LIS1 a Critical Regulator of Dynein Complex Formation and Function
Background - Dynein Complex Structure
Movement of cargo towards the minus-end of microtubules is performed by cytoplasmic dynein-1 that is in a complex with the regulatory factor dynactin and cargo adaptor proteins(reviewed in 1). Mutations in dynein, its regulatory proteins, and cargo adaptor proteins have all been associated with severe neurologic disorders, and recent studies showed that the impact the mutation has on the molecular pathology directly correlates with the severity of the neurologic disease2. DDA complexes are very large, which has traditionally made it difficult to study, and deciphering critical functions each component has on the complex has also proven challenging. The advancement in single-molecule imaging and structural studies via cryo-electron microscopy (cryo-EM) produced a flurry of information regarding the DDA complex structure and function. In 2014, two studies showed that mammalian dynein had minimal movement along microtubules on its own, which was striking relative to much of the earlier studies that were commonly performed with yeast dynein3 or on solid surfaces (glass coverslips)4; importantly, upon mammalian dynein binding to mammalian dynactin and cargo adaptors, significant movement was observed5,6. Cryo-EM studies were performed to identify the autoinhibitory state of dynein, which is known as the phi particle7. In 2018, the Carter group utilized elegant Cryo-EM studies to show that dynactin can bind one or two dynein proteins depending on the adaptor protein that is in the DDA complex; importantly, the double-bound dynein had much higher force and speed providing insight into how different adaptors produce profound regulatory effects on cargo movement8. More recently, it was shown that DDA complexes with two dynein proteins contained two adaptors, which were asymmetrically bound to dynactin9. Collectively, these and other studies point to a DDA complex that is highly regulated and involves multiple precise interactions for proper function. In addition to understanding the structure and function of the DDA complex, there has been significant work in understanding how regulatory factors like the LIS1 protein affect dynein motor movement. This newsletter will discuss some of these recent findings and how they expand our understanding of LIS1 in the DDA complex and beyond.
LIS1 Regulation of the DDA and Movement on Tubulin
LIS1 is a critical accessory factor that was first identified as the protein responsible for the neurodevelopmental disorder, Lissencephaly (smooth brain). It was shown to interact with dynein over two decades ago(reviewed in 10), and it’s the only dynein accessory factor that can bind directly to dynein’s motor domain; yet, its precise effect on dynein has remained largely unknown. In 2020, several groups identified a key function for LIS1, which was to bind to auto-inhibited dynein and orient it in an open confirmation to promote its binding to dynactin11-13. While these studies were performed in different organisms they similarly showed that LIS1 bound directly to dynein and destabilized the phi particle to promote a DDA complex that showed enhanced movement11-13. More recently, it was determined that LIS1 binding to dynein produced an intermediate state known as “chi”, which exposed specific dynein regions to promote dynactin binding14.
Importantly, the previous LIS1 studies also investigated whether the protein remained bound while the DDA complex moved along microtubules. The two reports where mammalian dynein was used showed that LIS1 did not need to remain bound, and in cases where the complex still contained LIS1, it moved with lower velocities11,12. Similar studies in yeast support these findings that when LIS1 remains bound to tubulin it causes the dynein to move slower15. Ton et al. confirmed these previous studies by showing that LIS1 preferentially bound to DDA that was not also bound to tubulin, and utilized cryo-EM studies showing differences of DDA bound to microtubules in the presence or absence of LIS116. The Carter group recently solved a 4 MDa structure of LIS1 with a dynein, dynactin, and JIP3 (adaptor) complex associated with microtubules17. The findings support several of the previously published papers while providing key insight into several conflicting reports. Specifically, in addition to LIS1’s function to move dynein into an open state, it also binds both dynein and dynactin to help facilitate interaction between these two proteins17. They also provided information on LIS1 binding to dynein in the presence of microtubules. Collectively, these new structural studies shed light on the many functions that LIS1 performs in the forming DDA complex and its impact on cargo movement along microtubules.
