Cargo adapter-mediated dynein activation needs LIS1 in Aspergillus nidulans

R. Qiu1 , J. Zhang1 , A. Osmani2 , S. Osmani2 , X. Xiang1

Author address: 

1) Department of Biochemistry and Molecular Biology, Uniformed Services University - F. Edward Hébert School of Medicine, Bethesda, Maryland; 2) Department of Molecular Genetics, The Ohio State University, Columbus, Ohio.

Abstract: 

 Cytoplasmic dynein-mediated intracellular cargo transport needs the dynactin complex and cargo adapters, and recent work showed that cargo adapters and dynactin activate the in vitro motility of mammalian dynein. Studies are needed to examine this process in vivo and how other proteins may affect it. In the filamentous fungus Aspergillus nidulans, HookA is an early endosomal adapter for dynein. Here we overexpressed HookA missing its C-terminal early endosome-binding site (?C-HookA), which allowed us to observe dynein-dynactin occupied by cytosolic adapters but not linked to the cargo. In contrast to normal cells exhibiting the microtubule plus-end comets formed by the accumulation of GFP-labeled dynein and dynactin, cells with overexpressed ?C-HookA do not exhibit plus-end comets. Instead, signals of GFP-labeled dynein or dynactin were seen to localize along microtubules, as a single dot on nuclei or concentrated on septa, which contain active microtubule-organizing centers. These observations are consistent with activated dynein being relocated from the plus ends to the minus ends of microtubules. We used this system to study the dynein regulator LIS1 (NudF in A. nidulans) whose mechanism of action is intriguing because it could be either a positive or a negative regulator of dynein motility in vitro depending on different factors in different systems including the nucleotide state of AAA3 of yeast dynein and whether cargo adapters are present for mammalian dynein. In A. nidulans and Ustilago maydis, LIS1 is not needed for the microtubule plus-end accumulation of dynein or dynactin, but it is critical for the initiation of dynein-mediated early endosome transport. By introducing three different nudF loss-of-function mutant alleles into the strain with overexpressed ?C-HookA, we found that loss of NudF/LIS1 function almost totally prevented dynein from leaving the microtubule plus ends, as evidenced by the bright plus-end comets in mutant cells. This effect on the cargo-free dynein in vivo is striking and somewhat surprising because LIS1 was thought to only affect the movement of heavy cargoes in cells. While we are in the process of dissecting whether NudF/LIS1 affects the HookA-dynein-dynactin complex formation, our current data indicate that NudF/LIS1 is critical for cargo adapterinitiated dynein departure from microtubule plus ends in vivo

2019

abstract No: 

128

Full conference title: 

30th Fungal Genetics Conference 2019
    • Fungal Genetics Conference 30th (2019)