Negative Regulation of Fibroblast Motility by Ena/VASP Proteins

Bear, James E.; Loureiro, Joseph; Libova, Irina; Fässler, Reinhard; Wehland, Jürgen; Gertler, Frank B.

doi:10.1016/s0092-8674(00)80884-3

Cited by 429 publications

(500 citation statements)

References 37 publications

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“…erns the supramolecular organization of actin filaments in motile cells and has a profound effect on motility and migration (13)(14)(15)(16)(17)(18)(19)(20). With EGFP-CALI, we recapitulate the loss-of-function phenotypes of Capping protein and Mena reported previously: CALI of capping protein creates a local increase in barbed ends and F-actin resulting in numerous protrusive structures (21), whereas CALI of Mena induces larger and more stable lamellipodial protrusions (17).…”

mentioning

confidence: 52%

“…1A). The LTR promoter drives modest expression levels (15), a detail that is important if a physiological level of the rescue protein is desired. This simultaneous knockdown/rescue system requires the complementing EGFP-fusion protein to be resistant to the RNAi because of mismatches in the RNAi target sequence.…”

Section: A Lentivirus System For Single-step Generation Of Deficientmentioning

confidence: 99%

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Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Vitriol

Uetrecht

Shen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Chromophore-assisted laser inactivation (CALI) is a light-mediatedtechnique that offers precise spatiotemporal control of protein inactivation, enabling better understanding of the protein's role in cell function. EGFP has been used effectively as a CALI chromophore, and its cotranslational attachment to the target protein avoids having to use exogenously added labeling reagents. A potential drawback to EGFP-CALI is that the CALI phenotype can be obscured by the endogenous, unlabeled protein that is not susceptible to light inactivation. Performing EGFP-CALI experiments in deficient cells rescued with functional EGFP-fusion proteins permits more complete loss of function to be achieved. Here, we present a modified lentiviral system for rapid and efficient generation of knockdown cell lines complemented with physiological levels of EGFP-fusion proteins. We demonstrate that CALI of EGFP-CapZ␤ increases uncapped actin filaments, resulting in enhanced filament growth and the formation of numerous protrusive structures. We show that these effects are completely dependent upon knocking down the endogenous protein. We also demonstrate that CALI of EGFP-Mena in Mena/VASP-deficient cells stabilizes lamellipodial protrusions.capping protein ͉ Mena/VASP ͉ spatiotemporal ͉ lentivirus

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mentioning

confidence: 52%

Section: A Lentivirus System For Single-step Generation Of Deficientmentioning

confidence: 99%

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Vitriol

Uetrecht

Shen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Sequestering the endogenous mena to the mitochondria by expressing a protein containing mena-binding sites and a mitochondrial targeting sequence causes fibroblasts to increase translocation speed. Targeting the endogenous mena to the cell membrane instead causes decreased motility (Bear et al 2000). To understand better how ena/VASP proteins promote bacterial motility, but inhibit mammalian cell motility, the effects of these proteins on actin organization were examined.…”

Section: Rho Gtpases: Organizers Of Actin Structuresmentioning

confidence: 99%

Signaling mechanisms that regulate actin‐based motility processes in the nervous system

Meyer

Feldman

2002

Journal of Neurochemistry

172

108

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Actin-based motility is critical for nervous system development. Both the migration of neurons and the extension of neurites require organized actin polymerization to push the cell membrane forward. Numerous extracellular stimulants of motility and axon guidance cues regulate actin-based motility through the rho GTPases (rho, rac, and cdc42). The rho GTPases reorganize the actin cytoskeleton, leading to stress fiber, filopodium, or lamellipodium formation. The activity of the rho GTPases is regulated by a variety of proteins that either stimulate GTP uptake (activation) or hydrolysis (inactivation). These proteins potentially link extracellular signals to the activation state of rho GTPases. Effectors downstream of the rho GTPases that directly influence actin polymerization have been identified and are involved in neurite development.The Arp2/3 complex nucleates the formation of new actin branches that extend the membrane forward. Ena/VASP proteins can cause the formation of longer actin filaments, characteristic of growth cone actin morphology, by preventing the capping of barbed ends. Actin-depolymerizing factor (ADF)/cofilin depolymerizes and severs actin branches in older parts of the actin meshwork, freeing monomers to be re-incorporated into actively growing filaments. The signaling mechanisms by which extracellular cues that guide axons to their targets lead to direct effects on actin filament dynamics are becoming better understood. Keywords: actin-depolymerizing factor (ADF)/cofilin, actinrelated protein Arp2/3, ena/VASP, LIM kinase, rho GTPase.

