Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras.

Olson, Keith R.; McIntosh, J. Richard; Olmsted, J.B.

doi:10.1083/jcb.130.3.639

Cited by 184 publications

(135 citation statements)

References 70 publications

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“…(data not shown) showed that disruption of the cytoskeleton did not mobilize the peripheral punctate spots and tubules and that therefore the cytoskeleton probably does not play a role in anchorage of foci at the plasma membrane. The fluorescent marker proteins GFP-MPD (the microtubule-binding domain of the microtubule-associated Olson et al, 1995) and YFP-talin (Pfaff et al, 1998), labelling microtubules and actin filaments, respectively, were used to show that the inhibitors did indeed disrupt the cytoskeleton under these conditions (data not shown), as previously described (Pouwels et al, 2002b).…”

Section: Resultsmentioning

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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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: Resultsmentioning

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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“…Expression of GFP on its own results in a diffuse distribution throughout the cytoplasm, in many cases including the nucleus [13][14][15]. Pioneering work by Wang and Hazelrigg [6] has demonstrated that GFP can be used as a fluorescent tag for the N-or C-termini of proteins.…”

Section: Gfp-tagged Proteins and Their Subcellular Localisationmentioning

confidence: 99%

“…This protein is involved in mRNA-localisation during oogenesis. Subsequently tubulin [20], Map4 [15], and tau [21] were tagged successfully. In a recent study a recombinant tobacco mosaic virus, expressing the viral movement protein (MP) fused to GFP, was used to infect plant leaves [22].…”

Section: Cytoskeletonmentioning

confidence: 99%

Green fluorescent protein: applications in cell biology

1996

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The green fluorescent protein (GFP) of Aequorea victoria is a unique in vivo reporter for monitoring dynamic processes in cells or organisms. As a fusion tag GFP can be used to localize proteins, to follow their movement or to study the dynamics of the subcellular compartments to which these proteins are targeted. Recent studies where GFP technology has revealed new insights regarding physiological activities of living cells are discussed.

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“…4G). Because the level of gene expression between different cells differed in transient assays, we searched for cells expressing high levels of GFP-MBD, which is known to result in microtubule bundling (Olson et al, 1995;Marc et al, 1998). In these cells peroxisome movement was not affected despite the presence of pronounced microtubule bundles.…”

Section: Covisualization Of Peroxisomes and Cytoskeletal Elements Revmentioning

confidence: 99%

Simultaneous Visualization of Peroxisomes and Cytoskeletal Elements Reveals Actin and Not Microtubule-Based Peroxisome Motility in Plants,

2002

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Peroxisomes were visualized in living plant cells using a yellow fluorescent protein tagged with a peroxisomal targeting signal consisting of the SKL motif. Simultaneous visualization of peroxisomes and microfilaments/microtubules was accomplished in onion (Allium cepa) epidermal cells transiently expressing the yellow fluorescent protein-peroxi construct, a green fluorescent protein-mTalin construct that labels filamentous-actin filaments, and a green fluorescent proteinmicrotubule-binding domain construct that labels microtubules. The covisualization of peroxisomes and cytoskeletal elements revealed that, contrary to the reports from animal cells, peroxisomes in plants appear to associate with actin filaments and not microtubules. That peroxisome movement is actin based was shown by pharmacological studies. For this analysis we used onion epidermal cells and various cell types of Arabidopsis including trichomes, root hairs, and root cortex cells exhibiting different modes of growth. In transient onion epidermis assay and in transgenic Arabidopsis plants, an interference with the actin cytoskeleton resulted in progressive loss of saltatory movement followed by the aggregation and a complete cessation of peroxisome motility within 30 min of drug application. Microtubule depolymerization or stabilization had no effect.

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Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras.

Cited by 184 publications

References 70 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

Green fluorescent protein: applications in cell biology

Simultaneous Visualization of Peroxisomes and Cytoskeletal Elements Reveals Actin and Not Microtubule-Based Peroxisome Motility in Plants,

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