The suitability and application of a GFP-actin fusion protein for long-term imaging of the organization and dynamics of the cytoskeleton in mammalian cells

Choidas, Axel; Jungbluth, Andreas; Sechi, Antonio; Murphy, John J.; Ullrich, Axel; Marriott, Gerard

doi:10.1016/s0171-9335(98)80075-7

Cited by 85 publications

(76 citation statements)

References 31 publications

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“…cDNAs, antibodies and chemical reagents YFP-actin and YFP-paxillin were constructed by re-cloning the published GFP constructs of these proteins (Choidas et al, 1998;Zamir et al, 2000) into the pEYFP vector (Clonetech, Palo-Alto, CA). Vinculin-GFP was re-cloned into pECFP (Clonetech).…”

Section: Methodsmentioning

confidence: 99%

Polarized downregulation of the paxillin-p130CAS-Rac1 pathway induced by shear flow

Zaidel-Bar

Kam

Geiger

2005

Journal of Cell Science

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Exposure of sparsely plated endothelial cells or a wounded monolayer to shear flow induces an instantaneous inhibition of `upstream' lamellipodial protrusion and suppresses cell migration against the flow. This phenomenon is caused by the inhibition of Rac1 activity in the upstream lamellae, as demonstrated by fluorescence resonance energy transfer experiments, and by the capacity of constitutively active Rac1 to abolish flow-induced cell polarization. The local inactivation of Rac1 coincides with rapid dephosphorylation of paxillin and the adapter protein p130CAS, which, in their phosphorylated state, participate in the activation of the Rac1 exchange factor complex DOCK180/ELMO. Indeed, overexpression of DOCK180 and ELMO rescue upstream protrusion in cells exposed to flow. Searching for the mechanosensors responsible for the polarized p130CAS dephosphorylation, we discovered that shear stress stimulates the turnover and overall growth of upstream focal adhesions, whereas downstream adhesions tend to shrink. We propose that polarized, shear stress-induced signaling from focal adhesions at the upstream lamellae, leads to the local inactivation of Rac1 by inhibiting paxillin and p130CAS phosphorylation, and consequently blocking the DOCK180/ELMO pathway.

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Section: Methodsmentioning

confidence: 99%

Polarized downregulation of the paxillin-p130CAS-Rac1 pathway induced by shear flow

Zaidel-Bar

Kam

Geiger

2005

Journal of Cell Science

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“…We investigated actin cytoskeleton dynamics in vivo using cells expressing GFP-actin (27). ATIs induced enhanced stress fiber formation in both HeLa cells and fibroblasts and dramatically decreased the dynamics of actin cytoskeleton (SI Movies 7 and 8 and Fig.…”

Section: Atis Interfere With Endocytosis and Endosomal Dynamics In Nomentioning

confidence: 99%

Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

Dhonukshe

Grigoriev

Fischer

et al. 2008

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

223

191

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“…Choidas et al (Choidas et al, 1998) found that GFP-actin co-assembled with endogenous actin into a variety of actin-based structures. GFP-actin has also been utilized to measure actin dynamics in microvilli (Tyska and Mooseker, 2002;Loomis et al, 2003) and stereocilia (Rzadzinska et al, 2004).…”

mentioning

confidence: 99%

Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells

Jerdeva

Yarber

et al. 2005

Journal of Cell Science

109

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The acinar epithelial cells of the lacrimal gland exocytose the contents of mature secretory vesicles containing tear proteins at their apical membranes in response to secretagogues. Here we use time-lapse confocal fluorescence microscopy and fluorescence recovery after photobleaching to investigate the changes in actin filaments located beneath the apical membrane during exocytosis evoked by the muscarinic agonist, carbachol (100 μM). Time-lapse confocal fluorescence microscopy of apical actin filaments in reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus containing GFP-actin revealed a relatively quiescent apical actin array in resting acini. Carbachol markedly increased apical actin filament turnover and also promoted transient actin assembly around apparent fusion intermediates. Fluorescence recovery after photobleaching measurements revealed significant (P≤0.05) increases and decreases, respectively, in mobile fraction (Mf) and turnover times (t½) for apical actin filaments in carbachol-stimulated acini relative to untreated acini. The myosin inhibitors, 2,3-butanedione monoxime (BDM, 10 mM, 15 minutes) and ML-7 (40 μM, 15 minutes), significantly decreased carbachol-stimulated secretion of bulk protein and the exogenous secretory vesicle marker, syncollin-GFP; these agents also promoted accumulation of actin-coated structures which were enriched, in transduced acini, in syncollin-GFP, confirming their identity as fusion intermediates. Actin-coated fusion intermediates were sized consistent with incorporation of multiple rather than single secretory vesicles; moreover, BDM and ML-7 caused a shift towards formation of multiple secretory vesicle aggregates while significantly increasing the diameter of actin-coated fusion intermediates. Our findings suggest that the increased turnover of apical actin filaments and the interaction of actin with non-muscle myosin II assembled around aggregates of secretory vesicles facilitate exocytosis in lacrimal acinar epithelial cells.

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The suitability and application of a GFP-actin fusion protein for long-term imaging of the organization and dynamics of the cytoskeleton in mammalian cells

Cited by 85 publications

References 31 publications

Polarized downregulation of the paxillin-p130CAS-Rac1 pathway induced by shear flow

Polarized downregulation of the paxillin-p130CAS-Rac1 pathway induced by shear flow

Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells

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