Lrrcc1 and Ccdc61 are conserved effectors of multiciliated cell function

AbstractCiliated epithelia perform a variety of essential functions across animal evolution, ranging from locomotion of marine organisms to mucociliary clearance of airways in mammals. These epithelia are composed of multiciliated cells (MCCs) harbouring myriads of motile cilia, which rest on modified centrioles called basal bodies (BBs), and beat coordinately to generate directed fluid flows. Thus, BB biogenesis and organization is central to MCC function. In basal eukaryotes, the coiled-coil domain proteins Lrrcc1 and Ccdc61 were shown to be required for proper BB construction and function. Here, we used the Xenopus embryonic ciliated epidermis to characterize Lrrcc1 and Ccdc61 in vertebrate MCCs. We found that they both encode BB components, with a prominent association to striated rootlets. Knocking down either gene caused defects in BB docking, spacing, and polarization. Moreover, their depletion impaired the apical cytoskeleton, and altered ciliary beating. Consequently, cilia-powered fluid flow was greatly reduced in morphant tadpoles, which displayed enhanced mortality when exposed to pathogenic bacteria. This work illustrates how integration across organizational scales make elementary BB components essential for the emergence of the physiological function of ciliated epithelia.

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Ciliary transcription factors and miRNAs precisely regulate Cp110 levels required for ciliary adhesions and ciliogenesis

eLife ◽

10.7554/elife.17557 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 30

Author(s):

Peter Walentek ◽

Ian K Quigley ◽

Dingyuan I Sun ◽

Umeet K Sajjan ◽

Christopher Kintner ◽

...

Keyword(s):

Cell Cycle ◽

Transcription Factors ◽

Cell Function ◽

Coiled Coil ◽

Basal Bodies ◽

Cell Cycle Exit ◽

Multiciliated Cells ◽

Cilia Formation ◽

Preferential Binding ◽

Post Transcriptional Regulation

Upon cell cycle exit, centriole-to-basal body transition facilitates cilia formation. The centriolar protein Cp110 is a regulator of this process and cilia inhibitor, but its positive roles in ciliogenesis remain poorly understood. Using Xenopus we show that Cp110 inhibits cilia formation at high levels, while optimal levels promote ciliogenesis. Cp110 localizes to cilia-forming basal bodies and rootlets, and is required for ciliary adhesion complexes that facilitate Actin interactions. The opposing roles of Cp110 in ciliation are generated in part by coiled-coil domains that mediate preferential binding to centrioles over rootlets. Because of its dual role in ciliogenesis, Cp110 levels must be precisely controlled. In multiciliated cells, this is achieved by both transcriptional and post-transcriptional regulation through ciliary transcription factors and microRNAs, which activate and repress cp110 to produce optimal Cp110 levels during ciliogenesis. Our data provide novel insights into how Cp110 and its regulation contribute to development and cell function.

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Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

The Journal of Cell Biology ◽

10.1083/jcb.200206113 ◽

2002 ◽

Vol 159 (6) ◽

pp. 993-1004 ◽

Cited By ~ 126

Author(s):

Christine L. Humphries ◽

Heath I. Balcer ◽

Jessica L. D'Agostino ◽

Barbara Winsor ◽

David G. Drubin ◽

...

Keyword(s):

Binding Protein ◽

Coiled Coil ◽

Actin Binding ◽

Complex Activity ◽

Actin Binding Protein ◽

Coiled Coil Domain ◽

Allele Specific ◽

And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

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Multimerization and tubulin binding are required for the SPIRAL2 protein to localize to and stabilize microtubule minus ends

10.1101/2021.10.05.463238 ◽

2021 ◽

Author(s):

YUANWEI FAN ◽

Natasha Bilkey ◽

Ram Dixit

Keyword(s):

Coiled Coil ◽

Spatial Arrangement ◽

In Planta ◽

Structural Determinants ◽

Coiled Coil Domain ◽

Tubulin Binding ◽

And Function ◽

Necessary And Sufficient ◽

Basic Region

Accruing evidence points to the control of microtubule minus-end dynamics as being crucial for the spatial arrangement and function of the microtubule cytoskeleton. In plants, the SPIRAL2 (SPR2) protein has emerged as a microtubule minus-end regulator that is structurally distinct from the animal minus-end regulators. Previously, SPR2 was shown to autonomously localize to microtubule minus ends and decrease their depolymerization rate. Here, we used in vitro and in planta experiments to identify the structural determinants required for SPR2 to recognize and stabilize microtubule minus ends. We show that SPR2 contains a single N-terminal TOG domain that binds to soluble tubulin. The TOG domain, a basic region, and coiled-coil domain are necessary and sufficient to target and stabilize microtubule minus ends. We demonstrate that the coiled-coil domain mediates multimerization of SPR2 that provides avidity for microtubule binding and is essential for binding to soluble tubulin. While TOG domain-containing proteins are traditionally thought to function as microtubule plus-end regulators, our results reveal that nature has repurposed the TOG domain of SPR2 to regulate microtubule minus ends.

