Vesicles driven by dynein and kinesin exhibit directional reversals without external regulators
Posted on: 25 January 2023
Preprint posted on 28 September 2022
Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-023-42605-8
To move or not to move: A novel in vitro assay successfully recreates intracellular bidirectional cargo transport, laying the foundations for further understanding
Selected by Divya PathakCategories: biochemistry, bioengineering, biophysics, cell biology
Background:
The cell is a very busy environment, wherein hundreds of molecules and vesicles are being transported at any given moment. Most intracellular cargoes show association with multiple types of motors raising the question– how is the directionality of cargo determined? In this preprint, D’Souza and colleagues try to answer this question.
Regulated intracellular transport is critical for proper cellular functioning, affecting processes like cell division, the positioning of organelles, and organelle transport. Intracellular transport can be long range using microtubules and short range using actin filaments. The microtubule-based motors Kinesin and Dynein carry cargo in opposite directions. What is surprising is that most intracellular cargo shows simultaneous association with multiple Kinesin and Dynein motors. Without a specific regulatory mechanism governing cargo transport, it is likely for the cargo to get stuck in limbo with both the motors engaged and undergoing futile ATPase cycles with no net movement. Alternatively, the cargo might randomly switch direction and end up in a place it’s not destined for. Intracellular cargoes like mitochondria, peroxisomes, endosomes, melanosomes and lipid droplets often move bidirectionally and regularly change direction en route to their final destinations.
Analyzing motor compositions on in vivo cargoes has been experimentally challenging not only due to difficulties in purifying a specific intracellular cargo type, but also the variation in motor-cargo associations over time. For most of the bidirectionally moving cargo, there are three models that can apply1. (1) Tug-of war, in which two oppositely directed motors simultaneously generate force and engage in a physical tug-of-war whose outcome decides the cargo’s direction2,3,4. In this model, changing the number of motors or their organization on cargo dictates the cargo’s net movement. (2) Exclusionary presence through motor association with and dissociation from the cargo. Adapter proteins on the cargo can interact with opposing motors and, depending on their interacting partner, the cargo either moves anterograde or retrograde5. This model prevents the futile use of ATP and ensures coordinated cargo movement without interference from the opposing motor. This model is also supported by the observation that motors exist in inactive states and that this autoinhibition is released upon cargo binding. (3) Activation by opposing motor, in which case the driving motor does not necessarily interfere, but rather activate the opposing motor by sheer stretching/force. It has been shown that inactivating one motor results in reduced motion by the opposing motor as well; disruption of either kinesin or dynein inhibits opposite-polarity vesicle transport6,7.
Until now, most of our insights into bidirectional transport have come from motors recruited to non-vesicular assemblies like DNA scaffolds, quantum dots, coverslips and beads4,8,13,14. These don’t recapitulate native cargo mostly due to the missing complexity of the lipid bilayer of the vesicular cargo9,10,11. Also, most of the reconstituted experiments couldn’t recapitulate the fast transport and directional reversals, and instead showed slowing down of motion and long stationary pauses. This suggested and provided increasing evidence for the exclusionary model in which dynein and kinesin are not active simultaneously on the same cargo. As most cargoes showed association with opposing motors, a model emerged in which external regulators or adaptors coordinate the activity of opposing motors to prevent their simultaneous activation.
Key findings:
- In this study D’souza et al for the very first time successfully reconstituted bidirectional transport in vitro on liposomes that resemble the majority of the intracellular cargo (Fig. 1). They targeted purified motors – Dynein-Dynactin-BicD2 (DDB) complex and kinesin (KIF 16B) – to 132nm liposomes and could recapitulate the in vivo transport of cargoes including the fast unidirectional runs, intermittent pauses and directional reversals in the absence of any external regulatory protein.
- Cargo (liposome) pausing was enhanced when opposing motors were present. Not all pauses were the result of a tug-of-war as vesicles with only one kind of motor – either kinesin or dynein – exhibited processive runs with frequent pauses. The researchers titrated increasing amounts of kinesin (KIF16B) for the same amount of dynein (DDB) and could shift the balance in favor of kinesin-driven runs (Fig. 2). Also, in the pause phase, they observed elongation of cargo along the microtubule as seen for purified endosomes from Dictyostelium. These observations support the tug-of-war model.
- The successful reconstitution of bidirectional transport reported in this preprint also suggests that the presence of opposing motors doesn’t hinder the velocity of active driving motors nor slow down transport, reflected by the smooth unidirectional runs seen on DDB-KIF16B liposomes. Instead, presence of opposing motors results in longer pauses marked by the motors engaging in a tug-of war, followed by stochastic fluctuations that can lead to directional reversals without the need of external regulators.
