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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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Acute inhibition of the Arp2/3 complex in astrocytes in brain slices. (A) Demonstration of a modified tissue clearance procedure, allowing deep-tissue antibody staining and imaging by confocal microscopy. Untreated cortical slice (top) in comparison to cleared tissue (bottom). (B) Z-projection of a control astrocyte 40 µm within the cortical slice, previously stained for DNA (Hoechst 33258), GFAP and S100β, as acquired by confocal microscopy after tissue clearance. Scale bars: 10 µm. (C) Filament tracing of GFAP- and S100β-positive processes in an individual astrocyte. Main processes were defined by GFAP immunoreactivity (top). Fine structures were determined by S100β immunoreactivity (center). Overlay of 3D models and confocal z-projections (bottom). Left panels: control astrocyte. Right panels: astrocyte from a slice treated with CK-548. Scale bars: 10 µm. (D) Quantification of longest processes in control and CK-548-treated astrocytes, based on GFAP immunoreactivity. n = 20 per condition, P>0.05 (unpaired Student's t-test). (E) Sholl analyzes on combined GFAP- and S100β-positive processes in control (blue) and CK-548-treated (red) astrocytes. n = 20 per condition, **P<0.005, ***P<0.0005 (unpaired Student's t-test and Sidak–Bonferroni method). (F) Quantification of soma volumes from control and CK-548-treated astrocytes. n = 20 per condition, ***P<0.0005 (unpaired Student's t-test). (G) Frequency of individual S100β-positive process volumes from control and CK-548-treated astrocytes. (90,000 S100β-positive processes from 20 cells per condition). Left graph: small processes up to 1.25 µm3. Right graph: larger processes greater than 1.5 µm3.
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f02: Acute inhibition of the Arp2/3 complex in astrocytes in brain slices. (A) Demonstration of a modified tissue clearance procedure, allowing deep-tissue antibody staining and imaging by confocal microscopy. Untreated cortical slice (top) in comparison to cleared tissue (bottom). (B) Z-projection of a control astrocyte 40 µm within the cortical slice, previously stained for DNA (Hoechst 33258), GFAP and S100β, as acquired by confocal microscopy after tissue clearance. Scale bars: 10 µm. (C) Filament tracing of GFAP- and S100β-positive processes in an individual astrocyte. Main processes were defined by GFAP immunoreactivity (top). Fine structures were determined by S100β immunoreactivity (center). Overlay of 3D models and confocal z-projections (bottom). Left panels: control astrocyte. Right panels: astrocyte from a slice treated with CK-548. Scale bars: 10 µm. (D) Quantification of longest processes in control and CK-548-treated astrocytes, based on GFAP immunoreactivity. n = 20 per condition, P>0.05 (unpaired Student's t-test). (E) Sholl analyzes on combined GFAP- and S100β-positive processes in control (blue) and CK-548-treated (red) astrocytes. n = 20 per condition, **P<0.005, ***P<0.0005 (unpaired Student's t-test and Sidak–Bonferroni method). (F) Quantification of soma volumes from control and CK-548-treated astrocytes. n = 20 per condition, ***P<0.0005 (unpaired Student's t-test). (G) Frequency of individual S100β-positive process volumes from control and CK-548-treated astrocytes. (90,000 S100β-positive processes from 20 cells per condition). Left graph: small processes up to 1.25 µm3. Right graph: larger processes greater than 1.5 µm3.

Mentions: To study whether Arp2/3 inhibition would evoke analogous changes in astrocytes in intact tissue, we exposed acute cortical slices from P14 rats to CK-548 (Fig. 2). To detect changes in astrocyte morphology within tissue, we established a new technique to combine whole-mount immunohistochemistry with a recently published method for tissue clearance (see Materials and Methods) (Fig. 2A) (Hama et al., 2011), allowing antibody labeling for astrocytic markers observable deep within the tissue by confocal microscopy (supplementary material Movie 5). We used antibodies specific for glial fibrillary acidic protein (GFAP), which labels the main astrocytic processes, and S100β, which localizes to the cytoplasm and, to a minor extent, to membranes (Sen and Belli, 2007) (Fig. 2B). S100β staining therefore acts as a useful marker to define the fine details of complex astrocyte morphology (Fig. 2B, supplementary material Movie 6). We used a filament-tracing algorithm in Imaris software, originally designed for quantifying dendritic spines on neurons. We defined GFAP-positive processes as ‘dendrites’ from where smaller processes (‘spines’) originate and branch (Fig. 2C). To study the effect of Arp2/3 inhibition on astrocytes, we incubated cortical slices with either CK-548 or DMSO in oxygenated artificial cerebrospinal fluid (aCSF) and performed quantitative analyzes of astrocyte morphology. Fig. 2D shows that CK-548 has no effect on the length of the longest GFAP-positive process. Astrocytes from control slices exhibit a highly complex arborization of fine, branched S100β-positive processes emerging from the main shafts. In contrast, astrocytes in CK-548-treated slices show fewer, thicker S100β-containing protrusions compared with the number in control cells (Fig. 2C). To quantify these changes in astrocyte complexity, we performed Sholl analyzes taking GFAP- as well as S100β-positive processes into account. Pharmacological inhibition of the Arp2/3 complex leads to a dramatic reduction in astrocyte complexity compared with that in control cells (Fig. 2E). In addition, we studied whether the volume of individual S100β-containing protrusions changes in response to Arp2/3 inhibition. We observed a decrease in the number of small protrusions, whereas bigger processes become more frequent (Fig. 2G). Furthermore, inhibition of Arp2/3 activity causes a marked increase in cell body volume (Fig. 2F). These experiments demonstrate that acute inhibition of the Arp2/3 complex also evokes the expansion of astrocytes within intact tissue.


