The dynein motor is recruited to the kinetochore to capture spindle microtubules and control the spindle assembly checkpoint. Gama et al. reveal the molecular mechanism of how the Rod–Zw10–Zwilch complex and Spindly mediate dynein recruitment in Caenorhabditis elegans and human cells.

All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo.

The induction of P-glycoprotein (P-gp), an ATP-dependent efflux pump, has been proposed as a strategy against the toxicity induced by P-gp substrates such as the herbicide paraquat (PQ). The aim of this study was to screen five newly synthetized thioxanthonic derivatives, a group known to interact with P-gp, as potential inducers of the pump's expression and/or activity and to evaluate whether they would afford protection against PQ-induced toxicity in Caco-2 cells. All five thioxanthones (20 µM) caused a significant increase in both P-gp expression and activity as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, it was demonstrated that the tested compounds, when present only during the efflux of rhodamine 123, rapidly induced an activation of P-gp. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, indicating that all derivatives acted as P-gp substrates. PQ cytotoxicity was significantly reduced in the presence of four thioxanthone derivatives, and this protective effect was reversed upon incubation with a specific P-gp inhibitor. In silico studies showed that all the tested thioxanthones fitted onto a previously described three-feature P-gp induction pharmacophore. Moreover, in silico interactions between thioxanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. Based on the in vitro activation results, a pharmacophore model for P-gp activation was built, which will be of further use in the screening for new P-gp activators. In conclusion, the study demonstrated the potential of the tested thioxanthonic compounds in protecting against toxic effects induced by P-gp substrates through P-gp induction and activation.

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Xanthones are a family of compounds with several known biological activities and therapeutic potential for which information on their interaction with membrane transporters is lacking. Knowing that P-glycoprotein (P-gp) acts as a cellular defense mechanism by effluxing its toxic substrates, the aim of this study was to investigate the potential of five dihydroxylated xanthones as inducers of P-gp expression and/or activity and to evaluate whether they could protect Caco-2 cells against the cytotoxicity induced by the toxic P-gp substrate paraquat (PQ). After 24 h of incubation, all tested xanthones caused a significant increase in both P-gp expression and activity, as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, after a short 45-min incubation, all the tested xanthones induced a rapid increase in P-gp activity, indicating direct pump activation without increased P-gp protein expression. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, demonstrating to be P-gp substrates. Moreover, when simultaneously incubated with PQ, all xanthones significantly reduced the cytotoxicity of the herbicide, and these protective effects were completely reversed upon incubation with a specific P-gp inhibitor. In silico studies evaluating the interactions between xanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. A quantitative structure-activity relationship model was developed and validated, and the maximal partial charge for an oxygen atom was the descriptor predicted as being implicated in P-gp activation by the dihydroxylated xanthones. These results disclose new perspectives in preventing PQ- and other P-gp substrates-induced poisonings.

The MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. How JIP3’s interaction with dynein and kinesin-1 contributes to organelle clearance is unclear. We show that human dynein light intermediate chain (DLIC) binds the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in RH1 abrogates DLIC binding without perturbing the interaction between JIP3’s RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3–bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3’s leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3–bound dynein and kinesin-1 participate in bidirectional organelle transport.

The microtubule-based motor dynein generates pulling forces for centrosome centration and mitotic spindle positioning in animal cells. How the essential dynein activator dynactin regulates these functions of the motor is incompletely understood. Here, we dissect the role of dynactin's microtubule binding activity, located in the p150 CAP-Gly domain and an adjacent basic patch, in the C. elegans zygote. Analysis of p150 mutants engineered by genome editing suggests that microtubule tip tracking of dynein-dynactin is dispensable for targeting the motor to the cell cortex and for generating robust cortical pulling forces. Instead, mutations in p150's CAP-Gly domain inhibit cytoplasmic pulling forces responsible for centration of centrosomes and attached pronuclei. The centration defects are mimicked by mutations of α-tubulin's C-terminal tyrosine, and both p150 CAP-Gly and tubulin tyrosine mutants decrease the frequency of early endosome transport from the cell periphery towards centrosomes during centration. Our results suggest that p150 GAP-Gly domain binding to tyrosinated microtubules promotes initiation of dynein-mediated organelle transport in the dividing one-cell embryo, and that this function of p150 is critical for generating cytoplasmic pulling forces for centrosome centration.

Abuse of synthetic drugs is widespread among young people worldwide. In this context, piperazine derived drugs recently appeared in the recreational drug market. Clinical studies and case-reports describe sympathomimetic effects including hypertension, tachycardia, and increased heart rate. Our aim was to investigate the cytotoxicity of N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl) piperazine (TFMPP), 1-(4-methoxyphenyl) piperazine (MeOPP), and 1-(3,4-methylenedioxybenzyl) piperazine (MDBP) in the H9c2 rat cardiac cell line. Complete cytotoxicity curves were obtained at a 0-20 mM concentration range after 24 h incubations with each drug. The EC50 values (μM) were 343.9, 59.6, 570.1, and 702.5 for BZP, TFMPP, MeOPP, and MDBP, respectively. There was no change in oxidative stress markers. However, a decrease in total GSH content was noted for MDBP, probably due to metabolic conjugation reactions. All drugs caused significant decreases in intracellular ATP, accompanied by increased intracellular calcium levels and a decrease in mitochondrial membrane potential that seems to involve the mitochondrial permeability transition pore. The cell death mode revealed early apoptotic cells and high number of cells undergoing secondary necrosis. Among the tested drugs, TFMPP seems to be the most potent cytotoxic compound. Overall, piperazine designer drugs are potentially cardiotoxic and support concerns on risks associated with the intake of these drugs.