Figure 6. The NRZ tether links TANGO1 to ERGIC membranes.

(A) Rings of TANGO1 (green) in RDEB/FB/C7 cells with RINT1 (red) and ERGIC-53 (blue). Deconvolved z-stacks of ten cells were used to quantify the location of the tether protein RINT1 relative to a ring of TANGO1. 90 rings of TANGO1 were manually scored, three adjacent slices in the image stack were used to identify signal from RINT1 in the vicinity of the ring of TANGO1. 23 rings showed RINT1 within the ring, 19 rings showed RINT1 on the circumference (at the edge) of the ring, 39 had RINT1 outside the ring, nine rings showed no detectable RINT1. (B) TANGO1, TANGO1Δ1255–1295, TANGO1Δ1296–1336 and TANGO1-Lum were expressed in HEK293T cells and immunoprecipitated. Samples were probed for NBAS, RINT1, cTAGE5 and ZW10. TANGO1 and TANGO1Δ1296–1336 immunoprecipitated all four proteins, but TANGO1Δ1255–1295 did not immunoprecipitate tether proteins. TANGO1-Lum did not pull down any of the four proteins. (C) RDEB/FB/C7 were transfected with siRNA (siCTRL, siNBAS, siRINT1 and siTANGO1) and immunostained for intracellular collagen VII (red) and calreticulin (green). (D) Quantification of fluorescence associated with intracellular collagen VII in (C). (E) Collagen VII secreted by RDEB/FB/C7 was looked at as the ratio of collagen in the medium to the lysate, quantified, and plotted as the average of values from at least three independent experiments. β-actin is a loading control. (F) siRINT1-, siNBAS- and siTANGO1-treated RDEB/FB/C7 were stained for collagen VII and ERGIC-53. (G) A plot of Manders’ overlap coefficient for ERGIC-53 and collagen VII from (F) used to quantify ERGIC-53 localisation to collagen accumulations. (H) Representative blots showing the efficiency of knockdown of NBAS, RINT1 and ZW10, quantified and plotted as the average ±s.d. from at least three independent experiments. ***p<0.001; **p<0.01. Scale bars (A) 200 nm, (C, F) 10 μm, (C) inset) 5 μm.

Figure 6—figure supplement 1. RINT1 is recruited to exit sites at collagen accumulations.

Collagen accumulations in RDEB/FB/C7 cells were visualised for Sec31 (red), TANGO1 (green) and RINT1 (blue). Colocalisation at these patches was measured by the Manders’ overlap coefficient of 0.691 ± 0.124 (s.d.) of RINT1 with TANGO1. Scale bars 5 μm.

Figure 6—figure supplement 2. RINT1 localises to one or two puncta in a TANGO1 ring.

RDEB/FB/C7 cells were visualised for TANGO1 (green) and RINT1 (red) Fourteen examples are shown here. Scale bar 200 nm.