A doublecortin containing microtubule-associated protein is implicated in mechanotransduction in Drosophila sensory cilia.
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ABSTRACT:
SUBMITTER: Bechstedt S
PROVIDER: S-EPMC2892299 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The protein doublecortin (DCX) is expressed in post-mitotic migrating and differentiating neurons in the developing vertebrate brain and, as a part of the microtubule machinery, is required for neuronal migration. DCX expression is generally maximal during embryonic and early post-natal life but decreases markedly and almost disappears in older animals in parallel with the major decrease or cessation of neurogenesis. In several seasonally breeding songbird species such as canaries, the volume of several song control nuclei in the brain varies seasonally such that the largest nuclei are observed in the late spring and early summer. This variation is based on changes in cell size, dendritic branching, and, in nucleus HVC, on the incorporation of neurons newly born in adulthood. Because songbirds maintain an active neurogenesis and neuronal incorporation in their telencephalon throughout their lives, we investigated here the distribution of DCX-immunoreactive (ir) structures in the brain of adult male canaries. Densely stained DCX-ir cells were found exclusively in parts of the telencephalon that are known to incorporate new neurons in adulthood, in particular the nidopallium. Within this brain region, the boundaries of the song control nucleus HVC could be clearly distinguished from surrounding structures by a higher density of DCX-ir structures. In most telencephalic areas, about two thirds of these cells displayed a uni- or bipolar fusiform morphology suggesting they were migrating neurons. The rest of the DCX-ir cells in the telencephalon were larger and had a round multipolar morphology. No such staining was found in the rest of the brain. The broad expression of DCX specifically in adult brain structures that exhibit the characteristic of active incorporation of new neurons suggests that DCX plays a key role in the migration of new neurons in the brain of adult songbirds as it presumably does during ontogeny.
| S-EPMC2040224 | biostudies-literature
Project description:Proper regulation of microtubule (MT) stability and dynamics is vital for essential cellular processes, including axonal transportation and synaptic growth and remodeling in neurons. In the present study, we demonstrate that the Drosophila ankyrin repeat and KH domain-containing protein Mask negatively affects MT stability in both larval muscles and motor neurons. In larval muscles, loss-of-function of mask increases MT polymer length, and in motor neurons, loss of mask function results in overexpansion of the presynaptic terminal at the larval neuromuscular junctions (NMJs). mask genetically interacts with stathmin (stai), a neuronal modulator of MT stability, in the regulation of axon transportation and synaptic terminal stability. Our structure-function analysis of Mask revealed that its ankyrin repeats domain-containing N-terminal portion is sufficient to mediate Mask's impact on MT stability. Furthermore, we discovered that Mask negatively regulates the abundance of the MT-associated protein Jupiter in motor neuron axons, and that neuronal knocking down of Jupiter partially suppresses mask loss-of-function phenotypes at the larval NMJs. Taken together, our studies demonstrate that Mask is a novel regulator for MT stability, and such a role of Mask requires normal function of Jupiter.
| S-EPMC8572007 | biostudies-literature
Project description:We recently reported that mutations in the widely expressed nuclear protein TOPORS (topoisomerase I-binding arginine/serine rich) are associated with autosomal dominant retinal degeneration. However, the precise localization and a functional role of TOPORS in the retina remain unknown. Here, we demonstrate that TOPORS is a novel component of the photoreceptor sensory cilium, which is a modified primary cilium involved with polarized trafficking of proteins. In photoreceptors, TOPORS localizes primarily to the basal bodies of connecting cilium and in the centrosomes of cultured cells. Morpholino-mediated silencing of topors in zebrafish embryos demonstrates in another species a comparable retinal problem as seen in humans, resulting in defective retinal development and failure to form outer segments. These defects can be rescued by mRNA encoding human TOPORS. Taken together, our data suggest that TOPORS may play a key role in regulating primary cilia-dependent photoreceptor development and function. Additionally, it is well known that mutations in other ciliary proteins cause retinal degeneration, which may explain why mutations in TOPORS result in the same phenotype.
