Doublecortin and JIP3 are neural-specific counteracting regulators of dynein-mediated retrograde trafficking
Abstract
Mutations in the microtubule (MT)-binding protein doublecortin (DCX) or in the MT-based molecular motor dynein result in lissencephaly. However, a functional link between DCX and dynein has not been defined. Here, we demonstrate that DCX negatively regulates dynein-mediated retrograde transport in neurons from Dcx-/y or Dcx-/y;Dclk1-/- mice by reducing dynein's association with MTs and by disrupting the composition of the dynein motor complex. Previous work showed an increased binding of the adaptor protein C-Jun-amino-terminal kinase-interacting protein 3 (JIP3) to dynein in the absence of DCX. Using purified components, we demonstrate that JIP3 forms an active motor complex with dynein and its cofactor dynactin with two dyneins per complex. DCX competes with the binding of the second dynein, resulting in a velocity reduction of the complex. We conclude that DCX negatively regulates dynein-mediated retrograde transport through two critical interactions by regulating dynein binding to MTs and by regulating the composition of the dynein motor complex.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for figure 2, 3, 4, figure 2-figure supplement 1 and table 1.
-
Doublecortin and JIP3 are neural-specific counteracting regulators of dynein-mediated retrograde traffickingDryad Digital Repository, doi:10.5061/dryad.vmcvdncwt.
Article and author information
Author details
Funding
National Natural Science Foundation of China (81971425)
- Xiaoqin Fu
National Natural Science Foundation of China (81871035)
- Peijun Li
Natural Science Foundation of Zhejiang Province (LZ09H090001)
- Peijun Li
Natural Science Foundation of Zhejiang Province (LY20H040002)
- Xiaoqin Fu
National Institutes of Health (R01GM098469)
- Arne Gennerich
National Institutes of Health (R01NS114636)
- Arne Gennerich
National Institutes of Health (RO1NS104428-01)
- Judy Shih-Hwa Liu
Brain and Behavior Research Foundation
- Judy Shih-Hwa Liu
Whitehall Foundation
- Judy Shih-Hwa Liu
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal procedures were approved by the Committee on the Ethics of Animal Experiments of Wenzhou Medical University (Permit number: wydw2019-0723).
Copyright
© 2022, Rao 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
-
- 1,256
- views
-
- 215
- downloads
-
- 10
- 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
-
- Cell Biology
Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an ‘all in one’ solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.
-
- Cell Biology
Meiotic progression requires coordinated assembly and disassembly of protein complexes involved in chromosome synapsis and meiotic recombination. Mouse TRIP13 and its ortholog Pch2 are instrumental in remodeling HORMA domain proteins. HORMAD proteins are associated with unsynapsed chromosome axes but depleted from the synaptonemal complex (SC) of synapsed homologs. Here we report that TRIP13 localizes to the synapsed SC in early pachytene spermatocytes and to telomeres throughout meiotic prophase I. Loss of TRIP13 leads to meiotic arrest and thus sterility in both sexes. Trip13-null meiocytes exhibit abnormal persistence of HORMAD1 and HOMRAD2 on synapsed SC and chromosome asynapsis that preferentially affects XY and centromeric ends. These major phenotypes are consistent with reported phenotypes of Trip13 hypomorph alleles. Trip13 heterozygous mice exhibit meiotic defects that are less severe than the Trip13-null mice, showing that TRIP13 is a dosage-sensitive regulator of meiosis. Localization of TRIP13 to the synapsed SC is independent of SC axial element proteins such as REC8 and SYCP2/SYCP3. Terminal FLAG-tagged TRIP13 proteins are functional and recapitulate the localization of native TRIP13 to SC and telomeres. Therefore, the evolutionarily conserved localization of TRIP13/Pch2 to the synapsed chromosomes provides an explanation for dissociation of HORMA domain proteins upon synapsis in diverse organisms.