Publications by authors named "Guangshuo Ou"

63 Publications

COVID-19, cilia, and smell.

FEBS J 2020 09 6;287(17):3672-3676. Epub 2020 Aug 6.

Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, McGovern Institute for Brain Research, School of Life Sciences, MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China.

The novel coronavirus SARS-CoV-2 is the causative agent of the global coronavirus disease 2019 (COVID-19) outbreak. In addition to pneumonia, other COVID-19-associated symptoms have been reported, including loss of smell (anosmia). However, the connection between infection with coronavirus and anosmia remains enigmatic. It has been reported that defects in olfactory cilia lead to anosmia. In this Viewpoint, we summarize transmission electron microscopic studies of cilia in virus-infected cells. In the human nasal epithelium, coronavirus infects the ciliated cells and causes deciliation. Research has shown that viruses such as influenza and Sendai attach to the ciliary membrane. The Sendai virus enters cilia by fusing its viral membrane with the ciliary membrane. A recent study on SARS-CoV-2-human protein-protein interactions revealed that the viral nonstructural protein Nsp13 interacts with the centrosome components, providing a potential molecular link. The mucociliary escalator removes inhaled pathogenic particles and functions as the first line of protection mechanism against viral infection in the human airway. Thus, future investigation into the virus-cilium interface will help further the battle against COVID-19.
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http://dx.doi.org/10.1111/febs.15491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426555PMC
September 2020

The spectrin-based membrane skeleton is asymmetric and remodels during neural development in .

J Cell Sci 2020 08 5;133(15). Epub 2020 Aug 5.

Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, McGovern Institute for Brain Research, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing 100084, China

Perturbation of spectrin-based membrane mechanics causes hereditary elliptocytosis and spinocerebellar ataxia, but the underlying cellular basis of pathogenesis remains unclear. Here, we introduced conserved disease-associated spectrin mutations into the genome and studied the contribution of spectrin to neuronal migration and dendrite formation in developing larvae. The loss of spectrin resulted in ectopic actin polymerization outside of the existing front and secondary membrane protrusions, leading to defective neuronal positioning and dendrite morphology in adult animals. Spectrin accumulated in the lateral region and rear of migrating neuroblasts and redistributes from the soma into the newly formed dendrites, indicating that the spectrin-based membrane skeleton is asymmetric and remodels to regulate actin assembly and cell shape during development. We affinity-purified spectrin from and showed that its binding partner ankyrin functions with spectrin. Asymmetry and remodeling of the membrane skeleton might enable spatiotemporal modulation of membrane mechanics for distinct developmental events.
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http://dx.doi.org/10.1242/jcs.248583DOI Listing
August 2020

Spatial confinement of receptor activity by tyrosine phosphatase during directional cell migration.

Proc Natl Acad Sci U S A 2020 06 8;117(25):14270-14279. Epub 2020 Jun 8.

Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China;

Directional cell migration involves signaling cascades that stimulate actin assembly at the leading edge, and additional pathways must inhibit actin polymerization at the rear. During neuroblast migration in , the transmembrane protein MIG-13/Lrp12 acts through the Arp2/3 nucleation-promoting factors WAVE and WASP to guide the anterior migration. Here we show that a tyrosine kinase, SRC-1, directly phosphorylates MIG-13 and promotes its activity on actin assembly at the leading edge. In GFP knockin animals, SRC-1 and MIG-13 distribute along the entire plasma membrane of migrating cells. We reveal that a receptor-like tyrosine phosphatase, PTP-3, maintains the F-actin polarity during neuroblast migration. Recombinant PTP-3 dephosphorylates SRC-1-dependent MIG-13 phosphorylation in vitro. Importantly, the endogenous PTP-3 accumulates at the rear of the migrating neuroblast, and its extracellular domain is essential for directional cell migration. We provide evidence that the asymmetrically localized tyrosine phosphatase PTP-3 spatially restricts MIG-13/Lrp12 receptor activity in migrating cells.
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http://dx.doi.org/10.1073/pnas.2003019117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321996PMC
June 2020

Optimal sidestepping of intraflagellar transport kinesins regulates structure and function of sensory cilia.

EMBO J 2020 06 27;39(12):e103955. Epub 2020 Apr 27.

Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China.

Cytoskeletal-based molecular motors produce force perpendicular to their direction of movement. However, it remains unknown whether and why motor proteins generate sidesteps movement along their filamentous tracks in vivo. Using Hessian structured illumination microscopy, we located green fluorescent protein (GFP)-labeled intraflagellar transport (IFT) particles inside sensory cilia of live Caenorhabditis elegans with 3-6-nanometer accuracy and 3.4-ms resolution. We found that IFT particles took sidesteps along axoneme microtubules, demonstrating that IFT motors generate torque in a living animal. Kinesin-II and OSM-3-kinesin collaboratively drive anterograde IFT. We showed that the deletion of kinesin-II, a torque-generating motor protein, reduced sidesteps, whereas the increase of neck flexibility of OSM-3-kinesin upregulated sidesteps. Either increase or decrease of sidesteps of IFT kinesins allowed ciliogenesis to the regular length, but changed IFT speeds, disrupted axonemal ninefold symmetry, and inhibited sensory cilia-dependent animal behaviors. Thus, an optimum level of IFT kinesin sidestepping is associated with the structural and functional fidelity of cilia.
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http://dx.doi.org/10.15252/embj.2019103955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298308PMC
June 2020

Theme series: Cilia and ciliopathies.

Authors:
Wei Li Guangshuo Ou

Biol Cell 2019 12;111(12):293

China Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.

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http://dx.doi.org/10.1111/boc.201900079DOI Listing
December 2019

Chemoproteomic Profiling of O-GlcNAcylation in .

Biochemistry 2020 09 4;59(34):3129-3134. Epub 2019 Nov 4.

Tsinghua-Peking Center for Life Sciences, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing 100084, China.

Genetic studies have revealed essential functions of O-linked -acetylglucosamine (O-GlcNAc) modification in . However, large-scale identification of O-GlcNAcylated proteins and mapping the modification sites in remain relatively unexplored. By using a chemoproteomic strategy, we herein report the identification of 108 high-confidence O-GlcNAcylated proteins and 64 modification sites in . Furthermore, quantitative proteomics upon altering O-GlcNAcylation show that the abundance of a large number of proteins are affected by O-GlcNAc. These proteins are involved in regulating reproduction and lifespan, which may correlate with the previously observed phenotypes in genetic studies. The data set in this study reveals the O-GlcNAc modification landscape in and provides a valuable resource for dissecting the biological function of O-GlcNAcylation.
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http://dx.doi.org/10.1021/acs.biochem.9b00622DOI Listing
September 2020

Spectrin-based membrane skeleton supports ciliogenesis.

PLoS Biol 2019 07 12;17(7):e3000369. Epub 2019 Jul 12.

Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China.

Cilia are remarkable cellular devices that power cell motility and transduce extracellular signals. To assemble a cilium, a cylindrical array of 9 doublet microtubules push out an extension of the plasma membrane. Membrane tension regulates cilium formation; however, molecular pathways that link mechanical stimuli to ciliogenesis are unclear. Using genome editing, we introduced hereditary elliptocytosis (HE)- and spinocerebellar ataxia (SCA)-associated mutations into the Caenorhabditis elegans membrane skeletal protein spectrin. We show that these mutations impair mechanical support for the plasma membrane and change cell shape. RNA sequencing (RNA-seq) analyses of spectrin-mutant animals uncovered a global down-regulation of ciliary gene expression, prompting us to investigate whether spectrin participates in ciliogenesis. Spectrin mutations affect intraflagellar transport (IFT), disrupt axonemal microtubules, and inhibit cilium formation, and the endogenous spectrin periodically distributes along cilia. Mammalian spectrin also localizes in cilia and regulates ciliogenesis. These results define a previously unrecognized yet conserved role of spectrin-based mechanical support for cilium biogenesis.
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http://dx.doi.org/10.1371/journal.pbio.3000369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6655744PMC
July 2019

Ciliopathy-associated proteins are involved in vesicle distribution in sensory cilia.

J Genet Genomics 2019 05 15;46(5):269-271. Epub 2019 May 15.

Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China. Electronic address:

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http://dx.doi.org/10.1016/j.jgg.2019.03.012DOI Listing
May 2019

The kinases male germ cell-associated kinase and cell cycle-related kinase regulate kinesin-2 motility in Caenorhabditis elegans neuronal cilia.

Traffic 2018 07 17;19(7):522-535. Epub 2018 May 17.

Tsinghua-Peking Center for Life Sciences, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China.

Kinesin-2 motors power anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. However, it remains elusive how kinesin-2 motors are regulated in vivo. Here, we performed forward genetic screens to isolate suppressors that rescue the ciliary defects of OSM-3-kinesin (homolog of mammalian homodimeric kinesin-2 KIF17) mutants in Caenorhabditis elegans. We identified the C. elegans dyf-5 and dyf-18, which encode the homologs of mammalian male germ cell-associated kinase and cell cycle-related kinase, respectively. Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate elongated cilia and ectopic localization of the heterotrimeric kinesin-2 (kinesin-II) at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are important for stabilizing the interaction between IFT particles and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.
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http://dx.doi.org/10.1111/tra.12572DOI Listing
July 2018

Migration of Q Cells in Caenorhabditis elegans.

Methods Mol Biol 2018 ;1749:239-255

Tsinghua-Peking Center for Life Sciences, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China.

During C. elegans larval development, the Q neuroblasts produce their lineage by three rounds of divisions along with continuous cell migrations. Their neuronal progeny is dispersed from the pharynx to the anus. This in vivo system to study cell migration is appealing for several reasons. The lineage development is stereotyped; functional analysis and genomic screens are rendered easy and powerful thanks to powerful tools; transgenic manipulations and genome engineering are efficient and can be conveniently combined with live-cell imaging. Here we describe a series of protocols in Q cell migration studies, including quantifications of progeny position, genetic screening strategies, preparation of migration mutants or transgenic worms expressing related fluorescent proteins, multipositional time-lapse tracking of Q cell migration using confocal microscopy and image analyses of single cell movements and dynamics.
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http://dx.doi.org/10.1007/978-1-4939-7701-7_18DOI Listing
January 2019

Microtubule-binding protein FOR20 promotes microtubule depolymerization and cell migration.

Cell Discov 2017 5;3:17032. Epub 2017 Sep 5.

Department of Cell Biology and Program in Molecular Cell Biology, Zhejiang University School of Medicine, Yuhangtang Road, Hangzhou, Zhejiang, China.

Microtubules are highly dynamic filaments assembled from αβ-tubulin heterodimers and play important roles in many cellular processes, including cell division and migration. Microtubule dynamics is tightly regulated by microtubule-associated proteins (MAPs) that function by binding to microtubules or free tubulin dimers. Here, we report that FOR20 (FOP-related protein of 20 kDa), a conserved protein critical for ciliogenesis and cell cycle progression, is a previously uncharacterized MAP that facilitates microtubule depolymerization and promotes cell migration. FOR20 not only directly binds to microtubules but also regulates microtubule dynamics by decreasing the microtubule growth rate and increasing the depolymerization rate and catastrophe frequency. In the microtubule dynamics assays, FOR20 appears to preferentially interact with free tubulin dimers over microtubules. Depletion of FOR20 inhibits microtubule depolymerization and promotes microtubule regrowth after the nocodazole treatment in HeLa cells. In addition, FOR20 knockdown significantly inhibits both individual and collective migration of mammalian cells. Taken together, these data suggest that FOR20 functions as a MAP to promote microtubule depolymerization and cell migration.
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http://dx.doi.org/10.1038/celldisc.2017.32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5583970PMC
September 2017

WASP-Arp2/3-dependent actin polymerization influences fusogen localization during cell-cell fusion in embryos.

Biol Open 2017 Sep 15;6(9):1324-1328. Epub 2017 Sep 15.

