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.
Similar content being viewed by others
Availability of data and material
All data generated or analyzed during this study are included in this published article and its supplementary information files.
References
Allaire PD, Ritter B, Thomas S et al (2006) Connecdenn, a novel DENN domain-containing protein of neuronal clathrin-coated vesicles functioning in synaptic vesicle endocytosis. J Neurosci 26:13202–13212. https://doi.org/10.1523/JNEUROSCI.4608-06.2006
Chesneau L, Dambournet D, MacHicoane M et al (2012) An ARF6/Rab35 GTPase cascade for endocytic recycling and successful cytokinesis. Curr Biol 22:147–153. https://doi.org/10.1016/j.cub.2011.11.058
Chevallier J, Koop C, Srivastava A et al (2009) Rab35 regulates neurite outgrowth and cell shape. FEBS Lett 583:1096–1101. https://doi.org/10.1016/j.febslet.2009.03.012
Dambournet D, MacHicoane M, Chesneau L et al (2011) Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis. Nat Cell Biol 13:981–988. https://doi.org/10.1038/ncb2279
Dutta D, Donaldson JG (2015) Sorting of clathrin-independent cargo proteins depends on Rab35 delivered by clathrin-mediated endocytosis. Traffic 16:994–1009. https://doi.org/10.1111/tra.12302
Egami Y, Fukuda M, Araki N (2011) Rab35 regulates phagosome formation through recruitment of ACAP2 in macrophages during FcγR-mediated phagocytosis. J Cell Sci 124:3557–3567. https://doi.org/10.1242/jcs.083881
Egami Y, Fujii M, Kawai K et al (2015) Activation-inactivation cycling of Rab35 and ARF6 is required for phagocytosis of zymosan in RAW264 macrophages. J Immunol Res 2015:429439. https://doi.org/10.1155/2015/429439
Fujii M, Kawai K, Egami Y, Araki N (2013) Dissecting the roles of Rac1 activation and deactivation in macropinocytosis using microscopic photo-manipulation. Sci Rep 3:2385. https://doi.org/10.1038/srep02385
Fukata M, Watanabe T, Noritake J et al (2002) Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170. Cell. https://doi.org/10.1016/S0092-8674(02)00800-0
Kato T, Kawai K, Egami Y et al (2014) Rac1-dependent lamellipodial motility in prostate cancer PC-3 cells revealed by optogenetic control of Rac1 activity. PLoS ONE 9:e97749. https://doi.org/10.1371/journal.pone.0097749
Klinkert K, Echard A (2016) Rab35 GTPase: a central regulator of phosphoinositides and F-actin in endocytic recycling and beyond. Traffic 17:1063–1077. https://doi.org/10.1111/tra.12422
Kulasekaran G, Nossova N, Marat AL et al (2015) Phosphorylation-dependent regulation of connecdenn/DENND1 guanine nucleotide exchange factors. J Biol Chem. https://doi.org/10.1074/jbc.M115.636712
Marat AL, McPherson PS (2010) The connecdenn family, Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery. J Biol Chem 285:10627–10637. https://doi.org/10.1074/jbc.M109.050930
Marat AL, Dokainish H, McPherson PS (2011) DENN domain proteins: regulators of Rab GTPases. J Biol Chem 286:13791–13800
Marat AL, Ioannou MS, McPherson PS (2012) Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton. Mol Biol Cell 23:163–175. https://doi.org/10.1091/mbc.E11-05-0474
Minowa-Nozawa A, Nozawa T, Okamoto-Furuta K et al (2017) Rab35 GTPase recruits NDP52 to autophagy targets. EMBO J 36:2790–2807. https://doi.org/10.15252/embj.201796463
Pollard TD, Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112:453–465
Riveline D, Zamir E, Balaban NQ et al (2001) Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. J Cell Biol 153:1175–1185. https://doi.org/10.1083/jcb.153.6.1175
Shah M, Baterina OY, Taupin V, Farquhar MG (2013) ARH directs megalin to the endocytic recycling compartment to regulate its proteolysis and gene expression. J Cell Biol 202:113–127. https://doi.org/10.1083/jcb.201211110
Sleiman PMA, Flory J, Imielinski M et al (2010) Variants of DENND1B associated with asthma in children. N Engl J Med 362:36–44. https://doi.org/10.1056/NEJMoa0901867
Walseng E, Bakke O, Roche PA (2008) Major histocompatibility complex class II-peptide complexes internalize using a clathrin- and dynamin-independent endocytosis pathway. J Biol Chem 283:14717–14727. https://doi.org/10.1074/jbc.M801070200
Wu YI, Frey D, Lungu OI et al (2009) A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461:104–108. https://doi.org/10.1038/nature08241
Yang C-W, Hojer CD, Zhou M et al (2016) Regulation of T cell receptor signaling by DENND1B in TH2 Cells and allergic disease. Cell 164:141–155. https://doi.org/10.1016/j.cell.2015.11.052
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.
Funding
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).
Author information
Authors and Affiliations
Contributions
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.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that there are no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
Ethical approval
Non-human study.
Consent to participate
Non-human study.
Consent for publication
Non-human study.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
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)
Rights and permissions
About this article
Cite this article
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
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-020-01935-0