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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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Identification of Arp2/3 regulators in astrocytes. (A) Astrocytes were transfected with control siRNA (siControl) or WAVE2-specific siRNA (siWAVE2). WAVE2 and GAPDH expression were analyzed by western blotting. (B) Confocal images of siControl- and siWAVE2-transfected astrocytes, subjected to by serum starvation, forskolin treatment and phalloidin staining. Scale bars: 10 µm. (C) Frequency analysis of the astrocyte complexity in WAVE2-knockdown and control cells after forskolin treatment. For frequency analysis, n = 300 cells per condition from three independent experiments. (D) Quantification of the proportion of polygonal astrocytes of siControl- and siWAVE2-transfected cells, *P<0.05 (unpaired t-test). (E) Sholl analysis on processes of astrocytes transfected with either control siRNA (siControl, blue) or siWAVE2-specific siRNA (siWAVE2, red). n = 108 (siControl), n = 140 (siWAVE2), *P<0.05 (unpaired t-test and Sidak-Bonferroni method). (F) Astrocytes were transfected with control siRNA (siControl) or N-WASP-specific siRNA (siN-WASP). N-WASP and GAPDH expression were analyzed by western blotting. (G) Confocal images of astrocytes after transfection with control siRNA (siControl) or N-WASP-specific siRNA (siN-WASP), followed by serum starvation, forskolin treatment and phalloidin staining. Scale bars: 10 µm. (H) Frequency analysis of astrocyte complexity of N-WASP-knockdown and control cells after forskolin treatment. For frequency analysis, n = 300 cells per condition from three independent experiments. (I) Quantification of the proportion of polygonal astrocytes from control (siControl) and N-WASP-depleted cells (siN-WASP), as shown in H. **P<0.005 (unpaired Student's t-test). (J) Astrocytes were transfected with control siRNA (siControl) or PICK1-specific siRNAs (siPICK1). PICK1 and GAPDH expression were analyzed by western blotting. (K) Confocal images of astrocytes after transfection with control siRNA (left) and PICK1-specific siRNA (right), stained with actin and PICK1-specific antibodies. Before fixation and immunocytochemistry, cells were serum-starved and treated with forskolin. Scale bars: 10 µm. (L) Frequency analysis of astrocyte complexity of PICK1-knockdown and control cells after forskolin treatment (300 cells per condition from three independent experiments in each frequency analysis). (M) Sholl analysis on processes of astrocytes transfected with either control siRNA (siControl, blue) or PICK1-specific siRNA (siPICK1, red). n = 108 (siControl), n = 82 (siPICK1), *P<0.05 (unpaired Student's t-test and Sidak–Bonferroni method).
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f04: Identification of Arp2/3 regulators in astrocytes. (A) Astrocytes were transfected with control siRNA (siControl) or WAVE2-specific siRNA (siWAVE2). WAVE2 and GAPDH expression were analyzed by western blotting. (B) Confocal images of siControl- and siWAVE2-transfected astrocytes, subjected to by serum starvation, forskolin treatment and phalloidin staining. Scale bars: 10 µm. (C) Frequency analysis of the astrocyte complexity in WAVE2-knockdown and control cells after forskolin treatment. For frequency analysis, n = 300 cells per condition from three independent experiments. (D) Quantification of the proportion of polygonal astrocytes of siControl- and siWAVE2-transfected cells, *P<0.05 (unpaired t-test). (E) Sholl analysis on processes of astrocytes transfected with either control siRNA (siControl, blue) or siWAVE2-specific siRNA (siWAVE2, red). n = 108 (siControl), n = 140 (siWAVE2), *P<0.05 (unpaired t-test and Sidak-Bonferroni method). (F) Astrocytes were transfected with control siRNA (siControl) or N-WASP-specific siRNA (siN-WASP). N-WASP and GAPDH expression were analyzed by western blotting. (G) Confocal images of astrocytes after transfection with control siRNA (siControl) or N-WASP-specific siRNA (siN-WASP), followed by serum starvation, forskolin treatment and phalloidin staining. Scale bars: 10 µm. (H) Frequency analysis of astrocyte complexity of N-WASP-knockdown and control cells after forskolin treatment. For frequency analysis, n = 300 cells per condition from three independent experiments. (I) Quantification of the proportion of polygonal astrocytes from control (siControl) and N-WASP-depleted cells (siN-WASP), as shown in H. **P<0.005 (unpaired Student's t-test). (J) Astrocytes were transfected with control siRNA (siControl) or PICK1-specific siRNAs (siPICK1). PICK1 and GAPDH expression were analyzed by western blotting. (K) Confocal images of astrocytes after transfection with control siRNA (left) and PICK1-specific siRNA (right), stained with actin and PICK1-specific antibodies. Before fixation and immunocytochemistry, cells were serum-starved and treated with forskolin. Scale bars: 10 µm. (L) Frequency analysis of astrocyte complexity of PICK1-knockdown and control cells after forskolin treatment (300 cells per condition from three independent experiments in each frequency analysis). (M) Sholl analysis on processes of astrocytes transfected with either control siRNA (siControl, blue) or PICK1-specific siRNA (siPICK1, red). n = 108 (siControl), n = 82 (siPICK1), *P<0.05 (unpaired Student's t-test and Sidak–Bonferroni method).

