scholarly journals Rapid Movements of Vimentin on Microtubule Tracks: Kinesin-dependent Assembly of Intermediate Filament Networks

1998 ◽  
Vol 143 (1) ◽  
pp. 159-170 ◽  
Author(s):  
Veena Prahlad ◽  
Miri Yoon ◽  
Robert D. Moir ◽  
Ronald D. Vale ◽  
Robert D. Goldman
Keyword(s):  
Green Fluorescent Protein ◽  
Indirect Immunofluorescence ◽  
Intermediate Filament ◽  
Fluorescent Protein ◽  
Model System ◽  
Local Regulation ◽  
Filament Networks ◽  
Conventional Kinesin ◽  
Green Fluorescent ◽  
Membranous Protein

The assembly and maintenance of an extended intermediate filament (IF) network in fibroblasts requires microtubule (MT) integrity. Using a green fluorescent protein–vimentin construct, and spreading BHK-21 cells as a model system to study IF–MT interactions, we have discovered a novel mechanism involved in the assembly of the vimentin IF cytoskeleton. This entails the rapid, discontinuous, and MT-dependent movement of IF precursors towards the peripheral regions of the cytoplasm where they appear to assemble into short fibrils. These precursors, or vimentin dots, move at speeds averaging 0.55 ± 0.24 μm/s. The vimentin dots colocalize with MT and their motility is inhibited after treatment with nocodazole. Our studies further implicate a conventional kinesin in the movement of the vimentin dots. The dots colocalize with conventional kinesin as shown by indirect immunofluorescence, and IF preparations from spreading cells are enriched in kinesin. Furthermore, microinjection of kinesin antibodies into spreading cells prevents the assembly of an extended IF network. These studies provide insights into the interactions between the IF and MT systems. They also suggest a role for conventional kinesin in the distribution of non-membranous protein cargo, and the local regulation of IF assembly.

Novel features of intermediate filament dynamics revealed by green fluorescent protein chimeras

1998 ◽  
Vol 111 (13) ◽  
pp. 1767-1778 ◽  
Author(s):  
C.L. Ho ◽  
J.L. Martys ◽  
A. Mikhailov ◽  
G.G. Gundersen ◽  
R.K. Liem
Keyword(s):  
Green Fluorescent Protein ◽  
Dynamic Behavior ◽  
Cell Lines ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Fluorescent Protein ◽  
Nih3t3 Cells ◽  
Green Fluorescent ◽  
Filament Network ◽  
Perinuclear Area

In order to study the dynamic behavior of intermediate filament networks in living cells, we have prepared constructs fusing green fluorescent protein to intermediate filament proteins. Vimentin fused to green fluorescent protein labeled the endogenous intermediate filament network. We generated stable SW13 and NIH3T3 cell lines that express an enhanced green fluorescent protein fused to the N-terminus of full-length vimentin. We were able to observe the dynamic behavior of the intermediate filament network in these cells for periods as long as 4 hours (images acquired every 2 minutes). In both cell lines, the vimentin network constantly moves in a wavy manner. In the NIH3T3 cells, we observed extension of individual vimentin filaments at the edge of the cell. This movement is dependent on microtubules, since the addition of nocodazole stopped the extension of the intermediate filaments. Injection of anti-IFA causes the redistribution or ‘collapse’ of intermediate filaments. We injected anti-IFA antibodies into NIH3T3 cells stably expressing green fluorescent protein fused to vimentin and found that individual intermediate filaments move slowly towards the perinuclear area without obvious disassembly. These results demonstrate that individual intermediate filaments are translocated during the collapse, rather than undergoing disassembly-induced redistribution. Injections of tubulin antibodies disrupt the interactions between intermediate filaments and stable microtubules and cause the collapse of the vimentin network showing that these interactions play an important role in keeping the intermediate filament network extended. The nocodazole inhibition of intermediate filament extension and the anti-IFA microinjection experiments are consistent with a model in which intermediate filaments exhibit an extended distribution when tethered to microtubules, but are translocated to the perinuclear area when these connections are severed.

