PubMed Health⌕ Search

Biomedical subjects

Anne K Kenworthy

Publications and source records attributed to Anne K Kenworthy.

9 recordsLinked to original sources
PubMed2006

The DNA binding activity of p53 displays reaction-diffusion kinetics.

The tumor suppressor protein p53 plays a key role in maintaining the genomic stability of mammalian cells and preventing malignant transformation. In this study, we investigated the intracellular diffusion of a p53-GFP fusion protein using confocal fluorescence recovery after photobleaching. We show that the diffusion of p53-GFP within the nucleus is well described by a mathematical model for diffusion of particles that bind temporarily to a spatially homogeneous immobile structure with binding and release rates k1 and k2, respectively. The diffusion constant of p53-GFP was estimated to be Dp53-GFP=15.4 microm2 s-1, significantly slower than that of GFP alone, DGFP=41.6 microm2 s-1. The reaction rates of the binding and unbinding of p53-GFP were estimated as k1=0.3 s-1 and k2=0.4 s-1, respectively, values suggestive of nonspecific binding. Consistent with this finding, the diffusional mobilities of tumor-derived sequence-specific DNA binding mutants of p53 were indistinguishable from that of the wild-type protein. These data are consistent with a model in which, under steady-state conditions, p53 is latent and continuously scans DNA, requiring activation for sequence-specific DNA binding.

Active Transport, Cell Nucleus↗
PubMed2006

Antibody-specific detection of caveolin-1 in subapical compartments of MDCK cells.

Caveolin-1 is the major structural component of caveolae and is also found in the Golgi complex of many cell types. Occasionally, caveolin-1 has been observed in additional intracellular compartments, including recycling endosomes. Why caveolin-1 expression is detected at these sites only infrequently is not clear. In this study, we test the hypothesis that non-caveolar, non-Golgi pools of caveolin-1 display unique and/or fixation-dependent epitopes. We compared the ability of a panel of antibodies raised against various domains of caveolin-1 to detect distinct subcellular pools of the protein by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells, a cell line where the subcellular localization of caveolin-1 has been extensively characterized. We show that three antibodies directed to the N-terminus of caveolin-1 recognize a previously undetected pool of caveolin-1 in the subapical region of MDCK cells, a localization characteristic of endosomal recycling compartments. The antibodies vary in their ability to label caveolin-1 at the cell surface, and the epitopes detected by each are highly fixation dependent. Our findings suggest that no single caveolin antibody or staining condition is capable of detecting all the caveolin-1 in a cell simultaneously. Consequently, the subcellular distribution of caveolin-1 may be much broader than currently believed.

Animals↗
PubMed2006

Fluorescence-based methods to image palmitoylated proteins.

A well known function of palmitoylation is to promote protein binding to cell membranes. Until recently, it was unclear what additional roles, if any, palmitoylation has in controlling protein localization in cells. Recent studies of palmitoylated forms of the small GTPase Ras have now revealed that palmitoylation plays multiple roles in the regulation of protein trafficking, including targeting proteins into the secretory pathway and recycling proteins between the plasma membrane and Golgi complex. We here describe how quantitative fluorescence microscopy and photobleaching approaches can be used to study the intracellular targeting and trafficking of GFP-tagged palmitoylated proteins in living cells. We discuss (1) general considerations for fluorescence recovery after photobleaching (FRAP) measurements of GFP-tagged proteins; (2) FRAP-based assays to test the strength of binding of palmitoylated proteins to cell membranes; (3) methods to establish the kinetics and mechanisms of recycling of palmitoylated proteins between the Golgi complex and the plasma membrane; (4) the use of the palmitoylation inhibitor 2-bromo-palmitate as a tool to study the dynamic regulation of protein targeting and trafficking by palmitate turnover.

Acylation↗
PubMed2005

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway.

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.

Animals↗
PubMed2005

Ras diffusion is sensitive to plasma membrane viscosity.

The cell surface contains a variety of barriers and obstacles that slow the lateral diffusion of glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins below the theoretical limit imposed by membrane viscosity. How the diffusion of proteins residing exclusively on the inner leaflet of the plasma membrane is regulated has been largely unexplored. We show here that the diffusion of the small GTPase Ras is sensitive to the viscosity of the plasma membrane. Using confocal fluorescence recovery after photobleaching, we examined the diffusion of green fluorescent protein (GFP)-tagged HRas, NRas, and KRas in COS-7 cells loaded with or depleted of cholesterol, a well-known modulator of membrane bilayer viscosity. In cells loaded with excess cholesterol, the diffusional mobilities of GFP-HRas, GFP-NRas, and GFP-KRas were significantly reduced, paralleling the behavior of the viscosity-sensitive lipid probes DiIC(16) and DiIC(18). However, the effects of cholesterol depletion on protein and lipid diffusion in cell membranes were highly dependent on the depletion method used. Cholesterol depletion with methyl-beta-cyclodextrin slowed Ras diffusion by a viscosity-independent mechanism, whereas overnight cholesterol depletion slightly increased both protein and lipid diffusion. The ability of Ras to sense membrane viscosity may represent a general feature of proteins residing on the cytoplasmic face of the plasma membrane.

Animals↗
PubMed2005

Photobleaching approaches to investigate diffusional mobility and trafficking of Ras in living cells.

Recent advances in our understanding of the intracellular trafficking, membrane microenvironment, and subcellular sites of signaling of Ras have been driven by observations of GFP-tagged Ras in living cells. Here, we describe methods to gain further insight into the regulation of these events through the use of quantitative fluorescence microscopy. We focus on three techniques, fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and selective photobleaching. While all of these techniques exploit photobleaching as a tool to monitor protein dynamics, they each provide a unique subset of information. In particular, FRAP provides measurements of protein mobility via lateral diffusion by monitoring recovery of fluorescence into a region following a single photobleaching event. FLIP assesses the level of continuity and communication between subcellular compartments by repetitively photobleaching a region of interest and following concomitant loss of fluorescence from other areas in the cell. Selective photobleaching reveals kinetic information about active and passive transport of proteins into organelles such as the Golgi complex or between areas of protein enrichment such as caveolae. We describe how to implement these techniques using commercially available confocal microscopes and outline methods for data analysis. Finally, we discuss how these approaches are being used to provide new insights into the mechanisms of membrane microdomain localization, vesicular versus non-vesicular transport, and kinetics of exchange of Ras on and off of cell membranes.

Animals↗
PubMed2005

Fleeting glimpses of lipid rafts: how biophysics is being used to track them.

Cell membranes are fluid but can exhibit local order that gives rise to lateral inhomogeneities, often referred to as membrane microdomains. Among the best studied yet least well understood of these microdomains are lipid rafts. Lipid rafts are hypothesized to participate in a variety of physiologic and pathologic pathways important to human health by causing the spatial segregation of proteins and lipids within the plane of the membrane. Despite the widespread implications of the raft model, major questions remain about raft size, composition, and life span. This article discusses how recent biophysical measurements of the dynamic properties of rafts and putative raft-associated proteins and lipids are being used to test the hypothesis that confinement of proteins in rafts slows and/or impairs their ability to sample their microenvironment by lateral diffusion.

Animals↗
PubMed2004

Dynamics of putative raft-associated proteins at the cell surface.

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (> 4 microm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.

Animals↗