Journal: bioRxiv

Article Title: A mechanism that integrates microtubule motors of opposite polarity at the kinetochore corona

doi: 10.1101/2023.04.25.538277

Figure Lengend Snippet: ( A ) Organization of the human kinetochore and corona. ( B ) Drawing depicting the hierarchical organization of outer kinetochore and kinetochore corona components. Thick arrows indicate recruitment of a protein to the protein indicated by the arrowhead. Thin arrows indicate phosphorylation. The white arrow indicates polymerization. ( C ) Representative images of the localization of CENP-E after depletion of Zwilch and BUBR1. Zwilch RNAi treatment was performed with 100 nM siRNA for 72 h. 48 h after Zwilch RNAi treatment cells were transfected with 100 nM BUBR1 siRNA. 8 h after transfection, cells were synchronized in G2 phase with 9 μM RO3306 for 15 h and then released into mitosis. Subsequently, cells were immediately treated with 3.3 μM Nocodazole, 10 μM MG132 for an additional hour. CENP-C was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 5 μm. ( D ) Quantification of CENP-E levels at kinetochores of the experiment shown in panel C. n refers to individually measured kinetochores. ( E ) Organization of CENP-E with coiled-coil prediction. ( F ) Representative images showing the localization of different EGFP CENP-E constructs in prometaphase after depletion of CENP-E with 60 nM siRNA. 13 h after RNAi treatment cells were electroporated with electroporation buffer or recombinant EGFP CENP-E constructs as indicated. Following an 8 h recovery, cells were synchronized in G2 phase with 9 μM RO3306 for 15 h and then released into mitosis. Subsequently, cells were immediately treated with 3.3 μM Nocodazole for an additional hour. CENP-C was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 5 μm. ( G ) Quantification of EGFP levels at kinetochores of the experiment shown in panel F. n refers to individually measured kinetochores. ( H ) mCH RZZ mCh S ring-binding assays showing the recruitment of various EGFP CENP-E truncations (10 nM concentration) to mCH RZZ mCh S rings (approx. 40 nM concentration). Scale bar: 5 μm. ( I ) RZZS ring-binding assays showing the recruitment of EGFP CENP-E 2070C to mCH RZZ mCh S rings is unaffected by increasing concentrations of BUBR1 KD . Scale bar: 5 μm.

Article Snippet: After blocking with 5% boiled goat serum (BGS) in PHEM buffer for 1 hour, cells were incubated for 2 hours at room temperature with the following primary antibodies: BUB1 (mouse, Abcam, ab54893, 1:400), BUBR1 (rabbit, Thermo Scientific #720297, 1:1000), CENP-C (guinea pig, MBL, #PD030, 1:1000), CENP-E (mouse, Abcam, ab5093, 1:200), CENP-F (rabbit, Novus NB500–101, 1:300), CREST/anti-centromere antibodies (human, Antibodies, Inc., 1:200), Dynactin-p150 glued (mouse, BD Trans.

Techniques: Transfection, Staining, Construct, Electroporation, Recombinant, Binding Assay, Concentration Assay