Green Fluorescent Protein-ExpressingEscherichiacolias a Model System for Investigating the Antimicrobial Activities of Silver Nanoparticles

Langmuir ◽  
2006 ◽  
Vol 22 (22) ◽  
pp. 9322-9328 ◽  
Author(s):  
Sonit Kumar Gogoi ◽  
P. Gopinath ◽  
Anumita Paul ◽  
A. Ramesh ◽  
Siddhartha Sankar Ghosh ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Antimicrobial Activities ◽  
Model System ◽  
Green Fluorescent

scholarly journals Motile Properties of Vimentin Intermediate Filament Networks in Living Cells

1998 ◽  
Vol 143 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Miri Yoon ◽  
Robert D. Moir ◽  
Veena Prahlad ◽  
Robert D. Goldman
Keyword(s):  
Intermediate Filament ◽  
Cell Shape ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
A Cell ◽  
Filament Networks ◽  
Green Fluorescent ◽  
Vimentin Intermediate Filament

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.

Agrobacterium tumefaciens-mediated transformation of wheat using suspension cells as a model system and green fluorescent protein as a visual marker

2001 ◽  
Vol 28 (8) ◽  
pp. 807 ◽  
Author(s):  
Brian Weir ◽  
Xu Gu ◽  
Mingbo Wang ◽  
Narayana Upadhyaya ◽  
Adrian R. Elliott ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Agrobacterium Tumefaciens ◽  
Fluorescent Protein ◽  
Model System ◽  
35S Promoter ◽  
Suspension Cells ◽  
Agrobacterium Strain ◽  
Agrobacterium Mediated Transformation ◽  
Green Fluorescent ◽  
Visual Marker

Conditions for Agrobacterium-mediated transformation of wheat (Triticum aestivum L.) were defined using wheat suspension cells as a model system and green fluorescent protein (GFP) as a visual marker. Different strains of Agrobacterium tumefaciens were compared using established wheat cell suspension cultures, where the frequency of cell clusters showing transient activity of GFP ranged from 2 to 52%. High levels of transient GFP activity and stable transformed callus lines were obtained with plasmid pTO134 containing a gfp gene with an enhanced CaMV 35S promoter and a bar gene with a 35S promoter in combination with Agrobacterium strain AGL0. These results suggest that the important variables in Agrobacterium-mediated transformation of wheat cells include media composition, Agrobacterium strain, plasmid vector and the addition of virulence-inducing agents such as acetosyringone. The conditions deemed optimal for transformation of wheat suspension cell lines were applied to scutella isolated from immature embryos and scutella-derived calli. Transient GFP expression in these tissues ranged from 10 to 75% and, while quite variable among and within cultivars, stably transformed scutellum-derived callus was obtained. Further studies with scutellum-derived calli suggested that variables such as duration of pre-inoculation culture and co-cultivation, as well as co-cultivation temperature, were also important. Optimisation of these variables resulted in the recovery of transformed wheat plants at a transformation frequency of 1.8%, which is comparable with other reports.

scholarly journals Infection of Ixodes scapularis ticks with Rickettsia monacensis expressing green fluorescent protein: A model system

2007 ◽  
Vol 94 (3) ◽  
pp. 163-174 ◽  
Author(s):  
Gerald D. Baldridge ◽  
Timothy J. Kurtti ◽  
Nicole Burkhardt ◽  
Abigail S. Baldridge ◽  
Curtis M. Nelson ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ixodes Scapularis ◽  
Protein A ◽  
Model System ◽  
Rickettsia Monacensis ◽  
Green Fluorescent

scholarly journals Accumulation of Cytoplasmic Dynein and Dynactin at Microtubule Plus Ends in Aspergillus nidulans Is Kinesin Dependent

2003 ◽  
Vol 14 (4) ◽  
pp. 1479-1488 ◽  
Author(s):  
Jun Zhang ◽  
Shihe Li ◽  
Reinhard Fischer ◽  
Xin Xiang
Keyword(s):  
Green Fluorescent Protein ◽  
Aspergillus Nidulans ◽  
Filamentous Fungus ◽  
Fluorescent Protein ◽  
Cytoplasmic Dynein ◽  
Loss Of Function ◽  
Conventional Kinesin ◽  
Green Fluorescent

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end–directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF.

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