TIRF time-lapse imaging of myosin-GFP (left) and mCherry-tubulin (right) in an S2 cell depleted of kinesin-6 (Pav) by RNAi. Myosin-GFP localization to the equator and loss from the poles does not occur after RNAi kinesin-6; microtubules tend to be more homogeneous throughout the cortex and less bundled. Video corresponds to Fig 3B and video 6 from J Cell Bio 186:727-738, 2009. Note that the wild type control is available as another video in the series. Total internal reflection microscopy was performed using a microscope (Perfect-focus TE2000; Nikon) with a 100×, 1.45 NA objective (Nikon) and illumination from either a 488-nm argon laser (100 mW) or a 491-nm solid-state laser (100 mW) and a 561-nm solid-state laser (50 mW). For dual-color TIRF microscopy, we used a triplepass dichroic filter (z491/561/633rpc) and changed the emission filter (ET525/50 or ET595/50; both from Chroma Technology Corp.) with a filter wheel placed before the camera. In some cases, an excitation notch filter was used in the filter cube (NF01-405/488/561/635; Semrock, Inc.). Images were typically captured every 2-3 s with a 50-200 ms exposure with an EM charge-coupled device camera (iXon; Andor Technology). The microscope was controlled and images were acquired using open source MicroManager software (http://www.micro-manager.org).
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