JCS00637 Supplemental Data

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• Fig S1 - F-Dex is internalized into to endosomes devoid of Cy3mBSA at 45 seconds.(A-C) Hemocytes were pulsed for 45 seconds with F-Dex (A, green in C) and Cy3mBSA (B, red in C) and subsequently chased for 5 minutes. Note that the two probes are extensively co-localized in centrally located endosomes (arrows). D-F) Hemocytes incubated with F-Dex (D, green in F) and Cy3mBSA (E, red in F) for 2 minutes at 21°C show endosomes that do not co-localize with Cy3mBSA (arrowheads) and some that do co-localize (arrows). In a 2 minutes pulse, there is increasing co-localization of Cy3mBSA and F-Dex compared with 45 seconds pulse (see Fig. 3). Bar, 5 mm.

• Fig S2 - Hemocytes directly placed into minimal buffered saline and embryonic macrophages also display dDyn-independent fluid-phase uptake. (A-F) Hemocytes from shi^ts1 (A-C) or wild-type (D-E) animals were dissected into HEPES-buffered saline with BSA (2 mg/ml) and glucose (1 mg/ml) and immediately preincubated at 31°C for 5 minutes. After incubation with F-Dex (B,E; green in C,F) and Cy3mBSA (A,D; red in C,F) for an additional 5 minutes at 31°C, cells were washed at the same temperature, fixed and imaged on a wide field microsocope. (G-I) Macrophages derived from embryonic stage 11 shi^ts1 animals were preincubated at 31°C for 5 minutes and with F-Dex (H; green in I) and Cy3mBSA (G; red in I) for an additional 5 minutes at the same temperature, and examined on a wide-field microscope. Note that cells from shi animals do not endocytose Cy3mBSA at the restrictive temperature whereas F-Dex uptake is unaffected in all culture conditions. Bar, 5 mm.

• Fig S3 - ALBR is arrested in a-adaptin-marked sites at the plasma membrane at the restrictive temperature. Immunolocalization of a-adaptin (B, E; green in C, F) in cells from shi^ts1 (A-C) or shi^ts2 (D-F) animals incubated with Cy3mBSA (A, D; red in C, F) for 5 minutes at 31°C as described in Fig. S1. Note co-localization of surface-accessible Cy3mBSA with a-adaptin in discrete puncta (see insets). Insets show magnified views of the area marked by an asterisk. Bar, 5 mm; inset, 1 mm.