Single-molecule imaging of cytoplasmic dynein in cellulo reveals the mechanism of motor activation and cargo capture

Tirumala, Nireekshit Addanki; Redpath, Gregory; Kapoor‐Kaushik, Natasha; Ariotti, Nicholas; Dolai, Pritha; Kumar, K. Vinod; Ananthanarayanan, Vaishnavi

doi:10.1016/j.bpj.2021.11.2703

Cited by 2 publications

(8 citation statements)

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“…During several minutes of filming, the vast majority of the dual-labelled punctate structures exhibited short, oscillatory movements (Movie S1). This behaviour is reminiscent of the behaviour of early endosomal cargoes for microtubule motors in HeLa cells, which can take tens of minutes to traverse the cytoplasm (Flores-Rodriguez et al, 2011; Zajac et al, 2013; Tirumala et al, 2022). However, a small fraction of AP1σ1 puncta exhibited unidirectional movements of between 1.5 and 10 μm both towards and away from the perinuclear region with a mean instantaneous velocity of 260 ± 4 nm/s (± SEM; 22 particles) (Figure 3D and Movie S2).…”

Section: Resultsmentioning

confidence: 85%

HEATR5B associates with dynein-dynactin and selectively promotes motility of AP1-bound endosomal membranes

Madan

Albacete

et al. 2023

Preprint

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The dynein motor complex mediates polarised trafficking of a wide variety of organelles, intracellular vesicles and macromolecules. These functions are dependent on the dynactin complex, which helps recruit cargoes to dynein's tail region and activates motor movement. How dynein and dynactin orchestrate trafficking of diverse cargoes is unclear. Here, we identify HEATR5B, an interactor of the AP1 clathrin adaptor complex, as a novel player in dynein-dynactin function. HEATR5B is one of several proteins recovered in a biochemical screen for proteins whose association with the human dynein tail complex is augmented by dynactin. We show that HEATR5B binds directly to the dynein tail and dynactin and stimulates motility of AP1-associated endosomal membranes in human cells. We also demonstrate that the HEATR5B homologue in Drosophila is an essential gene that promotes dynein-based transport of AP1-bound membranes to the Golgi apparatus. As HEATR5B lacks the coiled-coil architecture typical of dynein adaptors, our data point to a non-canonical process orchestrating motor function on a specific cargo. We additionally show that HEATR5B promotes association of AP1 with endosomal membranes in a dynein-independent manner. Thus, HEATR5B co ordinates multiple events in AP1-based trafficking.

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Section: Resultsmentioning

confidence: 85%

HEATR5B associates with dynein-dynactin and selectively promotes motility of AP1-bound endosomal membranes

Madan

Albacete

et al. 2023

Preprint

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“…The use of an endogenous HaloTag on dynein thus allows us to image much longer runs than observed previously 4,6 . However, with this setup we were unable to conclusively determine if the dyneins we observed had travelled the whole length of the axon.…”

Section: Visualising Single Dynein and Dynactin Molecules In Ineuronsmentioning

confidence: 95%

“…To ask whether our labelled dyneins are present as isolated molecules or as clusters we performed a photobleaching analysis (Fig 2A , B, C, Video 5,6). At first this proved difficult due the movement of the dynein spots in and out of the focal plane.…”

Section: Visualising Single Dynein and Dynactin Molecules In Ineuronsmentioning

confidence: 99%

“…In contrast, a recent study in HeLa cells, used highly inclined and laminated optical sheet (HILO) imaging to visualise single molecules of GFP-tagged dynein heavy chain. This suggested dynein has a short residence time on microtubules and only undergoes short-range (1-2 µm) movements 6 , leading to the conclusion that long-range transport is achieved by a constant exchange of motile dynein complexes. These studies raise the question of how far dynein motors move in the axon.…”

Section: Introductionmentioning

confidence: 99%

“…This enabled us to endogenously tag dynein and its associated proteins, avoiding any potential artefacts of overexpression 8 . We used HILO imaging 6,9 combined with SNAP-tag and HaloTag-linked fluorophores for improved photostability. This allowed live imaging of dynein molecules in human neurons at a near-single molecule regime.…”

Section: Introductionmentioning

confidence: 99%

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Dynein and dynactin move long-range but are delivered separately to the axon tip

Fellows

Bruntraeger

Burgold

et al. 2023

Preprint

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The microtubule motor dynein drives retrograde transport from the axon tip back to the cell body and is essential for neuronal survival. Its function requires its cofactor dynactin and regulators LIS1 and NDEL1. However, it is unclear if all dynein components can travel along the axon, how far they move and whether they do so together. Here, we use neuron-inducible (NGN2-OPTi-OX) human-stem-cell lines to endogenously tag dynein components and track them under a near single molecule regime. In the retrograde direction dynein and dynactin can move the entire > 500 um length of the axon in one go. Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with initiation of transport. Intriguingly, in the anterograde direction dynein/LIS1 move faster than dynactin/NDEL1 implying they can travel on different cargos. Therefore, neurons ensure efficient transport by keeping dynein/dynactin on cargos over long distances, but keeping them separate until their function is required.

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Single-molecule imaging of cytoplasmic dynein in cellulo reveals the mechanism of motor activation and cargo capture

Cited by 2 publications

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HEATR5B associates with dynein-dynactin and selectively promotes motility of AP1-bound endosomal membranes

HEATR5B associates with dynein-dynactin and selectively promotes motility of AP1-bound endosomal membranes

Dynein and dynactin move long-range but are delivered separately to the axon tip

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