| S-EPMC3033188 | biostudies-literature
Project description:Neurons, like all cells, face the problem that tubulin forms microtubules with too many or too few protofilaments (pfs). Cells overcome this heterogeneity with the γ-tubulin ring complex, which provides a nucleation template for 13-pf microtubules. Doublecortin (DCX), a protein that stabilizes microtubules in developing neurons, also nucleates 13-pf microtubules in vitro. Using fluorescence microscopy assays, we show that the binding of DCX to microtubules is optimized for the lateral curvature of the 13-pf lattice. This sensitivity depends on a cooperative interaction wherein DCX molecules decrease the dissociation rate of their neighbors. Mutations in DCX found in patients with subcortical band heterotopia weaken these cooperative interactions. Using assays with dynamic microtubules, we discovered that DCX binds to polymerization intermediates at growing microtubule ends. These results support a mechanism for stabilizing 13-pf microtubules that allows DCX to template new 13-pf microtubules through associations with the sides of the microtubule lattice.
| S-EPMC3951992 | biostudies-literature
Project description:Chromosome segregation during mitosis and meiosis relies on the spindle and the functions of numerous microtubule-associated proteins (MAPs). One of the best-studied spindle MAPs is the highly conserved TPX2, which has been reported to have characteristic intracellular dynamics and molecular activities, such as nuclear localisation in interphase, poleward movement in the metaphase spindle, microtubule nucleation, microtubule stabilisation, microtubule bundling, Aurora A kinase activation, kinesin-5 binding, and kinesin-12 recruitment. This protein has been shown to be essential for spindle formation in every cell type analysed so far. However, as yet, TPX2 homologues have not been found in the Drosophila genome. In this study, I found that the Drosophila protein Ssp1/Mei-38 has significant homology to TPX2. Sequence conservation was limited to the putative spindle microtubule-associated region of TPX2, and intriguingly, D-TPX2 (Ssp1/Mei-38) lacks Aurora A- and kinesin-5-binding domains, which are highly conserved in other animal and plant species, including many insects such as ants and bees. D-TPX2 uniformly localised to kinetochore microtubule-enriched regions of the metaphase spindle in the S2 cell line, and it had microtubule binding and bundling activities in vitro. In comparison with other systems, the contribution of D-TPX2 to cell division seems to be minor; live cell imaging of microtubules and chromosomes after RNAi knockdown identified significant delay in chromosome congression in only 18% of the cells. Thus, while this conserved spindle protein is present in Drosophila, other mechanisms may largely compensate for its spindle assembly and chromosome segregation functions.
| S-EPMC3227607 | biostudies-literature
Project description:Doublecortin is a neuronal microtubule-stabilising protein, mutations of which cause mental retardation and epilepsy in humans. How doublecortin influences microtubule dynamics, and thereby brain development, is unclear. We show here by video microscopy that purified doublecortin has no effect on the growth rate of microtubules. However, it is a potent anti-catastrophe factor that stabilises microtubules by linking adjacent protofilaments and counteracting their outward bending in depolymerising microtubules. We show that doublecortin-stabilised microtubules are substrates for kinesin translocase motors and for depolymerase kinesins. In addition, doublecortin does not itself oligomerise and does not bind to tubulin heterodimers but does nucleate microtubules. In cells, doublecortin is enriched at the distal ends of neuronal processes and our data raise the possibility that the function of doublecortin in neurons is to drive assembly and stabilisation of non-centrosomal microtubules in these doublecortin-enriched distal zones. These distinct properties combine to give doublecortin a unique function in microtubule regulation, a role that cannot be compensated for by other microtubule-stabilising proteins and nucleating factors.
| S-EPMC1590004 | biostudies-literature
Project description:Background & aimsNeonatal sclerosing cholangitis (NSC) is a severe neonatal-onset cholangiopathy commonly leading to liver transplantation (LT) for end-stage liver disease in childhood. Liver biopsy findings histopathologically resemble those in biliary atresia (BA); however, in NSC extrahepatic bile ducts are patent, whilst in BA their lumina are obliterated. NSC is commonly seen in consanguineous kindreds, suggesting autosomal recessive inheritance.MethodsFrom 29 NSC patients (24 families) identified, DNA was available in 24 (21 families). Thirteen (7 male) patients (12 families) of consanguineous parentage were selected for whole exome sequencing. Sequence variants were filtered for homozygosity, pathogenicity, minor allele frequency, quality score, and encoded protein expression pattern.ResultsFour of 13 patients were homozygous and two were compound heterozygous for mutations in the doublecortin domain containing 2 gene (DCDC2), which encodes DCDC2 protein and is expressed in cholangiocyte cilia. Another 11 patients were sequenced: one (with one sibling pair) was compound heterozygous for DCDC2 mutations. All mutations were protein-truncating. In available liver tissue from patients with DCDC2 mutations, immunostaining for human DCDC2 and the ciliary protein acetylated alpha-tubulin (ACALT) showed no expression (n=6) and transmission electron microscopy found that cholangiocytes lacked primary cilia (n=5). DCDC2 and ACALT were expressed in NSC patients without DCDC2 mutations (n=22). Of the patients carrying DCDC2 mutations, one died awaiting LT; five came to LT, of whom one died 2years later. The other 4 are well.ConclusionAmong 24 NSC patients with available DNA, 7 had mutations in DCDC2 (6 of 19 families). NSC patients in substantial proportion harbour mutations in DCDC2. Their disease represents a novel liver-based ciliopathy.Lay summaryNeonatal sclerosing cholangitis (NSC) is a rare genetic form of liver disease presenting in infancy. Through next generation sequencing we identified mutations in the gene encoding for doublecortin domain containing 2 (DCDC2) protein in a group of NSC children. DCDC2 is a signalling and structural protein found in primary cilia of cholangiocytes. Cholangiocytes are the cells forming the biliary system which is the draining system of the liver.