Tsinghua-Peking Center for Life Sciences, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing 100084, China

Cell-cell fusion is essential for development and physiology. Actin polymerization was implicated in the fusogen EFF-1 engagement in a reconstituted cell culture system, and the actin-binding protein spectraplakin links EFF-1 to the actin cytoskeleton and promotes cell-cell fusions in larvae. However, it remains unclear whether and how fusogens and the actin cytoskeleton are coordinated in embryos. Here, we used live imaging analysis of GFP knock-in and RNAi embryos to study the embryonic cell-cell fusions in Our results show that the inhibition of WASP-Arp2/3-dependent actin polymerization delays cell-cell fusions. EFF-1 is primarily distributed in intracellular vesicles in embryonic fusing cells, and we find that the perturbation of actin polymerization reduces the number of EFF-1-postive vesicles. Thus, the actin cytoskeleton differently promotes cell-cell fusion by regulating fusogen localization to the fusing plasma membrane in larvae or to intracellular vesicles in embryos.
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http://dx.doi.org/10.1242/bio.026807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612239PMC
September 2017

Centriole translocation and degeneration during ciliogenesis in neurons.

EMBO J 2017 09 25;36(17):2553-2566. Epub 2017 Jul 25.

Tsinghua-Peking Center for Life Sciences, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China

Neuronal cilia that are formed at the dendritic endings of sensory neurons are essential for sensory perception. However, it remains unclear how the centriole-derived basal body is positioned to form a template for cilium formation. Using fluorescence time-lapse microscopy, we show that the centriole translocates from the cell body to the dendrite tip in the sensory neurons. The centriolar protein SAS-5 interacts with the dynein light-chain LC8 and conditional mutations of cytoplasmic dynein-1 block centriole translocation and ciliogenesis. The components of the central tube are essential for the biogenesis of centrioles, which later drive ciliogenesis in the dendrite; however, the centriole loses these components at the late stage of centriole translocation and subsequently recruits transition zone and intraflagellar transport proteins. Together, our results provide a comprehensive model of ciliogenesis in sensory neurons and reveal the importance of the dynein-dependent centriole translocation in this process.
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http://dx.doi.org/10.15252/embj.201796883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579362PMC
September 2017

CED-10-WASP-Arp2/3 signaling axis regulates apoptotic cell corpse engulfment in C. elegans.

Dev Biol 2017 08 9;428(1):215-223. Epub 2017 Jun 9.

School of Medicine, Tsinghua University, Beijing 100084, China. Electronic address:

Efficient clearance of apoptotic cells is essential for tissue homeostasis in metazoans. Genetic studies in Caenorhabditis elegans have identified signaling cascades that activate CED-10/Rac1 GTPase and promote actin cytoskeletal rearrangement during apoptotic cell engulfment. However, the molecular connection between CED-10 activation and actin reorganization remains elusive. Here, we provide evidence that CED-10 binds to the Arp2/3 nucleation promoting factor WASP; CED-10 recruits WASP and Arp2/3 to apoptotic cell corpses in the phagocytes. The loss of WASP and Arp2/3 impaired cell corpse engulfment. Furthermore, we uncover that a WASP-activating factor SEM-5/GRB2 functions in the phagocytes to promote cell corpse clearance. Together, our results suggest CED-10 reorganizes the actin cytoskeleton by recruiting the WASP-Arp2/3 actin nucleation factors during apoptotic cell engulfment.
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http://dx.doi.org/10.1016/j.ydbio.2017.06.005DOI Listing
August 2017

Dynein-Driven Retrograde Intraflagellar Transport Is Triphasic in C. elegans Sensory Cilia.

Curr Biol 2017 May 4;27(10):1448-1461.e7. Epub 2017 May 4.

Tsinghua-Peking Center for Life Sciences, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing 100084, China. Electronic address:

Cytoplasmic dynein-2 powers retrograde intraflagellar transport that is essential for cilium formation and maintenance. Inactivation of dynein-2 by mutations in DYNC2H1 causes skeletal dysplasias, and it remains unclear how the dynein-2 heavy chain moves in cilia. Here, using the genome-editing technique to produce fluorescent dynein-2 heavy chain in Caenorhabditis elegans, we show by high-resolution live microscopy that dynein-2 moves in a surprising way along distinct ciliary domains. Dynein-2 shows triphasic movement in the retrograde direction: dynein-2 accelerates in the ciliary distal region and then moves at maximum velocity and finally decelerates adjacent to the base, which may represent a physical obstacle due to transition zone barriers. By knocking the conserved ciliopathy-related mutations into the C. elegans dynein-2 heavy chain, we find that these mutations reduce its transport speed and frequency. Disruption of the dynein-2 tail domain, light intermediate chain, or intraflagellar transport (IFT)-B complex abolishes dynein-2's ciliary localization, revealing their important roles in ciliary entry of dynein-2. Furthermore, our affinity purification and genetic analyses show that IFT-A subunits IFT-139 and IFT-43 function redundantly to promote dynein-2 motility. These results reveal the molecular regulation of dynein-2 movement in sensory cilia.
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http://dx.doi.org/10.1016/j.cub.2017.04.015DOI Listing
May 2017

