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The EMBO Journal Oct 2023The localization of RNAs in cells is critical for many cellular processes. Whereas motor-driven transport of ribonucleoprotein (RNP) condensates plays a prominent role...
The localization of RNAs in cells is critical for many cellular processes. Whereas motor-driven transport of ribonucleoprotein (RNP) condensates plays a prominent role in RNA localization in cells, their study remains limited by the scarcity of available tools allowing to manipulate condensates in a spatial manner. To fill this gap, we reconstitute in cellula a minimal RNP transport system based on bioengineered condensates, which were functionalized with kinesins and dynein-like motors, allowing for their positioning at either the cell periphery or centrosomes. This targeting mostly occurs through the active transport of the condensate scaffolds, which leads to localized nucleation of phase-separated condensates. Then, programming the condensates to recruit specific mRNAs is able to shift the localization of these mRNAs toward the cell periphery or the centrosomes. Our method opens novel perspectives for examining the role of RNA localization as a driver of cellular functions.
Topics: Microtubules; Ribonucleoproteins; RNA; RNA, Messenger; Dyneins
PubMed: 37724036
DOI: 10.15252/embj.2023114106 -
Disease Models & Mechanisms Feb 2024Motile cilia on ependymal cells that line brain ventricular walls beat in concert to generate a flow of laminar cerebrospinal fluid (CSF). Dyneins and kinesins are...
Motile cilia on ependymal cells that line brain ventricular walls beat in concert to generate a flow of laminar cerebrospinal fluid (CSF). Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how kinesin motors contribute to cilia motility. Here, we show that Kif6 is a slow processive motor (12.2±2.0 nm/s) on microtubules in vitro and localizes to both the apical cytoplasm and the axoneme in ependymal cells, although it does not display processive movement in vivo. Using a mouse mutant that models a human Kif6 mutation in a proband displaying macrocephaly, hypotonia and seizures, we found that loss of Kif6 function causes decreased ependymal cilia motility and, subsequently, decreases fluid flow on the surface of brain ventricular walls. Disruption of Kif6 also disrupts orientation of cilia, formation of robust apical actin networks and stabilization of basal bodies at the apical surface. This suggests a role for the Kif6 motor protein in the maintenance of ciliary homeostasis within ependymal cells.
Topics: Humans; Brain; Cilia; Dyneins; Ependyma; Kinesins
PubMed: 38235522
DOI: 10.1242/dmm.050137 -
Nature Structural & Molecular Biology Sep 2023The lissencephaly-related protein LIS1 is a critical regulator of cytoplasmic dynein that governs motor function and intracellular localization (for example, to...
The lissencephaly-related protein LIS1 is a critical regulator of cytoplasmic dynein that governs motor function and intracellular localization (for example, to microtubule plus-ends). Although LIS1 binding is required for dynein activity, its unbinding prior to initiation of cargo transport is equally important, since preventing dissociation leads to dynein dysfunction. To understand whether and how dynein-LIS1 binding is modulated, we engineered dynein mutants locked in a microtubule-bound (MT-B) or microtubule-unbound (MT-U) state. Whereas the MT-B mutant exhibits low LIS1 affinity, the MT-U mutant binds LIS1 with high affinity, and as a consequence remains almost irreversibly associated with microtubule plus-ends. We find that a monomeric motor domain is sufficient to exhibit these opposing LIS1 affinities, and that this is evolutionarily conserved between yeast and humans. Three cryo-EM structures of human dynein with and without LIS1 reveal microtubule-binding induced conformational changes responsible for this regulation. Our work reveals key biochemical and structural insight into LIS1-mediated dynein activation.
Topics: Humans; Dyneins; Microtubule-Associated Proteins; Microtubules; Saccharomyces cerevisiae; Protein Binding
PubMed: 37322240
DOI: 10.1038/s41594-023-01010-x -
Scientific Reports Jun 2023Dynein is a minus-end-directed motor that generates oscillatory motion in eukaryotic flagella. Cyclic beating, which is the most significant feature of a flagellum,...
