Fig. 1. Expression of myosin VI tail isoforms in different tissues and polarized and unpolarized cells. (A) Schematic representation of myosin VI showing the position and sequence of the tail inserts. (B) The myosin VI tail isoform with the large insert is expressed in rat tissues containing many polarized cells having microvilli (liver, lane 1; kidney, lane 2; and small intestine, lane 3) and in polarized Caco-2 cells (lane 8), whereas unpolarized Caco-2 cells (lane 7) and rat tissues containing few polarized cells with microvilli on their apical surface (brain, lane 4; testis, lane 5; and lung, lane 6) contain myosin VI with either the small or no insert in the tail domain. mRNA prepared from rat tissues and Caco-2 cells was used in RT–PCR reactions with primers flanking the region of the tail containing the two inserts (∼300 bp). (C) Expression levels of myosin VI tail isoforms (large insert, closed squares; small or no insert, closed triangles) during polarization of Caco-2 cells plotted as percent of total isoforms. Polarization was monitored by measuring the TEER (open circle). Caco-2 cells were grown on filters and harvested for RT–PCR reactions at different times after seeding.

Fig. 2. Endogenous myosin VI is localized to clathrin-coated vesicles in the apical domain of polarized Caco-2 cells. (A) Caco-2 cells, grown on filters for 14 days, were (a) labelled for immunofluorescence with rhodamine-phalloidin to localize actin or (b) with antibody to myosin VI. Confocal vertical X–Z optical sections are shown. (B) Myosin VI co-localizes with clathrin-coated pits/vesicles at the apical domain of Caco-2 cells. Immunofluorescence (a) and (c) with antibodies to whole myosin VI tail or (b) and (d) with antibodies to AP-2. The polyclonal antibody to the whole tail of myosin VI has been described previously (Buss et al., 1998). A section through the apical domain above the nucleus is shown. (c) and (d) show enlarged images of co-localization of myosin VI (c) and AP-2 (d). Arrows identify a few of the spots showing co-localization of myosin VI and AP-2. (A) Bar: 10 µm; (B) bar: 3 µm.

Fig. 3. Schematic representation of whole myosin VI and different myosin VI deletion mutants tagged with GFP. (a) Whole myosin VI; (b) whole tail (tail); (c) myosin VI without the ΔGT; (d) GT with the large insert (GT + LI); (e) GT without the large insert (GT – LI); (f) CCT with the large insert (CCT + LI) or (g) CCT without the large insert (CCT – LI). All constructs carried a GFP tag at their N-termini for expression in NRK cells.

Fig. 4. Co-localization of myosin VI with AP-2 and clathrin in NRK cells. (A) An untransfected cell stained for endogenous myosin VI with an antibody to the whole tail. Endogenous myosin VI not containing the large insert (A) shows partial co-localization with clathrin (B) in NRK cells. NRK cells transiently expressing whole myosin VI from chicken brush border containing the large insert (C) or only the tail containing the large insert, both tagged with GFP (E, G and I), were stained with antibodies to AP-2 (K) or clathrin (D, F and H). The GFP–myosin VI and the tail show co-localization with clathrin and AP-2 at the plasma membrane. Bar: 20 µm.

Fig. 5. Localization of myosin VI in clathrin-coated pits/vesicles. For immuno-EM, stable NRK cells lines expressing GFP–GT were labelled (A, B and C) with antibodies to GFP (Molecular Probes, Leiden) followed by protein-A 15 nm gold as described by Buss et al. (1998). (D, E and F) The cryosections were double labelled for GFP–GT (5 nm gold, arrow heads) and for clathrin (15 nm gold). Bar: 100 nm.

Fig. 6. Localization of GFP–myosin VI and GFP–myosin VI deletion mutants in NRK cells. (A) An untransfected cell stained for endo genous myosin VI with an antibody to the whole tail (a-myosin VI). Myosin VI is enriched in the perinuclear area (small arrows), in ruffles at the edge of the cell (large arrows) and in a vesicular staining pattern in the thin leading lamella (arrowheads). (B–H) NRK cells transiently expressing GFP-tagged constructs: (B) whole myosin VI tagged with GFP [symbols as in (A)]; (C) GFP–tail; (D) GFP–myosin VI without the GT (GFP ΔGT); (E) the GT containing the large insert (GFP GT + LI); (F) the GT without the large insert (GFP GT – LI); (G) the coiled-coil domain of the tail tagged with GFP containing the large insert (GFP CCT + LI); and (H) the coiled-coil domain without the large insert (GFP CCT – LI). The GT together with the large insert are important for targeting to vesicular structures [see (B), (C), (E) and (F) compared with (D), (G) and (H)]. Only expression constructs including the motor domain were found in ruffles [compare (B) and (D) with (C), (E) and (F)]. Bar: 15 µm.

