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The NF2 tumor suppressor regulates microtubule-based vesicle trafficking via a novel Rac, MLK and p38(SAPK) pathway.

Hennigan RF, Moon CA, Parysek LM, Monk KR, Morfini G, Berth S, Brady S, Ratner N - Oncogene (2012)

Bottom Line: Conversely, an activated Rac mutant decreased Rab6 vesicle velocity.Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38(SAPK) kinase.Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA. Robert.Hennigan@cchmc.org

ABSTRACT
Neurofibromatosis type 2 patients develop schwannomas, meningiomas and ependymomas resulting from mutations in the tumor suppressor gene, NF2, encoding a membrane-cytoskeleton adapter protein called merlin. Merlin regulates contact inhibition of growth and controls the availability of growth factor receptors at the cell surface. We tested if microtubule-based vesicular trafficking might be a mechanism by which merlin acts. We found that schwannoma cells, containing merlin mutations and constitutive activation of the Rho/Rac family of GTPases, had decreased intracellular vesicular trafficking relative to normal human Schwann cells. In Nf2-/- mouse Schwann (SC4) cells, re-expression of merlin as well as inhibition of Rac or its effector kinases, MLK and p38(SAPK), each increased the velocity of Rab6 positive exocytic vesicles. Conversely, an activated Rac mutant decreased Rab6 vesicle velocity. Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38(SAPK) kinase. Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.

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Rab6 vesicle velocity is regulated by a Merlin-Rac pathwayThe velocities of anterograde Rab6-mStrawberry exocytic vesicles in transfected SC4 cells displayed as both frequency histograms (left) and a bar graph of mean velocities with the 95% confidence interval (right). A) SC4 cells co-transfected with Rab6-mStrawberry and either a merlin expressing plasmid or empty vector. B) SC4 cells treated for 2–3 hours with 100 μM of the Rac inhibitor NSC23766. C) SC4 cells co-transfected with Rab6-mStrawberry and either an active, fast cycling F28L-Rac mutant expressing plasmid or empty vector. D) SC4 cells co-transfected with Rab6-mStrawberry and either a constitutively GTP-bound Q61L-Rac mutant expressing plasmid or empty vector. E) SC4 cells treated for 2–3 hours with 100 nM of the MLK inhibitor CEP11004. F) SC4 cells treated for 2–3 hours with 20 μM of the p38SAPK inhibitor SB203580.
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Figure 5: Rab6 vesicle velocity is regulated by a Merlin-Rac pathwayThe velocities of anterograde Rab6-mStrawberry exocytic vesicles in transfected SC4 cells displayed as both frequency histograms (left) and a bar graph of mean velocities with the 95% confidence interval (right). A) SC4 cells co-transfected with Rab6-mStrawberry and either a merlin expressing plasmid or empty vector. B) SC4 cells treated for 2–3 hours with 100 μM of the Rac inhibitor NSC23766. C) SC4 cells co-transfected with Rab6-mStrawberry and either an active, fast cycling F28L-Rac mutant expressing plasmid or empty vector. D) SC4 cells co-transfected with Rab6-mStrawberry and either a constitutively GTP-bound Q61L-Rac mutant expressing plasmid or empty vector. E) SC4 cells treated for 2–3 hours with 100 nM of the MLK inhibitor CEP11004. F) SC4 cells treated for 2–3 hours with 20 μM of the p38SAPK inhibitor SB203580.

Mentions: To test whether merlin loss affects Rab6 vesicle velocity, Nf2−/− SC4 cells were co-transfected with plasmids expressing Rab6-mStrawberry and either merlin or empty vector then velocities were measured as described above. The average velocities of 200 to 500 vesicle tracks per condition were calculated. Re-expression of merlin caused a reproducible shift to a faster moving population of Rab6 vesicles compared to empty vector (Figure 5A). Inhibition of Rac using 100 μM NSC23766 increased vesicle velocity to a similar degree that merlin transfection did (Figure 5B). This suggests that merlin expression in live cells suppresses Rac activity, thereby controlling Rab6 vesicle velocity. Conversely, transfection with a hyperactive fast cycling Rac mutant, F28L, caused a significant decrease in Rab6 vesicle velocity (Figure 5C), consistent with Rac activity acting as a brake on exocytic transport. Interestingly, a constitutively GTP-bound, active Rac mutant, Q61L, did not affect Rab6 velocity (Figure 5D), suggesting that the ability of Rac to cycle from the GDP to the GTP bound states is critical to this regulation. Interestingly, we found that Rac-GTP co-localized with microtubules in SC4 cells (Supplemental Figure 1), consistent with previous reports that Rac-GTP binds to tubulin polymers (32). These data place active Rac in a position to influence exocytic vesicle transport.


