Figure 1

Hook2 co-localizes with aggresomes at the centrosome and promotes aggresome formation. Vero cells were transfected to express Hook2. (A) Immunostaining revealed Hook2 either diffusely distributed (type I) or enriched at centrosomes (types II-IV) and [21]. Type IV refers to large aggresomes (> 4 μm). (B) Cells containing distinct Hook2 patterns were counted after the indicated days of expression. The fraction of cells with some hook2 at centrosomes (type II-IV combined) only modestly increased from 1 to 3 days of expression. The fraction of cells in which co-expressed CFTR accumulated in large juxtanuclear aggresomes (type IV) increased about ten-fold from day 1 to 3. (C-D) Cells were transfected to express hook2 and ΔF508-CFTR or (E) hook2 and CFTR-GFP. After 24 hours, 10 μM lactacystin was added and cells were incubated for an additional 16 hours before either being double-stained (C-D) for hook2 (red in merged images) and co-expressed CFTR (green) or triple stained (E) for hook2, CFTR and endogenous hsc70. (F-J) Hook2 was expressed in the absence of CFTR and lactacystin and tested for co-localization with the endogenous aggresome markers hsc-70, ubiquitin (FK1), the 20S proteasome subunit or cytoplasmic dynein, as indicated. Merged images in (I and J) indicate the accumulation of dynein in hook2-induced type II (I) and type IV (J) aggresomes.