type alpha chain, and the derived transfectants were analyzed biochemically. Using an endosomal isolation technique, we have quantified the level of class II molecules expressed in endocytic compartments and found that in the absence of Ii, approximately 15% of total cellular class II molecules can be isolated from endosomal compartments. Mutation at position 80 enhances this localization, while changes at positions 81 and 82 ablate class II expression in endosomal compartments. In addition, we have evaluated whether the induced changes in intracellular distribution of class II molecules were due to alterations in early biosynthetic events, indicative of misfolding of the molecules, or to modulation of later trafficking events more likely to be a consequence of the modulation of a specific transport event. Despite the dramatic effects on endosomal localization induced by the mutations, early biosynthetic events and maturation of class II were unaffected by the mutations. Collectively, our data argue that late trafficking events that control the ability of the class II molecule to access antigens is regulated by the 80-82 segment of the MHC class II beta chains.

Animals↗

PubMed1996

Remodeling the shape of the skeleton in the intact red cell.

The role of the membrane skeleton in determining the shape of the human red cell was probed by weakening it in situ with urea, a membrane-permeable perturbant of spectrin. Urea by itself did not alter the biconcave disk shape of the red cell; however, above threshold conditions (1.5 M, 37 degrees C, 10 min), it caused an 18% reduction in the membrane elastic shear modulus. It also potentiated the spiculation of cells by lysophosphatidylcholine. These findings suggest that the contour of the resting cell is not normally dependent on the elasticity of or tension in the membrane skeleton. Rather, the elasticity of the skeleton stabilizes membranes against deformation. Urea treatment also caused the projections induced both by micropipette aspiration and by lysophosphatidylcholine to become irreversible. Furthermore, urea converted the axisymmetric conical spicules induced by lysophosphatidylcholine into irregular, curved and knobby spicules; i.e., echinocytosis became acanthocytosis. Unlike controls, the ghosts and membrane skeletons obtained from urea-generated acanthocytes were imprinted with spicules. These data suggest that perturbing interprotein associations with urea in situ allowed the skeleton to evolve plastically to accommodate the contours imposed upon it by the overlying membrane.

Biomechanical Phenomena↗

PubMed1995

The pegs on the decorated tubules of the contractile vacuole complex of Paramecium are proton pumps.

Our previous study has shown that the decorated tubules (collectively known as the decorated spongiome) of the contractile vacuole complex (CVC) in Paramecium are the site of fluid segregation, as the binding of microinjected monoclonal antibody (mAb) DS-1 to the tubules reduced the CVC's fluid output. In this study, we showed by immunogold labeling on cryosections that the antigenic sites for mAb DS-1 were located on the 15 nm 'pegs' protruding from the cytosolic surface of the decorated tubules. In immunofluorescence studies, both polyclonal antibodies against the subunits of the V-ATPase of Dictyostelium discoideum and against the 57 kDa B-subunit of the V-ATPase of chromaffin granules gave identical labeling patterns to that produced by mAb DS-1. On cryosections, all three antigens were located most consistently near or on the pegs of the decorated tubules. These data support the notion that the pegs on the membrane of the decorated tubules represent the V1 complex of a proton pump. Concanamycin B, a potent inhibitor of V-ATPase activity and of acidification of lysosomes and endosomes, strongly and reversibly inhibited fluid output from the CVC but had minimal effect on the integrity of the decorated spongiome as observed by immunofluorescence. Such inhibition suggests that a V-ATPase is intimately involved in fluid segregation. Exposing Paramecium to 12 degrees C or 1 degrees C for 30 minutes resulted in the dissociation of the decorated tubules from the smooth spongiome that borders the collecting canals; thus the DS-1-reactive A4 antigen, the 75 kDa and 66 kDa antigens were all found dispersed in the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Cholesterol homeostasis. Modulation by amphiphiles.

