The highly conserved FOXJ1 target CFAP161 is dispensable for motile ciliary function in mouse and Xenopus

AbstractCilia are protrusions of the cell surface and composed of hundreds of proteins many of which are evolutionary and functionally well conserved. In cells assembling motile cilia the expression of numerous ciliary components is under the control of the transcription factor FOXJ1. Here, we analyse the evolutionary conserved FOXJ1 target CFAP161 in Xenopus and mouse. In both species Cfap161 expression correlates with the presence of motile cilia and depends on FOXJ1. Tagged CFAP161 localises to the basal bodies of multiciliated cells of the Xenopus larval epidermis, and in mice CFAP161 protein localises to the axoneme. Surprisingly, disruption of the Cfap161 gene in both species did not lead to motile cilia-related phenotypes, which contrasts with the conserved expression in cells carrying motile cilia and high sequence conservation. In mice mutation of Cfap161 stabilised the mutant mRNA making genetic compensation triggered by mRNA decay unlikely. However, genes related to microtubules and cilia, microtubule motor activity and inner dyneins were dysregulated, which might buffer the Cfap161 mutation.

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Scoring a backstage pass: Mechanisms of ciliogenesis and ciliary access

The Journal of Cell Biology ◽

10.1083/jcb.201111146 ◽

2012 ◽

Vol 197 (6) ◽

pp. 697-709 ◽

Cited By ~ 153

Author(s):

Francesc R. Garcia-Gonzalo ◽

Jeremy F. Reiter

Keyword(s):

Cell Surface ◽

Primary Cilia ◽

Basal Bodies ◽

Protein Traffic ◽

Ciliary Function ◽

Kidney Cysts ◽

Motile Cilia ◽

Cilia And Flagella ◽

Inborn Defects

Cilia are conserved, microtubule-based cell surface projections that emanate from basal bodies, membrane-docked centrioles. The beating of motile cilia and flagella enables cells to swim and epithelia to displace fluids. In contrast, most primary cilia do not beat but instead detect environmental or intercellular stimuli. Inborn defects in both kinds of cilia cause human ciliopathies, diseases with diverse manifestations such as heterotaxia and kidney cysts. These diseases are caused by defects in ciliogenesis or ciliary function. The signaling functions of cilia require regulation of ciliary composition, which depends on the control of protein traffic into and out of cilia.

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Cytoplasmic E2f4 forms organizing centres for initiation of centriole amplification during multiciliogenesis

Nature Communications ◽

10.1038/ncomms15857 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 22

Author(s):

Munemasa Mori ◽

Renin Hazan ◽

Paul S. Danielian ◽

John E. Mahoney ◽

Huijun Li ◽

...

Keyword(s):

Transcription Factor ◽

Large Scale ◽

Abnormal Development ◽

Basal Bodies ◽

Transcriptional Program ◽

Mutant Proteins ◽

Multiprotein Complexes ◽

Airway Diseases ◽

Multiciliated Cells ◽

Cytoplasmic Structures

Abstract Abnormal development of multiciliated cells is a hallmark of a variety of human conditions associated with chronic airway diseases, hydrocephalus and infertility. Multiciliogenesis requires both activation of a specialized transcriptional program and assembly of cytoplasmic structures for large-scale centriole amplification that generates basal bodies. It remains unclear, however, what mechanism initiates formation of these multiprotein complexes in epithelial progenitors. Here we show that this is triggered by nucleocytoplasmic translocation of the transcription factor E2f4. After inducing a transcriptional program of centriole biogenesis, E2f4 forms apical cytoplasmic organizing centres for assembly and nucleation of deuterosomes. Using genetically altered mice and E2F4 mutant proteins we demonstrate that centriole amplification is crucially dependent on these organizing centres and that, without cytoplasmic E2f4, deuterosomes are not assembled, halting multiciliogenesis. Thus, E2f4 integrates nuclear and previously unsuspected cytoplasmic events of centriole amplification, providing new perspectives for the understanding of normal ciliogenesis, ciliopathies and cancer.

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Mcidas mutant mice reveal a two-step process for the specification and differentiation of multiciliated cells in mammals

10.1101/439703 ◽

2018 ◽

Author(s):

Hao Lu ◽

Priyanka Anujan ◽

Feng Zhou ◽

Yiliu Zhang ◽

Yan Ling Chong ◽

...

