Fig 1.
Astrocytes treated with hyaluronidase acquire the stellate-like morphology.
A. Hyaluronan digestion by hyaluronidase was evaluated by staining with hyaluronan binding protein (HABP) (red). Scale: 30 μm. B. Measurement of fluorescence intensity. One way ANOVA test was performed, F(2.57) = 53.169; p<0.001, Dunnett’s C post hoc. C. Representative images of astrocytes transfected with β-actin GFP and either untreated (control) or treated with hyaluronidase or heat inactivated hyaluronidase for 48h. Cell nuclei were visualized with DAPI staining. Scale: 30 μm.D. Morphometric analysis of shape-describing parameters of cells treated as described in C. One way ANOVA test was performed, area: F(2.57) = 2.658; p>0.05, solidity: F(2.57) = 16.814; p<0.001, circularity: F(2.57) = 13.799; p<0.001, branching: F(2.57) = 16.774; p<0,001 Dunnett’s C post hoc test.
Fig 2.
CD44 regulates astrocyte morphology.
A: Validation of CD44shRNA and CD44-GFP constructs. Astrocytes were transfected with pSuper, CD44shRNA or CD44-GFP constructs (together with β-actin-GFP plasmid) and then immunostained with anti-CD44 antibody (red). The level of CD44 expression was evaluated by measuring CD44 immunofluorescence (IF) signal intensity with the use of ImageJ program. One way ANOVA, F(2.71) = 71.187, p<0.001, Dunnett C post hoc tests. Scale: 30 μm. B: Morphological analysis of shape-describing parameters of astrocytes in 2D cultures co-transfected with pSuper, CD44shRNA or CD44shRNA/CD44Rescue constructs together with β-actin-GFP plasmid. One way ANOVA test was performed, area: F(3.150) = 8.169; p<0.001, solidity: F(3.153) = 21.454; p<0.001, circularity: F(3.153) = 18.873; p<0.001, Dunnett’s C post hoc tests, branching: F(3.151) = 33,478; p<0.001, Sidak post hoc test. Scale: 30 μm. C: The morphological analysis of shape-describing parameters of astrocytes in 3D cultures transfected with pSuper or CD44shRNA constructs (together with β-actin-GFP plasmid) or CD44-GFP. One way ANOVA test was performed, area: F(2.92) = 12.311; p<0.001, Sidak post hoc test; solidity: F(2.95) = 42.208; p<0.001, Dunnett’s C post hoc test, circularity: F(2.94) = 20.609; p<0.001, Dunnett’s C post hoc test, branching: F(2.95) = 17.703; p<0.001, Sidak post hoc test. Scale: 30 μm.
Fig 3.
Hyaluronidase treatment and CD44-knockdown leads to enhanced Rac1 activity.
A: Cells were transfected with FRET based biosensor pRaichu-Rac1/1011X and then treated or not with hyaluronidase for 24h. YFP-CFP ratio was calculated as a readout of Rac1 activity. T-Student test, t(589) = 3.212; p<0,001. B: Cells were co-transfected with pRaichu-Rac1/1011X and pSuper/CD44shRNA/CD44-RFP constructs. YFP-CFP ratio was calculated as a readout of Rac1 activity. One way ANOVA, F(2.527) = 39.998; p<0.001, Sidak post hoc test.
Fig 4.
Deactivation of Rac1 activity rescues CD44 knockdown and hyaluronidase-induced morphological changes of astrocytes.
A: Representative images of astrocytes transfected with CD44shRNA/pSuper or co-transfected with pcDNA3-EGFP-Rac1-T17N (Rac1-DN) constructs. The β-actin-RFP construct was used for cell visualization. Scale: 20 μm. B: Morphometric analysis of shape-describing parameters of cells treated as in A. One way ANOVA, area: F(3.112) = 2.456, p>0,05, solidity: F(3.114) = 30.173, p<0.001 Sidak post hoc test, circularity: F(3.114) = 13.834, p<0.001, branching: F(3.114) = 51, 825, p<0,001. Dunnett’s C post hoc test. C: Representative images of astrocytes transfected with pcDNA3-EGFP-Rac1-T17N (Rac1-DN) and β-actin-RFP constructs and treated with hyaluronidase. Scale: 20 μm. D: Morphometric analysis of shape-describing parameters of cells treated as in C. One way ANOVA, area: F(2.147) = 1.520, p>0.05, solidity: F(2.147) = 106.292, p<0.001, circularity: F(2.147) = 96.843, p<0.001, branching: F(2.147) = 135.932; p<0,001, Dunnett’s C post hoc tests.