Motor- and Tail-Dependent Targeting of Dynein to Microtubule Plus Ends and the Cell Cortex

Abstract

Summary Background Cytoplasmic dynein mediates spindle positioning in budding yeast by powering sliding of microtubules along the cell cortex. Although previous studies have demonstrated cortical and plus-end targeting of dynein heavy chain (Dyn1/HC), the regulation of its recruitment to these sites remains elusive. Results Here we show that separate domains of Dyn1/HC confer differential localization to the dynein complex. The N-terminal tail domain targets Dyn1/HC to cortical Num1 receptor sites, whereas the C-terminal motor domain targets Dyn1/HC to microtubule plus-ends in a Bik1/CLIP-170 and Pac1/LIS1 dependent manner. Surprisingly, the isolated motor domain blocks plus-end targeting of Dyn1/HC, leading to a dominant negative effect on dynein function. Overexpression of Pac1/LIS1, but not Bik1/CLIP-170, rescues the dominant negativity by restoring Dyn1/HC to plus-ends. In contrast, the isolated tail domain has no inhibitory effect on Dyn1/HC targeting and function. However, cortical targeting of the tail construct is more robust than full-length Dyn1/HC, and occurs independently of Bik1/CLIP-170 or Pac1/LIS1. Conclusions Our results suggest that the cortical association domain is normally masked in the full-length dynein molecule. We propose that targeting of dynein to plus-ends unmasks the tail, priming the motor for off-loading to cortical Num1 sites.

Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

“…This inability to move is consistent with the in vivo microtubule decoration phenotype ( Figure 1B; Figure 2A) and the nucleotide-insensitive microtubule-binding phenotypes ( Figure 6, A and B). Our studies were the first to examine the AAA3 Walker B E/Q mutation in a whole dynein molecule, and our results are in general agreement with previous studies that showed increased MT-binding affinity and altered dynein localization in fruit fly and budding yeast (Silvanovich et al 2003;Cho et al 2008;Markus et al 2009). For example, one study showed that UV-vanadate cleavage occurred at levels equal to wild-type strains (Silvanovich et al 2003).…”

Identification of a Novel Site in the Tail of Dynein Heavy Chain Important for Dynein Function in Vivo

“…Exactly how a tail mutation would affect dynein motor function is unclear at this point. It has been postulated that the budding yeast dynein tail may interact with the motor domain in a way that the tail would be "masked" in the presence of the motor domain (78,79). Interestingly, although the dynein regulator LIS1 binds to the motor domain at AAA3/AAA4 (26), removal of the tail appears to enhance dynein-LIS1 interaction (26,31,78).…”

“…It has been postulated that the budding yeast dynein tail may interact with the motor domain in a way that the tail would be "masked" in the presence of the motor domain (78,79). Interestingly, although the dynein regulator LIS1 binds to the motor domain at AAA3/AAA4 (26), removal of the tail appears to enhance dynein-LIS1 interaction (26,31,78). Given that the dynein HC is involved in multiple protein interactions (14 -18, 21-26), it remains to be tested whether the HC tail affects dynein function directly through regulating the motor domain or indirectly through coordinating the functions of other dynein regulators.…”

Mechanism of Nuclear Movements in a Multinucleated Cell

“…These phenotypes and the observed onset of pulling, when a cMT slides along the hyphal cortex, described by the same authors, support a mechanism for cortical pulling known from studies in S. cerevisiae. The dynein/dynactin complex is believed to be inactive while transported either by the kinesin Kip2 toward the cMT plus ends or directly recruited to plus ends from the cytoplasm (Markus et al, 2009;Roberts et al, 2014). When the cMT plus end associates with a cortical Num1 patch during sliding at the plasma membrane, the dynein/dynactin complex is off-loaded, anchored with the amino-terminal tail of Dyn1, the dynein heavy chain, to the amino-terminal binding domain of Num1, and the dynein motor is activated to eventually pull on the cMT on which it was previously hitchhiking (Lee et al, 2003;Moore et al, 2009;.…”