Abstract
Cytoplasmic dynein is a minus-end-directed microtubule-based motor that acts at diverse subcellular sites. During mitosis, dynein localizes simultaneously to the mitotic spindle, spindle poles, kinetochores and the cell cortex. However, it is unclear what controls the relative targeting of dynein to these locations. As dynein is heavily post-translationally modified, we sought to test a role for these modifications in regulating dynein localization. We find that dynein rapidly and strongly accumulates at mitotic spindle poles following treatment with NSC697923, a small molecule that inhibits the ubiquitin E2 enzyme, Ubc13, or treatment with PYR-41, a ubiquitin E1 inhibitor. Subsets of dynein regulators such as Lis1, ZW10 and Spindly accumulate at the spindle poles, whereas others do not, suggesting that NSC697923 differentially affects specific dynein populations. We additionally find that dynein relocalization induced by NSC697923 or PYR-41 can be suppressed by simultaneous treatment with the non-selective deubiquitinase inhibitor, PR-619. However, we did not observe altered dynein localization following treatment with the selective E1 inhibitor, TAK-243. Although it is possible that off-target effects of NSC697923 and PYR-41 are responsible for the observed changes in dynein localization, the rapid relocalization upon drug treatment highlights the highly dynamic nature of dynein regulation during mitosis.
Highlights
Dynein is a AAAþ ATPase that uses the energy from ATP hydrolysis to walk along a microtubule [1]
Treatment with a low dose of nocodazole resulted in a similar disruption of the mitotic spindle, but did not induce dynein accumulation at the spindle poles, suggesting that the NSC697923-induced effects on dynein localization are independent of its effects on the mitotic spindle
Co-staining for NuMA, one of the outermost proteins at the spindle pole [20], revealed that NuMA localization is unaffected by NSC697923 treatment and that the spindle pole-localized dynein was slightly internal to the localization of NuMA
Summary
Dynein is a AAAþ ATPase that uses the energy from ATP hydrolysis to walk along a microtubule [1]. Dynein localizes to diverse sites within the cell, including the mitotic spindle, the centrosomes, the nuclear envelope, the kinetochores and the cell cortex, where it performs numerous roles in interphase and mitosis [2]. This subcellular targeting is accomplished in part by the association of the dynein complex with various regulatory and adaptor proteins, including the dynactin complex, NuMA, Spindly, Bicaudal D2, Nde ( known as NudE) and others [3]. We use a small molecule inhibitor of Ubc, as well as inhibitors targeting other components of the ubiquitin conjugation machinery to reveal the highly dynamic nature of dynein localization in mitotic cells
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