Mentions: We used a published siRNA sequence (Danson et al., 2007), which reduces endogenous WAVE2 expression to 27.4%±7.2 in cultured astrocytes (Fig. 4A). Most WAVE2-depleted cells acquired a stellate morphology following forskolin treatment, and the proportion of polygonal cells was not significantly changed from controls (Fig. 4B–D). To further investigate whether the depletion of WAVE2 affected astrocyte arborization, we employed Sholl analyzes, and specifically analyzed astrocytic processes of stellate astrocytes (with a cell-outline:cell-area ratio greater than 0.2) not including cell bodies. Despite a high variance, we were able to determine a significantly decreased complexity in WAVE2-depleted astrocytes (Fig. 4E). These findings demonstrate that WAVE2 is involved in organizing astrocytic processes, however knocking down WAVE2 did not completely block changes in astrocyte morphology, as observed upon Arp2/3 inhibition (see Fig. 1).


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)

Identification of Arp2/3 regulators in astrocytes. (A) Astrocytes were transfected with control siRNA (siControl) or WAVE2-specific siRNA (siWAVE2). WAVE2 and GAPDH expression were analyzed by western blotting. (B) Confocal images of siControl- and siWAVE2-transfected astrocytes, subjected to by serum starvation, forskolin treatment and phalloidin staining. Scale bars: 10 µm. (C) Frequency analysis of the astrocyte complexity in WAVE2-knockdown and control cells after forskolin treatment. For frequency analysis, n = 300 cells per condition from three independent experiments. (D) Quantification of the proportion of polygonal astrocytes of siControl- and siWAVE2-transfected cells, *P<0.05 (unpaired t-test). (E) Sholl analysis on processes of astrocytes transfected with either control siRNA (siControl, blue) or siWAVE2-specific siRNA (siWAVE2, red). n = 108 (siControl), n = 140 (siWAVE2), *P<0.05 (unpaired t-test and Sidak-Bonferroni method). (F) Astrocytes were transfected with control siRNA (siControl) or N-WASP-specific siRNA (siN-WASP). N-WASP and GAPDH expression were analyzed by western blotting. (G) Confocal images of astrocytes after transfection with control siRNA (siControl) or N-WASP-specific siRNA (siN-WASP), followed by serum starvation, forskolin treatment and phalloidin staining. Scale bars: 10 µm. (H) Frequency analysis of astrocyte complexity of N-WASP-knockdown and control cells after forskolin treatment. For frequency analysis, n = 300 cells per condition from three independent experiments. (I) Quantification of the proportion of polygonal astrocytes from control (siControl) and N-WASP-depleted cells (siN-WASP), as shown in H. **P<0.005 (unpaired Student's t-test). (J) Astrocytes were transfected with control siRNA (siControl) or PICK1-specific siRNAs (siPICK1). PICK1 and GAPDH expression were analyzed by western blotting. (K) Confocal images of astrocytes after transfection with control siRNA (left) and PICK1-specific siRNA (right), stained with actin and PICK1-specific antibodies. Before fixation and immunocytochemistry, cells were serum-starved and treated with forskolin. Scale bars: 10 µm. (L) Frequency analysis of astrocyte complexity of PICK1-knockdown and control cells after forskolin treatment (300 cells per condition from three independent experiments in each frequency analysis). (M) Sholl analysis on processes of astrocytes transfected with either control siRNA (siControl, blue) or PICK1-specific siRNA (siPICK1, red). n = 108 (siControl), n = 82 (siPICK1), *P<0.05 (unpaired Student's t-test and Sidak–Bonferroni method).
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f04: Identification of Arp2/3 regulators in astrocytes. (A) Astrocytes were transfected with control siRNA (siControl) or WAVE2-specific siRNA (siWAVE2). WAVE2 and GAPDH expression were analyzed by western blotting. (B) Confocal images of siControl- and siWAVE2-transfected astrocytes, subjected to by serum starvation, forskolin treatment and phalloidin staining. Scale bars: 10 µm. (C) Frequency analysis of the astrocyte complexity in WAVE2-knockdown and control cells after forskolin treatment. For frequency analysis, n = 300 cells per condition from three independent experiments. (D) Quantification of the proportion of polygonal astrocytes of siControl- and siWAVE2-transfected cells, *P<0.05 (unpaired t-test). (E) Sholl analysis on processes of astrocytes transfected with either control siRNA (siControl, blue) or siWAVE2-specific siRNA (siWAVE2, red). n = 108 (siControl), n = 140 (siWAVE2), *P<0.05 (unpaired t-test and Sidak-Bonferroni method). (F) Astrocytes were transfected with control siRNA (siControl) or N-WASP-specific siRNA (siN-WASP). N-WASP and GAPDH expression were analyzed by western blotting. (G) Confocal images of astrocytes after transfection with control siRNA (siControl) or N-WASP-specific siRNA (siN-WASP), followed by serum starvation, forskolin treatment and phalloidin staining. Scale bars: 10 µm. (H) Frequency analysis of astrocyte complexity of N-WASP-knockdown and control cells after forskolin treatment. For frequency analysis, n = 300 cells per condition from three independent experiments. (I) Quantification of the proportion of polygonal astrocytes from control (siControl) and N-WASP-depleted cells (siN-WASP), as shown in H. **P<0.005 (unpaired Student's t-test). (J) Astrocytes were transfected with control siRNA (siControl) or PICK1-specific siRNAs (siPICK1). PICK1 and GAPDH expression were analyzed by western blotting. (K) Confocal images of astrocytes after transfection with control siRNA (left) and PICK1-specific siRNA (right), stained with actin and PICK1-specific antibodies. Before fixation and immunocytochemistry, cells were serum-starved and treated with forskolin. Scale bars: 10 µm. (L) Frequency analysis of astrocyte complexity of PICK1-knockdown and control cells after forskolin treatment (300 cells per condition from three independent experiments in each frequency analysis). (M) Sholl analysis on processes of astrocytes transfected with either control siRNA (siControl, blue) or PICK1-specific siRNA (siPICK1, red). n = 108 (siControl), n = 82 (siPICK1), *P<0.05 (unpaired Student's t-test and Sidak–Bonferroni method).
Mentions: We used a published siRNA sequence (Danson et al., 2007), which reduces endogenous WAVE2 expression to 27.4%±7.2 in cultured astrocytes (Fig. 4A). Most WAVE2-depleted cells acquired a stellate morphology following forskolin treatment, and the proportion of polygonal cells was not significantly changed from controls (Fig. 4B–D). To further investigate whether the depletion of WAVE2 affected astrocyte arborization, we employed Sholl analyzes, and specifically analyzed astrocytic processes of stellate astrocytes (with a cell-outline:cell-area ratio greater than 0.2) not including cell bodies. Despite a high variance, we were able to determine a significantly decreased complexity in WAVE2-depleted astrocytes (Fig. 4E). These findings demonstrate that WAVE2 is involved in organizing astrocytic processes, however knocking down WAVE2 did not completely block changes in astrocyte morphology, as observed upon Arp2/3 inhibition (see Fig. 1).

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