Spectraplakin Induces Positive Feedback between Fusogens and the Actin Cytoskeleton to Promote Cell-Cell Fusion.

Dev Cell 2017 04;41(1):107-120.e4

Tsinghua-Peking Center for Life Sciences, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing 100084, China. Electronic address:

Cell-cell fusion generally requires cellular fusogenic proteins and actin-propelled membrane protrusions. However, the molecular connections between fusogens and the actin cytoskeleton remain unclear. Here, we show that the Caenorhabditis elegans fusogen EFF-1 and F-actin are enriched at the cortex of the post-embryonic fusing cells, and conditional mutations of WASP and Arp2/3 delay cell-cell fusion by impairing EFF-1 localization. Our affinity purification and mass spectrometry analyses determined that an actin-binding protein, spectraplakin/VAB-10A, binds to EFF-1. VAB-10A promotes cell-cell fusion by linking EFF-1 to the actin cytoskeleton. Conversely, EFF-1 enhanced the F-actin bundling activity of VAB-10A in vitro, and actin dynamics in the cortex were reduced in eff-1 or vab-10a mutants. Thus, cell-cell fusion is promoted by a positive feedback loop in which actin filaments that are crosslinked by spectraplakin to recruit fusogens to fusion sites are reinforced via fusogens, thereby increasing the probability of further fusogen accumulation to form fusion synapses.
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http://dx.doi.org/10.1016/j.devcel.2017.03.006DOI Listing
April 2017

Hippo kinases maintain polarity during directional cell migration in Caenorhabditis elegans.

EMBO J 2017 02 23;36(3):334-345. Epub 2016 Dec 23.

Tsinghua-Peking Center for Life Sciences, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China

Precise positioning of cells is crucial for metazoan development. Despite immense progress in the elucidation of the attractive cues of cell migration, the repulsive mechanisms that prevent the formation of secondary leading edges remain less investigated. Here, we demonstrate that Caenorhabditis elegans Hippo kinases promote cell migration along the anterior-posterior body axis via the inhibition of dorsal-ventral (DV) migration. Ectopic DV polarization was also demonstrated in gain-of-function mutant animals for C. elegans RhoG MIG-2. We identified serine 139 of MIG-2 as a novel conserved Hippo kinase phosphorylation site and demonstrated that purified Hippo kinases directly phosphorylate MIG-2 Live imaging analysis of genome-edited animals indicates that MIG-2 phosphorylation impedes actin assembly in migrating cells. Intriguingly, Hippo kinases are excluded from the leading edge in wild-type cells, while MIG-2 loss induces uniform distribution of Hippo kinases. We provide evidence that Hippo kinases inhibit RhoG activity locally and are in turn restricted to the cell body by RhoG-mediated polarization. Therefore, we propose that the Hippo-RhoG feedback regulation maintains cell polarity during directional cell motility.
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http://dx.doi.org/10.15252/embj.201695734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286363PMC
February 2017

The glial actin cytoskeleton regulates neuronal ciliogenesis.

Cell Res 2017 03 11;27(3):448-451. Epub 2016 Nov 11.

Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.

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http://dx.doi.org/10.1038/cr.2016.131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339830PMC
March 2017

Functional Coordination of WAVE and WASP in C. elegans Neuroblast Migration.

