Increased Diffusional Mobility of CFTR at the Plasma Membrane after Deletion of Its C-terminal PDZ Binding Motif

Haggie, Peter M.; Stanton, Bruce A.; Verkman, A. S.

doi:10.1074/jbc.m312445200

Cited by 84 publications

(88 citation statements)

References 51 publications

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“…animal cells (>90 %; Adams et al, 1998;Haggie et al, 2003;Umenishi et al, 2000). The mobile fraction was comparable in both the plasma membrane surrounding the tubule and the plasma membrane outside the tubule, showing that the small mobile fractions were due to an intrinsic property of the plasma membrane interaction domain and not due to the presence of the tubule.…”

Section: Mp Molecules Interact Within the Tubulementioning

confidence: 92%

“…Currently, we cannot explain why YFP-Zm7hvr and 110-YFP-Zm7hvr had such relatively small mobile fractions. In spite of their small mobile fractions, YFP-Zm7hvr and 110-YFP-Zm7hvr could be used in this experiment, since calculation of the diffusion coefficient of a mobile fraction is independent of the size of this fraction (see Methods), and the diffusion coefficients of YFP-Zm7hvr and 110-YFP-Zm7hvr in the plasma membrane outside the tubules (Table 1) were comparable to those of other plasma membrane-associated proteins (Adams et al, 1998;Haggie et al, 2003;Jans et al, 1990;Meissner & Haberlein, 2003;Tardin et al, 2003;Umenishi et al, 2000).…”

Section: Mp Molecules Interact Within the Tubulementioning

confidence: 99%

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Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus

Pouwels¹,

Velden²,

Willemse³

et al. 2004

Journal of General Virology

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Cowpea mosaic virus (CPMV) moves from cell to cell by transporting virus particles via tubules formed through plasmodesmata by the movement protein (MP). On the surface of protoplasts, a fusion between the MP and the green fluorescent protein forms similar tubules and peripheral punctate spots. Here it was shown by time-lapse microscopy that tubules can grow out from a subset of these peripheral punctate spots, which are dynamic structures that seem anchored to the plasma membrane. Fluorescence resonance energy transfer experiments showed that MP subunits interacted within the tubule, where they were virtually immobile, confirming that tubules consist of a highly organized MP multimer. Fluorescence recovery after photobleaching experiments with protoplasts, transiently expressing fluorescent plasma membrane-associated proteins of different sizes, indicated that tubules made by CPMV MP do not interact directly with the surrounding plasma membrane. These experiments indicated an indirect interaction between the tubule and the surrounding plasma membrane, possibly via a host plasma membrane protein. INTRODUCTIONFor successful systemic infection, a plant virus must spread throughout the plant, a process that starts with transport from the initially infected cell to neighbouring uninfected cells (cell-to-cell movement) and is followed by transport through the phloem into roots and young developing leaves (systemic movement). Since plant viruses cannot pass through the rigid cell wall, they have evolved ways of exploiting plasmodesmata, the naturally occurring transport channels present between plant cells (Haywood et al., 2002). Normally, only small molecules are able to pass through plasmodesmata, but plant viruses encode one or more proteins, the so-called movement proteins (MPs), that modify the structure of plasmodesmata in such a way that viral transport is enabled. So far, two basic principles for cell-to-cell movement of plant viruses have been described: tubule-guided movement of virus particles and movement as ribonucleoprotein complexes (Lazarowitz & Beachy, 1999).Cowpea mosaic virus (CPMV), a positive-stranded, bipartite RNA virus belonging to the family Comoviridae, moves from cell to cell by transporting virus particles using tubular structures, which connect the infected cell to the neighbouring uninfected cell (Pouwels et al., 2002a). Immunogold labelling has shown that the CPMV MP is present in these tubular structures (van Lent et al., 1990). On the surface of CPMV-infected protoplasts, similar tubular structures are formed, which protrude up to 20 mm into the culture medium, are tightly surrounded by the plasma membrane and have the same ultrastructure as tubules in plant tissue (van Lent et al., 1991). Remarkably, tubules are also formed on protoplasts transiently expressing MP , showing that MP is the only viral protein required for tubule formation. So far, protoplasts have proved extremely useful as a model system for studying targeting and assembly of both wt and mutant MPs (Bertens et al., 20...

