Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody Dynab
Abstract
Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (1, 2). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (3): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that the GTP energy is primarily spent in constriction.
Article and author information
Author details
Funding
Human Frontier Science Program (CDA-0061-08)
- Aurélien Roux
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Grant 31003A_130520)
- Aurélien Roux
H2020 European Research Council (Starting Grant 311536 (2011 call))
- Aurélien Roux
Seventh Framework Programme (Marie Curie ITN grant #264399)
- Aurélien Roux
Agence Nationale de la Recherche (ANR-12-BSV2-0003-01)
- Franck Perez
Centre National de la Recherche Scientifique
- Franck Perez
Fondation pour la Recherche Médicale (DEQ20120323723)
- Franck Perez
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Christien Merrifield, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, France
Version history
- Received: January 17, 2017
- Accepted: October 11, 2017
- Accepted Manuscript published: October 12, 2017 (version 1)
- Version of Record published: October 26, 2017 (version 2)
Copyright
© 2017, Galli et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,224
- views
-
- 407
- downloads
-
- 17
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cancer Biology
- Cell Biology
Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.
-
- Cell Biology
Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+–adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD’s asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates.