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The antagonistic modulation of Arp2/3 activity by N-WASP, WAVE2 and PICK1 defines dynamic changes in astrocyte morphology.

Murk K, Blanco Suarez EM, Cockbill LM, Banks P, Hanley JG - J. Cell. Sci. (2013)

Bottom Line: This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices.Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes.Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.

ABSTRACT
Astrocytes exhibit a complex, branched morphology, allowing them to functionally interact with numerous blood vessels, neighboring glial processes and neuronal elements, including synapses. They also respond to central nervous system (CNS) injury by a process known as astrogliosis, which involves morphological changes, including cell body hypertrophy and thickening of major processes. Following severe injury, astrocytes exhibit drastically reduced morphological complexity and collectively form a glial scar. The mechanistic details behind these morphological changes are unknown. Here, we investigate the regulation of the actin-nucleating Arp2/3 complex in controlling dynamic changes in astrocyte morphology. In contrast to other cell types, Arp2/3 inhibition drives the rapid expansion of astrocyte cell bodies and major processes. This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices. We show that this expansion requires functional myosin II downstream of ROCK and RhoA. Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes. By contrast, knockdown of the Arp2/3 inhibitor PICK1 increases astrocyte branching complexity. Furthermore, astrocyte expansion induced by ischemic conditions is delayed by PICK1 knockdown or N-WASP overexpression. Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes. The Arp2/3 regulators PICK1, and N-WASP and WAVE2 function antagonistically to control the complexity of astrocyte branched morphology, and this mechanism underlies the morphological changes seen in astrocytes during their response to pathological insult.

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Inhibition of formins and Myosin II counteracts Arp2/3 inhibition and is associated with changes in small GTPase activation. Images (left) and frequency analysis (right) for astrocytes after serum starvation, forskolin and subsequent incubation with DMSO (A), CK-548 (B), CK-548 plus the formin inhibitor SMIFH2 (C), CK-548 plus blebbistatin (D) and CK-548 plus the ROCK inhibitor Y-27632 (E). Scale bars: 10 µm. Cell morphology is visualized by Alexa-546–phalloidin staining. Graphs show quantification of astrocyte morphology following the drug treatments (n = 300 cells per condition from three independent experiments). (F) Quantification of the proportion of polygonal astrocytes after the treatments shown in A–E. *P<0.05, **P<0.005 (ANOVA followed by Bonferroni's correction). (G) Determination of RhoA activation in astrocytes after forskolin treatment followed by CK-548 treatment for 1 h. Upper blots indicate total RhoA levels, lower blots show the GTP-bound fraction. The graph shows a quantification of the relative proportion of active RhoA, as shown in the western blots. n = 5, *P<0.05 (unpaired Student's t-test). (H) Determination of Rac1 activation in astrocytes after forskolin treatment, followed by CK-548 treatment for 1 h. Upper blots indicate total Rac1 levels, lower blots show the GTP-bound fraction. The graph shows a quantification of the relative proportion of active Rac1, as shown in the western blots. n = 4. *P>0.05 (unpaired Student's t-test).
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f03: Inhibition of formins and Myosin II counteracts Arp2/3 inhibition and is associated with changes in small GTPase activation. Images (left) and frequency analysis (right) for astrocytes after serum starvation, forskolin and subsequent incubation with DMSO (A), CK-548 (B), CK-548 plus the formin inhibitor SMIFH2 (C), CK-548 plus blebbistatin (D) and CK-548 plus the ROCK inhibitor Y-27632 (E). Scale bars: 10 µm. Cell morphology is visualized by Alexa-546–phalloidin staining. Graphs show quantification of astrocyte morphology following the drug treatments (n = 300 cells per condition from three independent experiments). (F) Quantification of the proportion of polygonal astrocytes after the treatments shown in A–E. *P<0.05, **P<0.005 (ANOVA followed by Bonferroni's correction). (G) Determination of RhoA activation in astrocytes after forskolin treatment followed by CK-548 treatment for 1 h. Upper blots indicate total RhoA levels, lower blots show the GTP-bound fraction. The graph shows a quantification of the relative proportion of active RhoA, as shown in the western blots. n = 5, *P<0.05 (unpaired Student's t-test). (H) Determination of Rac1 activation in astrocytes after forskolin treatment, followed by CK-548 treatment for 1 h. Upper blots indicate total Rac1 levels, lower blots show the GTP-bound fraction. The graph shows a quantification of the relative proportion of active Rac1, as shown in the western blots. n = 4. *P>0.05 (unpaired Student's t-test).

Mentions: To investigate the molecular mechanisms responsible for the Arp2/3-dependent expansion of astrocytes, we incubated stellated astrocyte cultures with CK-548 in conjunction with small inhibitors specific for other cytoskeletal components (Fig. 3A–E). Cells treated with CK-548 or DMSO alone served as controls (Fig. 3A,B). The formin inhibitor SMIFH2 (Rizvi et al., 2009) appeared to cause a small reduction in the effect of CK-548 on cell expansion (Fig. 3C). However, statistical analysis indicated that there was no significant difference in the proportion of polygonal cells in astrocytes treated with CK-548 and SMIFH2 compared with in astrocytes treated with CK-548 alone (Fig. 3F). The myosin II inhibitor blebbistatin more completely blocked astrocyte expansion (Fig. 3D,F). This result led us to test upstream regulators of myosin II in the presence of CK-548. We used Y-27632 to inhibit the Rho-dependent kinase (ROCK), which activates myosin II (Vicente-Manzanares et al., 2009). The efficiency of Y-27632 in counteracting the effects of Arp2/3 inhibition on astrocyte morphology is similar to that of blebbistatin (Fig. 3D–F).