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Optimized filopodia formation requires myosin tail domain cooperation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1901527116 ◽

2019 ◽

Vol 116 (44) ◽

pp. 22196-22204

Author(s):

Ashley L. Arthur ◽

Livia D. Songster ◽

Helena Sirkia ◽

Akash Bhattacharya ◽

Carlos Kikuti ◽

...

Keyword(s):

Coiled Coil ◽

Tail Domain ◽

Tail Region ◽

Lever Arm ◽

Coiled Coil Domain ◽

And Function

Filopodia are actin-filled protrusions employed by cells to interact with their environment. Filopodia formation in Amoebozoa and Metazoa requires the phylogenetically diverse MyTH4-FERM (MF) myosins DdMyo7 and Myo10, respectively. While Myo10 is known to form antiparallel dimers, DdMyo7 lacks a coiled-coil domain in its proximal tail region, raising the question of how such divergent motors perform the same function. Here, it is shown that the DdMyo7 lever arm plays a role in both autoinhibition and function while the proximal tail region can mediate weak dimerization, and is proposed to be working in cooperation with the C-terminal MF domain to promote partner-mediated dimerization. Additionally, a forced dimer of the DdMyo7 motor is found to weakly rescue filopodia formation, further highlighting the importance of the C-terminal MF domain. Thus, weak dimerization activity of the DdMyo7 proximal tail allows for sensitive regulation of myosin activity to prevent inappropriate activation of filopodia formation. The results reveal that the principles of MF myosin-based filopodia formation are conserved via divergent mechanisms for dimerization.

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The Vfl1 Protein in Chlamydomonas Localizes in a Rotationally Asymmetric Pattern at the Distal Ends of the Basal Bodies

The Journal of Cell Biology ◽

10.1083/jcb.153.1.63 ◽

2001 ◽

Vol 153 (1) ◽

pp. 63-74 ◽

Cited By ~ 64

Author(s):

Carolyn D. Silflow ◽

Matthew LaVoie ◽

Lai-Wa Tam ◽

Susan Tousey ◽

Mark Sanders ◽

...

Keyword(s):

Basal Body ◽

Rotational Symmetry ◽

Coiled Coil ◽

Mutant Phenotype ◽

Basal Bodies ◽

Repeat Sequences ◽

Coiled Coil Domain ◽

Cell Biol ◽

Asymmetric Pattern ◽

Rotational Orientation

In the unicellular alga Chlamydomonas, two anterior flagella are positioned with 180° rotational symmetry, such that the flagella beat with the effective strokes in opposite directions (Hoops, H.J., and G.B. Witman. 1983. J. Cell Biol. 97:902–908). The vfl1 mutation results in variable numbers and positioning of flagella and basal bodies (Adams, G.M.W., R.L. Wright, and J.W. Jarvik. 1985. J. Cell Biol. 100:955–964). Using a tagged allele, we cloned the VFL1 gene that encodes a protein of 128 kD with five leucine-rich repeat sequences near the NH2 terminus and a large α-helical–coiled coil domain at the COOH terminus. An epitope-tagged gene construct rescued the mutant phenotype and expressed a tagged protein (Vfl1p) that copurified with basal body flagellar apparatuses. Immunofluorescence experiments showed that Vfl1p localized with basal bodies and probasal bodies. Immunogold labeling localized Vfl1p inside the lumen of the basal body at the distal end. Distribution of gold particles was rotationally asymmetric, with most particles located near the doublet microtubules that face the opposite basal body. The mutant phenotype, together with the localization results, suggest that Vfl1p plays a role in establishing the correct rotational orientation of basal bodies. Vfl1p is the first reported molecular marker of the rotational asymmetry inherent to basal bodies.

