EB1 Comet Tracking in Drosophila Egg Chambers.

ubi-EB1-GFP time-lapse movies of stage 6 to 8 egg chambers in either control or msps-RNAi were acquired on a Nikon W1 spinning disk confocal microscope (Yokogawa CSU with pinhole size 50 µm) with a Hamamatsu ORCA-Fusion Digital CMOS Camera, and a 40× 1.25 N.A. silicone oil lens, at a frame rate of every 2 s for a total of 2 min, controlled by Nikon Elements software. Images were processed in FIJI and analyzed in DiaTrack 3.04 Pro (88), with a maximal particle jump distance of 0.57 μm/s, a minimal speed limit of 0.01 μm/s, and a minimal lifetime of 6 s. The sum of the length of EB1-GFP tracks was considered to be the total length of newly polymerized microtubules. For each sample, both the number of EB1 comets and the length of newly polymerized microtubules are normalized by the cell area size to get the EB1 density (#/µm²) and the new microtubule length per min (µm/µm²).