Dev Cell 2016 10;39(2):224-238

Tsinghua-Peking Center for Life Sciences, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing 100084, China. Electronic address:

Directional cell migration is critical for metazoan development. We define two molecular pathways that activate the Arp2/3 complex during neuroblast migration in Caenorhabditis elegans. The transmembrane protein MIG-13/Lrp12 is linked to the Arp2/3 nucleation-promoting factors WAVE or WASP through direct interactions with ABL-1 or SEM-5/Grb2, respectively. WAVE mutations partially impaired F-actin organization and decelerated cell migration, and WASP mutations did not inhibit cell migration but enhanced migration defects in WAVE-deficient cells. Purified SEM-5 and MIG-2 synergistically stimulated the F-actin branching activity of WASP-Arp2/3 in vitro. In GFP knockin animals, WAVE and WASP were largely organized into separate clusters at the leading edge, and the amount of WASP was less than WAVE but could be elevated by WAVE mutations. Our results indicate that the MIG-13-WAVE pathway provides the major force for directional cell motility, whereas MIG-13-WASP partially compensates for its loss, underscoring their coordinated activities in facilitating robust cell migration.
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http://dx.doi.org/10.1016/j.devcel.2016.09.029DOI Listing
October 2016

Stox1 as a novel transcriptional suppressor of Math1 during cerebellar granule neurogenesis and medulloblastoma formation.

Cell Death Differ 2016 12 26;23(12):2042-2053. Epub 2016 Aug 26.

State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.

Cerebellar granule neuronal progenitors (GNPs) are the precursors of cerebellar granule cells (CGCs) and are believed to be the cell of origin for medulloblastoma (MB), yet the molecular mechanisms governing GNP neurogenesis are poorly elucidated. Here, we demonstrate that storkhead box 1 (Stox1), a forkhead transcriptional factor, has a pivotal role in cerebellar granule neurogenesis and MB suppression. Expression of Stox1 is upregulated along with GNP differentiation and repressed by activation of sonic hedgehog (SHH) signaling. Stox1 exerts its neurogenic and oncosuppressing effect via direct transcriptional repression of Math1, a basic helix-loop-helix transcription activator essential for CGC genesis. This study illustrates a SHH-Stox1-Math1 regulatory axis in normal cerebellar development and MB formation.
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http://dx.doi.org/10.1038/cdd.2016.85DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5136492PMC
December 2016

Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length.

Nat Commun 2016 06 16;7:11874. Epub 2016 Jun 16.

Beijing Advanced Innovation Center for Structural Biology, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China.

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAP(F375A), with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAP(EE343RR) that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a 'clutch-like' mechanism.
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http://dx.doi.org/10.1038/ncomms11874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912634PMC
June 2016

The application of somatic CRISPR-Cas9 to conditional genome editing in Caenorhabditis elegans.

Authors:
Wei Li Guangshuo Ou

Genesis 2016 Apr 14;54(4):170-81. Epub 2016 Apr 14.

Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.

Forward and reverse genetic approaches have been well developed in the nematode Caenorhabditis elegans; however, efficient genetic tools to generate conditional gene mutations are still in high demand. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system for genome modification has provided an additional tool for C. elegans researchers to achieve simple and efficient conditional targeted mutagenesis. Here, we review recent advances in the somatic expression of Cas9 endonuclease for conditional gene editing. We present some practical considerations for improving the efficiency and reducing the off-target effects of somatic CRISPR-Cas9 and highlight a strategy to analyze somatic mutation at single-cell resolution. Finally, we outline future applications and consider challenges for this emerging genome editing platform that will need to be addressed in the future.
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http://dx.doi.org/10.1002/dvg.22932DOI Listing
April 2016

EGL-20/Wnt and MAB-5/Hox Act Sequentially to Inhibit Anterior Migration of Neuroblasts in C. elegans.

PLoS One 2016 10;11(2):e0148658. Epub 2016 Feb 10.

Programs in Genetics and Molecular, Cellular and Developmental Biology, Department of Molecular Biosciences, University of Kansas, Lawrence, KS, 66045, United States of America.

