CIISB Research Highlights Archive

Tau cooperativity by local microtubule lattice compaction

a, Schematics of the assay geometry. b, Quantification of cooperative binding of tau to taxol-lattice microtubules (mean ± s.d., n = 652 microtubules, 60 experiments, 95% confidence bounds, r² = 0.9633, gray), Hill–Langmuir equation fit (green). AU, arbitrary units. c, Fluorescence time-lapse micrographs showing microtubule lattice straightening (yellow arrow) upon formation of tau envelopes (green). Twenty nanomolar tau-mCherry was added at t = 0 s. Scale bar, 2 μm.

Zdeněk Lánský Research Group

Significance

Tau is an intrinsically disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive ‘envelopes’ around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau envelope disassembly. Investigating other members of the tau family, we find that MAP2 similarly forms envelopes governed by lattice spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct regions. We conclude that the interdependent allostery between lattice spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2.

Valerie Siahaan, Ruensern Tan, Tereza Humhalova, Lenka Libusova, Samuel E. Lacey, Tracy Tan, Mariah Dacy, Kassandra M. Ori-McKenney, Richard J. McKenney, Marcus Braun & Zdenek Lansky

Microtubule lattice spacing governs cohesive envelope formation of tau family proteins

Na.t Chem. Biol., 18, 1224-1235 (2022): | https://doi.org/10.1038/s41589-022-01096-2

a Front and side views of the composite model with both monomers in rotational state 1. The two F₁/c₁₀-ring complexes, each augmented by three copies of the phylum-specific p18 subunit, are tied together at a 60°-angle. The membrane-bound F_o region displays a unique architecture and is composed of both conserved and phylum-specific subunits.
b Side view of the F_o region showing the lumenal interaction of the ten-stranded β-barrel of the c-ring (grey) with ATPTB12 (pale blue). The lipid-filled peripheral F_o cavity is indicated.
c Close-up view of the bound lipids within the peripheral F_o cavity with cryo-EM density shown.
d Top view into the decameric c-ring with a bound pyrimidine ribonucleoside triphosphate, assigned as UTP, although not experimentally detected. Map density shown in transparent blue, interacting residues shown.

Alena Zíková and Alexey Amunts Research Group

Significance

Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g together with subunit-eform an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.

Gahura, O., Mühleip, A., Hierro-Yap, C., Panicucci, B, Jain, M., Hollaus, D., Slapničková, M., Zíková, A. & Amunts, A.: An ancestral interaction module promotes oligomerization in divergent mitochondrial ATP synthases, Nature Comm. (2022) 13:5989,https://doi.org/10.1038/s41467-022-33588-z

Cryo-EM structures of mouse full-length Dicer alone and in complex with Dicerdpre-miRNA reveal the molecular basis of locking Dicer in the closed state
(A) Domain architecture of full-length mouse Dicer numbered at boundaries.
(B) Overall structure of the full-length mouse Dicer, shown as 3.8-A ̊ cryo-EM density map and ribbon representations in two orthogonal views. Interface between the HEL1 and RNase IIIb domains is highlighted by a box.
(C) Overall structure of the full-length mouse Dicer-RNA complex, shown as 4.2-A ̊ cryo-EM density map and ribbon representations in two orthogonal views.

Richard Štefl and Petr Svoboda Research Groups

Significance

MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucle- ases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is spe- cifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer’s DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the com- plete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicerd–miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleav- age-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.

Zapletal, D., Taborska, E., Pasulka, J., Malik, R., Kubicek, K., Zanova, M., Much, Ch., Sebesta, M. Buccheri, V., Horvat, F., Jenickova, I., Prochazkova, M., Prochazka, J., Pinkas, M., Novacek, J., Joseph, D. F., Sedlacek R., Bernecky C., O’Carrol, D., Stefl. R., and Svoboda, P.: Structural and functional basis of mammalian microRNA biogenesis by Dicer

Mol. Cell 2022, 82, 4064–4079, https://doi.org/10.1016/j.molcel.2022.10.010

Dendrite-shaped hematite microrobots have been developed as an effective GSH depletion agent for photodynamic therapy (PDT) of prostate cancer cells. These single-component microrobots with dual light/magnetic field actuation can induce GSH depletion, greatly enhancing in vitro PDT performance and accomplishing the non-contact transportation of micro-sized objects.

Martin Pumera Research Group

Significance

Photocatalytic micromotors that exhibit wireless and controllable motion by light have been extensively explored for cancer treatment by photodynamic therapy (PDT). However, overexpressed glutathione (GSH) in the tumor microenvironment can down-regulate the reactive oxygen species (ROS) level for cancer therapy. Herein, we present dendrite-shaped light-powered hematite microrobots as an effective GSH depletion agent for PDT of prostate cancer cells. These hematite microrobots can display negative phototactic motion under light irradiation and flexible actuation in a defined path controlled by an external magnetic field. Non-contact transportation of micro-sized cells can be achieved by manipulating the microrobot's motion. In addition, the biocompatible microrobots induce GSH depletion and greatly enhance PDT performance. The proposed dendrite-shaped hematite microrobots contribute to developing dual light/magnetic field-powered micromachines for the biomedical field.

