scholarly journals Protein turnover dynamics suggest a diffusion to capture mechanism for peri-basal body recruitment and retention of intraflagellar transport proteins

2020 ◽  
Author(s):  
Jaime V.K. Hibbard ◽  
Neftali Vazquez ◽  
Rohit Satija ◽  
John B. Wallingford
Keyword(s):  
Protein Dynamics ◽  
Basal Body ◽  
Fluorescence Recovery After Photobleaching ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Transport Proteins ◽  
Basal Bodies ◽  
Multiciliated Cells ◽  
Capture Mechanism ◽  
Body Components

ABSTRACTIntraflagellar transport (IFT) is essential for construction and maintenance of cilia. IFT proteins concentrate at the basal body, where they are thought to assemble into trains and bind cargoes for transport. To study the mechanisms of IFT recruitment to this peri-basal body pool, we quantified protein dynamics of eight IFT proteins, as well as five other basal body localizing proteins, using fluorescence recovery after photobleaching in vertebrate multiciliated cells. We found that members of the IFT-A and IFT-B protein complexes show distinct turnover kinetics from other basal body components. Additionally, known IFT sub-complexes displayed shared dynamics, and these dynamics were not altered during cilia regeneration as compared to homeostasis. Finally, we evaluated the mechanisms of basal body recruitment by depolymerizing cytosolic MTs, which suggested that IFT proteins are recruited to basal bodies through a diffusion-to-capture mechanism. Our survey of IFT protein dynamics provides new insights into IFT recruitment to basal bodies, a crucial step in ciliogenesis.

scholarly journals Protein turnover dynamics suggest a diffusion-to-capture mechanism for peri-basal body recruitment and retention of intraflagellar transport proteins

2021 ◽  
pp. mbc.E20-11-0717
Author(s):  
Jaime V.K. Hibbard ◽  
Neftali Vazquez ◽  
Rohit Satija ◽  
John B. Wallingford
Keyword(s):  
Protein Dynamics ◽  
Basal Body ◽  
Fluorescence Recovery After Photobleaching ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Basal Bodies ◽  
Multiciliated Cells ◽  
Capture Mechanism ◽  
Body Components ◽  
Ciliary Signaling

Intraflagellar transport (IFT) is essential for construction and maintenance of cilia. IFT proteins concentrate at the basal body, where they are thought to assemble into trains and bind cargoes for transport. To study the mechanisms of IFT recruitment to this peri-basal body pool, we quantified protein dynamics of eight IFT proteins, as well as five other basal body localizing proteins, using fluorescence recovery after photobleaching in vertebrate multiciliated cells. We found that members of the IFT-A and IFT-B protein complexes show distinct turnover kinetics from other basal body components. Additionally, known IFT sub-complexes displayed shared dynamics, suggesting shared basal body recruitment and/or retention mechanisms. Finally, we evaluated the mechanisms of basal body recruitment by depolymerizing cytosolic MTs, which suggested that IFT proteins are recruited to basal bodies through a diffusion-to-capture mechanism. Our survey of IFT protein dynamics provides new insights into IFT recruitment to basal bodies, a crucial step in ciliogenesis and ciliary signaling.

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

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.

scholarly journals The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells

2015 ◽  
Vol 211 (5) ◽  
pp. 963-973 ◽  
Author(s):  
Takayuki Yasunaga ◽  
Sylvia Hoff ◽  
Christoph Schell ◽  
Martin Helmstädter ◽  
Oliver Kretz ◽  
...  
Keyword(s):  
Fluid Flow ◽  
Xenopus Laevis ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Structural Defects ◽  
Basal Bodies ◽  
Actin Network ◽  
Multiciliated Cells ◽  
Associated Proteins ◽  
Molecular Machinery

Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.

scholarly journals The Distal Appendage Protein CEP164 Is Essential for Efferent Duct Multiciliogenesis and Male Fertility

