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A RhoC biosensor reveals differences in the activation kinetics of RhoA and RhoC in migrating cells.

Zawistowski JS, Sabouri-Ghomi M, Danuser G, Hahn KM, Hodgson L - PLoS ONE (2013)

Bottom Line: To understand these differences, we developed and validated a biosensor of RhoC activation (RhoC FLARE).The two isoforms differed markedly in the kinetics of activation.During macropinocytosis, differences were observed during vesicle closure and in the area surrounding vesicle formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Lineberger Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America.

ABSTRACT
RhoA and RhoC GTPases share 92% amino acid sequence identity, yet play different roles in regulating cell motility and morphology. To understand these differences, we developed and validated a biosensor of RhoC activation (RhoC FLARE). This was used together with a RhoA biosensor to compare the spatio-temporal dynamics of RhoA and RhoC activity during cell protrusion/retraction and macropinocytosis. Both GTPases were activated similarly at the cell edge, but in regions more distal from the edge RhoC showed higher activation during protrusion. The two isoforms differed markedly in the kinetics of activation. RhoC was activated concomitantly with RhoA at the cell edge, but distally, RhoC activation preceded RhoA activation, occurring before edge protrusion. During macropinocytosis, differences were observed during vesicle closure and in the area surrounding vesicle formation.

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Construction and validation of the RhoC FLARE biosensor.(A) Design of the biosensor (B) Analysis of cell suspensions expressing biosensor mutants. RhoC G14V but not Q63L is susceptible to Rho GDI. Data shown with S.E.M. n=3. (C) RhoC biosensor cell suspensions co-expressing Rho GEFs (Dbs, Dbl), Rac-specific GEF Tiam1 and Cdc42-specific GEF intersectin. Results were normalized to wildtype=1, mean of at least three independent experiments. Error bars represent standard error of the mean, ** = P < 0.001 and * = P < 0.05 (t-test, one-tailed), for B and for C. (D) 293T cell suspensions expressing RhoC biosensor with (dashed curve) or without (solid curve) RhoGDI coexpression (excitation at 433 nm). (E) RhoA and RhoC biosensor cell suspensions co-expressing Rho GDI and the RhoA/B-specific GEF XPLN. The normalized emission ratio of a negative control biosensor harboring p21 binding domain (PBD) of p21 activated kinase 1 (PAK1) instead of the Rho binding domain (RBD) from ROCK is shown in the absence of GDI overexpression. * = p < 0.001 (t-test, one-tailed), n=3, data shown with SEM.(F) Raw emission ratios of cells stably expressing wildtype RhoC FLARE.sc or control RhoC-PBD biosensors following serum stimulation for the indicated timepoints. * = p < 0.05 compared to t=0 (t-test, one-tailed), n=10 at each time point, data shown with SEM.
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pone-0079877-g001: Construction and validation of the RhoC FLARE biosensor.(A) Design of the biosensor (B) Analysis of cell suspensions expressing biosensor mutants. RhoC G14V but not Q63L is susceptible to Rho GDI. Data shown with S.E.M. n=3. (C) RhoC biosensor cell suspensions co-expressing Rho GEFs (Dbs, Dbl), Rac-specific GEF Tiam1 and Cdc42-specific GEF intersectin. Results were normalized to wildtype=1, mean of at least three independent experiments. Error bars represent standard error of the mean, ** = P < 0.001 and * = P < 0.05 (t-test, one-tailed), for B and for C. (D) 293T cell suspensions expressing RhoC biosensor with (dashed curve) or without (solid curve) RhoGDI coexpression (excitation at 433 nm). (E) RhoA and RhoC biosensor cell suspensions co-expressing Rho GDI and the RhoA/B-specific GEF XPLN. The normalized emission ratio of a negative control biosensor harboring p21 binding domain (PBD) of p21 activated kinase 1 (PAK1) instead of the Rho binding domain (RBD) from ROCK is shown in the absence of GDI overexpression. * = p < 0.001 (t-test, one-tailed), n=3, data shown with SEM.(F) Raw emission ratios of cells stably expressing wildtype RhoC FLARE.sc or control RhoC-PBD biosensors following serum stimulation for the indicated timepoints. * = p < 0.05 compared to t=0 (t-test, one-tailed), n=10 at each time point, data shown with SEM.

Mentions: The design of the new RhoC biosensor was similar to that of our previously published biosensor for RhoA [9], but incorporating RhoC , a different set of linkers, and a binding domain from ROCK1 (RBD, amino acids 905-1046) that preferentially binds to GTP-loaded RhoC [18]. The domain, at the amino terminus of the biosensor, was fused to monomeric Cerulean fluorescent protein [11], followed by an optimized linker, monomeric Venus [13], and finally full-length RhoC. Upon GTP-loading, the RBD bound to the RhoC, increasing FRET (Figure 1A). The two fluorophores were placed on the internal portion of the biosensor chain, leaving the C-terminus of the GTPase intact for binding and regulation by Rho guanine nucleotide dissociation inhibitor (RhoGDI). Consistent with recent nomenclature we have introduced to differentiate biosensor designs, the new biosensor is named RhoC FLARE.sc (sc denotes a single chain design) [19,20].


