Abstract
Rab35 is a small G protein involved in various cellular events including clathrin-dependent endocytosis, phagocytosis, and autophagy. DENND1B, a DENN family member, acts as a guanine nucleotide exchange factor (GEF) for Rab35 to convert it to the GTP-bound active form from the GDP-bound inactive form. DENND1B contains the DENN domain which harbors GEF activity for Rab35 in the N-terminus, while the clathrin binding motif and adaptor protein-2-interaction motif are at the C-terminus. In this study, we investigated the intracellular localization of DENN1B in various cell types and found novel DENND1B-localized gathered line structures in BS-C-1 cells and in some other cell types. The localization of DENND1B to gathered line structures was dependent on a specific region located in the C-terminus of DENND1B protein. DENND1B-localized gathered lines were partially associated with microtubules but not with F-actin; instead, F-actin bundles surrounded the assembly of gathered lines. We also show that the gathered line structures appeared at the bottom of spreading lamellipodia and disappeared at the retracting site during cell motility in EGF-stimulated BS-C-1 cells. These results shed light on a new role for DENND1B in the regulation of cell migration.
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Acknowledgements
We are grateful to Prof. Joel A. Swanson for providing plasmids; and to Mr. Toshitaka Nakagawa, Mr. Kazuhiro Yokoi and Ms. Yukiko Iwabu for their skillful assistance.
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This study was supported by the Japan Society for the Promotion of Science (Grant Numbers: 19K07248 to KK, 16K08468 and 20K07245 to YE, 18K06831 to NA).
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Conceived and designed the experiments: EP, KK, and NA. Performed the experiments: EP, KK, YE. Analyzed the data: EP, KK, and NA. Drafting and revising the manuscript: EP, and NA. All authors have read and approved the final submission.
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Supplementary file2. Supplementary Movie 1 Live-cell movie of BS-C-1 cells co-expressing EGFP-fused full-length DENND1B (green) and mCherry-Lifeact were observed by fluorescence confocal laser microscopy. An assembly of DENND1B-localized gathered lines is found at the base of F-actin-enriched lamellipodium in BS-C-1 cells. Scale bar = 10 μm, × 200 speed (MP4 1554 kb)
Supplementary file3. Supplementary Movie 2 Time-lapse movie showing the effect of cytochalasin D on EGFP-DENND1B-localized gathered line structures in BS-C-1 cells. Cytochalasin D (3.3 μm) was added to the cells at the start of recording. Scale bar = 10 μm, × 600 speed (MP4 1039 kb)
Supplementary file4. Supplementary Movie 3 Time-lapse movie showing the effect of nocodazole on EGFP-DENND1B-localized gathered line structures in BS-C-1 cells. Nocodazole (10 μm) was added to the cells at the start of recording. Scale bar = 10 μm, × 600 speed (MP4 562 kb)
Supplementary file5. Supplementary Movie 4 Time-lapse movie of BS-C-1 cells expressing EGFP-DENND1B after stimulation with 100 ng/ml EGF. The gathered line structures moved toward the extended direction of movement of the lamellipodium and disappeared at cell retraction. Scale bar = 10 μm, × 200 speed (MP4 1380 kb)
Supplementary file6. Supplementary Movie 5 Time-lapse movie of BS-C-1 cells expressing EGFP-DENND1B and PA-Rac1. The red rectangle area was irradiated with a blue laser to photoactivate PA-Rac1. Scale bars = 10 μm, × 200 speed (MP4 1383 kb)
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Park, E.W., Kawai, K., Egami, Y. et al. A novel DENND1B-localized structure found at the basal side of adherent cells. Histochem Cell Biol 155, 9–18 (2021). https://doi.org/10.1007/s00418-020-01935-0
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DOI: https://doi.org/10.1007/s00418-020-01935-0