2020 ◽  
Author(s):  
Mohammed Hoque ◽  
Danny Chen ◽  
Rex A. Hess ◽  
Feng-Qian Li ◽  
Ken-Ichi Takemaru
Keyword(s):  
Basal Body ◽  
Male Fertility ◽  
Apical Cell ◽  
Proper Function ◽  
Seminiferous Tubules ◽  
Basal Bodies ◽  
Epithelial Surface ◽  
Efferent Ducts ◽  
Multiciliated Cells

AbstractCilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. Multiciliated cells (MCCs) are a specialized cell type with hundreds of motile multicilia, lining the brain ventricles, airways, and reproductive tracts to propel fluids/substances across the epithelial surface. In the male reproductive tract, MCCs in efferent ducts (EDs) move in a whip-like motion to stir the luminal contents and prevent sperm agglutination. Previously, we demonstrated that the essential distal appendage protein CEP164 recruits Chibby1 (Cby1), a small coiled-coil-containing protein, to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in MCCs (FoxJ1-Cre;CEP164fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility, however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. Consistent with these findings, multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164fl/fl mice. In FoxJ1-Cre;CEP164fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that deletion of CEP164 in the MCCs of EDs causes basal body docking defects and loss of multicilia, leading to sperm agglutination, obstruction of EDs, and male infertility. Our study therefore unravels an essential role of the distal appendage protein CEP164 in male fertility.Author SummaryMulticilia are tinny hair-like microtubule-based structures that beat in a whip-like pattern to generate a fluid flow on the apical cell surface. Multiciliated cells are essential for the proper function of major organs such as brain, airway, and reproductive tracts. In the male reproductive system, multiciliated cells are present in the efferent ducts, which are small tubules that connect the testis to the epididymis. However, the importance of multiciliated cells in male fertility remains poorly understood. Here, we investigated the role of the critical ciliary protein CEP164 in male fertility using a mouse model lacking CEP164 in multiciliated cells. Male mice are infertile with reduced sperm counts. We demonstrate that, in the absence of CEP164, multiciliated cells are present in the efferent ducts but fail to extend multicilia due to basal body docking defects. Consistent with this, the recruitment of key ciliary proteins is perturbed. As a result, these mice show sperm agglutination, obstruction of sperm transport, and degeneration of germ cells in the testis, leading to infertility. Our study therefore reveals essential roles of CEP164 in the formation of multicilia in the efferent ducts and male fertility.

scholarly journals Emerging Picture of Deuterosome-Dependent Centriole Amplification in MCCs

Cells ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. 152 ◽  
Author(s):  
Umama Shahid ◽  
Priyanka Singh
Keyword(s):  
Basal Body ◽  
De Novo ◽  
Super Resolution ◽  
Basal Bodies ◽  
Parallel Pathways ◽  
Multiciliated Cells ◽  
Dependent Pathway ◽  
Microtubule Structure

Multiciliated cells (MCCs) have several hair-like structures called cilia, which are required to propel substances on their surface. A cilium is organized from a basal body which resembles a hollow microtubule structure called a centriole. In terminally differentiated MCCs, hundreds of new basal bodies/centrioles are formed via two parallel pathways: the centriole- and deuterosome-dependent pathways. The deuterosome-dependent pathway is also referred to as “de novo” because unlike the centriole-dependent pathway which requires pre-existing centrioles, in the de novo pathway multiple new centrioles are organized around non-microtubule structures called deuterosomes. In the last five years, some deuterosome-specific markers have been identified and concurrent advancements in the super-resolution techniques have significantly contributed to gaining insights about the major stages of centriole amplification during ciliogenesis. Altogether, a new picture is emerging which also challenges the previous notion that deuterosome pathway is de novo. This review is primarily focused on studies that have contributed towards the better understanding of deuterosome-dependent centriole amplification and presents a developing model about the major stages identified during this process.

scholarly journals WDR5 stabilizes actin architecture to promote multiciliated cell formation

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.