Directed neuroblast and neuronal migration is important in the proper development of nervous systems. In C. elegans the bilateral Q neuroblasts QR (on the right) and QL (on the left) undergo an identical pattern of cell division and differentiation but migrate in opposite directions (QR and descendants anteriorly and QL and descendants posteriorly). EGL-20/Wnt, via canonical Wnt signaling, drives the expression of MAB-5/Hox in QL but not QR. MAB-5 acts as a determinant of posterior migration, and mab-5 and egl-20 mutants display anterior QL descendant migrations. Here we analyze the behaviors of QR and QL descendants as they begin their anterior and posterior migrations, and the effects of EGL-20 and MAB-5 on these behaviors. The anterior and posterior daughters of QR (QR.a/p) after the first division immediately polarize and begin anterior migration, whereas QL.a/p remain rounded and non-migratory. After ~1 hour, QL.a migrates posteriorly over QL.p. We find that in egl-20/Wnt, bar-1/β-catenin, and mab-5/Hox mutants, QL.a/p polarize and migrate anteriorly, indicating that these molecules normally inhibit anterior migration of QL.a/p. In egl-20/Wnt mutants, QL.a/p immediately polarize and begin migration, whereas in bar-1/β-catenin and mab-5/Hox, the cells transiently retain a rounded, non-migratory morphology before anterior migration. Thus, EGL-20/Wnt mediates an acute inhibition of anterior migration independently of BAR-1/β-catenin and MAB-5/Hox, and a later, possible transcriptional response mediated by BAR-1/β-catenin and MAB-5/Hox. In addition to inhibiting anterior migration, MAB-5/Hox also cell-autonomously promotes posterior migration of QL.a (and QR.a in a mab-5 gain-of-function).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148658PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4749177PMC
July 2016

Anillin Regulates Neuronal Migration and Neurite Growth by Linking RhoG to the Actin Cytoskeleton.

Curr Biol 2015 May 2;25(9):1135-45. Epub 2015 Apr 2.

Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China. Electronic address:

Neuronal migration and neurite growth are essential events in neural development, but it remains unclear how guidance cues are transduced through receptors to the actin cytoskeleton, which powers these processes. We report that a cytokinetic scaffold protein, Anillin, is redistributed to the leading edge of the C. elegans Q neuroblast during cell migration and neurite growth. To bypass the requirement for Anillin in cytokinesis, we used the somatic CRISPR-Cas9 technique to generate conditional mutations in Anillin. We demonstrate that Anillin regulates cell migration and growth cone extension by stabilizing the F-actin network at the leading edge. Our biochemical analysis shows that the actin-binding domain of Anillin is sufficient to stabilize F-actin by antagonizing the F-actin severing activity of Cofilin. We further uncover that the active form of RhoG/MIG-2 directly binds to Anillin and recruits it to the leading edge. Our results reveal a novel pathway in which Anillin transduces the RhoG signal to the actin cytoskeleton during neuronal migration and neurite growth.
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http://dx.doi.org/10.1016/j.cub.2015.02.072DOI Listing
May 2015

Somatic CRISPR-Cas9-induced mutations reveal roles of embryonically essential dynein chains in Caenorhabditis elegans cilia.

J Cell Biol 2015 Mar;208(6):683-92

Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China

Cilium formation and maintenance require intraflagellar transport (IFT). Although much is known about kinesin-2-driven anterograde IFT, the composition and regulation of retrograde IFT-specific dynein remain elusive. Components of cytoplasmic dynein may participate in IFT; however, their essential roles in cell division preclude functional studies in postmitotic cilia. Here, we report that inducible expression of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system in Caenorhabditis elegans generated conditional mutations in IFT motors and particles, recapitulating ciliary defects in their null mutants. Using this method to bypass the embryonic requirement, we show the following: the dynein intermediate chain, light chain LC8, and lissencephaly-1 regulate retrograde IFT; the dynein light intermediate chain functions in dendrites and indirectly contributes to ciliogenesis; and the Tctex and Roadblock light chains are dispensable for cilium assembly. Furthermore, we demonstrate that these components undergo biphasic IFT with distinct transport frequencies and turnaround behaviors. Together, our results suggest that IFT-dynein and cytoplasmic dynein have unique compositions but also share components and regulatory mechanisms.
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http://dx.doi.org/10.1083/jcb.201411041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362450PMC
March 2015

Transcription factor STOX1 regulates proliferation of inner ear epithelial cells via the AKT pathway.

Cell Prolif 2015 Apr 10;48(2):209-20. Epub 2015 Feb 10.

State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 210027, China.

