Fig. 5. Dis2.NEGFP in endocytosis. dis2.NEGFP (IH2908; A), dis2.NEGFP sla2.Cherry (IH5205; B), dis2.NEGFP sla2. (IH4722; C), a mix of dis2.NEGFP (IH2908) and crn1.GFP (IH3528) cells (D,E) and a mix of dis2.NEGFP sla2.Cherry (IH5205) and act1.GFP (IH4266) cells (F) were processed as for Fig. 1 with the exception that DMSO containing Lat A, to a final concentration of 50 µM, was included in the culture medium for mounting in E and F and an equivalent amount of DMSO added to the culture in D. (A) Kymograph of the area between the green lines shows the internalisation movement of Dis2.NEGFP. (B) Kymograph of the area in the rectangles shows the colocalisation of Dis2.NEGFP and Sla2.Cherry. Note that Sla2.Cherry signal appears at the cell cortex before Dis2.NEGFP (C) Kymograph showing that the internalisation of Dis2.NEGFP is avoided in sla2. cells grown at 33°C. (D,E) Whereas Dis2.NEGFP distribution was not affected by the addition of DMSO (D), disruption of the actin cytoskeleton (verified by the lack of any Crn1.GFP signals in the control crn1.GFP cells – asterisks) severely reduced the number of Dis2.NEGFP dots seen away from the cell tip, but did not affect the association of Dis2.NEGFP with cell tips (compare inset in D and E). Bars, 5 µm. (F) Kymograph showing that internalisation of Dis2.NEGFP and Sla2.CH foci was abolished by depolymerisation of the F-actin cytoskeleton.