scholarly journals Differential Dynamics of α5 Integrin, Paxillin, and α-Actinin during Formation and Disassembly of Adhesions in Migrating Cells

2001 ◽  
Vol 153 (7) ◽  
pp. 1427-1440 ◽  
Author(s):  
Christina M. Laukaitis ◽  
Donna J. Webb ◽  
Karen Donais ◽  
Alan F. Horwitz
Keyword(s):  
Green Fluorescent Protein ◽  
Transferrin Receptor ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Perinuclear Region ◽  
Membrane Protrusions ◽  
Green Fluorescent ◽  
Endocytic Vesicles ◽  
Migrating Cells

To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed α5 integrin, α-actinin, and paxillin as green fluorescent protein (GFP) fusions. All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. α5-GFP also rescued the adhesive defects in CHO B2 cells, which are α5 integrin deficient. In ruffling cells, α5-GFP and α-actinin–GFP localized prominently at the leading edge in membrane protrusions. Of the three GFP fusion proteins that we examined, paxillin was the first component to appear visibly organized in protrusive regions of the cell. When a new protrusion formed, the paxillin appeared to remodel from older to newer adhesions at the leading edge. α-Actinin subsequently entered adhesions, which translocated toward the cell center, and inhibited paxillin turnover. The new adhesions formed from small foci of α-actinin–GFP and paxillin-GFP, which grew in size. Subsequently, α5 integrin entered the adhesions to form visible complexes, which served to stabilize the adhesions. α5-GFP also resided in endocytic vesicles that emanated from the leading edge of protrusions. Integrin vesicles at the cell rear moved toward the cell body. As cells migrated, α5 vesicles also moved from a perinuclear region to the base of the lamellipodium. The α5 vesicles colocalized with transferrin receptor and FM 4-64 dye. After adhesions broke down in the rear, α5-GFP was found in fibrous structures behind the cell, whereas α-actinin–GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dissipated.

scholarly journals Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

2000 ◽  
Vol 74 (23) ◽  
pp. 11339-11346 ◽  
Author(s):  
Vitaly Boyko ◽  
Jessica van der Laak ◽  
Jacqueline Ferralli ◽  
Elena Suslova ◽  
Myoung-Ok Kwon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Leading Edge ◽  
Intercellular Transport ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

scholarly journals Dynamics of NF κB and IκBα Studied with Green Fluorescent Protein (GFP) Fusion Proteins

2000 ◽  
Vol 275 (22) ◽  
pp. 17035-17042 ◽  
Author(s):  
Johannes A. Schmid ◽  
Andreas Birbach ◽  
Renate Hofer-Warbinek ◽  
Margarete Pengg ◽  
Ursula Burner ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals In vivo localisation and stability of human Mcl-1 using green fluorescent protein (GFP) fusion proteins

FEBS Letters ◽  
2000 ◽  
Vol 478 (1-2) ◽  
pp. 72-76 ◽  
Author(s):  
Cahit Akgul ◽  
Dale A Moulding ◽  
M.R.H White ◽  
Steven W Edwards
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals M153R Mutation in a pH-Sensitive Green Fluorescent Protein Stabilizes Its Fusion Proteins

PLoS ONE ◽  
2011 ◽  
Vol 6 (5) ◽  
pp. e19598 ◽  
Author(s):  
Yusuke V. Morimoto ◽  
Seiji Kojima ◽  
Keiichi Namba ◽  
Tohru Minamino
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Ph Sensitive ◽  
Green Fluorescent

scholarly journals A Yeast Platform for the Production of Single-Chain Antibody-Green Fluorescent Protein Fusions

2006 ◽  
Vol 72 (12) ◽  
pp. 7748-7759 ◽  
Author(s):  
Dagang Huang ◽  
Eric V. Shusta
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Protein Secretion ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Expression System ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Fusion Construct ◽  
Green Fluorescent

ABSTRACT Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 μg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)3 linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.

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