Search PubMed⌕ Search

Biomedical subjects

W Rodgers

Publications and source records attributed to W Rodgers.

16 recordsLinked to original sources
PubMed2001

Glycolipid-enriched membrane domains are assembled into membrane patches by associating with the actin cytoskeleton.

Nonionic detergent lysates of cells contain a glycolipid-enriched membrane (GEM) fraction. It has been proposed that the GEM fraction represents poorly solubilized GEM microdomains, or lipid rafts. However, the properties of GEM domains in intact cells remain controversial. To study the properties of a GEM-associated protein using confocal microscopy, GFP was targeted to GEM domains using the N-terminal domain of p56(lck) (LckNT). Imaging of HeLa cells expressing LckNT-GFP showed that it was targeted to large actin-rich patches in the plasma membrane that contained up to a fivefold enrichment of protein. Double-labeling experiments showed that the patches were selectively enriched with other GEM-associated molecules. Furthermore, the patches were resistant to extraction by TX-100, and disrupting GEM domains by extracting cholesterol also disrupted colocalization of LckNT-GFP with F-actin. Analogous to the actin-rich patches in HeLa cells, LckNT-GFP colocalized with actin-rich membrane caps in stimulated T cells. Furthermore, disrupting the GEM-targeting signal of LckNT-GFP also inhibited its targeting to membrane caps. Altogether, these findings extend previous studies by showing that association of GEM domains with the actin cytoskeleton provides a mechanism for targeting signaling molecules to membrane patches and caps.

Actins↗
PubMed2001

Visualization of negative signaling in B cells by quantitative confocal microscopy.

Numerous biochemical experiments have invoked a model in which B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G (FcgammaRII) coclustering provides a dominant negative signal that blocks B-cell activation. Here, we tested this model using quantitative confocal microscopic techniques applied to ex vivo splenic B cells. We found that FcgammaRII and BCR colocalized with intact anti-Ig and that the SH2 domain-containing inositol 5'-phosphatase (SHIP) was recruited to the same site. Colocalization of BCR and SHIP was inefficient in FcgammaRII-/- but not gamma chain-/- splenic B cells. We also examined the subcellular location of a variety of enzymes and adapter proteins involved in signal transduction. Several proteins (CD19, CD22, SHP-1, and Dok) and a lipid raft marker were co-recruited to the BCR, regardless of the presence or absence of FcgammaRII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin) displayed reduced colocalization with BCR in the presence of FcgammaRII and SHIP. Colocalization of BCR and F-actin required phosphatidylinositol (PtdIns) 3-kinase and was inhibited by SHIP, because the block in BCR/F-actin colocalization was not seen in B cells of SHIP-/- animals. Furthermore, BCR internalization was inhibited with intact anti-Ig stimulation or by expression of a dominant-negative mutant form of Rac. From these results, we propose that SHIP recruitment to BCR/FcgammaRII and the resulting hydrolysis of PtdIns-3,4,5-trisphosphate prevents the appropriate spatial redistribution and activation of enzymes distal to PtdIns 3-kinase, including those that promote Rac activation, actin polymerization, and receptor internalization.

Actins↗
PubMed1998

A plasma membrane localization signal in the HIV-1 envelope cytoplasmic domain prevents localization at sites of vesicular stomatitis virus budding and incorporation into VSV virions.

Previous studies showed that the HIV-1 envelope (Env) protein was not incorporated into vesicular stomatitis virus (VSV) virions unless its cytoplasmic tail was replaced with that of the VSV glycoprotein (G). To determine whether the G tail provided a positive incorporation signal for Env, or if sequences in the Env tail prevented incorporation, we generated mutants of Env with its 150-amino-acid tail shortened to 29, 10, or 3 amino acids (Envtr mutants). Cells infected with VSV recombinants expressing these proteins or an Env-G tail hybrid showed similar amounts of Env protein at the surface. The Env-G tail hybrid or the Envtr3 mutant were incorporated at the highest levels into budding VSV virions. In contrast, the Envtr29 or Envtr10 mutants were incorporated poorly. These results defined a signal preventing incorporation within the 10 membrane-proximal amino acids of the Env tail. Confocal microscopy revealed that this signal functioned by causing localization of human immunodeficiency virus type 1 Env to plasma membrane domains distinct from the VSV budding sites, where VSV proteins were concentrated.