Diverse amphiphiles act on cellular cholesterol metabolism as if signaling regulatory sites. One class (oxysterols) mimics the homeostatic effects of excess cell cholesterol, inhibiting cholesterol biosynthesis and stimulating plasma membrane cholesterol esterification. A second class of amphiphiles has effects precisely opposite to the oxysterols, i.e. they immediately inhibit plasma membrane cholesterol esterification and progressively induce 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and cholesterol biosynthesis. This second class of agents includes steroids, hydrophobic amines, phenothiazines, ionophores, colchicine, cytochalasins, and lysophosphatides, most of which interact with P-glycoproteins. These data support a general hypothesis describing cellular cholesterol homeostasis. (a) Proteins regulating sterol metabolism are embedded in intracellular membranes where their activities are governed by the local level of cholesterol. (b) Excess plasma membrane and lysosomal cholesterol circulates through those intracellular membranes and sets the homeostatic activities therein. (c) The two classes of agents mentioned above affect cholesterol homeostasis by increasing or decreasing, respectively, the ambient level of cholesterol at the sites of regulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

A general method for plasma membrane isolation by colloidal gold density shift.

A general method for isolating plasma membranes is described. Vegetative amoebae of Dictyostelium discoideum were allowed to directly adsorb raw colloidal gold of particle diameter 10-20 nm. After quenching the gold surface, the cells were lysed and the lysates were diluted in 60% sucrose and centrifuged through a 65% sucrose cushion to selectively pellet the gold-laden membranes. Three generally applicable exogenous cell surface markers were used to follow the plasma membranes: intercalated [3H]cholesterol, octadecylrhodamine, and the adsorbed gold colloid itself. The isolates routinely contained approximately 60% of these tags, enriched approximately 15-fold with respect to protein. The recovery and degree of enrichment of contaminating markers in the plasma membrane fraction were lysosomes (3% and 1-fold); mitochondria (11% and 3-fold); rough endoplasmic reticulum, as reflected by RNA (3% and 0.7-fold); and DNA (9% and 4-fold). Membrane proteins and lipids were quantitatively solubilized from the gold by detergents. We conclude that this methodology provides an approach to the isolation of plasma membranes which compares favorably to existing techniques with respect to yield, purity, and ease.

Cell Membrane↗

PubMed1994

Analysis of successive endocytic compartments isolated from Dictyostelium discoideum by magnetic fractionation.

A colloidal iron probe was fed to the amoeba Dictyostelium discoideum and chased for different intervals. Successive segments of the endocytic pathway were then isolated magnetically at high yield and purity. There were approx. 500 endocytic vacuoles per cell; their diameters increased from approx. 0.1-0.2 microns after 3 min of feeding to approx. 2 microns after 15 min of feeding and 60 min of chase. The wave-like progression of ingested probes along the endocytic pathway suggested that the transfer of cargo involved a maturation mechanism rather than the shuttling of cargo between stable compartments. The lifetime of primary pinosomes was calculated to be approx. 1 s. Multivesicular bodies were common in the 3 min fraction and abundant in 15 min lysosomes. alpha- and beta-adaptins of molecular masses of approx. 89 and 83 kDa were richer in the 3 min vesicles than in plasma membranes and later endocytic vacuoles. Acid phosphatase, intrinsic vacuole acidity, the vacuolar proton pump protein and pump activity were present at all endocytic stages but rose between the 3 min and 15 min vacuoles and declined thereafter. Bis(monoacyglycero)phosphate or BMP, a lipid characteristic of lysosomes, followed a similar time course; it contributed up to half of the total lipid in lysosomal vacuoles. We conclude that there is both continuity and differentiation along this endocytic pathway.

Animals↗

PubMed1994

Characterization of lysosomal membrane proteins of Dictyostelium discoideum. A complex population of acidic integral membrane glycoproteins, Rab GTP-binding proteins and vacuolar ATPase subunits.