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)

Acute inhibition of the Arp2/3 complex in astrocytes in brain slices. (A) Demonstration of a modified tissue clearance procedure, allowing deep-tissue antibody staining and imaging by confocal microscopy. Untreated cortical slice (top) in comparison to cleared tissue (bottom). (B) Z-projection of a control astrocyte 40 µm within the cortical slice, previously stained for DNA (Hoechst 33258), GFAP and S100β, as acquired by confocal microscopy after tissue clearance. Scale bars: 10 µm. (C) Filament tracing of GFAP- and S100β-positive processes in an individual astrocyte. Main processes were defined by GFAP immunoreactivity (top). Fine structures were determined by S100β immunoreactivity (center). Overlay of 3D models and confocal z-projections (bottom). Left panels: control astrocyte. Right panels: astrocyte from a slice treated with CK-548. Scale bars: 10 µm. (D) Quantification of longest processes in control and CK-548-treated astrocytes, based on GFAP immunoreactivity. n = 20 per condition, P>0.05 (unpaired Student's t-test). (E) Sholl analyzes on combined GFAP- and S100β-positive processes in control (blue) and CK-548-treated (red) astrocytes. n = 20 per condition, **P<0.005, ***P<0.0005 (unpaired Student's t-test and Sidak–Bonferroni method). (F) Quantification of soma volumes from control and CK-548-treated astrocytes. n = 20 per condition, ***P<0.0005 (unpaired Student's t-test). (G) Frequency of individual S100β-positive process volumes from control and CK-548-treated astrocytes. (90,000 S100β-positive processes from 20 cells per condition). Left graph: small processes up to 1.25 µm3. Right graph: larger processes greater than 1.5 µm3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f02: Acute inhibition of the Arp2/3 complex in astrocytes in brain slices. (A) Demonstration of a modified tissue clearance procedure, allowing deep-tissue antibody staining and imaging by confocal microscopy. Untreated cortical slice (top) in comparison to cleared tissue (bottom). (B) Z-projection of a control astrocyte 40 µm within the cortical slice, previously stained for DNA (Hoechst 33258), GFAP and S100β, as acquired by confocal microscopy after tissue clearance. Scale bars: 10 µm. (C) Filament tracing of GFAP- and S100β-positive processes in an individual astrocyte. Main processes were defined by GFAP immunoreactivity (top). Fine structures were determined by S100β immunoreactivity (center). Overlay of 3D models and confocal z-projections (bottom). Left panels: control astrocyte. Right panels: astrocyte from a slice treated with CK-548. Scale bars: 10 µm. (D) Quantification of longest processes in control and CK-548-treated astrocytes, based on GFAP immunoreactivity. n = 20 per condition, P>0.05 (unpaired Student's t-test). (E) Sholl analyzes on combined GFAP- and S100β-positive processes in control (blue) and CK-548-treated (red) astrocytes. n = 20 per condition, **P<0.005, ***P<0.0005 (unpaired Student's t-test and Sidak–Bonferroni method). (F) Quantification of soma volumes from control and CK-548-treated astrocytes. n = 20 per condition, ***P<0.0005 (unpaired Student's t-test). (G) Frequency of individual S100β-positive process volumes from control and CK-548-treated astrocytes. (90,000 S100β-positive processes from 20 cells per condition). Left graph: small processes up to 1.25 µm3. Right graph: larger processes greater than 1.5 µm3.
Mentions: To study whether Arp2/3 inhibition would evoke analogous changes in astrocytes in intact tissue, we exposed acute cortical slices from P14 rats to CK-548 (Fig. 2). To detect changes in astrocyte morphology within tissue, we established a new technique to combine whole-mount immunohistochemistry with a recently published method for tissue clearance (see Materials and Methods) (Fig. 2A) (Hama et al., 2011), allowing antibody labeling for astrocytic markers observable deep within the tissue by confocal microscopy (supplementary material Movie 5). We used antibodies specific for glial fibrillary acidic protein (GFAP), which labels the main astrocytic processes, and S100β, which localizes to the cytoplasm and, to a minor extent, to membranes (Sen and Belli, 2007) (Fig. 2B). S100β staining therefore acts as a useful marker to define the fine details of complex astrocyte morphology (Fig. 2B, supplementary material Movie 6). We used a filament-tracing algorithm in Imaris software, originally designed for quantifying dendritic spines on neurons. We defined GFAP-positive processes as ‘dendrites’ from where smaller processes (‘spines’) originate and branch (Fig. 2C). To study the effect of Arp2/3 inhibition on astrocytes, we incubated cortical slices with either CK-548 or DMSO in oxygenated artificial cerebrospinal fluid (aCSF) and performed quantitative analyzes of astrocyte morphology. Fig. 2D shows that CK-548 has no effect on the length of the longest GFAP-positive process. Astrocytes from control slices exhibit a highly complex arborization of fine, branched S100β-positive processes emerging from the main shafts. In contrast, astrocytes in CK-548-treated slices show fewer, thicker S100β-containing protrusions compared with the number in control cells (Fig. 2C). To quantify these changes in astrocyte complexity, we performed Sholl analyzes taking GFAP- as well as S100β-positive processes into account. Pharmacological inhibition of the Arp2/3 complex leads to a dramatic reduction in astrocyte complexity compared with that in control cells (Fig. 2E). In addition, we studied whether the volume of individual S100β-containing protrusions changes in response to Arp2/3 inhibition. We observed a decrease in the number of small protrusions, whereas bigger processes become more frequent (Fig. 2G). Furthermore, inhibition of Arp2/3 activity causes a marked increase in cell body volume (Fig. 2F). These experiments demonstrate that acute inhibition of the Arp2/3 complex also evokes the expansion of astrocytes within intact tissue.

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