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Section: Mp Molecules Interact Within the Tubulementioning

confidence: 92%

Section: Mp Molecules Interact Within the Tubulementioning

confidence: 99%

Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus

Pouwels¹,

Velden²,

Willemse³

et al. 2004

Journal of General Virology

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“…Plasmid constructs expressing GFP fused to the N terminus of wild-type CFTR (GFP-CFTR) and CFTR mutated to remove the C-terminal PDZ-binding domain (GFP-CFTR-⌬TRL) have been described (24,25). A second N terminus GFP-CFTR construct with a shorter 16-amino acid linker was also studied (GFP 16aa -CFTR, provided by Dr. G. Lukacs).…”

Section: Methodsmentioning

confidence: 99%

Monomeric CFTR in Plasma Membranes in Live Cells Revealed by Single Molecule Fluorescence Imaging

Haggie

Verkman

2008

Journal of Biological Chemistry

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel. There is indirect and conflicting evidence about whether CFTR exists in cell membranes as monomers, dimers, or higher order oligomers. We measured fluorescence intensities and photobleaching dynamics of distinct fluorescent spots in cells expressing functional CFTR-green fluorescent protein (GFP) chimeras. Intensity analysis of GFP-labeled CFTR in live cells showed singlecomponent distributions with mean intensity equal to that of purified monomeric GFP, indicating monomeric CFTR in cell membranes. Fluorescent spots showed single-step photobleaching, independently verifying that CFTR is monomeric. Results did not depend on whether GFP was added to the CFTR N terminus or fourth extracellular loop or on whether CFTR chloride conductance was stimulated by cAMP agonists. Control measurements with a CFTR chimera containing two GFPs showed two-step photobleaching and a single-component intensity distribution with mean intensity twice that of monomeric GFP. These results provide direct evidence for monomeric CFTR in live cells.The cystic fibrosis transmembrane conductance regulator (CFTR) 2 is a member of the ATP-binding cassette protein family that forms cAMP-regulated chloride channels (1). CFTR is expressed in epithelial cells in the airways, pancreas, intestine, and other tissues (2). Loss-of-function mutations in CFTR cause the hereditary lethal disease cystic fibrosis, in which chronic lung infection produces morbidity and mortality (1, 2). Excessive CFTR activity in the intestine in response to bacterial enterotoxins produces secretory diarrheas (3, 4). There is considerable interest in CFTR structure and assembly in cell membranes as CFTR is an important drug target for therapy of cystic fibrosis, secretory diarrheas, and polycystic kidney disease (3, 5-7).The assembly state of CFTR has been controversial, with indirect evidence reported for CFTR monomers, dimers, and mixed monomers/dimers. Patch clamp analysis of constructs containing linked wild-type (WT) CFTRs or WT and mutant CFTRs suggested that two CFTR polypeptides form a single chloride conductance pathway (8). Conflicting data from reconstituted membranes containing WT and mutant CFTRs did not reveal intermediary conductance states, consistent with independently functioning CFTR monomers (9). Electron crystallography has indicated that CFTR is a monomer with two conformations, likely the open and closed channel states (10). These data are in accord with high resolution crystal structures of bacterial ATP-binding cassettetype transporters showing unit cells containing two transmembrane-nucleotide binding domains (11). Biochemical approaches including velocity-gradient centrifugation, coimmunoprecipitation, gel filtration, and cross-linking have generated conflicting data suggesting monomeric CFTR (9, 12), dimeric CFTR (13), and mixed monomeric/dimeric CFTR (14, 15). Data supporting dimeric CFTR have also come from patch clamp of CFTR in the presence of...

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“…The diffusion of plasma membrane proteins is regulated by the cytoskeleton, especially actin filaments (27,28). We hypothesized that the slow diffusion of IP 3 R1 was caused by an interaction with the cytoskeleton and tested this possibility by disturbing microtubules and actin filaments pharmacologically.…”

Section: Gfp-ip 3 R1 Diffusion Is Sensitive To Actin Filament-destabimentioning

confidence: 99%

Lateral Diffusion of Inositol 1,4,5-Trisphosphate Receptor Type 1 Is Regulated by Actin Filaments and 4.1N in Neuronal Dendrites

Fukatsu

Bannai

Zhang³

et al. 2004

Journal of Biological Chemistry

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Increased Diffusional Mobility of CFTR at the Plasma Membrane after Deletion of Its C-terminal PDZ Binding Motif

Cited by 84 publications

References 51 publications

Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus

Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus

Monomeric CFTR in Plasma Membranes in Live Cells Revealed by Single Molecule Fluorescence Imaging

Lateral Diffusion of Inositol 1,4,5-Trisphosphate Receptor Type 1 Is Regulated by Actin Filaments and 4.1N in Neuronal Dendrites

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