| S-EPMC5116266 | biostudies-literature
Project description:Many microtubule (MT) functions are mediated by a diverse class of proteins (+TIPs) at growing MT plus ends that control intracellular MT interactions and dynamics and depend on end-binding proteins (EBs) [1]. Cryoelectron microscopy has recently identified the EB binding site as the interface of four tubulin dimers that undergoes a conformational change in response to β-tubulin GTP hydrolysis [2, 3]. Doublecortin (DCX), a MT-associated protein (MAP) required for neuronal migration during cortical development [4, 5], binds to the same site as EBs [6], and recent in vitro studies proposed DCX localization to growing MT ends independent of EBs [7]. Because this conflicts with observations in neurons [8, 9] and the molecular function of DCX is not well understood, we revisited intracellular DCX dynamics at low expression levels. Here, we report that DCX is not a +TIP in cells but, on the contrary, is excluded from the EB1 domain. In addition, we find that DCX-MT interactions are highly sensitive to MT geometry. In cells, DCX binding was greatly reduced at MT segments with high local curvature. Remarkably, this geometry-dependent binding to MTs was completely reversed in the presence of taxanes, which reconciles incompatible observations in cells [9] and in vitro [10]. We propose a model explaining DCX specificity for different MT geometries based on structural changes induced by GTP hydrolysis that decreases the spacing between adjacent tubulin dimers [11]. Our data are consistent with a unique mode of MT interaction in which DCX specifically recognizes this compacted GDP-like MT lattice.
| S-EPMC5023073 | biostudies-literature
Project description:Microtubule-associated 1B (MAP1B) proteins are expressed at the nervous system level where they control cytoskeleton activity and regulate neurotransmitter release. Here, we report about the identification of a planarian MAP1B factor (DjMap1B) that is enriched in cephalic ganglia and longitudinal nerve cords but not in neoblasts, the plentiful population of adult stem cells present in planarians, thanks to which these animals can continuously cell turnover and regenerate any lost body parts. DjMap1B knockdown induces morphological anomalies in the nervous system and affects neoblast commitment. Our data put forward a correlation between a MAP1B factor and stem cells and suggest a function of the nervous system in non-cell autonomous control of planarian stem cells.
| S-EPMC9744283 | biostudies-literature
Project description:17p13.3 microduplication syndrome is a newly identified genetic disorder characterized by duplications in the 17p13.3 chromosome locus, resulting in a variety of disorders including autism spectrum disorder (ASD). Importantly, a minimum duplication region has been defined, and this region exclusively contains the gene encoding 14-3-3?. Furthermore, duplication of this minimum region is strongly associated with the appearance of ASD in human patients, thus implicating the overexpression of 14-3-3? in ASD. Using in vitro and in vivo techniques, we have found that 14-3-3? binds to the microtubule binding protein doublecortin preventing its degradation. We also found that 14-3-3? overexpression disrupts neurite formation by preventing the invasion of microtubules into primitive neurites, which can be rescued by the knockdown of doublecortin. To analyse the function of 14-3-3? in neurite formation, we used 14-3-3? flox mice and found that 14-3-3? deficiency results in an increase in neurite formation. Our findings provide the first evidence of cellular pathology in 17p13.3 microduplication syndrome.
| S-EPMC6078596 | biostudies-literature