- The researchers simulated different motor configurations to explain the bidirectional cargo transport observed experimentally. Their simulation predicts that 3 active motors drive a processive run and that the cargo pauses when 2 of those active motors detach along with the simultaneous attachment of one opposing motor. Pauses were characterized by a force balance between two active motors of opposing polarity and 4 inactive opposing motors (2 of each kind) to stabilize it. Transitioning from a pause to an active run in either direction is initiated by the stochastic detachment of the active motor of the opposing polarity or the attachment of two active motors. For cargo being driven by a low number of engaged motors, their simulation predicts that stochastic attachment or detachment of single motors determines the direction of cargo.
Why I chose this preprint?
Richard Feynman once said “What I cannot create, I do not understand”. Developing, or rather creating, this assay to recapitulate bidirectional motility marks the first attempt at improving our understanding of intracellular transport. This assay provides a foundation to build complexity in the form of adapter proteins, bilayer lipid composition etc. to dissect their role in intracellular transport. This is the first time that bidirectional motility has been reconstituted on liposomes which recapitulates the endogenous intracellular cargo. This assay marks the beginning of our ability to understand in vivo transport by trying to create it in vitro.
References:
- Hancock, W. Bidirectional cargo transport: moving beyond tug of war. Nat Rev Mol Cell Biol (2014); 15, 615–628
- Soppina V, Rai AK, Ramaiya AJ, Barak P, Mallik R. Tug-of-war between dissimilar teams of microtubule motors regulates transport and fission of endosomes. PNAS (2009); 106(46):19381-6
- Rezaul, K., Gupta, D., Semenova, I., Ikeda, K., Kraikivski, P., Yu, J., Cowan, A., Zaliapin, I. and Rodionov, V. Engineered Tug-of-War Between Kinesin and Dynein Controls Direction of Microtubule Based Transport In Vivo. Traffic (2016); 17: 475-486
- Derr ND, Goodman BS, Jungmann R, Leschziner AE, Shih WM, Reck-Peterson SL. Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold. (2012); 338(6107):662-5
- Fenton, A.R., Jongens, T.A. & Holzbaur, E.L.F. Mitochondrial adaptor TRAK2 activates and functionally links opposing kinesin and dynein motors. Nat Commun (2021); 12: 4578
- Steven P. Gross, Michael A. Welte, Steven M. Block, Eric F. Wieschaus; Coordination of opposite-polarity microtubule motors . J Cell Biol (2002); 156 (4): 715–724
- Shabeen Ally, Adam G. Larson, Kari Barlan, Sarah E. Rice, Vladimir I. Gelfand; Opposite-polarity motors activate one another to trigger cargo transport in live cells. J Cell Biol (2009); 187 (7): 1071–1082
- Toba S, Watanabe TM, Yamaguchi-Okimoto L, Toyoshima YY, Higuchi H. Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein. PNAS (2006);103(15):5741-5
- Grover R, Fischer J, Schwarz FW, Walter WJ, Schwille P, Diez S. Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity. PNAS (2016);113(46):E7185-E7193
- Li Q, Tseng KF, King SJ, Qiu W, Xu J. A fluid membrane enhances the velocity of cargo transport by small teams of kinesin-1. J Chem Phys. (2018);148(12):123318
- Nelson SR, Trybus KM, Warshaw DM. Motor coupling through lipid membranes enhances transport velocities for ensembles of myosin Va. PNAS (2014);111, 3986–3995
- Gina A. Monzon, Lara Scharrel, Ashwin D’Souza, Verena Henrichs, Ludger Santen, Stefan Diez; Stable tug-of-war between kinesin-1 and cytoplasmic dynein upon different ATP and roadblock concentrations. J Cell Sci (2020); 133 (22): jcs249938
- Belyy, V., Schlager, M., Foster, H. et al. The mammalian dynein–dynactin complex is a strong opponent to kinesin in a tug-of-war competition. Nat Cell Biol (2016);18, 1018–1024
- Allison M Gicking, Tzu-Chen Ma, Qingzhou Feng, Rui Jiang, Somayesadat Badieyan, Michael A Cianfrocco, William O Hancock Kinesin-1, -2, and -3 motors use family-specific mechanochemical strategies to effectively compete with dynein during bidirectional transport. eLife(2022); 11:e82228
doi: https://doi.org/10.1242/prelights.33529
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