Supplementary Figure 5: The katanin p60/p80 heterodimer decorates and bends microtubule ends.
From: Microtubule minus-end regulation at spindle poles by an ASPM–katanin complex
(a) TIRFM live cell imaging and kymographs of colocalization of GFP-p60/p80C and EB3-TagRFP in MRC5 cells. Scale bars: horizontal, 2 μm; vertical, 10 s. (b) Live cell images of a single microtubule plus end bound to GFP-p60/p80C and EB3-TagRFP in MRC5 cells. White arrow, breaking of a bent microtubule end. Scale bar, 1 μm. (c) Live imaging of MRC5 cells expressing individual katanin subunits together with EB3-TagRFP. Scale bar, 2 μm. (d) TIRFM time lapse images showing the complete severing of dynamic microtubules by p60/SNAP-Alexa647-p80C at 750 nM in a flow-in experiment. Time represented as min:sec. Scale bar, 2 μm. (e–f) Maximum intensity projections of time lapse images of GFP-p60/p80 WT or mutants in HeLa cells. Imaging was performed using TIRFM with 500 ms exposure in a stream mode. 100–200 images representing consecutive frames were used to make projections. Mutations of R615A, K618A, G607A, V608A, D609A and I610A in p80 completely abolish the end-binding activity of p60/p80 in cells. Scale bar, 2 μm. (g) Protein sequence alignment of p60N and p80C from Caenorhabditis elegans, Chlamydomonas reinhardtii, Tetrahymena thermophila, Arabidopsis thaliana, Drosophila melanogaster , Danio rerio, Xenopus tropicalis, Homo sapiens and Mus musculus. The residues that are essential for microtubule end binding are indicated with red dots. Mutating charged residues denoted by black dots to alanine had no effect on end binding in vivo. Red triangles indicate the residues interacting with ASPM, which are conserved in vertebrates (red dashed rectangle). Numbers displayed on top of the sequence alignment are based on mouse katanin sequences.
