We next evaluated the effect of GFP-M6tail expression on the early endosomes and other endocytic organelles. transient association with endocytic vesicles and is released upon early endosome fusion. Green fluorescent protein (GFP) fused to myo6 as well as the cargo-binding tail Tretinoin (M6tail) alone targeted to the nascent endocytic vesicles. Overexpression of GFP-M6tail had no effect on a variety of organelle markers; however, GFP-M6tail displaced the endogenous myo6 from nascent vesicles and resulted in a significant delay in transferrin uptake. Pulse-chase experiments revealed that transferrin accumulated in uncoated vesicles within the peripheries of transfected cells and that Rab5 was recruited to the surface of these vesicles. Given sufficient time, the transferrin did traffic to the perinuclear sorting endosome. These data suggest that myo6 is an accessory protein required for the efficient transportation of nascent endocytic vesicles from the actin-rich peripheries of epithelial cells, allowing for timely fusion of endocytic vesicles with the early endosome. INTRODUCTION Myosins are a large family of structurally diverse molecular motors. Myosins bind to F-actin and hydrolyze ATP to produce unidirectional movement along the filament. Actin filaments are polarized, and myosins traditionally travel toward the barbed or plus end of the actin filament. Tretinoin An exception to this rule is the unconventional myosin, myosin-VI (myo6), which travels backwards Tretinoin toward the pointed or minus end of actin filaments (Wells (1994 ) with the following modifications. Extracts were prepared from ARPE-19 cells plated at 80% confluence. Cells were solubilized in extraction buffer (RIPA buffer with 300 mM sucrose, 5 mM ATP, containing 2 g/ml aprotinin, 10 g/ml leupeptin, 20 g/ml chymostatin, 10 g/ml pepstatin A, 0.2 mM pefabloc, and 1 phosphatase inhibitor cocktails I and II (Sigma-Aldrich)). After spinning at 20,800 (2001 ) as essential for endocytic function. Fluorescence microscopy revealed that when expressed at low levels, the GFP-M6tail fusion protein targeted to punctae on actin cables at the cell peripheries (Figure 5B). The GFP-M6tail punctae overlapped with GIPC (Figure 6A) and the TsfnR (Figure 6B), as judged by confocal microscopy. Therefore, the tail domain is sufficient to target myo6 the nascent endocytic vesicles. If the myo6-associated vesicles were indeed an endocytic intermediate, we predicted that they would be accessible to the fluid phase uptake marker, Rabbit polyclonal to CUL5 rhodamine-conjugated dextran (Rhod-dextran). As shown in Figure 6D, after a 10-min incubation with Rhod-dextran there was significant overlap with the GFP-M6tail-decorated endocytic vesicles. Therefore, the tail of myo6 is targeting the motor protein to endocytic vesicles. Removal of Myo6 from the Nascent Endocytic Vesicles Disrupts Trafficking Tretinoin of Transferrin The immunolocalization, GFP-fusion, and uptake data presented thus far support a model whereby myo6 serves as the bridge between the TsfnR-containing nascent endocytic vesicle and the actin cytoskeleton and that myo6 is linked to the endocytic vesicle solely by its tail domain. Therefore, we predicted that overexpression of the tail domain would displace the endogenous myo6 from the vesicle. As shown in Figure 7, A and B, overexpression of Tretinoin GFP alone had no effect on the association of endogenous myo6 with peripheral vesicles, as judged by an antibody directed to the motor domain of myo6. In contrast, overexpression of GFP-M6tail resulted in a selective removal of myo6 stain from the peripheries of transfected cells, as judged by the motor-specific antibody (Figure 7, CCE). GFP-M6tail staining of peripheral vesicles was evident in these transfected cells, supporting the hypothesis that the endogenous myo6 is competed off the endocytic vesicles when the myo6 tail domain is overexpressed. Open in a separate window Figure 7. GFP-M6tail displaces myo6 from peripheral endocytic vesicles. Immunofluorescence microscopy of ARPE-19 cells transiently transfected with GFP (A and B) or GFP-M6tail (CCE) and stained with antibodies to the motor domain of myo6. GFP fluorescence is shown in B, D, and E (green), and myo6-motor antibodies staining is shown in A, C, and E (red). The region of the cell where peripheral vesicles are found is demarcated by filled arrows in transfected cells and by open arrows in untransfected cells. Bar, 10 m. Presumably, myo6 was being recruited to endocytic vesicles to allow transportation through the actin meshwork. We predicted that, after overexpression of the M6tail domain, because no myo6 motor was present, the nascent endocytic vesicles would no longer be able to negotiate the actin-meshwork of the cell peripheries, precluding efficient fusion with the more centrally located early endosome. To test this theory, we evaluated whether overexpression of the GFP-m6tail construct would cause a block in transferrin uptake. ARPE-19 cells were transfected with GFP, GFP-M6full, and GFP-M6tail.