rabbit anti camsap1  (Bethyl)

Journal: bioRxiv

Article Title: Transient lagging chromosomes cause primary microcephaly

doi: 10.1101/2024.05.02.592199

Figure Lengend Snippet: (A) Immunofluorescence of RPE-1 cells treated with siCTRL or siCAMSAP1 stained for CAMSAP1 and DAPI. (B) Quantification of normalized CAMSAP1 level in RPE-1 cells treated with siCTRL (n=55 cells) and siCAMSAP1 (n=47 cells). N = 3, n = 47-55 cells, **** p < 0.0001, student t- test. (C) Scheme of photoactivation assay for microtubules poleward flux measurement. (D) Quantification of microtubule poleward flux speed in RPE-1 PAGFP-αtubulin cells treated with siCTRL and siCAMSAP1 . N= 3, n = 33-39 cells, **** p < 0.0001, student t-test. (E) Stills of RPE-1 GFP-Centrin1/GFP-CENPA cells in a normal anaphase or an anaphase with a transient lagging kinetochore. (F) Quantification of transient lagging chromosomes in 15 minutes live cell movies of RPE1 GFP-Centrin1/GFP-CENPA cells treated with siCTRL or siCAMSAP1 , n = 66-71 cells, *** p = 0.0009, Fisher’s exact test. (G) Immunofluorescence images of RPE-1 cells treated with siCTRL , siCAMSAP1 , siWDR62 , or siCAMSAP1 + siWDR62, stained for CAMSAP1 (left panels), WDR62 (right panels) and DAPI. (H and I) Quantification of normalized CAMSAP1 (H) and WDR62 (I) level in RPE-1 cells treated with indicated siRNAs. N=3, n = 66-95 cells, **** p < 0.0001; One-way Anova test. (J) Live cell images RPE-1 PAGFP-αtubulin cells treated with siCTRL , siCAMSAP1 , siWDR62, siCAMSAP1+siWDR62 before and after photoactivation (K) Quantification of microtubule poleward flux speeds in RPE-1 PAGFP-αtubulin cells treated with siCTRL , siCAMSAP1, siWDR62, siCAMSAP1+siWDR62. N = 3, n = 28-47 cells, **** p < 0.0001; One-way Anova test. (L) Percentage of RPE-1 GFP-Centrin1/GFP-CENPA cells with transient lagging chromosomes after indicated siRNA treatments. Lagging chromosomes were quantified as shown in (E); n = 29-48 cells; siCTRL vs siCAMSAP1 p = 0.0040, siCTRL vs siWDR62 p = 0.0317, siWDR62 vs siCAMSAP1+siWDR62 p = 0.0098; Fisher’s exact test. All scale bars = 5 µm.

Article Snippet: The following primary antibodies were used: recombinant human anti-α-tubulin (1:500; ( )), rabbit anti-CAMSAP1(1:1500; Novus Biologicals NBP1-26645), rabbit anti-WDR62 (1:1000; Bethyl A301- 560A), rabbit anti-MCAK (1:1000; ( )), rabbit anti-53BP1 (1:1000; Cell Signalling Technology 4937), mouse anti-p21 (1:1000; Cell Signalling Technology 2947), mouse anti-yH2Ax (1:2000; EMD Millipore, 05-636), mouse anti-AuroraB (1:2000; BD Biosciences 611083), rabbit anti-NPL4(1:500; Novus Biologicals NBP1-82166), rabbit anti-UBASH3B (1:500; Proteintech 19563-1-AP).

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Transient lagging chromosomes cause primary microcephaly

