Figure 5.
RUFY interacts with Syd, Arl8, and Rab2 and is required for axonal DCV transport. (A and B) Western blots of the indicated Co-IP eluates (∼40% eluate volume) and HEK cell lysates (∼1% reaction volume). A, HA-tagged RUFY co-immunoprecipitates myc-tagged full-length Syd and truncated Syd-C1 (Syd530-1226) containing only the C-terminal WD40 domain but not truncated Syd-N2 (Syd1-529) that lacks the WD40 domain. B, HA-RUFY co-immunoprecipitates V5-tagged Arl8, and co-expression of V5-Arl8 enhances Co-IP of myc-Rab2Q65L with HA-RUFY. (C) Representative kymographs showing transport of ILP2-GFP–positive DCVs in motor axons of control larvae and larvae subjected to motor neuron-specific knockdown of RUFY, driven by OK6-Gal4. Scale bar: 10 µm. (D) Top, directional distributions derived from C, averaged from the following number of larvae: control for RUFY-KD(KK) 9, RUFY-KD(KK) 7; control for RUFY-KD(VAL20) 10, RUFY-KD(VAL20) 12. Bottom, the logarithmic ratio of retrograde to anterograde peak amplitude and the relative static peak amplitude for the directional distributions at the top. In C and D, “KK” and “VAL20” refer to UAS-RNAi lines from the KK collection and the VALIUM20 vector-based collection, respectively. For simplicity, only KK line data are illustrated in C. (E) Representative kymographs showing transport of ANF-GFP–positive DCVs in motor axons of control and Blos1 larvae. Scale bar: 10 µm. (F) Top, directional distributions derived from E. Averages from 11 larvae are shown for both Control and Blos1. Bottom, the logarithmic ratio of retrograde to anterograde peak amplitude and the relative static peak amplitude for the directional distributions at the top. (G) Kymographs of transport of ILP2-GFP–positive DCVs in motor axons of control, sydz4/Df single mutant, and Rab2; sydz4/Df double mutant larvae. Scale bar: 10 µm. (H) Directional distributions derived from G, averaged from 16 sydz4/Df and 9 Rab2; sydz4/Df larvae. (I) Flux of dynamic vesicles, counts of static vesicles in the central unbleached region, and speed of dynamic vesicles in the sydz4/Df and Rab2; sydz4/Df mutants also shown in H. (J) Hypothetical model of the DCV dynein–dynactin recruitment complex, consisting of Syd and RUFY anchored to the vesicle by Rab2 and Arl8. Kinesin-1 bound by Syd and kinesin-3 regulated by Arl8-BORC are omitted. It is uncertain if RUFY can recruit and activate dynein–dynactin. Bar graphs in D, F, and I represent the mean + SEM and were analyzed with Student’s t test. For conversion of angles in D, F, and H to DCV velocities, see Fig. 3 B. All data are from third instar larvae. Results in E and F represent reanalysis of data published earlier (Lund et al., 2021). Source data are available for this figure: SourceData F5.

RUFY interacts with Syd, Arl8, and Rab2 and is required for axonal DCV transport. (A and B) Western blots of the indicated Co-IP eluates (∼40% eluate volume) and HEK cell lysates (∼1% reaction volume). A, HA-tagged RUFY co-immunoprecipitates myc-tagged full-length Syd and truncated Syd-C1 (Syd530-1226) containing only the C-terminal WD40 domain but not truncated Syd-N2 (Syd1-529) that lacks the WD40 domain. B, HA-RUFY co-immunoprecipitates V5-tagged Arl8, and co-expression of V5-Arl8 enhances Co-IP of myc-Rab2Q65L with HA-RUFY. (C) Representative kymographs showing transport of ILP2-GFP–positive DCVs in motor axons of control larvae and larvae subjected to motor neuron-specific knockdown of RUFY, driven by OK6-Gal4. Scale bar: 10 µm. (D) Top, directional distributions derived from C, averaged from the following number of larvae: control for RUFY-KD(KK) 9, RUFY-KD(KK) 7; control for RUFY-KD(VAL20) 10, RUFY-KD(VAL20) 12. Bottom, the logarithmic ratio of retrograde to anterograde peak amplitude and the relative static peak amplitude for the directional distributions at the top. In C and D, “KK” and “VAL20” refer to UAS-RNAi lines from the KK collection and the VALIUM20 vector-based collection, respectively. For simplicity, only KK line data are illustrated in C. (E) Representative kymographs showing transport of ANF-GFP–positive DCVs in motor axons of control and Blos1 larvae. Scale bar: 10 µm. (F) Top, directional distributions derived from E. Averages from 11 larvae are shown for both Control and Blos1. Bottom, the logarithmic ratio of retrograde to anterograde peak amplitude and the relative static peak amplitude for the directional distributions at the top. (G) Kymographs of transport of ILP2-GFP–positive DCVs in motor axons of control, sydz4/Df single mutant, and Rab2; sydz4/Df double mutant larvae. Scale bar: 10 µm. (H) Directional distributions derived from G, averaged from 16 sydz4/Df and 9 Rab2; sydz4/Df larvae. (I) Flux of dynamic vesicles, counts of static vesicles in the central unbleached region, and speed of dynamic vesicles in the sydz4/Df and Rab2; sydz4/Df mutants also shown in H. (J) Hypothetical model of the DCV dynein–dynactin recruitment complex, consisting of Syd and RUFY anchored to the vesicle by Rab2 and Arl8. Kinesin-1 bound by Syd and kinesin-3 regulated by Arl8-BORC are omitted. It is uncertain if RUFY can recruit and activate dynein–dynactin. Bar graphs in D, F, and I represent the mean + SEM and were analyzed with Student’s t test. For conversion of angles in D, F, and H to DCV velocities, see Fig. 3 B. All data are from third instar larvae. Results in E and F represent reanalysis of data published earlier (Lund et al., 2021). Source data are available for this figure: SourceData F5.

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