Figure Legend: Schematic diagram of Lis1/dynein complex interacting with F-actin and a microtubule. (Orange) – Lis1, (Red/Pink) – Dynein, (Green) – F-actin filament, (Blue/Purple) – Microtubule.
LIS1 Regulation by Nde1/Ndel1
Given the importance of LIS1 in DDA complex formation, there has also been an interest in elucidating the role of the LIS1 binding protein Ndel1. Ndel1 is a LIS1 and dynein binding protein that was shown to both inhibit and activate dynein depending on the cellular conditions(reviewed in 18). While the loss of LIS1 suppressed Ndel1 effects on dynein, the depletion of Ndel1 did not suppress LIS1 effects on dynein which suggested that Ndel1 effects on dynein were LIS1 dependent. Live cell studies suggested that Ndel1 functions by tethering the LIS1 and dynein proteins together19. In recent work by the Yildiz group, Ndel1 recruits LIS1 to the autoinhibited dynein and helps to promote DDA formation20. In concert with this finding the McKenney group showed that the intermediate chain N-terminal of dynein specifically interacts with Ndel1 as an important step in promoting the interaction between LIS1 and dynein21. Understanding the interplay between LIS1 and Ndel1 is critical as it has been shown to regulate critical biological processes like mitochondrial movement in neurons22 and FGF receptor intracellular trafficking23.
LIS1 Regulation by Other Mechanisms
Understanding alternative mechanisms of LIS1 is of significant interest and several post-transcriptional and post-translational mechanisms have emerged. Alonso et al. showed that the yeast homolog of LIS1 can directly interact with the SUMOylation machinery and could be both SUMOylated or ubiquitinated in their studies24. More recently, the CUL4B-based E3 ubiquitin ligase was shown to regulate mitosis and mechanistically was shown to directly interact with LIS125. MicroRNA post-transcriptional regulation has also been shown to regulate LIS1 levels. Both MiR-38026 and MiR-14427 have been shown to regulate cholangiocarcinoma cellular function through LIS1 silencing. As LIS1 plays such a critical role in dynein regulation, it will be interesting to see if subtle, yet impactful post-translational regulation can fine-tune cargo movement through LIS1 regulation.
Future Perspectives
The flurry of recent studies has identified the multi-faceted role that LIS1 plays in the formation of the DDA, and how it impacts attachment and movement along the microtubules. These studies highlight LIS1 and other adaptors produce unique structures that produce distinct movement of cargo along microtubules, and it will be interesting to determine why certain adaptors form their preferred DDA formations, how this affects the cargo it's carrying, and what are the physiologic factors that determine which adaptors associate in the DDAs. Also of great interest are novel roles that have emerged for LIS1. For example, a recent study by Kshirsagar et al. identified LIS1 as an RNA and RNA-binding protein28. It was determined that LIS1 affected embryonic stem cell development through its ability to interact with RNA-binding proteins like argonaut. Cytoskeleton Inc.’s purified tubulin proteins were used in several of these critical cryo-EM studies to better understand how the LIS1-DDA complex interacts with tubulin, and we are excited to see the next breakthroughs.
References
1. Canty, J.T., et al., Structure and Mechanics of Dynein Motors. Annu Rev Biophys, 2021. 50: p. 549-574.
2. Marzo, M.G., et al., Molecular basis for dyneinopathies reveals insight into dynein regulation and dysfunction. Elife, 2019. 8.
3. Reck-Peterson, S.L., et al., Single-molecule analysis of dynein processivity and stepping behavior. Cell, 2006. 126(2): p. 335-48.
4. Paschal, B.M., et al., Isolated flagellar outer arm dynein translocates brain microtubules in vitro. Nature, 1987. 330(6149): p. 672-4.
5. McKenney, R.J., et al., Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexes. Science, 2014. 345(6194): p. 337-41.
6. Schlager, M.A., et al., In vitro reconstitution of a highly processive recombinant human dynein complex. EMBO J, 2014. 33(17): p. 1855-68.
