Direct Observation of Single Microtubule Nucleation by Mto1[bonsai] Puncta Reveals Protein Copy Number within Individual Puncta
(A) Time-lapse images (3.3 s interval) of mCherry-microtubule nucleation (mCh-Atb2) from Mto1[bonsai]-GFP puncta as well as from Mto2-GFP, Alp4-GFP, and Alp6-GFP puncta in an untagged mto1[bonsai] background. Panels show single z sections or maximum projections of two adjacent z sections. Upper panels show entire cell area. Lower panels correspond to regions indicated by yellow boxes and show time-lapse images of nucleating puncta (white arrows). See Movie S2 for corresponding movies.
(B) Fluorescent signal of individual Mto1[bonsai]-GFP, Mto2-GFP, Alp4-GFP, and Alp6-GFP puncta (±SEM), quantified at the time of microtubule nucleation. Calculation of molecules per punctum used Rlc1-GFP cytokinesis nodes as a fluorescence calibration standard [19]. Scale bar, 5 μm.