scholarly journals Basal Body Components Exhibit Differential Protein Dynamics during Nascent Basal Body Assembly

2009 ◽  
Vol 20 (3) ◽  
pp. 904-914 ◽  
Author(s):  
Chad G. Pearson ◽  
Thomas H. Giddings ◽  
Mark Winey
Keyword(s):  
Basal Body ◽  
Tetrahymena Thermophila ◽  
Light And Electron Microscopy ◽  
Protein Assembly ◽  
Basal Bodies ◽  
Body Formation ◽  
Unique Protein ◽  
Body Components ◽  
And Function ◽  
Stable Populations

Basal bodies organize cilia that are responsible for both mechanical beating and sensation. Nascent basal body assembly follows a series of well characterized morphological events; however, the proteins and their assembly dynamics for new basal body formation and function are not well understood. High-resolution light and electron microscopy studies were performed in Tetrahymena thermophila to determine how proteins assemble into the structure. We identify unique dynamics at basal bodies for each of the four proteins analyzed (α-tubulin, Spag6, centrin, and Sas6a). α-Tubulin incorporates only during new basal body assembly, Spag6 continuously exchanges at basal bodies, and centrin and Sas6a exhibit both of these patterns. Centrin loads and exchanges at the basal body distal end and stably incorporates during new basal body assembly at the nascent site of assembly and the microtubule cylinder. Conversely, both dynamic and stable populations of Sas6a are found only at a single site, the cartwheel. The bimodal dynamics found for centrin and Sas6a reveal unique protein assembly mechanisms at basal bodies that may reflect novel functions for these important basal body and centriolar proteins.

scholarly journals Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and is required for primary cilia assembly

2004 ◽  
Vol 166 (5) ◽  
pp. 637-643 ◽  
Author(s):  
Agata Jurczyk ◽  
Adam Gromley ◽  
Sambra Redick ◽  
Jovenal San Agustin ◽  
George Witman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Interference ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Transport Proteins ◽  
Immunogold Electron Microscopy ◽  
Basal Bodies ◽  
Motile Cilia ◽  
Cilia And Flagella ◽  
Centrosome Protein

Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. Here, we show that the centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.

In vitro reassembly of basal body components

1984 ◽  
Vol 66 (1) ◽  
pp. 147-154
Author(s):  
R.H. Gavin
Keyword(s):  
Basal Body ◽  
Basal Bodies ◽  
Body Components

Singlet microtubules and structures that have the morphology and dimensions of the basal body cartwheel complex self-assemble in extracts derived from Tetrahymena basal bodies.

scholarly journals IFT trains in different stages of assembly queue at the ciliary base for consecutive release into the cilium

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jenna L Wingfield ◽  
Ilaria Mengoni ◽  
Heather Bomberger ◽  
Yu-Yang Jiang ◽  
Jonathon D Walsh ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Cell Body ◽  
Basal Body ◽  
Open System ◽  
Tetrahymena Thermophila ◽  
Intraflagellar Transport ◽  
Basal Bodies ◽  
Fluorescence Protein ◽  
Successive Release ◽  
Multiple Stages

Intraflagellar transport (IFT) trains, multimegadalton assemblies of IFT proteins and motors, traffic proteins in cilia. To study how trains assemble, we employed fluorescence protein-tagged IFT proteins in Chlamydomonas reinhardtii. IFT-A and motor proteins are recruited from the cell body to the basal body pool, assembled into trains, move through the cilium, and disperse back into the cell body. In contrast to this ‘open’ system, IFT-B proteins from retrograde trains reenter the pool and a portion is reused directly in anterograde trains indicating a ‘semi-open’ system. Similar IFT systems were also observed in Tetrahymena thermophila and IMCD3 cells. FRAP analysis indicated that IFT proteins and motors of a given train are sequentially recruited to the basal bodies. IFT dynein and tubulin cargoes are loaded briefly before the trains depart. We conclude that the pool contains IFT trains in multiple stages of assembly queuing for successive release into the cilium upon completion.

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