Biomedical subjects

John W Murray

Publications and source records attributed to John W Murray.

10 recordsLinked to original sources

PubMed2025

Lymphoid gene expression supports neuroprotective microglia function.

Microglia, the innate immune cells of the brain, play a defining role in the progression of Alzheimer's disease (AD)1. The microglial response to amyloid plaques in AD can range from neuroprotective to neurotoxic2. Here we show that the protective function of microglia is governed by the transcription factor PU.1, which becomes downregulated following microglial contact with plaques. Lowering PU.1 expression in microglia reduces the severity of amyloid disease pathology in mice and is linked to the expression of immunoregulatory lymphoid receptor proteins, particularly CD28, a surface receptor that is critical for T cell activation3,4. Microglia-specific deficiency in CD28, which is expressed by a small subset of plaque-associated PU.1low microglia, promotes a broad inflammatory microglial state that is associated with increased amyloid plaque load. Our findings indicate that PU.1low CD28-expressing microglia may operate as suppressive microglia that mitigate the progression of AD by reducing the severity of neuroinflammation. This role of CD28 and potentially other lymphoid co-stimulatory and co-inhibitory receptor proteins in governing microglial responses in AD points to possible immunotherapy approaches for treating the disease by promoting protective microglial functions.

Microglia↗

PubMed2006

PKCzeta is required for microtubule-based motility of vesicles containing the ntcp transporter.

Intracellular trafficking regulates the abundance and therefore activity of transporters present at the plasma membrane. The transporter, Na+-taurocholate co-transporting polypeptide (ntcp), is increased at the plasma membrane upon treatment of cells with cAMP, for which microtubules (MTs) are required and the PI3K pathway and PKCzeta have been implicated. However, trafficking of ntcp on MTs has not been demonstrated directly and the regulation and intracellular localization of ntcp is not well understood. Here, we utilize in vitro and whole-cell immunofluorescence microscopy assays to demonstrate that ntcp is present on intracellular vesicles that bind MTs and move bidirectionally, using kinesin-1 and dynein. These vesicles co-localize with markers for recycling endosomes and early but not late endosomes. They frequently undergo fission, providing a mechanism for the exclusion of ntcp from late endosomes. PI(3,4,5)P3 activates PKCzeta and enhances motility of the ntcp vesicles and overcomes the partial inhibition produced by a PI3-kinase inhibitor. Specific inhibition of PKCzeta blocks the motility of ntcp-containing vesicles but has no effect on late vesicles as shown both in vitro and in living cells transfected with ntcp-GFP. These data indicate that PKCzeta is required specifically for the intracellular movement of vesicles that contain the ntcp transporter.

Animals↗

PubMed2006

Reconstitution of herpes simplex virus microtubule-dependent trafficking in vitro.

Microtubule-mediated anterograde transport of herpes simplex virus (HSV) from the neuronal cell body to the axon terminal is crucial for the spread and transmission of the virus. It is therefore of central importance to identify the cellular and viral factors responsible for this trafficking event. In previous studies, we isolated HSV-containing cytoplasmic organelles from infected cells and showed that they represent the first and only destination for HSV capsids after they emerge from the nucleus. In the present study, we tested whether these cytoplasmic compartments were capable of microtubule-dependent traffic. Organelles containing green fluorescent protein-labeled HSV capsids were isolated and found to be able to bind rhodamine-labeled microtubules polymerized in vitro. Following the addition of ATP, the HSV-associated organelles trafficked along the microtubules, as visualized by time lapse microscopy in an imaging microchamber. The velocity and processivity of trafficking resembled those seen for neurotropic herpesvirus traffic in living axons. The use of motor-specific inhibitors indicated that traffic was predominantly kinesin mediated, consistent with the reconstitution of anterograde traffic. Immunocytochemical studies revealed that the majority of HSV-containing organelles attached to the microtubules contained the trans-Golgi network marker TGN46. This simple, minimal reconstitution of microtubule-mediated anterograde traffic should facilitate and complement molecular analysis of HSV egress in vivo.

Biological Transport↗

PubMed2005

Interaction with PDZK1 is required for expression of organic anion transporting protein 1A1 on the hepatocyte surface.

Although many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function.

Amino Acid Sequence↗

PubMed2005

Assay of Rab4-dependent trafficking on microtubules.

We present an in vitro method to measure how Rab4 and other regulatory proteins affect microtubule-based organelle motility. The protocols utilize small-volume, disposable "microchambers" designed for epifluorescence, confocal, or other microscope platforms and into which microtubules, organelles, and primary and fluorescent secondary antibodies are added. Our work has focused on the isolation and use of endocytic vesicles from rat liver, and we present these protocols. However, the techniques can be adapted for other organelles or cell types. Multiple fluorescent probes, rapid image capture, and immunofluorescence under non-fixation conditions allow for measurements of the location and intensity changes of endogenous proteins upon addition of ATP or upon addition of other proteins or regulatory factors. We review measurements of microtubule-based motility as well as measurements for protein localization and protein segregation in vitro.

Animals↗

PubMed2004

Microtubule-dependent movement of late endocytic vesicles in vitro: requirements for Dynein and Kinesin.

Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.

Animals↗

PubMed2003

Roles of the cytoskeleton and motor proteins in endocytic sorting.