Highly purified lysosomes, prepared by magnetic fractionation of homogenates from Dictyostelium discoideum cells fed colloidal iron, were lysed under hypoosmotic conditions, and the membrane-associated proteins were subjected to gel electrophoresis. Thirteen major membrane polypeptides, ranging in molecular weight from 25,000 to 100,000 were identified. The isoelectric points of these proteins ranged from below 3.8 to greater than 7.0. Most of these proteins were stripped from membranes exposed to a chaotropic agent, 3,5-diodo-2-hydroxybenzoic acid lithium salt, and were therefore classified as peripheral membrane proteins. Twenty five glycoprotein species were detected by lectin blot analysis; 19 were classified as integral membrane proteins, and were, in general, larger than 45 kDa and negatively charged due in part to the presence of mannose 6-sulfate. Western blot analysis also demonstrated that a Rab 4-like GTPase, a Rab 7-like GTPase, and at least three subunits of the vacuolar ATPase were associated with the lysosomal membrane; the ATPase subunits appeared to be major proteins in lysosomal membranes. Finally, based on N-terminal sequence analysis of a major 41-kDa lysosome-associated membrane protein, we cloned a cDNA that encodes a protein (DVA41) highly homologous to a yeast and a bovine vacuolar ATPase subunit of approximately 41 kDa. The D. discoideum DVA41 gene was apparently a single copy gene, expressed at constant levels during growth and development.

Adenosine Triphosphatases↗

PubMed1994

Isolation and initial characterization of the bipartite contractile vacuole complex from Dictyostelium discoideum.

The contractile vacuole complex serves to excrete excess cytosolic water from protists. In the amoeba, Dictyostelium discoideum, the organelle had a bipartite morphology: a large main vacuole (bladder) marked by lumenal alkaline phosphatase was surrounded by numerous satellite vacuoles (spongiomes). Bladders and spongiomes have now been purified for the first time. The spongiome membranes had a high density of surface projections identified as catalytically-active vacuolar proton pumps (V-H(+)-ATPase). Spongiomes were resolved from the pump-poor bladders by immunogold buoyant density shift with antibodies to the V-H(+)-ATPase; they contained little protein other than this pump. It appears that, following homogenization, most of the spongiome dissociated from bladders and populated the proton pump-rich membrane fraction called acidosomes. Isolated bladders were enriched > 40-fold in alkaline phosphatase and phosphodiesterase, the activities of which were > 85% latent. Bladders depleted of spongiomes bore several distinctive polypeptides; they also had an excess of the basepieces of the proton pump over the catalytic heads. Bladder membranes were also lipid-rich and had a distinctive lipid composition. We conclude that the contractile vacuole system in Dictyostelium is a complex of discrete, separable bladder and spongiome membranes. The V-H(+)-ATPase in the spongiome may catalyze the primary energy transduction step for pumping water out of the cytoplasm.

Acid Phosphatase↗

PubMed1993

Role of the plasma membrane in cholesterol esterification in rat hepatoma cells.

The source of the cholesterol used for ester synthesis by cultured rat hepatoma cells was examined. The activities synthesizing and esterifying cholesterol co-distributed with RNA at a high buoyant density, presumably in the rough endoplasmic reticulum (RER). Cholesterol mass was undetectable in the RER, and the transfer of cholesterol synthesized in the RER to the cell surface was more than 100 times greater than was its esterification. Similarly, essentially all of the cholesterol liberated from ingested intracellular lipoproteins was recovered at the cell surface. The plasma membranes, which contained approximately 87% of cell cholesterol, provided > 100 times more cholesterol for esterification in the RER than did nascent cholesterol. The supply of cholesterol was rate-limiting for esterification in cell homogenates. Prior oxidation of plasma membrane cholesterol in intact cells reduced the acyl-CoA:cholesterol acyltransferase activity in isolates proportionately. Finally, cholesterol in hepatoma plasma membranes was a far better substrate for in vitro esterification than was that in fibroblast plasma membranes, red blood cell ghosts, or liposomes. We conclude that the level of saturation of acyl-CoA:cholesterol acyltransferase, controlled principally through the bidirectional movement of the substrate between plasma membranes and RER, plays a major role in the regulation of cholesterol esterification.

Animals↗

PubMed1993

Characterization of lysosomes isolated from Dictyostelium discoideum by magnetic fractionation.