Fig. 7. Myosin VI interacts in vitro with AP-2 and clathrin. (A) The pull-down experiments shown demonstrated the binding of myosin VI (lane 2), GFP–tail (lane 4) and GFP–GT (lane 5) to the ear of the α-subunit of AP-2. Cytosol for these experiments was prepared from A431 (lanes 1–3) or NRK cells (lanes 4–6). The latter were stably transfected with GFP–tail (lane 4), GFP–GT (lane 5) or GFP (lane 6). Protein binding to the α-subunit of AP-2 was analysed by SDS–PAGE (lane 1) or by immunoblotting with anti-myosin VI serum (lanes 2 and 3) or with an antibody to GFP (Molecular Probes, Leiden, The Netherlands) (lanes 4–6). Lanes 3 and 6 show a blank control, in which instead of GST–α ear only GST was used. In lane 1 a Coomassie stained gel of a GST–α ear pull-down from A431 cytosol is shown. The bands seen in lane 1 in addition to the GST–α ear (∼50 kDa) are consistent with the expected size of EPS 15, AP 180, Ampiphysin 1, Ampiphysin 2 and Epsin (as marked by the asterisk). ( B) Co-immunoprecipitation of myosin VI with AP-2 and clathrin. AP-2 (lane 4), clathrin (lane 5) and myosin VI (lane 3) as a control were immunoprecipitated under native conditions from cytosol of A431 cells and analysed by western blotting using anti-myosin VI serum. Some myosin VI can be immunoprecipitated with the AP-2 complex (lane 4) and with clathrin (lane 5) but not with pre-immune serum used as a control (lane 6). Lane 1 shows a Coomassie-stained gel of an immunoprecipitate with anti-myosin VI antiserum. Lane 2 is the input lane showing 1/25 of the total cytosol used for one immuno precipitation as blotted with antibodies to myosin VI. (C) Immunoblot of purified clathrin-coated vesicles. Purified clathrin-coated vesicle proteins were separated by SDS–PAGE and stained with Coomassie Blue (lane 1) or blotted onto nitrocellulose and reacted with antibodies to clathrin (lane 2) or myosin VI (lane 3). Myosin VI was observed in purified clathrin-coated vesicles (lane 5). Blotting the same amounts of protein of a 100 000 g microsomal pellet (MP) (lane 4) from rat liver and purified clathrin-coated vesicles (CCV) (lane 5) showed that there was an enrichment of myosin VI in clathrin-coated vesicles similar to that observed for AP-1 and AP-2. Myosin II (MII) and myosin V (MV) were present as expected in the microsomal pellet and were barely detectable in the clathrin-coated vesicles.

Fig. 8. Over-expression of the whole myosin VI tail causes a marked reduction in clathrin-mediated transferrin uptake. (A–C) Immuno fluorescence of transferrin uptake [red, (B)] in transiently transfected NRK cells expressing GFP–tail (see Figure 1) [green, (A)]; (C) overlay. The effect of the tail in transiently transfected cells on transferrin uptake is shown in the bar graph (D). This quantitation is based on cells showing a reduction in transferrin uptake by >50% compared with untransfected control cells in four different experiments ± SD. In each experiment, at least 100 transfected and 100 non-transfected cells as a control were counted. To measure quantitatively transferrin uptake, stable NRK cell lines expressing myosin VI GFP–tail or GFP alone were used (see Figure 3). The effect of the tail on transferrin uptake is shown by the graph (E); open squares show untransfected cells; open diamonds show cells stably transfected with GFP alone; filled circles show cells stably transfected with myosin VI; filled triangles show cells stably transfected with the GT without the large insert and filled squares show a representative stable transfected cell line overexpressing GFP–myosin VI tail containing the large insert. Data show a representative experiment. Bar: 25 µm.