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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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Inactivation of the Arp2/3 complex in astrocytes results in an expanded destellated morphology. (A) Phase-contrast live-cell imaging of serum-starved cultured astrocytes in the presence of forskolin with or without the Arp2/3 inhibitor CK-548. Scale bars: 20 µm. (B) Western blot analysis of astrocytes transfected either with a control (siControl) or Arp3 (siArp3)-specific siRNA. Arp3 expression was determined using an Arp3-specific antibody, and GAPDH immunoreactivity was used as loading control. (C) Confocal images of astrocytes transfected with Arp3 siRNA or control siRNA followed by serum starvation and forskolin treatment. Arp3 expression was visualized by immunostaining for Arp3 (red) and F-actin by phalloidin staining (green). Arp3-depleted cells (arrowheads) do not have a stellated morphology, compared with Arp3-positive cells (arrows), which do. Scale bars: 10 µm. (D) Schematic example to illustrate representative differences in cell outlines and cell areas of polygonal (left) and stellate (right) astrocytes. (E) Frequency analysis of astrocyte complexity in Arp3-knockdown and control cells after forskolin treatment. Cells were analyzed regarding the ratio of cell outline and cell area. Cells with a cell outline to cell area ≤0.2 are defined as polygonal. High values for the cell-outline:cell-area ratios correspond to high levels of astrocyte complexity (n = 300 cells per condition from three independent experiments). (F) Quantification of the proportion of polygonal cells as shown in C and E. ***P<0.0005 (unpaired Student's t-test). (G) Phase contrast live-cell imaging of serum-starved astrocytes previously treated with forskolin and kept in serum-free medium in the absence or presence of CK-548. Scale bars: 10 µm. (H) Frequency analysis of astrocyte complexity of CK-548- and DMSO-treated cells (300 cells per condition from three independent experiments). (I) Quantification of the proportion of polygonal cells shown in G and I. **P<0.005 (Student's unpaired t-test). (J) Confocal images of stellated astrocytes before (upper panels), after 5 min of CK-548 incubation. Arp2/3 localization and actin filaments were visualized by immunostaining for Arp3 (red) and phalloidin staining for F-actin (green). Note that Arp3 is enriched along processes and the plasma membrane of stellated astrocytes (arrows). Scale bars: 10 µm.
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f01: Inactivation of the Arp2/3 complex in astrocytes results in an expanded destellated morphology. (A) Phase-contrast live-cell imaging of serum-starved cultured astrocytes in the presence of forskolin with or without the Arp2/3 inhibitor CK-548. Scale bars: 20 µm. (B) Western blot analysis of astrocytes transfected either with a control (siControl) or Arp3 (siArp3)-specific siRNA. Arp3 expression was determined using an Arp3-specific antibody, and GAPDH immunoreactivity was used as loading control. (C) Confocal images of astrocytes transfected with Arp3 siRNA or control siRNA followed by serum starvation and forskolin treatment. Arp3 expression was visualized by immunostaining for Arp3 (red) and F-actin by phalloidin staining (green). Arp3-depleted cells (arrowheads) do not have a stellated morphology, compared with Arp3-positive cells (arrows), which do. Scale bars: 10 µm. (D) Schematic example to illustrate representative differences in cell outlines and cell areas of polygonal (left) and stellate (right) astrocytes. (E) Frequency analysis of astrocyte complexity in Arp3-knockdown and control cells after forskolin treatment. Cells were analyzed regarding the ratio of cell outline and cell area. Cells with a cell outline to cell area ≤0.2 are defined as polygonal. High values for the cell-outline:cell-area ratios correspond to high levels of astrocyte complexity (n = 300 cells per condition from three independent experiments). (F) Quantification of the proportion of polygonal cells as shown in C and E. ***P<0.0005 (unpaired Student's t-test). (G) Phase contrast live-cell imaging of serum-starved astrocytes previously treated with forskolin and kept in serum-free medium in the absence or presence of CK-548. Scale bars: 10 µm. (H) Frequency analysis of astrocyte complexity of CK-548- and DMSO-treated cells (300 cells per condition from three independent experiments). (I) Quantification of the proportion of polygonal cells shown in G and I. **P<0.005 (Student's unpaired t-test). (J) Confocal images of stellated astrocytes before (upper panels), after 5 min of CK-548 incubation. Arp2/3 localization and actin filaments were visualized by immunostaining for Arp3 (red) and phalloidin staining for F-actin (green). Note that Arp3 is enriched along processes and the plasma membrane of stellated astrocytes (arrows). Scale bars: 10 µm.

Mentions: Rat astrocytes grow as polygonal cells in serum-rich medium, so we used a previously established protocol to drive astrocytes into a stellate morphology analogous to astrocytes in native tissue (Ramakers and Moolenaar, 1998). Forskolin stimulates actin bundle disassembly, cytoplasmic shrinkage and process outgrowth (Fig. 1A; supplementary material Movie 1). These morphological changes are completely blocked by CK-548 application (Fig. 1A; supplementary material Movie 2).


