CryoEM shows the active dynein complex on microtubules.

In a recent study, Chaaban and Carter use cryo-electron microscopy (cryo-EM) and an innovative data-processing pipeline to determine the first high-resolution structure of the dynein-dynactin-BICDR1 complex assembled on microtubules. The structure of the complex reveals novel stoichiometry and provides new mechanistic insight into dynein function and mechanism.

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

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.

Next-Generation Cell-Active Inhibitors of the Undrugged Oncogenic PTP4A3 Phosphatase.

Oncogenic protein tyrosine phosphatases (PTPs) are overexpressed in numerous human cancers but they have been challenging pharmacological targets. The emblematic oncogenic PTP4A tyrosine phosphatase family regulates many fundamental malignant processes. 7-Imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (JMS-053) is a novel, potent, and selective PTP4A inhibitor but its mechanism of action has not been fully elucidated, nor has the chemotype been fully investigated. Because tyrosine phosphatases are notoriously susceptible to oxidation, we interrogated JMS-053 and three newly synthesized analogs with specific attention on the role of oxidation. JMS-053 and its three analogs were potent in vitro PTP4A3 inhibitors, but 7-imino-5-methyl-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (NRT-870-59) appeared unique among the thienopyridinediones with respect to its inhibitory specificity for PTP4A3 versus both a PTP4A3 A111S mutant and an oncogenic dual specificity tyrosine phosphatase, CDC25B. Like JMS-053, NRT-870-59 was a reversible PTP4A3 inhibitor. All of the thienopyridinediones retained cytotoxicity against human ovarian and breast cancer cells grown as pathologically relevant three-dimensional spheroids. Inhibition of cancer cell colony formation by NRT-870-59, like JMS-053, required PTP4A3 expression. JMS-053 failed to generate significant detectable reactive oxygen species in vitro or in cancer cells. Mass spectrometry results indicated no disulfide bond formation or oxidation of the catalytic Cys104 after in vitro incubation of PTP4A3 with JMS-053 or NRT-870-59. Gene expression profiling of cancer cells exposed to JMS-053 phenocopied many of the changes seen with the loss of PTP4A3 and did not indicate oxidative stress. These data demonstrate that PTP4A phosphatases can be selectively targeted with small molecules that lack prominent reactive oxygen species generation and encourage further studies of this chemotype. SIGNIFICANCE STATEMENT: Protein tyrosine phosphatases are emerging as important contributors to human cancers. We report on a new class of reversible protein phosphatase small molecule inhibitors that are cytotoxic to human ovarian and breast cancer cells, do not generate significant reactive oxygen species in vitro and in cells, and could be valuable lead molecules for future studies of PTP4A phosphatases.

Ndel1 modulates dynein activation in two distinct ways.

Dynein is the primary minus-end-directed microtubule motor [1]. To achieve activation, dynein binds to the dynactin complex and an adaptor to form the "activated dynein complex" [2, 3]. The protein Lis1 aids activation by binding to dynein and promoting its association with dynactin and adaptor [4, 5]. Ndel1 and its orthologue Nde1 are dynein and Lis1 binding proteins that help control where dynein localizes within the cell [6]. Cell-based assays suggest that Ndel1/Nde1 also work with Lis1 to promote dynein activation, although the underlying mechanism is unclear [6]. Using purified proteins and quantitative binding assays, we found that Ndel1's C-terminal region contributes to binding to dynein 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 Ndel1's C-terminal domain 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. 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.

Nde1 and Ndel1: Outstanding Mysteries in Dynein-Mediated Transport.

Cytoplasmic dynein-1 (dynein) is the primary microtubule minus-end directed molecular motor in most eukaryotes. As such, dynein has a broad array of functions that range from driving retrograde-directed cargo trafficking to forming and focusing the mitotic spindle. Dynein does not function in isolation. Instead, a network of regulatory proteins mediate dynein's interaction with cargo and modulate dynein's ability to engage with and move on the microtubule track. A flurry of research over the past decade has revealed the function and mechanism of many of dynein's regulators, including Lis1, dynactin, and a family of proteins called activating adaptors. However, the mechanistic details of two of dynein's important binding partners, the paralogs Nde1 and Ndel1, have remained elusive. While genetic studies have firmly established Nde1/Ndel1 as players in the dynein transport pathway, the nature of how they regulate dynein activity is unknown. In this review, we will compare Ndel1 and Nde1 with a focus on discerning if the proteins are functionally redundant, outline the data that places Nde1/Ndel1 in the dynein transport pathway, and explore the literature supporting and opposing the predominant hypothesis about Nde1/Ndel1's molecular effect on dynein activity.

The KASH5 protein involved in meiotic chromosomal movements is a novel dynein activating adaptor.

Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis.