Biomedical subjects

Robert D Phair

Publications and source records attributed to Robert D Phair.

8 recordsLinked to original sources

PubMed2005

ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells.

Secretory protein trafficking relies on the COPI coat, which by assembling into a lattice on Golgi membranes concentrates cargo at specific sites and deforms the membranes at these sites into coated buds and carriers. The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear. Here, we use FRAP to monitor the membrane turnover of GFP-tagged versions of ArfGAP1, Arf1, and coatomer in living cells. ArfGAP1 underwent fast cytosol/Golgi exchange with approximately 40% of the exchange dependent on engagement of ArfGAP1 with coatomer and Arf1, and affected by secretory cargo load. Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner. These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assembly-disassembly cycle of the COPI coat in vivo.

ADP-Ribosylation Factor 1↗

PubMed2004

Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy.

We have described procedures for collecting, processing, and analyzing kinetic data obtained by photobleaching microscopy of GFP-tagged chromatin proteins in nuclei of cultured living cells. These procedures are useful for characterizing the in vivo binding of chromatin proteins to their natural template--unperturbed, native chromatin in an intact cell nucleus. These techniques have revealed several generalizations that significantly change our view of the nucleus. At the qualitative level, it has become clear that almost all chromatin proteins bind only transiently to their targets. More importantly, the combined use of in vivo microscopy and kinetic, computational analysis allows analysis of the kinetics of protein binding in vivo. These methods should prove useful in the further in vivo investigation of the molecular mechanisms involved in genome organization and expression.

Binding Sites↗

PubMed2004

Quantitation of in vivo human folate metabolism.

BACKGROUND: A quantitative understanding of human folate metabolism is needed. OBJECTIVE: The objective was to quantify and interpret human folate metabolism as it might occur in vivo. DESIGN: Adults (n = 13) received 0.5 nmol [(14)C]pteroylmonoglutamate (100 nCi radioactivity) plus 79.5 nmol pteroylmonoglutamate in water orally. (14)C was measured in plasma, erythrocytes, urine, and feces for >/=40 d. Kinetic modeling was used to analyze and interpret the data. RESULTS: According to the data, the population was healthy and had a mean dietary folate intake of 1046 nmol/d, and the apparent dose absorption of (14)C was 79%. The model predictions showed that only 0.25% of plasma folate was destined for marrow, mean bile folate flux was 5351 nmol/d, and the digestibility of the mix (1046 + 5351 nmol/d) was 92%. About 33% of visceral pteroylmonoglutamate was converted to the polyglutamate form, most of the body folate was visceral (>99%), most of the visceral folate was pteroylpolyglutamate (>98%), total body folate was 225 micromol, and pteroylpolyglutamate synthesis, recycling, and catabolism were 1985, 1429, and 556 nmol/d, respectively. Mean residence times were 0.525 d as visceral pteroylmonoglutamate, 119 d as visceral pteroylpolyglutamate, 0.0086 d as plasma folate, and 0.1 d as gastrointestinal folate. CONCLUSIONS: Across subjects, folate absorption, bile folate flux, and body folate stores were larger than prior estimates. Marrow folate uptake and pteroylpolyglutamate synthesis, recycling, and catabolism are saturable processes. Visceral pteroylpolyglutamate was an immediate precursor of plasma p-aminobenzoylglutamate. The model is a working hypothesis with derived features that are explicitly model-dependent. It successfully quantitated folate metabolism, encouraging further rigorous testing.

Adult↗

PubMed2004

Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins.

Genome structure and gene expression depend on a multitude of chromatin-binding proteins. The binding properties of these proteins to native chromatin in intact cells are largely unknown. Here, we describe an approach based on combined in vivo photobleaching microscopy and kinetic modeling to analyze globally the dynamics of binding of chromatin-associated proteins in living cells. We have quantitatively determined basic biophysical properties, such as off rate constants, residence time, and bound fraction, of a wide range of chromatin proteins of diverse functions in vivo. We demonstrate that most chromatin proteins have a high turnover on chromatin with a residence time on the order of seconds, that the major fraction of each protein is bound to chromatin at steady state, and that transient binding is a common property of chromatin-associated proteins. Our results indicate that chromatin-binding proteins find their binding sites by three-dimensional scanning of the genome space and our data are consistent with a model in which chromatin-associated proteins form dynamic interaction networks in vivo. We suggest that these properties are crucial for generating high plasticity in genome expression.

Animals↗

PubMed2003

A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis.

In mitosis, chromosome, cytoskeleton, and organelle dynamics must be coordinated for successful cell division. Here, we present evidence for a role for Arf1, a small GTPase associated with the Golgi apparatus, in the orchestration of mitotic Golgi breakdown, chromosome segregation, and cytokinesis. We show that early in mitosis Arf1 becomes inactive and dissociates from Golgi membranes. This is followed by the dispersal of numerous Arf1-dependent peripheral Golgi proteins and subsequent Golgi disassembly. If Arf1 is kept in an active state by treatment with the small molecule H89 or expression of its GTP-locked form, intact Golgi membranes with bound peripheral proteins persist throughout mitosis. These cells enter mitosis but exhibit gross defects in chromosome segregation and cytokinetic furrow ingression. These findings suggest that mitotic Golgi disassembly depends on Arf1 inactivation and is used by the cell to disperse numerous peripheral Golgi proteins for coordinating the behavior of Golgi membranes, chromosomes, and cytoskeleton during mitosis.

ADP-Ribosylation Factor 1↗

PubMed2002

A kinetic framework for a mammalian RNA polymerase in vivo.

We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.

Animals↗

PubMed2002

Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport.

Cytosolic coat proteins that bind reversibly to membranes have a central function in membrane transport within the secretory pathway. One well-studied example is COPI or coatomer, a heptameric protein complex that is recruited to membranes by the GTP-binding protein Arf1. Assembly into an electron-dense coat then helps in budding off membrane to be transported between the endoplasmic reticulum (ER) and Golgi apparatus. Here we propose and corroborate a simple model for coatomer and Arf1 activity based on results analysing the distribution and lifetime of fluorescently labelled coatomer and Arf1 on Golgi membranes of living cells. We find that activated Arf1 brings coatomer to membranes. However, once associated with membranes, Arf1 and coatomer have different residence times: coatomer remains on membranes after Arf1-GTP has been hydrolysed and dissociated. Rapid membrane binding and dissociation of coatomer and Arf1 occur stochastically, even without vesicle budding. We propose that this continuous activity of coatomer and Arf1 generates kinetically stable membrane domains that are connected to the formation of COPI-containing transport intermediates. This role for Arf1/coatomer might provide a model for investigating the behaviour of other coat protein systems within cells.

ADP-Ribosylation Factor 1↗

PubMedDate not supplied

John A. Jacquez.

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Biometry↗