The NF2 tumor suppressor regulates microtubule-based vesicle trafficking via a novel Rac, MLK and p38(SAPK) pathway.

Hennigan RF, Moon CA, Parysek LM, Monk KR, Morfini G, Berth S, Brady S, Ratner N - Oncogene (2012)

Rab6 vesicle velocity is regulated by a Merlin-Rac pathwayThe velocities of anterograde Rab6-mStrawberry exocytic vesicles in transfected SC4 cells displayed as both frequency histograms (left) and a bar graph of mean velocities with the 95% confidence interval (right). A) SC4 cells co-transfected with Rab6-mStrawberry and either a merlin expressing plasmid or empty vector. B) SC4 cells treated for 2–3 hours with 100 μM of the Rac inhibitor NSC23766. C) SC4 cells co-transfected with Rab6-mStrawberry and either an active, fast cycling F28L-Rac mutant expressing plasmid or empty vector. D) SC4 cells co-transfected with Rab6-mStrawberry and either a constitutively GTP-bound Q61L-Rac mutant expressing plasmid or empty vector. E) SC4 cells treated for 2–3 hours with 100 nM of the MLK inhibitor CEP11004. F) SC4 cells treated for 2–3 hours with 20 μM of the p38SAPK inhibitor SB203580.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4260777&req=5

Figure 5: Rab6 vesicle velocity is regulated by a Merlin-Rac pathwayThe velocities of anterograde Rab6-mStrawberry exocytic vesicles in transfected SC4 cells displayed as both frequency histograms (left) and a bar graph of mean velocities with the 95% confidence interval (right). A) SC4 cells co-transfected with Rab6-mStrawberry and either a merlin expressing plasmid or empty vector. B) SC4 cells treated for 2–3 hours with 100 μM of the Rac inhibitor NSC23766. C) SC4 cells co-transfected with Rab6-mStrawberry and either an active, fast cycling F28L-Rac mutant expressing plasmid or empty vector. D) SC4 cells co-transfected with Rab6-mStrawberry and either a constitutively GTP-bound Q61L-Rac mutant expressing plasmid or empty vector. E) SC4 cells treated for 2–3 hours with 100 nM of the MLK inhibitor CEP11004. F) SC4 cells treated for 2–3 hours with 20 μM of the p38SAPK inhibitor SB203580.
Mentions: To test whether merlin loss affects Rab6 vesicle velocity, Nf2−/− SC4 cells were co-transfected with plasmids expressing Rab6-mStrawberry and either merlin or empty vector then velocities were measured as described above. The average velocities of 200 to 500 vesicle tracks per condition were calculated. Re-expression of merlin caused a reproducible shift to a faster moving population of Rab6 vesicles compared to empty vector (Figure 5A). Inhibition of Rac using 100 μM NSC23766 increased vesicle velocity to a similar degree that merlin transfection did (Figure 5B). This suggests that merlin expression in live cells suppresses Rac activity, thereby controlling Rab6 vesicle velocity. Conversely, transfection with a hyperactive fast cycling Rac mutant, F28L, caused a significant decrease in Rab6 vesicle velocity (Figure 5C), consistent with Rac activity acting as a brake on exocytic transport. Interestingly, a constitutively GTP-bound, active Rac mutant, Q61L, did not affect Rab6 velocity (Figure 5D), suggesting that the ability of Rac to cycle from the GDP to the GTP bound states is critical to this regulation. Interestingly, we found that Rac-GTP co-localized with microtubules in SC4 cells (Supplemental Figure 1), consistent with previous reports that Rac-GTP binds to tubulin polymers (32). These data place active Rac in a position to influence exocytic vesicle transport.

Bottom Line: Conversely, an activated Rac mutant decreased Rab6 vesicle velocity.Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38(SAPK) kinase.Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA. Robert.Hennigan@cchmc.org

ABSTRACT
Neurofibromatosis type 2 patients develop schwannomas, meningiomas and ependymomas resulting from mutations in the tumor suppressor gene, NF2, encoding a membrane-cytoskeleton adapter protein called merlin. Merlin regulates contact inhibition of growth and controls the availability of growth factor receptors at the cell surface. We tested if microtubule-based vesicular trafficking might be a mechanism by which merlin acts. We found that schwannoma cells, containing merlin mutations and constitutive activation of the Rho/Rac family of GTPases, had decreased intracellular vesicular trafficking relative to normal human Schwann cells. In Nf2-/- mouse Schwann (SC4) cells, re-expression of merlin as well as inhibition of Rac or its effector kinases, MLK and p38(SAPK), each increased the velocity of Rab6 positive exocytic vesicles. Conversely, an activated Rac mutant decreased Rab6 vesicle velocity. Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38(SAPK) kinase. Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.

Show MeSH
Related in: MedlinePlus