AP-1 and Tyrp1 localize to subdomains of the recycling compartment adjacent to melanosomes. (A) MNT-1 cells that had internalized Tf-A488 for 45 min were analyzed by IFM after labeling for AP-1 (γ-adaptin; a–d), Rab11 (e–h), or EEA1 (i–l). Single labeling is shown in columns 1 and 2, merged images in 3, and magnified insets (of boxed areas) in 4. Note that AP-1 partially overlaps with Tf-A488 (a–d, arrows) and Rab11 (e–h, arrows) but not with EEA1 (i–l). (B) Quantification of colocalization between Tf and EEA1, Rab11, or γ-adaptin in IFM experiments as described in A. Data are presented as mean ± SD. (C) MNT-1 cells that had internalized Tf-biot for 45 min were processed for ultrathin cryosectioning and single immunogold labeled for biotin (PAG10; a) or double immunogold labeled for biotin (PAG10) and γ-adaptin (PAG15; b) or for Tyrp1 (PAG 15; c and d). After 45 min of internalization, Tf-biot localizes to a tubular network that is close to and surrounds melanosomes (a and inset, arrows). These Tf-positive tubules often bear AP-1 buds (b, arrows). Tyrp1 is present on the limiting membrane of pigmented melanosomes (M) and is also detected on the Tf-positive tubules (c and d, arrows). Bars: (A) 10 µm; (C) 200 nm.