On and off controls within dynein–dynactin on native cargoes

Sanghavi, Paulomi; Kumar, Pankaj; Roy, Ankit; Madhusudhan, M.S.; Mallik, Roop

doi:10.1073/pnas.2103383118

Cited by 16 publications

(9 citation statements)

References 53 publications

(120 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Taken together, incorporation of lipid membranes in in vitro motility assays provides an exciting opportunity to study different facets of multi-motor transport, including but not limited to, motor-membrane interactions 24–26,46,47 and motor-microtubule interactions 48 . In the future, it will be intriguing to extend the presented vesicle assay to systematically analyze the effects of MAPs, tubulin posttranslational-modifications and cargo adaptors on the various features of intracellular cargo transport.…”

Section: Discussionmentioning

confidence: 99%

Vesicles driven by dynein and kinesin exhibit directional reversals without external regulators

D’Souza¹,

Grover²,

Monzon³

et al. 2022

Preprint

View full text Add to dashboard Cite

Intracellular transport along cytoskeletal filaments propelled by molecular motors ensures the targeted delivery of cargoes to their destinations. Such transport is rarely unidirectional but rather bidirectional, including intermittent pauses and directional reversals owing to the simultaneous presence of opposite-polarity motors. It has been unclear whether such a complex motility pattern results from the sole mechanical interplay between opposite-polarity motors or requires external regulators. Here, we addressed this outstanding question by reconstituting cargo motility along microtubules in vitro by attaching purified Dynein-Dynactin-BICD2 (DDB) and kinesin-3 (KIF16B) to large unilamellar vesicles. Strikingly, we found that this minimal system is sufficient to recapitulate runs, pauses and reversals similar to in vivo cargo motility. In our experiments, reversals were always preceded by vesicle pausing and the transport directionality could be tuned by the relative numbers of opposite-polarity motors on the vesicles. Unexpectedly, during all runs the vesicle velocity was not influenced by the presence of the opposing motors. To gain mechanistic insight into bidirectional transport, we developed a mathematical model which predicts that low numbers of engaged motors are critical to transition between runs and pauses. Taken together, our results suggest that motors diffusively anchored to membranous cargo transiently engage in a tug-of-war during pauses where stochastic motor attachment and detachment events can lead to directional reversals without the necessity of external regulators.

show abstract

Section: Discussionmentioning

confidence: 99%

Vesicles driven by dynein and kinesin exhibit directional reversals without external regulators

D’Souza¹,

Grover²,

Monzon³

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Dynactin that was over expressed in cells was found to bind strongly to and bundle MTs (Quintyne et al , 1999; Vaughan et al , 2002). In fact, dynactin-MT interactions have recently been shown to be important for increasing the on-rate of dynein onto MTs (Sanghavi et al , 2021). We propose that dynactin may assist in anchoring cargoes to the MT, allowing rapid initiation of movement upon dynein binding and will investigate this in future studies.…”

Section: Discussionmentioning

confidence: 99%

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

Tirumala

Parton

2021

Preprint

View full text Add to dashboard Cite

Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells (1). Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin and a cargo adaptor (2-5). Thereupon, dynein transports cargo towards the minus ends of microtubules. How this process of motor activation occurs is unclear, since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualise and track fluorescently tagged dynein. First, we observed that dynein that bound to the microtubule engaged in minus end-directed movement only ~30% of the time and resided on the microtubule for a short duration. Next, using high-resolution imaging in live and fixed cells, we discovered that dynactin remained persistently attached to microtubules, and endosomal cargo remained in proximity to the microtubules and dynactin. Finally, we employed two-colour imaging to visualise cargo movement effected by single motor binding. Taken together, we discovered a search strategy that is facilitated by dynein's frequent microtubule binding-unbinding kinetics: (1) in a futile event when dynein does not encounter cargo anchored in proximity to the microtubule, dynein unbinds and diffuses into the cytoplasm, (2) when dynein encounters cargo and dynactin upon microtubule binding, it moves cargo in a short run. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on reduction of dimensionality to enable minus end-directed transport in vivo.

show abstract

“…In vitro , the p150 CAP-Gly domain strongly biases the landing of assembled dynein-dynactin-cargo adapter complexes onto tyrosinated, versus detyrosinated microtubules 33, 34 , but it is not required for sustained processive movement of these complexes after initiation of movement 33 . The p150 CAP-Gly domain also enhances the dynein-dynactin interaction with microtubules under load 35, 36 . Therefore, the p150 CAP-Gly domain directly affects the landing and force production properties of the cytoplasmic dynein-dynactin complex, perhaps representing largest effect of a tubulin PTM on molecular motor output measured to date.…”

Section: Introductionmentioning

confidence: 99%

Control of Motor Landing and Processivity by the CAP-Gly Domain in the KIF13B Tail

Fan

McKenney

2022

Preprint

View full text Add to dashboard Cite

Microtubules are a major component of the eukaryotic cytoskeleton that play crucial roles in diverse cellular process. Posttranslational modifications (PTMs) of tubulin dimers regulate the dynamics and organization of microtubules, as well as the interactions between microtubules and microtubule-associated proteins (MAPs). One unique PTM that occurs on microtubules is the cyclical removal and re-addition of the C-terminal tyrosine of α-tubulin. CAP-Gly (cytoskeleton-associated protein glycine-rich) domain containing proteins specifically recognize tyrosinated microtubules, a property exploited to regulate and spatially localize diverse microtubule effectors. KIF13B is a member of the long-distance transport kinesin-3 family, and the only kinesin motor that contains a conserved C-terminal CAP-Gly domain. What role the CAP-Gly domain plays in KIF13B's motility along microtubules is unknown. Here, we investigated the interaction between KIF13B's CAP-Gly domain, and the tyrosinated C-terminal tail domain of α-tubulin. We found that KIF13B's CAP-Gly domain strongly influences the initial motor-microtubule interaction, as well as the processive motility of KIF13B along microtubules. The effect of the CAP-Gly domain on kinesin-microtubule binding is specific to the nucleotide state of the motor domain, suggesting an interplay between the N-terminal motor domain and C-terminal CAP-Gly domain underlies the KIF13B-microtubule interaction. These results reveal that specialized kinesin tail domains play active roles in the initiation and continuation of motor movement.

show abstract

On and off controls within dynein–dynactin on native cargoes

Cited by 16 publications

References 53 publications

Vesicles driven by dynein and kinesin exhibit directional reversals without external regulators

Vesicles driven by dynein and kinesin exhibit directional reversals without external regulators

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

Control of Motor Landing and Processivity by the CAP-Gly Domain in the KIF13B Tail

Contact Info

Product

Resources

About