7. Zhang, K., et al., Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated. Cell, 2017. 169(7): p. 1303-1314 e18.
8. Urnavicius, L., et al., Cryo-EM shows how dynactin recruits two dyneins for faster movement. Nature, 2018. 554(7691): p. 202-206.
9. Chaaban, S. and A.P. Carter, Structure of dynein-dynactin on microtubules shows tandem adaptor binding. Nature, 2022. 610(7930): p. 212-216.
10. Lopez, J.C., A smoother path to LIS1. Nat Rev Neurosci, 2000. 1(3): p. 157.
11. Htet, Z.M., et al., LIS1 promotes the formation of activated cytoplasmic dynein-1 complexes. Nat Cell Biol, 2020. 22(5): p. 518-525.
12. Elshenawy, M.M., et al., Lis1 activates dynein motility by modulating its pairing with dynactin. Nat Cell Biol, 2020. 22(5): p. 570-578.
13. Marzo, M.G., J.M. Griswold, and S.M. Markus, Pac1/LIS1 stabilizes an uninhibited conformation of dynein to coordinate its localization and activity. Nat Cell Biol, 2020. 22(5): p. 559-569.
14. Karasmanis, E.P., et al., Lis1 relieves cytoplasmic dynein-1 autoinhibition by acting as a molecular wedge. Nat Struct Mol Biol, 2023. 30(9): p. 1357-1364.
15. Kusakci, E. and A. Yildiz, Studying Dynein Mechanochemistry with an Optical Trap. Methods Mol Biol, 2023. 2623: p. 201-219.
16. Ton, W.D., et al., Microtubule-binding-induced allostery triggers LIS1 dissociation from dynein prior to cargo transport. Nat Struct Mol Biol, 2023. 30(9): p. 1365-1379.
17. Singh, K., et al., Molecular mechanism of dynein-dynactin complex assembly by LIS1. Science, 2024. 383(6690): p. eadk8544.
18. Garrott, S.R., J.P. Gillies, and M.E. DeSantis, Nde1 and Ndel1: Outstanding Mysteries in Dynein-Mediated Transport. Front Cell Dev Biol, 2022. 10: p. 871935.
19. Zylkiewicz, E., et al., The N-terminal coiled-coil of Ndel1 is a regulated scaffold that recruits LIS1 to dynein. J Cell Biol, 2011. 192(3): p. 433-45.
20. Zhao, Y., S. Oten, and A. Yildiz, Nde1 promotes Lis1-mediated activation of dynein. Nat Commun, 2023. 14(1): p. 7221.
21. Okada, K., et al., Conserved roles for the dynein intermediate chain and Ndel1 in assembly and activation of dynein. Nat Commun, 2023. 14(1): p. 5833.
22. Pandey, J.P., et al., LIS1 and NDEL1 Regulate Axonal Trafficking of Mitochondria in Mature Neurons. Front Mol Neurosci, 2022. 15: p. 841047.
23. Liu, L., et al., The LIS1/NDE1 Complex Is Essential for FGF Signaling by Regulating FGF Receptor Intracellular Trafficking. Cell Rep, 2018. 22(12): p. 3277-3291.
24. Alonso, A., et al., The yeast homologue of the microtubule-associated protein Lis1 interacts with the sumoylation machinery and a SUMO-targeted ubiquitin ligase. Mol Biol Cell, 2012. 23(23): p. 4552-66.
25. Stier, A., et al., The CUL4B-based E3 ubiquitin ligase regulates mitosis and brain development by recruiting phospho-specific DCAFs. EMBO J, 2023. 42(17): p. e112847.
26. Wei, Z., et al., MiR-380 inhibits the proliferation and invasion of cholangiocarcinoma cells by silencing LIS1. Cancer Cell Int, 2024. 24(1): p. 129.
27. Yang, R., et al., MicroRNA-144 suppresses cholangiocarcinoma cell proliferation and invasion through targeting platelet activating factor acetylhydrolase isoform 1b. BMC Cancer, 2014. 14: p. 917.
28. Kshirsagar, A., et al., LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways. Nat Commun, 2023. 14(1): p. 3293.