Keyword(s):

Basal Body ◽

Target Genes ◽

Initial Step ◽

Mutant Mice ◽

Respiratory Disorder ◽

Basal Bodies ◽

Cell Cycle Regulators ◽

Multiciliated Cells ◽

Motile Cilia ◽

Body Production

ABSTRACTMotile cilia on multiciliated cells (MCCs) function in fluid clearance over epithelia. Studies with Xenopus embryos and patients with the congenital respiratory disorder reduced generation of multiple motile cilia, have implicated the nuclear protein MCIDAS (MCI), in the transcriptional regulation of MCC specification and differentiation. Recently, a paralogous protein, GMNC, was also shown to be required for MCC formation. Surprisingly, and in contrast to the presently held view, we find that Mci mutant mice can specify MCC precursors. However, these precursors cannot produce multiple basal bodies, and mature into single ciliated cells. We show that MCI is required specifically to induce deuterosome pathway components for the production of multiple basal bodies. Moreover, GMNC and MCI associate differentially with the cell-cycle regulators E2F4 and E2F5, which enables them to activate distinct sets of target genes (ciliary transcription factor genes versus genes for basal body generation). Our data establish a previously unrecognized two-step model for MCC development: GMNC functions in the initial step for MCC precursor specification. GMNC induces Mci expression, which then drives the second step of basal body production for multiciliation.SUMMARY STATEMENTWe show how two GEMININ family proteins function in mammalian multiciliated cell development: GMNC regulates precursor specification and MCIDAS induces multiple basal body formation for multiciliation.

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CAMSAP3 facilitates basal body polarity and the formation of the central pair of microtubules in motile cilia

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1907335117 ◽

2020 ◽

Vol 117 (24) ◽

pp. 13571-13579 ◽

Cited By ~ 2

Author(s):

Alan M. Robinson ◽

Satoe Takahashi ◽

Eva J. Brotslaw ◽

Aisha Ahmad ◽

Emma Ferrer ◽

...

Keyword(s):

Primary Ciliary Dyskinesia ◽

Basal Bodies ◽

Nasal Airway ◽

Directional Flow ◽

Multiple Phenotypes ◽

Multiciliated Cells ◽

Motile Cilia ◽

Ciliary Dyskinesia ◽

Different Tissues ◽

Proper Orientation

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),Camsap3tm1a/tm1a, exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination ofCamsap3tm1a/tm1amice 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, inCamsap3tm1a/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 ofXenopusembryos 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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WDR5 stabilizes actin architecture to promote multiciliated cell formation

10.1101/153361 ◽

2017 ◽

Author(s):

Saurabh S. Kulkarni ◽

John N. Griffin ◽

Karel F. Liem ◽

Mustafa K. Khokha

Keyword(s):

Cell Surface ◽

Actin Cytoskeleton ◽

Basal Body ◽

Chromatin Modification ◽

Cell Formation ◽

Basal Bodies ◽

Actin Network ◽

Tissue Morphogenesis ◽

Multiciliated Cells ◽

Intracellular Organelles

The actin cytoskeleton is critical to shape cells and pattern intracellular organelles to drive tissue morphogenesis. In multiciliated cells (MCCs), apical actin forms a lattice that drives expansion of the cell surface necessary to host hundreds of cilia. The actin lattice also uniformly distributes basal bodies across this surface. This apical actin network is dynamically remodeled, but the molecules that regulate its architecture remain poorly understood. We identify the chromatin modifier, WDR5, as a regulator of apical F-actin in multiciliated cells. Unexpectedly, WDR5 functions independently of chromatin modification in MCCs. Instead, we discover a scaffolding role for WDR5 between the basal body and F-actin. Specifically, WDR5 binds to basal bodies and migrates apically, where F-actin organizes around WDR5. Using a monomer trap for G-actin, we show that WDR5 stabilizes F-actin to maintain apical lattice architecture. In summary, we identify a novel, non-chromatin role for WDR5 in stabilizing F-actin in multiciliated cells.

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Inhibition of NGLY1 inactivates the transcription factor Nrf1 and potentiates proteasome inhibitor cytotoxicity

10.26434/chemrxiv.5388046.v1 ◽

2017 ◽

Author(s):

Carolyn Bertozzi ◽

Fred Tomlin ◽

Ulla Gerling-Driessen ◽

Yi-Chang Liu ◽

Ryan Flynn ◽

...

Keyword(s):

Transcription Factor ◽

Small Molecule ◽

Proteasome Inhibitor ◽

Activation Mechanism ◽

Cancer Drugs ◽

Small Molecule Library ◽

Library Screen ◽

In Cells

We discovered that the proteostasis modulating transcription factor Nrf1 requires cytosolic de-N-glycosylation by the N-glycanase NGly1 as part of its activation mechanism. Through a covalent small molecule library screen, we discovered an inhibitor of NGly1 that blocks Nrf1 activation in cells and potentiates the activity of proteasome inhibitor cancer drugs. The requirement of NGly1 for Nrf1 activity likely underlies several pathologies associated with a rare hereditary deficiency in NGly1.