Xia Peng, Urso, M., Balvan, J., Masařík, M., and Pumera, M.:

Self-Propelled Magnetic Dendrite-Shaped Microrobots for Photodynamic Prostate Cancer Therapy, Angew. Chem. Int. Ed. 2022, on-line version e202213505, https://doi.org/10.1002/anie.202213505

a Surface representation of composite cryo-EM map of virion of phage SU10 colored according to protein type. The major capsid protein (gp9) is shown in turquoise, portal protein (gp6) in magenta, adaptor protein (gp11) in yellow, long tail fibers (gp12) in violet, nozzle protein (gp17) in red, short tail fibers (gp16) in green, and tail needle (gp18) in light blue. The length of the virion is 1590 Å. For details on the construction of the composite map, please see the Materials and methods section. b The same as A, but the front half of the composite map of the SU10 head was removed to show the structure of the genome in grey. The inset shows a 2D class average of the SU10 virion. The scale bar indicates 45 nm. c Composite cryo-EM map of portal and tail complexes of SU10 virion. The length of the complex is 540 Å. d Cryo-EM reconstruction of portal and tail complexes from an SU10 genome release intermediate. e Composite cryo-EM map of genome-release intermediate of SU10. The front half of the head was removed to show the structure of the genome remaining in the capsid. The inset shows a 2D class average of the SU10 genome release intermediate. f Schematic representation of segment of SU10 genome encoding structural proteins color-coded the same as the proteins in panels a to e. Proteins shown in white are either non-structural or were not identified in the reconstructions.

Pavel Plevka Research Group

Significance

Escherichia coli phage SU10 belongs to the genus Kuravirus from the class Caudoviricetes of phages with short non-contractile tails. In contrast to other short-tailed phages, the tails of Kuraviruses elongate upon cell attachment. Here we show that the virion of SU10 has a prolate head, containing genome and ejection proteins, and a tail, which is formed of portal, adaptor, nozzle, and tail needle proteins and decorated with long and short fibers. The binding of the long tail fibers to the receptors in the outer bacterial membrane induces the straightening of nozzle proteins and rotation of short tail fibers. After the re-arrangement, the nozzle proteins and short tail fibers alternate to form a nozzle that extends the tail by 28 nm. Subsequently, the tail needle detaches from the nozzle proteins and five types of ejection proteins are released from the SU10 head. The nozzle with the putative extension formed by the ejection proteins enables the delivery of the SU10 genome into the bacterial cytoplasm. It is likely that this mechanism of genome delivery, involving the formation of the tail nozzle, is employed by all Kuraviruses.

Šiborová, M., Füzik, T., Procházková,M., Nováček, J., Benešík, M., Nilsson, A.S., and Plevka, P.:

Tail proteins of phage SU10 reorganize into the nozzle for genome delivery, Nature Comm. (2022)13:5622,https://doi.org/10.1038/s41467-022-33305-w

a, Kinome tree representation of the selectivity of OTS964 (at 1 µM concentration) in the Eurofins panel of 412 human kinases. The size of the red circles shows the percentage of inhibition of kinase activity. The green circle corresponds to CDK11. The percentage of CDK11 inhibition was derived from IVKAs presented in b and Fig. 3f The blue circle corresponds to TOPK. The percentage of TOPK inhibition was estimated from the published IC₅₀ = 353 nM and Extended Data Fig. 1b. b, Immunoblots of IVKAs of Flag-tagged (F) CDK11 WT, G579S and kinase dead (KD) mutants (left), or Flag-tagged CDK9 WT and KD mutant (right) phosphorylation of glutathione-S-transferase (GST)-tagged RNAPII^CTD substrate with the indicated concentrations of OTS964. c, The percentage of normalized NanoBRET ratio for CDK11/cyclin L2 or CDK9/cyclin T1 after OTS964 or control dinaciclib treatment. n = 2 biologically independent replicates; a representative replicate is shown. d, Immunoblot analysis of proteins after treatment of cells with OTS964 for 4 h. Short and long indicate short and long exposures of the film.

Dalibor Blažek Research Group

Significance

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA–protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex^. Splicing factor 3B subunit 1 (SF3B1) protein—a subunit of the U2 small nuclear ribonucleoprotein—is phosphorylated during spliceosome activation, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the B^act complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex B^act and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

Hluchý, M., Gajdušková, P., Ruiz de los Mozos, I., Rájecký, M., Kluge, M., Berger, B-T., Slabá, Z., Potěšil, D., Weiß, E., Ule, J., Zdráhal, Z., Knapp, S., Paruch, K., Friedel, C.C., and Blažek, D.:

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1, Nature. (2022) 609, 829-834. https://doi.org/10.1038/s41586-022-05204-z