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)

Inactivation of the Arp2/3 complex in astrocytes results in an expanded destellated morphology. (A) Phase-contrast live-cell imaging of serum-starved cultured astrocytes in the presence of forskolin with or without the Arp2/3 inhibitor CK-548. Scale bars: 20 µm. (B) Western blot analysis of astrocytes transfected either with a control (siControl) or Arp3 (siArp3)-specific siRNA. Arp3 expression was determined using an Arp3-specific antibody, and GAPDH immunoreactivity was used as loading control. (C) Confocal images of astrocytes transfected with Arp3 siRNA or control siRNA followed by serum starvation and forskolin treatment. Arp3 expression was visualized by immunostaining for Arp3 (red) and F-actin by phalloidin staining (green). Arp3-depleted cells (arrowheads) do not have a stellated morphology, compared with Arp3-positive cells (arrows), which do. Scale bars: 10 µm. (D) Schematic example to illustrate representative differences in cell outlines and cell areas of polygonal (left) and stellate (right) astrocytes. (E) Frequency analysis of astrocyte complexity in Arp3-knockdown and control cells after forskolin treatment. Cells were analyzed regarding the ratio of cell outline and cell area. Cells with a cell outline to cell area ≤0.2 are defined as polygonal. High values for the cell-outline:cell-area ratios correspond to high levels of astrocyte complexity (n = 300 cells per condition from three independent experiments). (F) Quantification of the proportion of polygonal cells as shown in C and E. ***P<0.0005 (unpaired Student's t-test). (G) Phase contrast live-cell imaging of serum-starved astrocytes previously treated with forskolin and kept in serum-free medium in the absence or presence of CK-548. Scale bars: 10 µm. (H) Frequency analysis of astrocyte complexity of CK-548- and DMSO-treated cells (300 cells per condition from three independent experiments). (I) Quantification of the proportion of polygonal cells shown in G and I. **P<0.005 (Student's unpaired t-test). (J) Confocal images of stellated astrocytes before (upper panels), after 5 min of CK-548 incubation. Arp2/3 localization and actin filaments were visualized by immunostaining for Arp3 (red) and phalloidin staining for F-actin (green). Note that Arp3 is enriched along processes and the plasma membrane of stellated astrocytes (arrows). Scale bars: 10 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f01: Inactivation of the Arp2/3 complex in astrocytes results in an expanded destellated morphology. (A) Phase-contrast live-cell imaging of serum-starved cultured astrocytes in the presence of forskolin with or without the Arp2/3 inhibitor CK-548. Scale bars: 20 µm. (B) Western blot analysis of astrocytes transfected either with a control (siControl) or Arp3 (siArp3)-specific siRNA. Arp3 expression was determined using an Arp3-specific antibody, and GAPDH immunoreactivity was used as loading control. (C) Confocal images of astrocytes transfected with Arp3 siRNA or control siRNA followed by serum starvation and forskolin treatment. Arp3 expression was visualized by immunostaining for Arp3 (red) and F-actin by phalloidin staining (green). Arp3-depleted cells (arrowheads) do not have a stellated morphology, compared with Arp3-positive cells (arrows), which do. Scale bars: 10 µm. (D) Schematic example to illustrate representative differences in cell outlines and cell areas of polygonal (left) and stellate (right) astrocytes. (E) Frequency analysis of astrocyte complexity in Arp3-knockdown and control cells after forskolin treatment. Cells were analyzed regarding the ratio of cell outline and cell area. Cells with a cell outline to cell area ≤0.2 are defined as polygonal. High values for the cell-outline:cell-area ratios correspond to high levels of astrocyte complexity (n = 300 cells per condition from three independent experiments). (F) Quantification of the proportion of polygonal cells as shown in C and E. ***P<0.0005 (unpaired Student's t-test). (G) Phase contrast live-cell imaging of serum-starved astrocytes previously treated with forskolin and kept in serum-free medium in the absence or presence of CK-548. Scale bars: 10 µm. (H) Frequency analysis of astrocyte complexity of CK-548- and DMSO-treated cells (300 cells per condition from three independent experiments). (I) Quantification of the proportion of polygonal cells shown in G and I. **P<0.005 (Student's unpaired t-test). (J) Confocal images of stellated astrocytes before (upper panels), after 5 min of CK-548 incubation. Arp2/3 localization and actin filaments were visualized by immunostaining for Arp3 (red) and phalloidin staining for F-actin (green). Note that Arp3 is enriched along processes and the plasma membrane of stellated astrocytes (arrows). Scale bars: 10 µm.
Mentions: Rat astrocytes grow as polygonal cells in serum-rich medium, so we used a previously established protocol to drive astrocytes into a stellate morphology analogous to astrocytes in native tissue (Ramakers and Moolenaar, 1998). Forskolin stimulates actin bundle disassembly, cytoplasmic shrinkage and process outgrowth (Fig. 1A; supplementary material Movie 1). These morphological changes are completely blocked by CK-548 application (Fig. 1A; supplementary material Movie 2).

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