The antagonistic modulation of Arp2/3 activity by N-WASP, WAVE2 and PICK1 defines dynamic changes in astrocyte morphology.

Murk K, Blanco Suarez EM, Cockbill LM, Banks P, Hanley JG - J. Cell. Sci. (2013)

Inhibition of formins and Myosin II counteracts Arp2/3 inhibition and is associated with changes in small GTPase activation. Images (left) and frequency analysis (right) for astrocytes after serum starvation, forskolin and subsequent incubation with DMSO (A), CK-548 (B), CK-548 plus the formin inhibitor SMIFH2 (C), CK-548 plus blebbistatin (D) and CK-548 plus the ROCK inhibitor Y-27632 (E). Scale bars: 10 µm. Cell morphology is visualized by Alexa-546–phalloidin staining. Graphs show quantification of astrocyte morphology following the drug treatments (n = 300 cells per condition from three independent experiments). (F) Quantification of the proportion of polygonal astrocytes after the treatments shown in A–E. *P<0.05, **P<0.005 (ANOVA followed by Bonferroni's correction). (G) Determination of RhoA activation in astrocytes after forskolin treatment followed by CK-548 treatment for 1 h. Upper blots indicate total RhoA levels, lower blots show the GTP-bound fraction. The graph shows a quantification of the relative proportion of active RhoA, as shown in the western blots. n = 5, *P<0.05 (unpaired Student's t-test). (H) Determination of Rac1 activation in astrocytes after forskolin treatment, followed by CK-548 treatment for 1 h. Upper blots indicate total Rac1 levels, lower blots show the GTP-bound fraction. The graph shows a quantification of the relative proportion of active Rac1, as shown in the western blots. n = 4. *P>0.05 (unpaired Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757329&req=5

f03: Inhibition of formins and Myosin II counteracts Arp2/3 inhibition and is associated with changes in small GTPase activation. Images (left) and frequency analysis (right) for astrocytes after serum starvation, forskolin and subsequent incubation with DMSO (A), CK-548 (B), CK-548 plus the formin inhibitor SMIFH2 (C), CK-548 plus blebbistatin (D) and CK-548 plus the ROCK inhibitor Y-27632 (E). Scale bars: 10 µm. Cell morphology is visualized by Alexa-546–phalloidin staining. Graphs show quantification of astrocyte morphology following the drug treatments (n = 300 cells per condition from three independent experiments). (F) Quantification of the proportion of polygonal astrocytes after the treatments shown in A–E. *P<0.05, **P<0.005 (ANOVA followed by Bonferroni's correction). (G) Determination of RhoA activation in astrocytes after forskolin treatment followed by CK-548 treatment for 1 h. Upper blots indicate total RhoA levels, lower blots show the GTP-bound fraction. The graph shows a quantification of the relative proportion of active RhoA, as shown in the western blots. n = 5, *P<0.05 (unpaired Student's t-test). (H) Determination of Rac1 activation in astrocytes after forskolin treatment, followed by CK-548 treatment for 1 h. Upper blots indicate total Rac1 levels, lower blots show the GTP-bound fraction. The graph shows a quantification of the relative proportion of active Rac1, as shown in the western blots. n = 4. *P>0.05 (unpaired Student's t-test).
Mentions: To investigate the molecular mechanisms responsible for the Arp2/3-dependent expansion of astrocytes, we incubated stellated astrocyte cultures with CK-548 in conjunction with small inhibitors specific for other cytoskeletal components (Fig. 3A–E). Cells treated with CK-548 or DMSO alone served as controls (Fig. 3A,B). The formin inhibitor SMIFH2 (Rizvi et al., 2009) appeared to cause a small reduction in the effect of CK-548 on cell expansion (Fig. 3C). However, statistical analysis indicated that there was no significant difference in the proportion of polygonal cells in astrocytes treated with CK-548 and SMIFH2 compared with in astrocytes treated with CK-548 alone (Fig. 3F). The myosin II inhibitor blebbistatin more completely blocked astrocyte expansion (Fig. 3D,F). This result led us to test upstream regulators of myosin II in the presence of CK-548. We used Y-27632 to inhibit the Rho-dependent kinase (ROCK), which activates myosin II (Vicente-Manzanares et al., 2009). The efficiency of Y-27632 in counteracting the effects of Arp2/3 inhibition on astrocyte morphology is similar to that of blebbistatin (Fig. 3D–F).

Bottom Line: This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices.Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes.Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.

ABSTRACT
Astrocytes exhibit a complex, branched morphology, allowing them to functionally interact with numerous blood vessels, neighboring glial processes and neuronal elements, including synapses. They also respond to central nervous system (CNS) injury by a process known as astrogliosis, which involves morphological changes, including cell body hypertrophy and thickening of major processes. Following severe injury, astrocytes exhibit drastically reduced morphological complexity and collectively form a glial scar. The mechanistic details behind these morphological changes are unknown. Here, we investigate the regulation of the actin-nucleating Arp2/3 complex in controlling dynamic changes in astrocyte morphology. In contrast to other cell types, Arp2/3 inhibition drives the rapid expansion of astrocyte cell bodies and major processes. This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices. We show that this expansion requires functional myosin II downstream of ROCK and RhoA. Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes. By contrast, knockdown of the Arp2/3 inhibitor PICK1 increases astrocyte branching complexity. Furthermore, astrocyte expansion induced by ischemic conditions is delayed by PICK1 knockdown or N-WASP overexpression. Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes. The Arp2/3 regulators PICK1, and N-WASP and WAVE2 function antagonistically to control the complexity of astrocyte branched morphology, and this mechanism underlies the morphological changes seen in astrocytes during their response to pathological insult.

Show MeSH
Related in: MedlinePlus