A RhoC biosensor reveals differences in the activation kinetics of RhoA and RhoC in migrating cells.

Zawistowski JS, Sabouri-Ghomi M, Danuser G, Hahn KM, Hodgson L - PLoS ONE (2013)

Construction and validation of the RhoC FLARE biosensor.(A) Design of the biosensor (B) Analysis of cell suspensions expressing biosensor mutants. RhoC G14V but not Q63L is susceptible to Rho GDI. Data shown with S.E.M. n=3. (C) RhoC biosensor cell suspensions co-expressing Rho GEFs (Dbs, Dbl), Rac-specific GEF Tiam1 and Cdc42-specific GEF intersectin. Results were normalized to wildtype=1, mean of at least three independent experiments. Error bars represent standard error of the mean, ** = P < 0.001 and * = P < 0.05 (t-test, one-tailed), for B and for C. (D) 293T cell suspensions expressing RhoC biosensor with (dashed curve) or without (solid curve) RhoGDI coexpression (excitation at 433 nm). (E) RhoA and RhoC biosensor cell suspensions co-expressing Rho GDI and the RhoA/B-specific GEF XPLN. The normalized emission ratio of a negative control biosensor harboring p21 binding domain (PBD) of p21 activated kinase 1 (PAK1) instead of the Rho binding domain (RBD) from ROCK is shown in the absence of GDI overexpression. * = p < 0.001 (t-test, one-tailed), n=3, data shown with SEM.(F) Raw emission ratios of cells stably expressing wildtype RhoC FLARE.sc or control RhoC-PBD biosensors following serum stimulation for the indicated timepoints. * = p < 0.05 compared to t=0 (t-test, one-tailed), n=10 at each time point, data shown with SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818223&req=5

pone-0079877-g001: Construction and validation of the RhoC FLARE biosensor.(A) Design of the biosensor (B) Analysis of cell suspensions expressing biosensor mutants. RhoC G14V but not Q63L is susceptible to Rho GDI. Data shown with S.E.M. n=3. (C) RhoC biosensor cell suspensions co-expressing Rho GEFs (Dbs, Dbl), Rac-specific GEF Tiam1 and Cdc42-specific GEF intersectin. Results were normalized to wildtype=1, mean of at least three independent experiments. Error bars represent standard error of the mean, ** = P < 0.001 and * = P < 0.05 (t-test, one-tailed), for B and for C. (D) 293T cell suspensions expressing RhoC biosensor with (dashed curve) or without (solid curve) RhoGDI coexpression (excitation at 433 nm). (E) RhoA and RhoC biosensor cell suspensions co-expressing Rho GDI and the RhoA/B-specific GEF XPLN. The normalized emission ratio of a negative control biosensor harboring p21 binding domain (PBD) of p21 activated kinase 1 (PAK1) instead of the Rho binding domain (RBD) from ROCK is shown in the absence of GDI overexpression. * = p < 0.001 (t-test, one-tailed), n=3, data shown with SEM.(F) Raw emission ratios of cells stably expressing wildtype RhoC FLARE.sc or control RhoC-PBD biosensors following serum stimulation for the indicated timepoints. * = p < 0.05 compared to t=0 (t-test, one-tailed), n=10 at each time point, data shown with SEM.
Mentions: The design of the new RhoC biosensor was similar to that of our previously published biosensor for RhoA [9], but incorporating RhoC , a different set of linkers, and a binding domain from ROCK1 (RBD, amino acids 905-1046) that preferentially binds to GTP-loaded RhoC [18]. The domain, at the amino terminus of the biosensor, was fused to monomeric Cerulean fluorescent protein [11], followed by an optimized linker, monomeric Venus [13], and finally full-length RhoC. Upon GTP-loading, the RBD bound to the RhoC, increasing FRET (Figure 1A). The two fluorophores were placed on the internal portion of the biosensor chain, leaving the C-terminus of the GTPase intact for binding and regulation by Rho guanine nucleotide dissociation inhibitor (RhoGDI). Consistent with recent nomenclature we have introduced to differentiate biosensor designs, the new biosensor is named RhoC FLARE.sc (sc denotes a single chain design) [19,20].

Bottom Line: To understand these differences, we developed and validated a biosensor of RhoC activation (RhoC FLARE).The two isoforms differed markedly in the kinetics of activation.During macropinocytosis, differences were observed during vesicle closure and in the area surrounding vesicle formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Lineberger Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America.

ABSTRACT
RhoA and RhoC GTPases share 92% amino acid sequence identity, yet play different roles in regulating cell motility and morphology. To understand these differences, we developed and validated a biosensor of RhoC activation (RhoC FLARE). This was used together with a RhoA biosensor to compare the spatio-temporal dynamics of RhoA and RhoC activity during cell protrusion/retraction and macropinocytosis. Both GTPases were activated similarly at the cell edge, but in regions more distal from the edge RhoC showed higher activation during protrusion. The two isoforms differed markedly in the kinetics of activation. RhoC was activated concomitantly with RhoA at the cell edge, but distally, RhoC activation preceded RhoA activation, occurring before edge protrusion. During macropinocytosis, differences were observed during vesicle closure and in the area surrounding vesicle formation.

Show MeSH
Related in: MedlinePlus