Using Budding Yeast as a Model to Understand Dynein-Mediated Cargo Transport

Ecklund, Kari H.; Marzo, Matthew G.; Markus, Steven M.

doi:10.1007/978-1-0716-2958-1_2

Cited by 2 publications

(5 citation statements)

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“…As a more sensitive readout for dynein function, we quantified dynein-mediated spindle movements (Fig. S2B) (Ecklund et al, 2023; Marzo et al, 2019). This revealed that cells lacking Ndl1 indeed possess some dynein activity, albeit to a lesser extent than WT cells.…”

Section: Resultsmentioning

confidence: 99%

“…A single z-stack of wide-field fluorescence images was acquired for mTurquoise2- or mRuby2-Tub1, as appropriate. For the spindle oscillation assay (Ecklund et al, 2023), cells were arrested with 200 mM hydroxyurea (HU) for 2.5 hours, and then mounted on agarose pads containing HU for fluorescence microscopy. GFP-labeled microtubules (GFP-Tub1) were imaged every 10 seconds for 10 minutes.…”

Section: Methodsmentioning

confidence: 99%

“…C - G Plots of indicated parameters for spindle dynamics in kar9Δ NDL1 and kar9Δ ndl1Δ strains. Briefly, the mitotic spindles were tracked in 3-dimensions using a custom written Matlab code (Ecklund et al, 2023; Marzo et al, 2019). Dynein-mediated spindle movements were manually selected from the tracking data, from which velocity C , displacement ( D , per event; or, E , per minute), and the number of dynein-mediated spindle movements per minute F were obtained.…”

Section: Supplementary Materialsmentioning

confidence: 99%

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Conserved Roles for the Dynein Intermediate Chain and Ndel1 in Assembly and Activation of Dynein

Okada

Iyer

Lammers

et al. 2023

Preprint

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Cytoplasmic dynein, the primary retrograde microtubule transport motor within cells, must be activated for processive motility through the regulated assembly of a dynein-dynactin-adapter (DDA) complex. The interaction between dynein and dynactin was initially ascribed to the N-terminus of the dynein intermediate chain (IC) and a coiled-coil of the dynactin subunit p150Glued. However, cryo-EM structures of DDA complexes have not resolve these regions of the IC and p150Glued, raising questions about the importance of this interaction. The IC N-terminus (ICN) also interacts with the dynein regulators Nde1/Ndel1, which compete with p150Glued for binding to ICN. Using a combination of approaches, we reveal that the ICN plays critical, evolutionarily conserved roles in DDA assembly by interacting with dynactin and Ndel1, the latter of which recruits the DDA assembly factor LIS1 to the dynein complex. In contrast to prior models, we find that LIS1 cannot simultaneously bind to Ndel1 and dynein, indicating that LIS1 must be handed off from Ndel1 to dynein in temporally discrete steps. Whereas exogenous Ndel1 or p150Glued disrupts DDA complex assembly in vitro, neither perturbs preassembled DDA complexes, indicating that the IC is stably bound to p150Glued within activated DDA complexes. Our study reveals previously unknown regulatory steps in the dynein activation pathway, and provides a more complete model for how the activities of LIS1/Ndel1 and dynactin/cargo-adapters are integrated to regulate dynein motor activity.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Supplementary Materialsmentioning

confidence: 99%

See 1 more Smart Citation

Conserved Roles for the Dynein Intermediate Chain and Ndel1 in Assembly and Activation of Dynein

Okada

Iyer

Lammers

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…As a more sensitive readout for dynein function, we quantified dynein-mediated spindle movements (Supplementary Fig. 2b ) 59 , 60 . This revealed that cells lacking Ndl1 indeed possess some dynein activity, albeit to a lesser extent than WT cells.…”

Section: Resultsmentioning

confidence: 99%

“…A single z-stack of wide-field fluorescence images was acquired for mTurquoise2- or mRuby2-Tub1, as appropriate. For the spindle oscillation assay 59 , cells were arrested with 200 mM hydroxyurea (HU) for 2.5 h, and then mounted on agarose pads containing HU for fluorescence microscopy. GFP-labeled microtubules (GFP-Tub1) were imaged every 10 s for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Conserved roles for the dynein intermediate chain and Ndel1 in assembly and activation of dynein

Okada,

Iyer,

Lammers

et al. 2023

Nat Commun

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Processive transport by the microtubule motor cytoplasmic dynein requires the regulated assembly of a dynein-dynactin-adapter complex. Interactions between dynein and dynactin were initially ascribed to the dynein intermediate chain N-terminus and the dynactin subunit p150Glued. However, recent cryo-EM structures have not resolved this interaction, questioning its importance. The intermediate chain also interacts with Nde1/Ndel1, which compete with p150Glued for binding. We reveal that the intermediate chain N-terminus is a critical evolutionarily conserved hub that interacts with dynactin and Ndel1, the latter of which recruits LIS1 to drive complex assembly. In additon to revealing that the intermediate chain N-terminus is likely bound to p150Glued in active transport complexes, our data support a model whereby Ndel1-LIS1 must dissociate prior to LIS1 being handed off to dynein in temporally discrete steps. Our work reveals previously unknown steps in the dynein activation pathway, and provide insight into the integrated activities of LIS1/Ndel1 and dynactin/cargo-adapters.

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Using Budding Yeast as a Model to Understand Dynein-Mediated Cargo Transport

Cited by 2 publications

References 10 publications

Conserved Roles for the Dynein Intermediate Chain and Ndel1 in Assembly and Activation of Dynein

Conserved Roles for the Dynein Intermediate Chain and Ndel1 in Assembly and Activation of Dynein

Conserved roles for the dynein intermediate chain and Ndel1 in assembly and activation of dynein

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