The minus-end-directed microtubule motor cytoplasmic dynein powers the retrograde movement of membranous cargoes such as early endosomes, but the mechanism of motor-cargo interaction is unclear. The objective of this proposal is to dissect the interaction between dynein and early endosomes using the filamentous fungus Aspergillus nidulans as a model system. In A. nidulans, dynein, dynactin and NUDF/LIS1 accumulate at the dynamic microtubule plus ends near the hyphal tip, where they engage early endosomes for their minus-end-directed transport. Loss of dynein, dynactin or NUDF/LIS1 impairs minus-end-directed transport, causing an abnormal buildup of early endosomes at the hyphal tip. Loss of NUDF/LIS1, which affects the movement of dynein-bound endosomes rather than dynein-endosome interaction, causes an obvious dynein-dynactin-early- endosome co-localization at the hyphal tip. Remarkably, deleting the gene encoding the p25 subunit of the dynactin complex in NUDF-null cells abolishes this localization. Moreover, p25 is unique among the analyzed dynactin components in that it is required for early endosome movement but not for dynein-mediated nuclear distribution, or the microtubule plus-end accumulation of dynein and dynactin. Based on these results, we hypothesize that p25 mediates the interaction between early endosomes and the dynactin-dynein supercomplex.
Specific Aim 1 is to determine the mechanism of p25 in binding dynactin-dynein to early endosomes. We will provide direct biochemical evidence that p25 is necessary for early-endosome-dynein interaction. We will determine whether p25 is sufficient for associating with early endosomes or if any other dynactin components are required to cooperate with p25 for its interaction with early endosomes. We will also perform a structure-function analysis on p25 to determine the amino acid residues required specifically for p25-dynactin interaction as well as those required for p25-early-endosome interaction.
Specific Aim 2 is to identify proteins that bridge and/or regulate the interaction between early endosomes and dynactin-dynein. We will perform a genome-wide screen for genes that are required for early-endosome-dynein interaction (eedi). The screen criteria prevent the re-isolation of genes in the dynein-mediated nuclear distribution pathway. To date, we have collected 20 eedi mutants, and the vast majority represents non-p25 alleles. We will organize the mutants in complementation groups and clone the eedi genes. We will also use imaging and biochemical approaches to further characterize the specific roles of these EEDI proteins.

Public Health Relevance

Dynein-mediated retrograde movement of early endosomes that carry signaling molecules towards the nucleus is crucial for neuronal survival. Deficiencies in the dynein complex and the dynactin complex that interacts with dynein cause devastating neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Perry syndrome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM097580-02
Application #
8321957
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Gindhart, Joseph G
Project Start
2011-09-01
Project End
2015-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
2
Fiscal Year
2012
Total Cost
$290,700
Indirect Cost
$100,700
Name
Henry M. Jackson Fdn for the Adv Mil/Med
Department
Type
DUNS #
144676566
City
Bethesda
State
MD
Country
United States
Zip Code
20817
Qiu, Rongde; Zhang, Jun; Xiang, Xin (2018) p25 of the dynactin complex plays a dual role in cargo binding and dynactin regulation. J Biol Chem 293:15606-15619
PeƱalva, Miguel A; Zhang, Jun; Xiang, Xin et al. (2017) Transport of fungal RAB11 secretory vesicles involves myosin-5, dynein/dynactin/p25, and kinesin-1 and is independent of kinesin-3. Mol Biol Cell 28:947-961
Gershoni-Emek, Noga; Mazza, Arnon; Chein, Michael et al. (2016) Proteomic Analysis of Dynein-Interacting Proteins in Amyotrophic Lateral Sclerosis Synaptosomes Reveals Alterations in the RNA-Binding Protein Staufen1. Mol Cell Proteomics 15:506-22
Wang, Betsy; Li, Kristin; Jin, Max et al. (2015) The Aspergillus nidulans bimC4 mutation provides an excellent tool for identification of kinesin-14 inhibitors. Fungal Genet Biol 82:51-5
Xiang, Xin; Qiu, Rongde; Yao, Xuanli et al. (2015) Cytoplasmic dynein and early endosome transport. Cell Mol Life Sci 72:3267-80
Yao, Xuanli; Arst Jr, Herbert N; Wang, Xiangfeng et al. (2015) Discovery of a vezatin-like protein for dynein-mediated early endosome transport. Mol Biol Cell 26:3816-27
Pantazopoulou, Areti; Pinar, Mario; Xiang, Xin et al. (2014) Maturation of late Golgi cisternae into RabE(RAB11) exocytic post-Golgi carriers visualized in vivo. Mol Biol Cell 25:2428-43
Yao, Xuanli; Wang, Xiangfeng; Xiang, Xin (2014) FHIP and FTS proteins are critical for dynein-mediated transport of early endosomes in Aspergillus. Mol Biol Cell 25:2181-9
Zhang, Jun; Qiu, Rongde; Arst Jr, Herbert N et al. (2014) HookA is a novel dynein-early endosome linker critical for cargo movement in vivo. J Cell Biol 204:1009-26
Zhang, Jun; Twelvetrees, Alison E; Lazarus, Jacob E et al. (2013) Establishing a novel knock-in mouse line for studying neuronal cytoplasmic dynein under normal and pathologic conditions. Cytoskeleton (Hoboken) 70:215-27

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