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1. Multivalency, autoinhibition, and protein disorder in the regulation of interactions of dynein intermediate chain with dynactin and the nuclear distribution protein

   https://doi.org/10.7554/eLife.80217  

   Jara, Kayla A; Loening, Nikolaus M; Reardon, Patrick N; Yu, Zhen; Woonnimani, Prajna; Brooks, Coban; Vesely, Cat H; Barbar, Elisar J (November 2022, eLife)

   Motor proteins are the freight trains of the cell, transporting large molecular cargo from one location to another using an array of ‘roads’ known as microtubules. These hollow tubes are oriented, with one extremity (the plus-end) growing faster than the other (the minus-end). While over 40 different motor proteins travel towards the plus-end of microtubules, just one is responsible for moving cargo in the opposite direction. This protein, called dynein, performs a wide range of functions which must be carefully regulated, often through changes in the shape and interactions of various dynein segments. The intermediate chain is one of the essential subunits that form dynein, and it acts as a binding site for a range of molecular actors. In particular, it connects the three other dynein subunits (known as the light chains) to the dynein heavy chain containing the motor domain. It also binds to two non-dynein proteins: NudE, which helps to organise microtubules, and the p150 Glued region of dynactin, a protein required for dynein activity. Despite their distinct roles, p150 Glued and NudE attach to the same region of the intermediate chain, a highly flexible ‘unstructured’ segment which is difficult to study. How the binding of p150 Glued and NudE is regulated has therefore remained unsolved. In response, Jara et al. decided to investigate how the three dynein light chains may help to control interactions between the intermediate chain and non-dynein proteins. They used more stable versions of dynein, NudE and dynactin (from a fungus that grows at high temperatures) to produce the various subcomplexes formed by the intermediate chain, the three dynein light chains, and parts of p150 Glued and NudE. A suite of biophysical techniques was applied to study these structures, as they are challenging to capture using traditional approaches. This revealed that the unstructured region of the intermediate chain can fold back on itself, bringing together its two extremities; such folding blocks the p150 Glued and NudE binding site. This obstruction is cleared when the light chains bind to the intermediate chain, demonstrating how these three subunits can regulate dynein activity. In humans, mutations in dynein are associated with a range of serious neurological and muscular diseases. The work by Jara et al. brings new insight into the way this protein works; more importantly, it describes how to combine several biophysical techniques to study non-structured proteins, offering a blueprint that is likely to be relevant for a wide range of scientists. 

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2. Cargo adaptor identity controls the mechanism and kinetics of dynein activation

   https://doi.org/10.1016/j.jbc.2025.108358  

   Gillies, John P; Little, Saffron R; Siva, Aravintha; Hancock, William O; DeSantis, Morgan E (April 2025, Journal of Biological Chemistry)

   Not AvailableCytoplasmic dynein-1 (dynein), the primary retrograde motor in most eukaryotes, supports the movement of hundreds of distinct cargos, each with specific trafficking requirements. To achieve this functional diversity, dynein must bind to the multi-subunit complex dynactin and one of a family of cargo adaptors to be converted into an active, processive motor complex. Very little is known about the dynamic processes that promote the formation of this complex. To delineate the kinetic steps that lead to dynein activation, we developed a single-molecule fluorescence assay to visualize the real-time formation of dynein-dynactin-adaptor complexes in vitro. We found that dynactin and adaptors bind dynein independently rather than cooperatively. We also found that different dynein adaptors promote dynein-dynactin-adaptor assembly with dramatically different kinetics, which results in complex formation occurring via different assembly pathways. Despite differences in association rates or mechanism of assembly, all adaptors tested can generate a population of tripartite complexes that are very stable. Our work provides a model for how modulating the kinetics of dynein-dynactin-adaptor binding can be harnessed to promote differential dynein activation and reveals a new facet of the functional diversity of the dynein motor. 