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The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00446.2015 ◽

2015 ◽

Vol 309 (12) ◽

pp. H2127-H2136 ◽

Cited By ~ 12

Author(s):

Daisuke Naito ◽

Takehiro Ogata ◽

Tetsuro Hamaoka ◽

Naohiko Nakanishi ◽

Kotaro Miyagawa ◽

...

Keyword(s):

Plasma Membrane ◽

Cardiac Function ◽

Membrane Trafficking ◽

Protein Level ◽

Interstitial Fibrosis ◽

Coiled Coil ◽

Cardiomyocyte Hypertrophy ◽

Coiled Coil Domain ◽

Cytoplasmic Proteins ◽

And Function

Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function.

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Requirement for the N-Terminal Coiled-Coil Domain for Expression and Function, but not Subunit Interaction of, the ADPR-Activated TRPM2 Channel

The Journal of Membrane Biology ◽

10.1007/s00232-009-9190-4 ◽

2009 ◽

Vol 230 (2) ◽

pp. 93-99 ◽

Cited By ~ 8

Author(s):

Zhu-Zhong Mei ◽

Lin-Hua Jiang

Keyword(s):

Coiled Coil ◽

Subunit Interaction ◽

Coiled Coil Domain ◽

Trpm2 Channel ◽

And Function

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Drosophila chibby is required for basal body formation and ciliogenesis but not for Wg signaling

The Journal of Cell Biology ◽

10.1083/jcb.201109148 ◽

2012 ◽

Vol 197 (2) ◽

pp. 313-325 ◽

Cited By ~ 50

Author(s):

Camille Enjolras ◽

Joëlle Thomas ◽

Brigitte Chhin ◽

Elisabeth Cortier ◽

Jean-Luc Duteyrat ◽

...

Keyword(s):

Wnt Signaling ◽

Basal Body ◽

Coiled Coil ◽

Cell Types ◽

Sensory Transduction ◽

Basal Bodies ◽

Neuronal Cilia ◽

Complex Process ◽

Specific Proteins ◽

And Function

Centriole-to–basal body conversion, a complex process essential for ciliogenesis, involves the progressive addition of specific proteins to centrioles. CHIBBY (CBY) is a coiled-coil domain protein first described as interacting with β-catenin and involved in Wg-Int (WNT) signaling. We found that, in Drosophila melanogaster, CBY was exclusively expressed in cells that require functional basal bodies, i.e., sensory neurons and male germ cells. CBY was associated with the basal body transition zone (TZ) in these two cell types. Inactivation of cby led to defects in sensory transduction and in spermatogenesis. Loss of CBY resulted in altered ciliary trafficking into neuronal cilia, irregular deposition of proteins on spermatocyte basal bodies, and, consequently, distorted axonemal assembly. Importantly, cby1/1 flies did not show Wingless signaling defects. Hence, CBY is essential for normal basal body structure and function in Drosophila, potentially through effects on the TZ. The function of CBY in WNT signaling in vertebrates has either been acquired during vertebrate evolution or lost in Drosophila.

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Unconventional conservation reveals structure-function relationships in the synaptonemal complex

eLife ◽

10.7554/elife.72061 ◽

2021 ◽

Vol 10 ◽

Author(s):

Lisa E Kursel ◽

Henry D Cope ◽

Ofer Rog

Keyword(s):

Sequence Analysis ◽

Synaptonemal Complex ◽

Protein Evolution ◽

Coiled Coil ◽

Structural Features ◽

Structure And Function ◽

Coiled Coils ◽

Functional Requirements ◽

Coiled Coil Domain ◽

And Function

Functional requirements constrain protein evolution, commonly manifesting in a conserved amino acid sequence. Here, we extend this idea to secondary structural features by tracking their conservation in essential meiotic proteins with highly diverged sequences. The synaptonemal complex (SC) is a ~100-nm-wide ladder-like meiotic structure present in all eukaryotic clades, where it aligns parental chromosomes and regulates exchanges between them. Despite the conserved ultrastructure and functions of the SC, SC proteins are highly divergent within Caenorhabditis. However, SC proteins have highly conserved length and coiled-coil domain structure. We found the same unconventional conservation signature in Drosophila and mammals, and used it to identify a novel SC protein in Pristionchus pacificus, Ppa-SYP-1. Our work suggests that coiled-coils play wide-ranging roles in the structure and function of the SC, and more broadly, that expanding sequence analysis beyond measures of per-site similarity can enhance our understanding of protein evolution and function.

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