Dynein is a minus-end-directed motor that generates oscillatory motion in eukaryotic flagella. Cyclic beating, which is the most significant feature of a flagellum, occurs by sliding spatiotemporal regulation by dynein along microtubules. To elucidate oscillation generated by dynein in flagellar beating, we examined its mechanochemical properties under three different axonemal dissection stages. By starting from the intact 9 + 2 structure, we reduced the number of interacting doublets and determined three parameters, namely, the duty ratio, dwell time and step size, of the generated oscillatory forces at each stage. Intact dynein molecules in the axoneme, doublet bundle and single doublet were used to measure the force with optical tweezers. The mean forces per dynein determined under three axonemal conditions were smaller than the previously reported stall forces of axonemal dynein; this phenomenon suggests that the duty ratio is lower than previously thought. This possibility was further confirmed by an in vitro motility assay with purified dynein. The dwell time and step size estimated from the measured force were similar. The similarity in these parameters suggests that the essential properties of dynein oscillation are inherent to the molecule and independent of the axonemal architecture, composing the functional basis of flagellar beating.
Topics: Axonemal Dyneins; Axoneme; Cilia
PubMed: 37386019
DOI: 10.1038/s41598-023-37242-6 -
Biochemical and Biophysical Research... Dec 2023Phagosomes are dynamic organelles formed by macrophages to capture and destroy microbial pathogens. Phagosome transport from the cell periphery to the perinuclear...
Phagosomes are dynamic organelles formed by macrophages to capture and destroy microbial pathogens. Phagosome transport from the cell periphery to the perinuclear region, is essential for fusion with lysosomes and the elimination of pathogens. Molecular motors, kinesin and dynein, generate opposing forces, transporting the phagosome away from and towards the lysosome, respectively. Luminal acidification plays a crucial role in determining the net directional movement of the phagosome. The mechanics of this regulation are not known. In this study, we used the sodium proton exchanger NHE9 to selectively modulate phagosomal acidification in macrophages. We then investigated its impact on the mechanical properties of kinesin and dynein motors through optical trapping experiments. We observed a negative correlation between the tenacity of dynein motors and pH under high resistive forces. Reduced luminal acidification impaired generation of dynein cooperative forces, which are crucial for transporting the phagosome to the lysosome. Conversely, the kinesin-powered motility of phagosomes is enabled by a decrease in phagosomal acidification. Given the various methods pathogens employ to limit phagosomal acidification, our findings are highly significant in the context of host-pathogen interactions.
Topics: Dyneins; Kinesins; Phagosomes; Lysosomes; Hydrogen-Ion Concentration
PubMed: 37979328
DOI: 10.1016/j.bbrc.2023.149236 -
Genome Biology Dec 2023Identifying host factors is key to understanding RNA virus pathogenicity. Besides proteins, RNAs can interact with virus genomes to impact replication.
BACKGROUND
Identifying host factors is key to understanding RNA virus pathogenicity. Besides proteins, RNAs can interact with virus genomes to impact replication.
RESULTS
Here, we use proximity ligation sequencing to identify virus-host RNA interactions for four strains of Zika virus (ZIKV) and one strain of dengue virus (DENV-1) in human cells. We find hundreds of coding and non-coding RNAs that bind to DENV and ZIKV viruses. Host RNAs tend to bind to single-stranded regions along the virus genomes according to hybridization energetics. Compared to SARS-CoV-2 interactors, ZIKV-interacting host RNAs tend to be downregulated upon virus infection. Knockdown of several short non-coding RNAs, including miR19a-3p, and 7SK RNA results in a decrease in viral replication, suggesting that they act as virus-permissive factors. In addition, the 3'UTR of DYNLT1 mRNA acts as a virus-restrictive factor by binding to the conserved dumbbell region on DENV and ZIKV 3'UTR to decrease virus replication. We also identify a conserved set of host RNAs that interacts with DENV, ZIKV, and SARS-CoV-2, suggesting that these RNAs are broadly important for RNA virus infection.
CONCLUSIONS
This study demonstrates that host RNAs can impact virus replication in permissive and restrictive ways, expanding our understanding of host factors and RNA-based gene regulation during viral pathogenesis.
Topics: Humans; Zika Virus; Zika Virus Infection; RNA, Viral; 3' Untranslated Regions; Dengue Virus; Virus Replication; Dengue; Antiviral Agents; Dyneins
PubMed: 38053173
DOI: 10.1186/s13059-023-03110-9 -
Nature Communications Apr 2024Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport...
Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.
Topics: Caenorhabditis elegans; Animals; Cilia; Caenorhabditis elegans Proteins; Kinesins; Flagella; Tubulin; Axoneme; Dyneins; Biological Transport; Single Molecule Imaging; Protein Transport
PubMed: 38658528
DOI: 10.1038/s41467-024-47807-2 -
Auris, Nasus, Larynx Jun 2024Primary ciliary dyskinesia (PCD) is a relatively rare genetic disorder that affects approximately 1 in 20,000 people. Approximately 50 genes are currently known to cause... (Review)
Review
OBJECTIVE
Primary ciliary dyskinesia (PCD) is a relatively rare genetic disorder that affects approximately 1 in 20,000 people. Approximately 50 genes are currently known to cause PCD. In light of differences in causative genes and the medical system in Japan compared with other countries, a practical guide was needed for the diagnosis and management of Japanese PCD patients.
METHODS
An ad hoc academic committee was organized under the Japanese Rhinologic Society to produce a practical guide, with participation by committee members from several academic societies in Japan. The practical guide including diagnostic criteria for PCD was approved by the Japanese Rhinologic Society, Japanese Society of Otolaryngology-Head and Neck Surgery, Japanese Respiratory Society, and Japanese Society of Pediatric Pulmonology.
RESULTS
The diagnostic criteria for PCD consist of six clinical features, six laboratory findings, differential diagnosis, and genetic testing. The diagnosis of PCD is categorized as definite, probable, or possible PCD based on a combination of the four items above. Diagnosis of definite PCD requires exclusion of cystic fibrosis and primary immunodeficiency, at least one of the six clinical features, and a positive result for at least one of the following: (1) Class 1 defect on electron microscopy of cilia, (2) pathogenic or likely pathogenic variants in a PCD-related gene, or (3) impairment of ciliary motility that can be repaired by correcting the causative gene variants in iPS cells established from the patient's peripheral blood cells.
CONCLUSION
This practical guide provides clinicians with useful information for the diagnosis and management of PCD in Japan.
Topics: Humans; Kartagener Syndrome; Genetic Testing; Diagnosis, Differential; Cilia; Japan; Axonemal Dyneins; Proteins
PubMed: 38537559
DOI: 10.1016/j.anl.2024.02.001 -
Biomolecules Sep 2023Dynein motors facilitate the majority of minus-end-directed transport events on microtubules. The dynein adaptor Bicaudal D2 (BicD2) recruits the dynein machinery to...
Dynein motors facilitate the majority of minus-end-directed transport events on microtubules. The dynein adaptor Bicaudal D2 (BicD2) recruits the dynein machinery to several cellular cargo for transport, including Nup358, which facilitates a nuclear positioning pathway that is essential for the differentiation of distinct brain progenitor cells. Previously, we showed that Nup358 forms a "cargo recognition α-helix" upon binding to BicD2; however, the specifics of the BicD2-Nup358 interface are still not well understood. Here, we used AlphaFold2, complemented by two additional docking programs (HADDOCK and ClusPro) as well as mutagenesis, to show that the Nup358 cargo-recognition α-helix binds to BicD2 between residues 747 and 774 in an anti-parallel manner, forming a helical bundle. We identified two intermolecular salt bridges that are important to stabilize the interface. In addition, we uncovered a secondary interface mediated by an intrinsically disordered region of Nup358 that is directly N-terminal to the cargo-recognition α-helix and binds to BicD2 between residues 774 and 800. This is the same BicD2 domain that binds to the competing cargo adapter Rab6, which is important for the transport of Golgi-derived and secretory vesicles. Our results establish a structural basis for cargo recognition and selection by the dynein adapter BicD2, which facilitates transport pathways that are important for brain development.
Topics: Dyneins; Microtubule-Associated Proteins; Microtubules; Biological Transport; Models, Structural
PubMed: 37892127
DOI: 10.3390/biom13101445 -
The Journal of Biological Chemistry Apr 2024Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different...
Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins-1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.
PubMed: 38677516
DOI: 10.1016/j.jbc.2024.107323