Amino Acid Sequence↗
PubMed1998

Making genes green: creating green fluorescent protein (GFP) fusions with blunt-end PCR products.

The jellyfish green fluorescent protein (GFP) has proven to be a useful tool in protein localization and trafficking studies. Fused to GFP, a protein of interest can be visualized and tracked in vivo through fluorescence microscopy. However, the process of making these fusion proteins is often tedious and painstaking. Here, we describe a simple and quick method for creating GFP fusion proteins using blunt-end PCR product ligation.

Base Sequence↗
PubMed1997

Recalling demonstrated and guided movements using imaginary and verbal rehearsal strategies.

This study investigated the recall of movement patterns presented either by demonstration or guided movement with vision eliminated. Participants were instructed to rehearse and remember each of the 12 patterns using one of four strategies: imagery, verbal labeling, imagery and verbal labeling, or no rehearsal strategy (i.e., control condition). Recall was better for patterns that were demonstrated than for those presented via guided movement. In addition, more patterns were remembered if a combination of imagery and verbal labeling were employed as a rehearsal strategy compared to using imagery alone. These results are discussed using Annett's (1994) model showing the relationships between action, language, and imagination in the acquisition of motor skills.

Analysis of Variance↗
PubMed1997

A comparative analysis of ADL questions in surveys of older people.

This article describes questions designed to assess limitations with respect to activities of daily living (ADLs) that were asked on the first wave of the AHEAD study, and it assesses their cross-sectional measurement properties. It also provides comparisons between those questions and parallel questions that have been asked on two other surveys of the elderly population in the United States: the 1984 Supplement on Aging (SOA) to the National Health Interview Survey and the screener for the 1982 National Long Term Care Survey (NLTCS). It also compares a single item from the 1990 Census. It then compares the ways in which the same individuals answer these different versions of ADL questions, using data from subsamples of the AHEAD respondents who were also asked the SOA, NLTCS, or Census questions. The analysis shows that there is a substantial amount of measurement error in the answers to ADL questions, and it suggests that this is a major contributor to apparent improvements and declines in functional health observed in longitudinal data.

Activities of Daily Living↗
PubMed1996

Exclusion of CD45 inhibits activity of p56lck associated with glycolipid-enriched membrane domains.

p56lck (Lck) is a lymphoid-specific Src family tyrosine kinase that is critical for T-cell development and activation. Lck is also a membrane protein, and approximately half of the membrane-associated Lck is associated with a glycolipid-enriched membrane (GEM) fraction that is resistant to solubilization by Triton X-100 (TX-100). To compare the membrane-associated Lck present in the GEM and TX-100-soluble fractions of Jurkat cells, Lck from each fraction was immunoblotted with antibody to phosphotyrosine. Lck in the GEM fraction was found to be hyperphosphorylated on tyrosine, and this correlated with a lower kinase specific activity relative to the TX-100-soluble Lck. Peptide mapping and phosphatase diagests showed that the hyperphosphorylation and lower kinase activity of GEM-associated Lck was due to phosphorylation of the regulatory COOH-terminal Tyr505. In addition, we determined that the membrane-bound tyrosine phosphatase CD45 was absent from the GEM fraction. Cells lacking CD45 showed identical phosphorylation of Lck in GEM and TX-100-soluble membranes. We propose that the GEM fraction represents a specific membrane domain present in T-cells, and that the hyperphosphorylation of tyrosine and lower kinase activity of GEM-associated Lck is due to exclusion of CD45 from these domains. Lck associated with the GEM domains may therefore consitute a reservoir of enzyme that can be readily activated.

Cell Fractionation↗
PubMed1994

Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction.

Glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins and certain protein tyrosine kinases associate with a Triton X-100-insoluble, glycolipid-enriched membrane fraction in MDCK cells. Also, certain protein tyrosine kinases have been shown to associate with GPI-anchored proteins in other cell types. To characterize the interaction between GPI-anchored proteins and protein tyrosine kinases, GPI-anchored proteins were coexpressed with p56lck in HeLa cells. Both proteins were shown to target independently to the glycolipid-enriched membranes. Coimmunoprecipitation of GPI-anchored proteins and p56lck occurred only when both proteins were located in the glycolipid-enriched membranes, and gentle disruption of these membranes abolished the interaction. The GPI anchor was found to be the targeting signal for this membrane fraction in GPI-anchored proteins. Analysis of mutants indicated that p56lck was nearly quantitatively palmitoylated at Cys-5 but not palmitoylated at Cys-3. The nonpalmitoylated cysteine at position 3 was very important for association of p56lck with the membrane fraction, while palmitoylation at Cys-5 promoted only a low level of interaction. Because other src family protein tyrosine kinases that are associated with GPI-anchored proteins always contain a Cys-3, we propose that this residue, in addition to the N-terminal myristate, is part of a common signal targeting these proteins to a membrane domain that has been linked to transmembrane signaling.

Amino Acid Sequence↗
PubMed1993

Distributions of proteins and lipids in the erythrocyte membrane.

Fluorescence digital imaging microscopy was used to characterize erythrocyte membrane domains. To investigate the role of specific proteins in forming lipid domains, erythrocyte ghosts were double-labeled with fluorescent phospholipids and a fluorescent label specific for band 3, the major integral membrane protein. Areas of enrichment, or domains, were observed for both the protein and lipid components. The size and enrichment of the protein and lipid domains in the membrane depended upon the ionic strength but not the temperature. Also, there was a higher correlation between the areas of band 3 and phosphatidylcholine enrichment than between band 3 and phosphatidylserine enrichment. This is consistent with band 3 playing a role in the formation of the phosphatidylcholine domains. Furthermore, areas of spectrin enrichment also were observed in immunofluorescently labeled ghosts, and the band 3 domains were found to coincide with the spectrin domains. Thus, the distribution of band 3 in ghosts is influenced by interactions with the cytoskeleton. Together, these experiments illustrate the type of membrane heterogeneity present in erythrocyte ghosts and the type of lipid and protein interactions that can affect the domains.

Animals↗
PubMed1991

Characterization of lipid domains in erythrocyte membranes.

Fluorescence digital imaging microscopy was used to study the lateral distribution of the lipid components in erythrocyte membranes. Intact erythrocytes labeled with phospholipids containing a fluorophore attached to one fatty acid chain showed an uneven distribution of the phospholipids in the membrane thereby demonstrating the presence of membrane domains. The enrichment of the lipotropic compound chlor-promazine in domains in intact erythrocytes also suggested that the domains are lipid-enriched regions. Similar membrane domains were present in erythrocyte ghosts. The phospholipid enrichment was increased in the domains by inducing membrane protein aggregation. Double-labeling experiments were done to determine the relative distributions of different phospholipids in the membrane. Vesicles made from extracted lipids did not show the presence of domains consistent with the conclusion that membrane proteins were responsible for creating the domains. Overall, it was found that large domains exist in the red blood cell membrane with unequal enrichment of the different phospholipid species.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Time course of vitamin E repletion in the premature infant.

Plasma and erythrocyte (RBC) tocopherol-isomer concentrations were determined serially in forty-two premature infants (25-35 weeks gestation) from birth to 8 weeks of age. For comparison purposes vitamin E status was also determined in six term infants over the first 8 d following birth and in a group of thirteen adult volunteers. Vitamin E intakes in term and preterm infants were calculated from recorded food intakes and blood transfusions. In term infants plasma vitamin E concentration rose from 1.9 mg/l (day 1) to 8.2 mg/l by day 8. In comparison preterm plasma vitamin E concentration, 0.3 mg/l (day 1), did not change appreciably by day 8 (0.7 mg/l). Likewise RBC vitamin E concentration increased in term infants from 1.3 mg/l (day 1) to 2.7 mg/l (day 8), while in preterm infants it remained unchanged, 1.5 mg/1 (day 1) v. 1.3 mg/l (day 8). Over the 3 weeks following birth, RBC vitamin E concentrations in the premature infants increased to adult values, while plasma vitamin E concentration did not reach the adult range until 8 weeks post-term. These slow changes in plasma vitamin E status occurred even though the vitamin E intake of these infants was similar to that proving adequate for term infants.

Adult↗