FM4-64 is a lipophylic dye used to determine membrane internalization; MK-0683 GFP-Snc1 is a reporter showing trafficking of a SNARE protein between the plasma membrane and endosomes; and analysis of Sla1-GFP allows the behaviour of individual endocytic sites to be assessed. As shown in figure 2B, in the presence of 25 mM Lat-A, Lucifer yellow was still internalized and trafficking to vacuoles was observed, while at 400 mM Lat-A uptake was completely abrogated. Analysis of lipid internalization using FM4-64 revealed that after 20 minutes incubation, 100% untreated cells internalized the dye and the majority of cells showed vacuolar staining. In the presence of 25 mM Lat-A 8964% cells internalized the dye showing endocytosis was functioning, however there was a reduced number of cells with predominant vacuolar staining indicating a role for Factin in post endosomal trafficking. A post-endosomal role for actin in yeast has previously been suggested. These two approaches indicate that bulk endocytosis is not affected following addition of 25 mM Lat-A. GFP-Snc1 is a fluorescently tagged SNARE protein that has been used as a reporter for endosomal uptake and recycling. High throughput screens analysing uptake and recycling of the tagged SNARE GFP-Snc1 have indicated the importance of the recognized clathrin mediated endocytic pathway for its uptake into cells. In wild type, untreated cells Snc1 is observed in puncta at the surface and also in discrete structures, presumed to be endosomes inside cells. In the presence of 25 mM Lat-A, uptake is inhibited and localization is only seen at the cell surface. Inhibition of GFPSnc1 uptake at levels of 25 mM Lat-A suggested that the CME pathway could be inhibited even when cortical patches are intact. In order to address whether the known endocytic route was disrupted, the behaviour of a well characterized endocytic reporter protein Sla1-GFP was analysed further. Cells expressing Sla1-GFP were incubated in the presence of DMSO or 25 mM Lat-A, and after 20 minutes cells were imaged to analyse the behaviour of membrane associated Sla1-GFP patches. It was already known that Sla1 is able to localize to the plasma membrane at discrete sites in the absence of F-actin. However, it was unexpected that the low level of Lat-A would inhibit movement of this protein such that it was not able to invaginate. Even in the Abp1-mRFP tagged cells, while patches were visible they were diffuse and difficult to discern above background, compared to the fluorescence signal in the untreated cells.