Objectives: Storkhead box 1 (STOX1) belongs to the forkhead family of transcription factors, and is reported to be involved in apoptosis of Caenorhabditis elegans. However, up to now the precise role of STOX1 in mammalian epithelial development has not been established. Here, we report that it plays an important role in regulation of proliferation of inner ear epithelial cells.

Materials And Methods: Immunohistochemistry and reverse transcription-PCR assays were used to determine expression pattern of STOX1 in the mouse inner ear. Furthermore, its overexpression and knockdown effects on mouse inner ear epithelial cells were studied using RT-PCR, immunofluorescence, MTT assay, BrdU labelling and western blotting.

Results: Storkhead box 1 was selectively expressed in epithelial cells, but not in stromal cells of the inner ear. Its over-expression enhanced cell proliferation and sphere formation, however, STOX1 knockdown inhibited cell proliferation and sphere formation in purified utricular epithelial cells in culture. Consistently, several cell cycle regulatory genes such as for PCNA, cyclin A and cyclin E, were up-regulated by STOX1 over-expression. Furthermore, biochemical analyses indicated that proliferation-promoting effects induced by STOX1 were mediated via phosphorylation of AKT in these cells.

Conclusions: Taken together, we demonstrate that STOX1 is a novel stimulatory factor for inner ear epithelial cell proliferation and might be an important target to be considered in regeneration or repair of inner ear epithelium.
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http://dx.doi.org/10.1111/cpr.12174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6495863PMC
April 2015

The heparan sulfate-modifying enzyme glucuronyl C5-epimerase HSE-5 controls Caenorhabditis elegans Q neuroblast polarization during migration.

Dev Biol 2015 Mar 19;399(2):306-14. Epub 2015 Jan 19.

Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.

Directional cell migration is fundamental for neural development, and extracellular factors are pivotal for this process. Heparan sulfate proteoglycans (HSPGs) that carry long chains of differentially modified sugar residues contribute to extracellular matrix; however, the functions of HSPG in guiding cell migration remain elusive. Here, we used the Caenorhabditis elegans mutant pool from the Million Mutation Project and isolated a mutant allele of the heparan sulfate-modifying enzyme glucuronyl C5-epimerase HSE-5. Loss-of-function of this enzyme resulted in defective Q neuroblast migration. We showed that hse-5 controlled Q cell migration in a cell non-autonomous manner. By performing live cell imaging in hse-5 mutant animals, we found that hse-5 controlled initial polarization during Q neuroblast migration. Furthermore, our genetic epistasis analysis demonstrated that lon-2 might act downstream of hse-5. Finally, rescue of the hse-5 mutant phenotype by expression of human and mouse hse-5 homologs suggested a conserved function for this gene in neural development. Taken together, our results indicated that proper HSPG modification in the extracellular matrix by HSE-5 is essential for neuroblast polarity during migration.
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http://dx.doi.org/10.1016/j.ydbio.2015.01.007DOI Listing
March 2015

Dynamic phosphorylation of CENP-A at Ser68 orchestrates its cell-cycle-dependent deposition at centromeres.

Dev Cell 2015 Jan 31;32(1):68-81. Epub 2014 Dec 31.

National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address:

The H3 histone variant CENP-A is an epigenetic marker critical for the centromere identity and function. However, the precise regulation of the spatiotemporal deposition and propagation of CENP-A at centromeres during the cell cycle is still poorly understood. Here, we show that CENP-A is phosphorylated at Ser68 during early mitosis by Cdk1. Our results demonstrate that phosphorylation of Ser68 eliminates the binding of CENP-A to the assembly factor HJURP, thus preventing the premature loading of CENP-A to the centromere prior to mitotic exit. Because Cdk1 activity is at its minimum at the mitotic exit, the ratio of Cdk1/PP1α activity changes in favor of Ser68 dephosphorylation, thus making CENP-A available for centromeric deposition by HJURP. Thus, we reveal that dynamic phosphorylation of CENP-A Ser68 orchestrates the spatiotemporal assembly of newly synthesized CENP-A at active centromeres during the cell cycle.
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http://dx.doi.org/10.1016/j.devcel.2014.11.030DOI Listing
January 2015
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