Katanin regulates dynamics of microtubules and biogenesis of motile cilia.
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ABSTRACT:
SUBMITTER: Sharma N
PROVIDER: S-EPMC2064628 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundLRRC6 is an assembly factor for dynein arms in the cytoplasm of motile ciliated cells, and when mutated, dynein arm components remained in the cytoplasm. Here, we demonstrate the role of LRRC6 in the active nuclear translocation of FOXJ1, a master regulator for cilia-associated gene transcription.MethodsWe generated Lrrc6 knockout (KO) mice, and we investigated the role of LRRC6 on ciliopathy development by using proteomic, transcriptomic, and immunofluorescence analysis. Experiments on mouse basal cell organoids confirmed the biological relevance of our findings.ResultsThe absence of LRRC6 in multi-ciliated cells hinders the assembly of ODA and IDA components of cilia; in this study, we showed that the overall expression of proteins related to cilia decreased as well. Expression of cilia-related transcripts, specifically ODA and IDA components, dynein axonemal assembly factors, radial spokes, and central apparatus was lower in Lrrc6 KO mice than in wild-type mice. We demonstrated that FOXJ1 was present in the cytoplasm and translocated into the nucleus when LRRC6 was expressed and that this process was blocked by INI-43, an importin α inhibitor.ConclusionsTaken together, these results hinted at the LRRC6 transcriptional regulation of cilia-related genes via the nuclear translocation of FOXJ1. Video Abstract.
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Project description:The cilium is a small cellular organelle with motility- and/or sensory-related functions that plays a crucial role during developmental and homeostatic processes. Although many molecules or signal transduction pathways that control cilia assembly have been reported, the mechanisms of ciliary length control have remained enigmatic. Here, we report that Smad2-dependent transforming growth factor ? (TGF-?) signaling impacts the length of motile cilia at the Xenopus left-right (LR) organizer, the gastrocoel roof plate (GRP), as well as at the neural tube and the epidermis. Blocking TGF-? signaling resulted in the absence of the transition zone protein B9D1/MSKR-1 from cilia in multi-ciliated cells (MCCs) of the epidermis. Interestingly, this TGF-? activity is not mediated by Mcidas, Foxj1, and RFX2, the known major regulators of ciliogenesis. These data indicate that TGF-? signaling is crucial for the function of the transition zone, which in turn may affect the regulation of cilia length.
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Project description:Katanin is a microtubule-severing complex whose catalytic activities are well characterized, but whose in vivo functions are incompletely understood. Human mutations in KATNB1, which encodes the noncatalytic regulatory p80 subunit of katanin, cause severe microlissencephaly. Loss of Katnb1 in mice confirms essential roles in neurogenesis and cell survival, while loss of zebrafish katnb1 reveals specific roles for katnin p80 in early and late developmental stages. Surprisingly, Katnb1 null mutant mouse embryos display hallmarks of aberrant Sonic hedgehog signaling, including holoprosencephaly. KATNB1-deficient human cells show defective proliferation and spindle structure, while Katnb1 null fibroblasts also demonstrate a remarkable excess of centrioles, with supernumerary cilia but deficient Hedgehog signaling. Our results reveal unexpected functions for KATNB1 in regulating overall centriole, mother centriole, and cilia number, and as an essential gene for normal Hedgehog signaling during neocortical development.
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Project description:Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a "9 + 2" microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), Camsap3 tm1a/tm1a , exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3 tm1a/tm1a mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in Camsap3 tm1a/tm1a , MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of Xenopus embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.
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Project description:The axoneme is the main extracellular part of cilia and flagella in eukaryotes. It consists of a microtubule cytoskeleton, which normally comprises nine doublets. In motile cilia, dynein ATPase motor proteins generate sliding motions between adjacent microtubules, which are integrated into a well-orchestrated beating or rotational motion. In primary cilia, there are a number of sensory proteins functioning on membranes surrounding the axoneme. In both cases, as the study of proteomics has elucidated, hundreds of proteins exist in this compartmentalized biomolecular system. In this article, we review the recent progress of structural studies of the axoneme and its components using electron microscopy and X-ray crystallography, mainly focusing on motile cilia. Structural biology presents snapshots (but not live imaging) of dynamic structural change and gives insights into the force generation mechanism of dynein, ciliary bending mechanism, ciliogenesis, and evolution of the axoneme.