After internalization, endocytic material is actively transported through the cytoplasm, predominantly by microtubule motor proteins. Microtubule-based endocytic transport facilitates sorting of endocytic contents, vesicle fusion and fission, delivery to lysosomes, cytosolic dispersal, as well as nuclear uptake and cytosolic egress of pathogens. Endosomes, like most organelles, move bidirectionally through the cytosol and regulate their cellular location by controlling the activity of motor proteins, and potentially by controlling microtubule and actin polymerization. Control of motor protein activity is manifest by increased microtubule "run lengths", and the binding of motor proteins to organelles can be regulated by motor protein receptors. A mechanistic understanding of how organelles control motor protein activity to allow for endocytic sorting presents an exciting avenue for future research.

Animals↗

PubMed2003

Regulation of early endocytic vesicle motility and fission in a reconstituted system.

We previously established conditions to reconstitute kinesin-dependent early endocytic vesicle motility and fission on microtubules in vitro. The present study examined the question whether motility and fission are regulated in this system. Screening for proteins by immunofluorescence microscopy revealed that the small G protein, Rab4, was associated with 80% of hepatocyte-derived early endocytic vesicles that contain the ligand asialoorosomucoid (ASOR). By contrast, other markers for early endocytic vesicles including clathrin, Rab5 and EEA1 were present in the preparation but did not colocalize with the ASOR vesicles. Guanine nucleotides exchanged into the Rab4 present on the vesicles as shown by solubilization of Rab4 by Rab-GDI; solubilization was inhibited by incubation with GTP-gamma-S and promoted by GDP. Pre-incubation of vesicles with GDP increased the number of vesicles moving on microtubules and markedly increased vesicle fission. This increase in motility from GDP was shown to be towards the minus end of microtubules, possibly through activation of the minus-end-directed kinesin, KIFC2. Pre-incubation of vesicles with GTP-gamma-S, by contrast, repressed motility. Addition of exogenous GST-Rab4- GTP-gamma-S led to a further repression of motility and fission. Repression was not seen with addition of GST-Rab4-GDP. Treatment of vesicles with Rab4 antibody also repressed motility, and repression was not seen when vesicles were pre-incubated with GDP. Based on these results we hypothesize that endogenous Rab4-GTP suppresses motility of ASOR-containing vesicles in hepatocytes and that conversion of Rab4-GTP to Rab4-GDP serves as a molecular switch that activates minus-end kinesin-based motility, facilitating early endosome fission and consequent receptor-ligand segregation.

Animals↗

PubMed2003

Feeding of benthic foraminifera on diatoms and sewage-derived organic matter: an experimental application of lipid biomarker techniques.

Foraminiferal ecology at sewage outfalls has been investigated in numerous field studies over the last 30 years. Foraminifera have been frequently used as biomonitors of sewage pollution since they are both abundant and ubiquitous. Sewage outfalls have been demonstrated to have both positive and negative effects on adjacent foraminiferal populations, but it has never been shown conclusively why sewage affects foraminifera in these ways. Such information on the impact mechanisms of sewage pollution is essential if foraminifera are to be used as sewage pollution biomonitors, and also to understand the ecology of these important protists. One possible cause of a positive effect is the direct consumption of sewage-derived particulate organic matter (POM) by the foraminifera themselves. However this hypothesis has never been tested experimentally. Here, lipid (fatty acid and sterol) biomarker techniques were applied to study the ingestion of two potential food items by the foraminiferan Haynesina germanica in the laboratory. An experiment was conducted to confirm that the laboratory conditions were conducive to the survival and feeding of the foraminifera. In this experiment, foraminifera were provided with the pennate diatom Phaeodactylum tricornutum, which was considered to be a suitable food source. After 2 weeks, a four-fold increase in the levels of the diatom fatty acid biomarker, 20:5(n-3), in the foraminifera suggested that they had fed actively on the diatoms and survived under the experimental conditions. These experimental conditions were used in the main experiment, where foraminifera were fed the POM from sewage. Lipid biomarker analysis indicated that H. germanica did not consume secondary treated sewage-derived POM. Neither fatty acid profiles in the sewage nor coprostanol, the diagnostic human faecal sterol, were detected in foraminifera after exposure to the potential sewage food source. However, foraminifera may have consumed bacteria associated with the sewage in the experiment. The findings are discussed in terms of current EU legislation on sewage treatment that has affected the composition of sewage discharges, and therefore possibly reduced the nutritive value of sewage to the marine benthos.

Animals↗

PubMed2002

Immunofluorescence microchamber technique for characterizing isolated organelles.

We describe a rapid technique for the localization and quantitation of specific proteins on organelles bound to microscope chambers. Disposable chambers are constructed from glass slides and provide a platform for the binding of organelles and subsequent immunofluorescence and biochemical assays. Several studies are presented to demonstrate the utility of this technique. Kinesin was visualized in postnuclear supernatants. Golgi and endoplasmic reticulum bound quantitatively to chambers. Endocytic vesicles prepared from rat liver that had been injected in situ with Texas red-labeled asialoorsomucoid allowed for simultaneous detection of asialoorosomucoid, asialoglycoprotein receptor, caveolin 1, and microtubules. Asialoglycoprotein receptor colocalized with asialoorosomucoid-containing vesicles, whereas many of the caveolin 1 structures had no asialoorosomucoid or asialoglycoprotein receptor. The microchambers were also used to measure the binding to endocytic vesicles of exogenously added Rab5 and to monitor the ATP-dependent acidification of endocytic vesicles using the fluorescent dye acridine orange.

Animals↗