doi: 10.1101/2024.05.02.592199

Figure Lengend Snippet: (A) Brains extracted from D. melanogaster larvae of w 1118 , PatroninRNAi , WDR62RNAi , and PatroninRNAi + WDR62RNAi , stained with DAPI. The dotted red circle represents the area of the right lobe from w 1118 brain superposed on PatroninRNAi , WDR62RNAi , PatroninRNAi+ WDR62RNAi brains. (B) Brain lobes area quantification of D. melanogaster larvae brains extracted from w 1118 (n= 73 lobes), PatroninRNAi (n= 56), WDR62RNAi (n= 76), PatroninRNAi+ WDR6RNAi (n= 23) larvae., w 1118 vs WDR62RNAi p < 0.0001, w 1118 vs PatroninRNAi + WDR62RNAi p < 0.0001, WDR62RNAi vs PatroninRNAi + WDR62RNAi p < 0.0001, one-way Anova test. (C) Quantification of neuroblast stained with Miranda antibodies in brains extracted from w 1118 (n= 9 brains), PatroninRNAi (n= 8), WDR62RNAi (n= 15), PatroninRNAi+ WDR6RNAi (n= 10) larvae, w 1118 vs WDR62RNAi p = 0.0041, WDR62RNAi vs PatroninRNAi + WDR62RNAi p = 0.0324, one-way Anova test. (D) Extracted D. melanogaster larvae brains from w 1118 , PatroninRNAi , WDR62RNAi , PatroninRNAi+ WDR6RNAi and stained for Miranda and DAPI. The dotted white line highlights the shape of lobes. (E) Immunofluorescence from a larva brain stained for Miranda and α-tubulin. Dotted lines represent the pole-to-pole axe and the cellular axe perpendicular to the Miranda signal (spindle orientation angle). The magenta line represents the angle between the 2 axes. (F) Quantification of the spindle orientation angles (as determined in ) in neuroblasts from D. melanogaster larvae brain lobes from w 1118 (n= 48 cells), PatroninRNAi (n= 20), WDR62RNAi (n= 44), PatroninRNAi+ WDR6RNAi (n= 35) larvae, all non-significant, Kruskal-Wallis test. (G) Scheme of larvae turning behaviour assay: the number of larva contraction before each change in direction was determined (see Supplementary Fig5C for examples of tracking). (H) Quantification of the contractions before change of direction in w 1118 (n=16 larvae), PatroninRNAi (n= 12), WDR62RNAi (n= 16), Patronin RNAi+ WDR6RNAi (n= 15) larvae. w 1118 vs PatroninRNAi , w 1118 vs WDR62 RNAi p = 0.0001, WDR62 RNAi vs PatroninRNAi + WDR62RNAi p < 0.0001, Kruskal-Wallis test. (I) Scheme of larvae fructose choice assay: the proportion of larvae on the half containing fructose was counted after 5 minutes of free movements (See Supplementary Fig5D for examples of tracking). (J) Quantification of larvae number on the Fructose- containing half after 5 minutes of movements of w 1118 (n=68 larvae), PatroninRNAi (n=58), WDR62RNAi (n=54), PatroninRNAi+ WDR6RNAi (n=53) larvae. w 1118 vs PatroninRNAi , w 1118 vs WDR62RNAi P = 0.0319, WDR62RNAi vs PatroninRNAi + WDR62RNAi P = 0.0390, Fisher’s exact test. (K) Proposed model for the origin of primary microcephaly origin: We postulate that cells can can detect lagging chromosomes via the Aurora B activity gradient. If these lagging chromosomes are only briefly in contact with Aurora B, as is the case in many wild-type or more frequently in CAMSAP1- depleted cells (fast poleward microtubule flux speed), this may lead to a transient activation of 53BP1, but not an activation of p21. If these lagging chromosomes remain longer, as is the case in WDR62- depleted cells (slow poleward microtubule flux speed) this will lead to a rapid 53BP1 activation and p21 induction resulting in a cell cycle delay which can exhaust the neuroprogenitor cell pool. Re- equilibrating poleward microtubule flux rates by co-depleting CAMSAP1 and WDR62 prevents transient lagging chromosomes and rescues the primary microcephaly phenotype.

Article Snippet: The following primary antibodies were used: recombinant human anti-α-tubulin (1:500; ( )), rabbit anti-CAMSAP1(1:1500; Novus Biologicals NBP1-26645), rabbit anti-WDR62 (1:1000; Bethyl A301- 560A), rabbit anti-MCAK (1:1000; ( )), rabbit anti-53BP1 (1:1000; Cell Signalling Technology 4937), mouse anti-p21 (1:1000; Cell Signalling Technology 2947), mouse anti-yH2Ax (1:2000; EMD Millipore, 05-636), mouse anti-AuroraB (1:2000; BD Biosciences 611083), rabbit anti-NPL4(1:500; Novus Biologicals NBP1-82166), rabbit anti-UBASH3B (1:500; Proteintech 19563-1-AP).

Techniques: Staining, Immunofluorescence, Activity Assay, Activation Assay