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3. Ndel1 disfavors dynein–dynactin–adaptor complex formation in two distinct ways

   https://doi.org/10.1016/j.jbc.2023.104735  

   Garrott, Sharon R.; Gillies, John P.; Siva, Aravintha; Little, Saffron R.; El Jbeily, Rita; DeSantis, Morgan E. (June 2023, Journal of Biological Chemistry)

   Dynein is the primary minus-end-directed microtubule motor protein. To achieve activation, dynein binds to the dynactin complex and an adaptor to form the "activated dynein complex." The protein Lis1 aids activation by binding to dynein and promoting its association with dynactin and the adaptor. Ndel1 and its paralog Nde1 are dynein- and Lis1-binding proteins that help control dynein localization within the cell. Cell-based assays suggest that Ndel1-Nde1 also work with Lis1 to promote dynein activation, although the underlying mechanism is unclear. Using purified proteins and quantitative binding assays, here we found that the C-terminal region of Ndel1 contributes to dynein binding and negatively regulates binding to Lis1. Using single-molecule imaging and protein biochemistry, we observed that Ndel1 inhibits dynein activation in two distinct ways. First, Ndel1 disfavors the formation of the activated dynein complex. We found that phosphomimetic mutations in the C-terminal domain of Ndel1 increase its ability to inhibit dynein-dynactin-adaptor complex formation. Second, we observed that Ndel1 interacts with dynein and Lis1 simultaneously and sequesters Lis1 away from its dynein-binding site. In doing this, Ndel1 prevents Lis1-mediated dynein activation. Together, our work suggests that in vitro, Ndel1 is a negative regulator of dynein activation, which contrasts with cellular studies where Ndel1 promotes dynein activity. To reconcile our findings with previous work, we posit that Ndel1 functions to scaffold dynein and Lis1 together while keeping dynein in an inhibited state. We speculate that Ndel1 release can be triggered in cellular settings to allow for timed dynein activation. 

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4. TRAK adaptors regulate the recruitment and activation of dynein and kinesin in mitochondrial transport

   https://doi.org/10.1038/s41467-023-36945-8  

   Canty, John T.; Hensley, Andrew; Aslan, Merve; Jack, Amanda; Yildiz, Ahmet (December 2023, Nature Communications)

   Abstract Mitochondrial transport along microtubules is mediated by Miro1 and TRAK adaptors that recruit kinesin-1 and dynein-dynactin. To understand how these opposing motors are regulated during mitochondrial transport, we reconstitute the bidirectional transport of Miro1/TRAK along microtubules in vitro. We show that the coiled-coil domain of TRAK activates dynein-dynactin and enhances the motility of kinesin-1 activated by its cofactor MAP7. We find that TRAK adaptors that recruit both motors move towards kinesin-1’s direction, whereas kinesin-1 is excluded from binding TRAK transported by dynein-dynactin, avoiding motor tug-of-war. We also test the predictions of the models that explain how mitochondrial transport stalls in regions with elevated Ca 2+ . Transport of Miro1/TRAK by kinesin-1 is not affected by Ca 2+ . Instead, we demonstrate that the microtubule docking protein syntaphilin induces resistive forces that stall kinesin-1 and dynein-driven motility. Our results suggest that mitochondrial transport stalls by Ca 2+ -mediated recruitment of syntaphilin to the mitochondrial membrane, not by disruption of the transport machinery. 

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5. NMR approaches to identify transient structure and interactions of intrinsically disordered dynein intermediate chain

   https://doi.org/10.1016/j.jmb.2025.169380  

   Loening, Nikolaus M; Jara, Kayla A; Barbar, Elisar J (August 2025, Journal of Molecular Biology)

   Nuclear magnetic resonance (NMR) spectroscopy is widely recognized for its ability to provide atomic-level resolution of structures and interactions in intrinsically disordered proteins (IDPs). However, its application is often limited when studying large proteins that contain both structured and disordered regions. This challenge arises due to the broad peaks exhibited by structured regions in such proteins, which result from local compaction and restricted motions, complicating spectral analysis. Additionally, broadening in IDP complexes caused by exchange between free and bound states and/or the large size of the bound state, further obscures NMR signals and hinders the mapping of interaction sites. Moreover, IDPs are highly sensitive to proteolytic cleavage, necessitating careful handling and optimization during expression, purification, and data collection. In this study, we demonstrate how we successfully overcame these hurdles using examples from our work on the N-terminal region of the dynein intermediate chain (IC), which contains both ɑ-helical and intrinsically disordered regions. By employing paramagnetic relaxation enhancement (PRE) NMR to probe conformational dynamics, water-amide chemical exchange to measure solvent accessibility, and saturation transfer difference (STD) NMR to map specific interactions with p150^Glued and Nudel, we identified novel transient structures and interaction networks within IC. Our findings highlight the utility of these advanced NMR techniques in elucidating the dynamic behavior of IDPs and their complexes, providing valuable insights into their structural and functional roles. 

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