Journal: Cerebral Cortex (New York, NY)
Article Title: Cytoskeletal Associated Filamin A and RhoA Affect Neural Progenitor Specification During Mitosis
Figure Lengend Snippet: FlnA physically interacts with RhoA and regulates RhoA activation to direct neural progenitor proliferation and differentiation. ( A ) Rho GTPase pull-down assays shows that only active (ACT) or WT-RhoA but not Cdc42 or Rac1 pulls down FlnA by Western blot (left panel) in Neuro2A cells. Conversely, RhoA is co-immunoprecipitated by FlnA C-terminal whereas WT or active Cdc42 and Rac1 did not show any significant binding interaction on Western blot (middle panel). Lastly, full-length FLNA can be co-immunoprecipitated by Flag-RhoA, whereas Flag-Rac1 and Cdc42 cannot (right panel). SUP = supernatant, LAD = molecular weight ladder. Control reflects empty FLAG vector lacking the Rho GTPase constructs followed by pull-down with anti-FLAG antibody and probing for FlnA. ( B ) Fluorescent photomicrographs of cerebral cortex demonstrate FlnA overlapping expression with RhoA in E14 brain cortex, especially, along the apical lining by co-immunostaining and at focal adhesion sites (arrows) of neuroblastoma cells. ( C ) FlnA loss impairs RhoA activation. At baseline, activated RhoA levels are diminished in null FlnA mice. Following laminin activation, RhoA GTPase dramatically increases in the FlnA y/− neural progenitors by 30 min but also declines more precipitously than WT progenitors at 60 min. WT and FlnA y/− neural progenitors are plated on uncoated (uncoated con) or laminin-coated dishes and RhoA activation is assessed at varied time points (30 and 60 min post plating). Upper band corresponds to activated RhoA, and lower band reflects total RhoA (T-RhoA) for loading control. The graph at the right represents mean ± SD values from 5 independent Western blot assays. ( D ) Fluorescent photomicrographs of E14 cerebral cortices 24 h after in utero electroporation of control GFP or dominant negative RhoA (DN-RhoA). Fewer neural cells expressing the DN-RhoA construct reach the cortical plate from the ventricular zone within this time frame, suggesting either impaired migration or diminished neurogenesis. The electroporations specifically target mitotic, proliferating progenitors at the ventricular lining. Progenitors which are therefore GFP + but Ki67- have exited the cell cycle. Higher magnification images and statistical analyses are seen to the right. Co-immunostaining was performed with anti-Ki-67 (marker of cells that remain in the cell cycle) and GFP (marker that initially captures proliferating progenitors) antibodies. The number of GFP + cells that are situated above the ventricular zone is increased in the electroporated GFP control mice (brackets in higher magnification), consistent with increased differentiation. ( E ) The GFP + ,Ki-67 + cells and total GFP + cells from same position-matched area of cortices were quantified. The statistical analysis ( n > 4) show that the ratio of the GFP + , Ki-67 + cells in total GFP + cells is increased approximately 25% in the DN-RhoA electroporation construct versus control. These observations mirror the FlnA loss of function cortical phenotypes and reinforce the likely functional interaction between FlnA and RhoA. Scale bar = 50 μm. ( F ) Fluorescent photomicrographs of GFP (fluorescein) expressing neural cells following electroporation of control GFP or DN-RhoA into lateral ventricles of E13.5 cerebral cortex and staining for early neuronal markers (Tuj1). Acute RhoA inhibition leads to reduction of early born neurons within 72 h, similar to that seen with loss of FlnA. ( G ) Quantification of neurogenic fates is graphically summarized. * = P
Article Snippet: The following antibodies with corresponding dilutions were used for the studies: mouse anti-BrdU (1:100, Calbiochem) and rat anti-BrdU (1:150, Serotec, cat. MCA2060), rabbit anti-Ki-67 monoclonal antibody (1:200, Epitomics, cat. 4203-1), rabbit anti-PH3 polyclonal antibody (1:250, Millipore cat: 06–570), rabbit anti-Sox2 (1:250, Epitomics, cat. 2683-1), rabbit anti-Tbr-1 and anti-Tbr-2 (1:150, Abcam, cat. ab31940, ab23345), mouse anti-E-cadherin and anti-N-cadherin (1:100, BD Biosciences, cat. 610181 and 610920), α-tubulin (Santa Cruz, cat. 32293), rat anti-β1 integrin (1:100, Millipore, cat: MAB 1997), FLNA (1:200, Epitomics cat. 2242-1), mouse ant-RhoA(1:50, Santa Cruz, clone:26C4, cat: sc-418) and rat anti-RhoA (clone: lulu51) was gifted from Dr Shigenobu Yonemura at RIKEN Center for Developmental Biology, mouse anti-Tuj1 (1:200, Covance cat: MMS-435P), mouse anti-aurora b (AIM-1, 1:100, BD Bioscience, cat. 611082), cdk1(1:1000, Calbiochem, pc25), Alexa fluor 488 or rhodamine-labeled phalloidin (1:50, Invitrogen cat. A12379 and R415).
Techniques: Activation Assay, Western Blot, Immunoprecipitation, Binding Assay, Molecular Weight, Plasmid Preparation, Construct, Expressing, Immunostaining, Mouse Assay, In Utero, Electroporation, Dominant Negative Mutation, Migration, Marker, Functional Assay, Staining, Inhibition