Superparamagnetic particles were prepared with iron oxide cores of congruent to 8 nm diameter and dextran coats. After feeding the probe to the amoeba, Dictyostelium discoideum, for 15 min and chasing for 15 min, a lysosome fraction was isolated magnetically. Isolates contained 76% of ingested iron, 82% of ingested fluorescent dextran, 1.3% of cell protein, 4% of the lipid, 28% of acid phosphatase, and 5% of the vacuolar H(+)-ATPase. Enrichment in endocytic markers was congruent to 60-fold; markers for other organelles were < 0.5%. The lysosomes were homogeneous, round (0.4-1.1 microns in diameter), and frequently adherent to one another through zones of intimate apposition. Cells were also fed the iron probe continuously for 3 h to fill their entire endocytic pathway; in this case, isolates contained 3.3% of cell protein, 11% of lipid, and 49% of cell acid phosphatase. Bis(monoacylglycerol)phosphate (BMP), a lipid characteristic of lysosomes in animal cells, comprised congruent to 6% of biosynthetically labelled cell lipids and up to half of the lipid in the endocytic pathway. Essentially all of the cellular BMP was recovered in isolates prepared after 3 h of feeding. The specificity and abundance of BMP in the endocytic organelles of this early diverged protist suggests that this phospholipid serves a universal and essential function in endocytosis.

Animals↗

PubMed1993

A post-lysosomal compartment in Dictyostelium discoideum.

Fluorescein isothiocyanate (FITC)-dextran and pyranine were fed to the social amoeba, Dictyostelium discoideum. These membrane-impermeable, pH-sensitive fluorophores initially entered a approximately equal to neutral endocytic compartment. They encountered maximal acidity (pH approximately equal to 5) about 15 min after ingestion, in what appeared to be digestive vacuoles (lysosomes). The environment of the probes returned to near neutrality by 30 min. At that time, the probes accumulated in a decreasing number of vacuoles of increasing size; ultimately, there were only a small number of vacuoles per cell with diameters of up to 3 microns. The late vacuoles sedimented more rapidly than did proton pumps, acid hydrolases, and recently ingested cargo. Unlike the vacuoles harvested immediately after the cells were fed FITC-dextran, the late vacuoles were not acidified by MgATP in vitro. Egestion of ingested FITC-dextran commenced after a lag of approximately equal to 45 min. A similar lag was observed for the resurfacing of two endocytosed bilayer-intercalated fluorophores. These results suggest that, in Dictyostelium, undigested endocytic cargo accumulates in and is returned to the cell surface through a distinctive compartment of large and nearly neutral post-lysosomal vacuoles. It will be important to determine the degree to which internalized plasma membrane components follow this post-lysosomal pathway.

Animals↗

PubMed1993

An immunocytochemical analysis of the vacuolar proton pump in Dictyostelium discoideum.

Antisera were generated in rabbits against the vacuolar proton pump (V-H(+)-ATPase) purified from Dictyostelium discoideum. The antisera inhibited V-H(+)-ATPase but not F1-ATPase activity and immunoprecipitated and immunoblotted only the polypeptide subunits of the V-H(+)-ATPase from cell homogenates. Immunocytochemical analysis of intact cells and subcellular fractions showed that the predominant immunoreactive organelles were clusters of empty, irregular vacuoles of various sizes and shapes, which corresponded to the acidosomes. The cytoplasmic surfaces of lysosomes, phagosomes and the tubular spongiome of the contractile vacuole also bore the pump antigen. The lumina of multivesicular bodies were often stained intensely; the internalized antigen may have been derived from acidosomes by autophagy. Antibodies against V-H(+)-ATPases from plant and animal cells cross-reacted with the proton pumps of Dictyostelium. Antisera directed against the V-H(+)-ATPase of Dictyostelium decorated a profusion of small vacuoles scattered throughout the cytoplasm of hepatocytes, epithelial cells, macrophages and fibroblasts. The pattern paralleled that of the endocytic and acidic spaces; there was no clear indication of discrete acidosomes in these mammalian cells. We conclude that the V-H(+)-ATPase in Dictyostelium is distributed among diverse endomembrane organelles and is immunologically cross-reactive with the proton pumps on endocytic vacuoles in mammalian cells.

Animals↗