The human cytoplasmic dynein interactome reveals novel activators of motility

Abstract

In human cells, cytoplasmic dynein-1 is essential for long-distance transport of many cargos, including organelles, RNAs, proteins, and viruses, towards microtubule minus ends. To understand how a single motor achieves cargo specificity, we identified the human dynein interactome by attaching a promiscuous biotin ligase (‘BioID’) to seven components of the dynein machinery, including a subunit of the essential cofactor dynactin. This method reported spatial information about the large cytosolic dynein/dynactin complex in living cells. To achieve maximal motile activity and to bind its cargos, human dynein/dynactin requires ‘activators’, of which only five have been described. We developed methods to identify new activators in our BioID data, and discovered that ninein and ninein-like are a new family of dynein activators. Analysis of the protein interactomes for six activators, including ninein and ninein-like, suggests that each dynein activator has multiple cargos.DOI: http://dx.doi.org/10.7554/eLife.28257.001

Parkinson’s Disease-linked LRRK2 structure and model for microtubule interaction

“…Microtubule association was monitored by measuring the effect of LRRK2 RCKW on the movement of microtubule-based motors. We used a truncated dimeric human kinesin-1, Kif5B ("kinesin" here) 32 , which moves towards the plus ends of microtubules, and activated human cytoplasmic dynein-1/ dynactin/ ninein-like complexes ("dynein" here) 33 , which move in the opposite direction. Using single-molecule in vitro motility assays (Fig.…”

Lis1 promotes the formation of maximally activated cytoplasmic dynein-1 complexes

“…pDyn1, pDyn2 and the pFastBac plasmid with codonoptimized human full-length Lis1 (PAFAH1B1) fused to an amino-terminal His-ZZ-TEV tag and pFastBac containing human dynein monomer (amino acids 1320-4646 of DYNC1H1) were gifts from Andrew Carter (LMB-MRC, Cambridge, UK). BicD2 constructs were amplified from a human cDNA library generated from RPE1 cells and the other activating adaptor constructs were obtained as described previously 34 . Activating adaptors were fused to a ZZ-TEV-HaloTag (Promega) on the amino-terminus and inserted into a pET28a expression vector.…”

“…Dynactin was purified from stable HEK293-T cell lines expressing p62-Halo-3xFlag as described previously 34 . Briefly, frozen pellets collected from 160 x 15cm plates were resuspended in 80 mL of Dynactin-lysis buffer (30 mM HEPES [pH 7.4], 50 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 1 mM DTT, 10% (v/v) glycerol) supplemented with 0.5 mM Mg-ATP, 0.2% Triton X-100 and 1 cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) per 50 mL and rotated slowly for 15 min.…”

“…To determine how Lis1 regulates activated human dynein complexes we purified human dynein 3 and Lis1 from insect cells, dynactin from human HEK-293T cells 34 , and the human activating adaptors BicD2, Hook3, and Ninl from E. coli ( Supplementary Fig. 1a).…”

“…1a). Since some activating adaptors are known to be autoinhibited 35 , we chose to use well-characterized carboxy-terminal truncations of BicD2, Hook3 and Ninl 3,4,34 (Fig. 1c).…”