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Parallel G-quadruplexes recruit the HSV-1 transcription factor ICP4 to promote viral transcription in herpes virus-infected human cells

Communications Biology ◽

10.1038/s42003-021-02035-y ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Ilaria Frasson ◽

Paola Soldà ◽

Matteo Nadai ◽

Sara Lago ◽

Sara N. Richter

Keyword(s):

Transcription Factor ◽

Early Gene ◽

Proximity Ligation Assay ◽

Gene Promoters ◽

Herpes Simplex Virus 1 ◽

Direct Binding ◽

Simplex Virus ◽

In Cells ◽

Hsv 1

AbstractG-quadruplexes (G4s) are four-stranded nucleic acid structures abundant at gene promoters. They can adopt several distinctive conformations. G4s have been shown to form in the herpes simplex virus-1 (HSV-1) genome during its viral cycle. Here by cross-linking/pull-down assay we identified ICP4, the major HSV-1 transcription factor, as the protein that most efficiently interacts with viral G4s during infection. ICP4 specific and direct binding and unfolding of parallel G4s, including those present in HSV-1 immediate early gene promoters, induced transcription in vitro and in infected cells. This mechanism was also exploited by ICP4 to promote its own transcription. Proximity ligation assay allowed visualization of G4-protein interaction at the single selected G4 in cells. G4 ligands inhibited ICP4 binding to G4s. Our results indicate the existence of a well-defined G4-viral protein network that regulates the productive HSV-1 cycle. They also point to G4s as elements that recruit transcription factors to activate transcription in cells.

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Clonal sector analysis and cell ablation confirm a function for DORNROESCHEN-LIKE in founder cells and the vasculature in Arabidopsis

Planta ◽

10.1007/s00425-020-03545-5 ◽

2021 ◽

Vol 253 (2) ◽

Author(s):

Dorothea Glowa ◽

Petra Comelli ◽

John W. Chandler ◽

Wolfgang Werr

Keyword(s):

Transcription Factor ◽

Functional Gene ◽

Gene Analysis ◽

Cell Ablation ◽

Sector Analysis ◽

Founder Cells ◽

Conventional Methods ◽

Functional Gene Analysis ◽

Lineage Analysis ◽

In Cells

Abstract Main conclusion Inducible lineage analysis and cell ablation via conditional toxin expression in cells expressing the DORNRÖSCHEN-LIKE transcription factor represent an effective and complementary adjunct to conventional methods of functional gene analysis. Abstract Classical methods of functional gene analysis via mutational and expression studies possess inherent limitations, and therefore, the function of a large proportion of transcription factors remains unknown. We have employed two complementary, indirect methods to obtain functional information for the AP2/ERF transcription factor DORNRÖSCHEN-LIKE (DRNL), which is dynamically expressed in flowers and marks lateral organ founder cells. An inducible, two-component Cre–Lox system was used to express beta-glucuronidase GUS in cells expressing DRNL, to perform a sector analysis that reveals lineages of cells that transiently expressed DRNL throughout plant development. In a complementary approach, an inducible system was used to ablate cells expressing DRNL using diphtheria toxin A chain, to visualise the phenotypic consequences. These complementary analyses demonstrate that DRNL functionally marks founder cells of leaves and floral organs. Clonal sectors also included the vasculature of the leaves and petals, implicating a previously unidentified role for DRNL in provasculature development, which was confirmed in cotyledons by closer analysis of drnl mutants. Our findings demonstrate that inducible gene-specific lineage analysis and cell ablation via conditional toxin expression represent an effective and informative adjunct to conventional methods of functional gene analysis.

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Cyto-friendly polymerization at cell surfaces modulates cell fate by clustering cell-surface receptors

Chemical Science ◽

10.1039/c9sc06385d ◽

2020 ◽

Vol 11 (16) ◽

pp. 4221-4225 ◽

Cited By ~ 1

Author(s):

Jing Qi ◽

Weishuo Li ◽

Xiaoling Xu ◽

Feiyang Jin ◽

Di Liu ◽

...

Keyword(s):

Cell Surface ◽

Cell Fate ◽

Cell Surface Receptors ◽

Cell Surfaces ◽

Receptor Clustering ◽

Surface Polymerization ◽

Surface Receptors ◽

Anti Cd20 ◽

In Cells

Cell-surface polymerization of anti-CD20 aptamer modified macromer to induce CD20 receptor clustering, and effectively initiate the apoptotic signals in cells.

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Export from Pericentriolar Endocytic Recycling Compartment to Cell Surface Depends on Stable, Detyrosinated (Glu) Microtubules and Kinesin

Molecular Biology of the Cell ◽

10.1091/mbc.01-05-0224 ◽

2002 ◽

Vol 13 (1) ◽

pp. 96-109 ◽

Cited By ~ 95

Author(s):

Sharron X. Lin ◽

Gregg G. Gundersen ◽

Frederick R. Maxfield

Keyword(s):

Elevated Temperature ◽

Cell Surface ◽

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Ovary Cell ◽

Temperature Sensitive ◽

Microtubule Organizing Center ◽

Endocytic Recycling ◽

In Cells

A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface.

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