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“…The N-terminal PID/PTB domain (PID1) of FE65 mediates the interaction of FE65 with the low density lipoprotein receptor-related protein, LRP (12), the histone acetyltransferase Tip60 (3), and the transcription factor CP2/LSF/LBP-1 (13), whereas the C-terminal PID/PTB domain of the FE65 protein family members binds APP (14 -16). In addition, the WW domain binds the mammalian homolog of Drosophila Enabled (17), implicated in cell motility (18). Biochemical complexes have been demonstrated for FE65⅐AICD⅐Tip60 (3) and FE65⅐APP⅐Mena (11) but only postulated for APP⅐FE65⅐LRP (12).…”

mentioning

confidence: 99%

Generation of the β-Amyloid Peptide and the Amyloid Precursor Protein C-terminal Fragment γ Are Potentiated by FE65L1

Chang

Tesco

Jeong

et al. 2003

Journal of Biological Chemistry

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Members of the FE65 family of adaptor proteins, FE65, FE65L1, and FE65L2, bind the C-terminal region of the amyloid precursor protein (APP). Overexpression of FE65 and FE65L1 was previously reported to increase the levels of ␣-secretase-derived APP (APPs␣). Increased ␤-amyloid (A␤) generation was also observed in cells showing the FE65-dependent increase in APPs␣. To understand the mechanism for the observed increase in both A␤ and APPs␣ given that ␣-secretase cleavage of a single APP molecule precludes A␤ generation, we examined the effects of FE65L1 overexpression on APP Cterminal fragments (APP CTFs). Our data show that FE65L1 potentiates ␥-secretase processing of APP CTFs, including the amyloidogenic CTF C99, accounting for the ability of FE65L1 to increase generation of APP Cterminal domain and A␤40. The FE65L1 modulation of these processing events requires binding of FE65L1 to APP and APP CTFs and is not because of a direct effect on ␥-secretase activity, because Notch intracellular domain generation is not altered by FE65L1. Furthermore, enhanced APP CTF processing can be detected in early endosome vesicles but not in endoplasmic reticulum or Golgi membranes, suggesting that the effects of FE65L1 occur at or near the plasma membrane. Finally, although FE65L1 increases APP C-terminal domain production, it does not mediate the APP-dependent transcriptional activation observed with FE65.Processing of the amyloid precursor protein (APP) 1 results in the generation of the amyloidogenic peptide, A␤, which plays a central role in the pathogenesis of Alzheimer's disease. Cleavage of C99, the APP C-terminal fragment derived from ␤-secretase processing of APP, by ␥-secretase generates the A␤ peptide. Furthermore, ␥-secretase cleavage of C99 and C83, the ␣-secretase derived APP C-terminal fragment (APP CTF), releases the APP C-terminal domain (AICD), a 6-kDa peptide also called CTF␥ or AID, that regulates transcription after translocation to the nucleus (1-4).The majority of proteins reported to bind the 47-amino acid intracellular region of APP (5-7), including the FE65 protein family members FE65, FE65L1, and FE65L2, bind the YENPTY sorting motif of APP via a phosphotyrosine interaction domain (PID/PTB). YENP is a clathrin-coated pit internalization domain required for trafficking of APP into the endocytic pathway (8, 9). Previous studies have shown that FE65 protein family members can alter the processing of APP by influencing APP trafficking. Increased maturation of APP and increased ␣-secretase-cleaved APP (APPs␣) secretion was observed in H4 neuroglioma cells induced for FE65L1 overexpression (10). Furthermore, enhanced secretion of APPs␣ and A␤ was reported in Madin-Darby canine kidney APP695 cells stably overexpressing FE65 (11). Cell surface APP levels were elevated in these cells, and increased routing of APP into the endocytic pathway from the plasma membrane was suggested to account for the observed increase in A␤ (11).The FE65 proteins are adaptor proteins that have three protein-protein interaction dom...

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Negative Regulation of Fibroblast Motility by Ena/VASP Proteins

Cited by 429 publications

References 37 publications

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins

Signaling mechanisms that regulate actin‐based motility processes in the nervous system

Generation of the β-Amyloid Peptide and the Amyloid Precursor Protein C-terminal Fragment γ Are Potentiated by FE65L1

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