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Project description:Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19) and p80 (pf15) subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (ε-tubulin) alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization.
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Project description:Among the many complex bioactuators functioning at different scales, the organelle cilium represents a fundamental actuating unit in cellular biology. Producing motions at submicrometer scales, dominated by viscous forces, cilia drive a number of crucial bioprocesses in all vertebrate and many invertebrate organisms before and after their birth. Artificially mimicking motile cilia has been a long-standing challenge while inspiring the development of new materials and methods. The use of magnetic materials has been an effective approach for realizing microscopic artificial cilia; however, the physical and magnetic properties of the magnetic material constituents and fabrication processes utilized have almost exclusively only enabled the realization of highly motile artificial cilia with dimensions orders of magnitude larger than their biological counterparts. This has hindered the development and study of model systems and devices with inherent size-dependent aspects, as well as their application at submicrometer scales. In this work, we report a magnetic elastomer preparation process coupled with a tailored molding process for the successful fabrication of artificial cilia with submicrometer dimensions showing unprecedented deflection capabilities, enabling the design of artificial cilia with high motility and at sizes equal to those of their smallest biological counterparts. The reported work crosses the barrier of nanoscale motile cilia fabrication, paving the way for maximum control and manipulation of structures and processes at micro- and nanoscales.
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Project description:Mechanoreceptor cells develop a specialized cytoskeleton that plays structural and sensory roles at the site of mechanotransduction. However, little is known about how the cytoskeleton is organized and formed. Using electron tomography and live-cell imaging, we resolve the 3D structure and dynamics of the microtubule-based cytoskeleton in fly campaniform mechanosensory cilia. Investigating the formation of the cytoskeleton, we find that katanin p60-like 1 (kat-60L1), a neuronal type of microtubule-severing enzyme, serves two functions. First, it amplifies the mass of microtubules to form the dense microtubule arrays inside the sensory cilia. Second, it generates short microtubules that are required to build the nanoscopic cytoskeleton at the mechanotransduction site. Additional analyses further reveal the functional roles of Patronin and other potential factors in the local regulatory network. In all, our results characterize the specialized cytoskeleton in fly external mechanosensory cilia at near-molecular resolution and provide mechanistic insights into how it is formed.
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Project description:The axonemal central pair (CP) are non-centrosomal microtubules critical for planar ciliary beat. How they form, however, is poorly understood. Here, we show that mammalian CP formation requires Wdr47, Camsaps, and microtubule-severing activity of Katanin. Katanin severs peripheral microtubules to produce central microtubule seeds in nascent cilia. Camsaps stabilize minus ends of the seeds to facilitate microtubule outgrowth, whereas Wdr47 concentrates Camsaps into the axonemal central lumen to properly position central microtubules. Wdr47 deficiency in mouse multicilia results in complete loss of CP, rotatory beat, and primary ciliary dyskinesia. Overexpression of Camsaps or their microtubule-binding regions induces central microtubules in Wdr47-/- ependymal cells but at the expense of low efficiency, abnormal numbers, and wrong location. Katanin levels and activity also impact the central microtubule number. We propose that Wdr47, Camsaps, and Katanin function together for the generation of non-centrosomal microtubule arrays in polarized subcellular compartments.
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Project description:Microtubules in the axon are more resistant to severing by katanin than microtubules elsewhere in the neuron. We have hypothesized that this is because of the presence of tau on axonal microtubules. When katanin is overexpressed in fibroblasts, the microtubules are severed into short pieces, but this phenomenon is suppressed by the coexpression of tau. Protection against severing is also afforded by microtubule-associated protein 2 (MAP2), which has a tau-like microtubule-binding domain, but not by MAP1b, which has a different microtubule-binding domain. The microtubule-binding domain of tau is required for the protection, but within itself, provides less protection than the entire molecule. When tau (but not MAP2 or MAP1b) is experimentally depleted from neurons, the microtubules in the axon lose their characteristic resistance to katanin. These results, which validate our hypothesis, also suggest a potential explanation for why axonal microtubules deteriorate in neuropathies involving the dissociation of tau from the microtubules.
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