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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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Arp2/3 stimulation by N-WASP overexpression inhibits OGD-dependent changes in astrocyte morphology. Astrocytes were transfected with GFP, GFP–N-WASP WT or constitutively active GFP–N-WASP-Δ227–267. (A) Confocal images of transfected, serum-starved and forskolin-treated astrocytes before OGD, stained with phalloidin–Alexa-546. Scale bars: 10 µm. (B) Confocal images of transfected astrocytes after 20 min OGD and stained for F-actin. Scale bars: 10 µm. (C) Frequency analysis of complexity of astrocytes transfected with GFP, N-WASP WT and N-WASP-Δ227–267 after OGD. For this analysis only cells with exogenous N-WASP in the cytosol and nuclei were taken into account (300 cells per condition from three independent experiments were used in each frequency analysis). (D) Quantification of the proportion of polygonal astrocytes, as shown in C. *P<0.05 (unpaired Student's t-test).
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f06: Arp2/3 stimulation by N-WASP overexpression inhibits OGD-dependent changes in astrocyte morphology. Astrocytes were transfected with GFP, GFP–N-WASP WT or constitutively active GFP–N-WASP-Δ227–267. (A) Confocal images of transfected, serum-starved and forskolin-treated astrocytes before OGD, stained with phalloidin–Alexa-546. Scale bars: 10 µm. (B) Confocal images of transfected astrocytes after 20 min OGD and stained for F-actin. Scale bars: 10 µm. (C) Frequency analysis of complexity of astrocytes transfected with GFP, N-WASP WT and N-WASP-Δ227–267 after OGD. For this analysis only cells with exogenous N-WASP in the cytosol and nuclei were taken into account (300 cells per condition from three independent experiments were used in each frequency analysis). (D) Quantification of the proportion of polygonal astrocytes, as shown in C. *P<0.05 (unpaired Student's t-test).

Mentions: To further explore the mechanism behind OGD-induced astrocyte expansion, we overexpressed GFP-tagged N-WASP variants. Compared with control cells transfected with GFP, N-WASP overexpressing astrocytes exhibit fewer stress fibers (supplementary material Fig. S3B). Moreover, a constitutively active mutant of N-WASP, Δ226-267 (Stamm et al., 2005), radically alters F-actin organization in polygonal astrocytes (supplementary material Fig. S3B). We assume that the differences in actin organization between astrocytes expressing N-WASP WT and N-WASP Δ226–267 are based on the fact that the activity of overexpressed N-WASP WT relies on upstream signaling pathways present in the transfected astrocytes. All transfected cells expressing either GFP or N-WASP variants acquire a stellate morphology by forskolin treatment (Fig. 6A). During OGD, 72.3%±6.1 of GFP-expressing control cells expand to a polygonal morphology (Fig. 6B–D). In astrocytes overexpressing GFP-tagged wild-type (WT) N-WASP, destellation is markedly inhibited, and only 43.3%±9 of cells exhibit a polygonal morphology after OGD (Fig. 6B–D). Before OGD, all GFP–N-WASP-Δ226–267-expressing astrocytes exhibit a stellate morphology and exhibit a frequency of polygonal cells, analogous to astrocytes overexpressing N-WASP-WT (Fig. 6D). However, stellate N-WASP-Δ226–267-expressing cells tend to be a higher cell-outline:cell-area ratio than astrocytes transfected with N-WASP WT (Fig. 6C).


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)

Arp2/3 stimulation by N-WASP overexpression inhibits OGD-dependent changes in astrocyte morphology. Astrocytes were transfected with GFP, GFP–N-WASP WT or constitutively active GFP–N-WASP-Δ227–267. (A) Confocal images of transfected, serum-starved and forskolin-treated astrocytes before OGD, stained with phalloidin–Alexa-546. Scale bars: 10 µm. (B) Confocal images of transfected astrocytes after 20 min OGD and stained for F-actin. Scale bars: 10 µm. (C) Frequency analysis of complexity of astrocytes transfected with GFP, N-WASP WT and N-WASP-Δ227–267 after OGD. For this analysis only cells with exogenous N-WASP in the cytosol and nuclei were taken into account (300 cells per condition from three independent experiments were used in each frequency analysis). (D) Quantification of the proportion of polygonal astrocytes, as shown in C. *P<0.05 (unpaired Student's t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757329&req=5

f06: Arp2/3 stimulation by N-WASP overexpression inhibits OGD-dependent changes in astrocyte morphology. Astrocytes were transfected with GFP, GFP–N-WASP WT or constitutively active GFP–N-WASP-Δ227–267. (A) Confocal images of transfected, serum-starved and forskolin-treated astrocytes before OGD, stained with phalloidin–Alexa-546. Scale bars: 10 µm. (B) Confocal images of transfected astrocytes after 20 min OGD and stained for F-actin. Scale bars: 10 µm. (C) Frequency analysis of complexity of astrocytes transfected with GFP, N-WASP WT and N-WASP-Δ227–267 after OGD. For this analysis only cells with exogenous N-WASP in the cytosol and nuclei were taken into account (300 cells per condition from three independent experiments were used in each frequency analysis). (D) Quantification of the proportion of polygonal astrocytes, as shown in C. *P<0.05 (unpaired Student's t-test).
Mentions: To further explore the mechanism behind OGD-induced astrocyte expansion, we overexpressed GFP-tagged N-WASP variants. Compared with control cells transfected with GFP, N-WASP overexpressing astrocytes exhibit fewer stress fibers (supplementary material Fig. S3B). Moreover, a constitutively active mutant of N-WASP, Δ226-267 (Stamm et al., 2005), radically alters F-actin organization in polygonal astrocytes (supplementary material Fig. S3B). We assume that the differences in actin organization between astrocytes expressing N-WASP WT and N-WASP Δ226–267 are based on the fact that the activity of overexpressed N-WASP WT relies on upstream signaling pathways present in the transfected astrocytes. All transfected cells expressing either GFP or N-WASP variants acquire a stellate morphology by forskolin treatment (Fig. 6A). During OGD, 72.3%±6.1 of GFP-expressing control cells expand to a polygonal morphology (Fig. 6B–D). In astrocytes overexpressing GFP-tagged wild-type (WT) N-WASP, destellation is markedly inhibited, and only 43.3%±9 of cells exhibit a polygonal morphology after OGD (Fig. 6B–D). Before OGD, all GFP–N-WASP-Δ226–267-expressing astrocytes exhibit a stellate morphology and exhibit a frequency of polygonal cells, analogous to astrocytes overexpressing N-WASP-WT (Fig. 6D). However, stellate N-WASP-Δ226–267-expressing cells tend to be a higher cell-outline:cell-area ratio than astrocytes transfected with N-WASP WT (Fig. 6C).

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