Browsing by Subject "Cilia"

Now showing 1 - 9 of 9

Control of intraflagellar transport : studies of the planar cell polarity effector Fuz, the small GTPase Rsg1, and the novel protein TTC29
(2014-05) Brooks, Eric Robert; Wallingford, John B.
Cilia are small microtubule based protrusions found on most cells of the vertebrate body. In humans, defects in the structure or function of cilia results in a large class of developmental and homeostatic diseases known collectively as the ciliopathies. Ciliogenesis is accomplished by the concerted action of a number of molecular pathways including the intraflagellar transport (IFT) system. IFT is a group of ~20 highly conserved proteins that assemble into large macromolecular complexes known as trains. These trains act to carry cargo bi-directionally between the cell body and ciliary tip, via interaction with the microtubule motors kinesin and dynein. IFT train dynamics are required for both cilia structure and function, however the controls on these dynamics are still incompletely understood. Here, I present the first platform for study of IFT dynamics within vertebrate multiciliated cells, an understudied population with critical functions in development and homeostasis. Using this platform, I demonstrate that the planar cell polarity effector protein Fuz is required for IFT dynamics via its control of the cytoplasmic localization of a subset of IFT proteins. Subsequently, I find that a Fuz binding partner, the putative small GTPase Rsg1, is also required for IFT protein localization and dynamics. Additionally, I describe a role for Rsg1 in basal body docking, one of the earliest events of ciliogenesis. Finally, I show that the poorly studied protein TTC29 is required for a specific subset of IFT dynamic behaviors. These data reveal novel regulatory motifs for ciliogenesis and demonstrate, specifically, the complexities of IFT regulation in the cytoplasm and within the cilium itself. Finally, they suggest that multiciliated cells provide a tractable platform for generating robust datasets for the investigation ciliary dynamics. Such studies are critical for informing our understanding of the molecular etiology of human ciliopathic diseases.
Discovery-driven proteomics provide novel insights into ciliary biology
(2023-08-15) Leggere, Janelle Colette; Wallingford, John B.; Marcotte, Edward M.; Paull, Tanya T; Vokes, Steven A; Xhemalce , Blerta
Cilia and flagella have been carefully observed and described since the dawn of the microscopy era. However, their constituent proteins were not widely studied by cell biologists until the pivotal discovery that mammalian primary cilia are not “vestigial organelles,” as was once believed, but instead are crucial regulators of human health. As the field of ciliary biology has grown in the last two decades, there has been a simultaneous rapid expansion in the capabilities of functional proteomics. High-throughput proteomic techniques have provided key insights into the proteins that build and maintain cilia. In Chapter 1, I provide an overview of some of the most impactful insights gained from ciliary proteomic studies. In Chapter 2, I describe my work using a new high-throughput proteomic technique to systematically identify changes in cellular proteomic organization when a gene required for proper ciliary function is lost. Finally, in Chapter 3, I provide an overview of the main contributions of my work, which has provided new insights into ciliary biology while also further developing robust and broadly applicable proteomic tools.
Fuz Regulates Craniofacial Development through Tissue Specific Responses to Signaling Factors
(Public Library of Science, 2011-09-14) Zhang, Zichao; Wlodarczyk, Bogdan J.; Niederreither, Karen; Venugopalan, Shankar; Florez, Sergio; Finnell, Richard H.; Amendt, Brad A.
The planar cell polarity effector gene Fuz regulates ciliogenesis and Fuz loss of function studies reveal an array of embryonic phenotypes. However, cilia defects can affect many signaling pathways and, in humans, cilia defects underlie several craniofacial anomalies. To address this, we analyzed the craniofacial phenotype and signaling responses of the Fuz−/− mice. We demonstrate a unique role for Fuz in regulating both Hedgehog (Hh) and Wnt/β-catenin signaling during craniofacial development. Fuz expression first appears in the dorsal tissues and later in ventral tissues and craniofacial regions during embryonic development coincident with cilia development. The Fuz−/− mice exhibit severe craniofacial deformities including anophthalmia, agenesis of the tongue and incisors, a hypoplastic mandible, cleft palate, ossification/skeletal defects and hyperplastic malformed Meckel's cartilage. Hh signaling is down-regulated in the Fuz null mice, while canonical Wnt signaling is up-regulated revealing the antagonistic relationship of these two pathways. Meckel's cartilage is expanded in the Fuz−/− mice due to increased cell proliferation associated with the up-regulation of Wnt canonical target genes and decreased non-canonical pathway genes. Interestingly, cilia development was decreased in the mandible mesenchyme of Fuz null mice, suggesting that cilia may antagonize Wnt signaling in this tissue. Furthermore, expression of Fuz decreased expression of Wnt pathway genes as well as a Wnt-dependent reporter. Finally, chromatin IP experiments demonstrate that β-catenin/TCF-binding directly regulates Fuz expression. These data demonstrate a new model for coordination of Hh and Wnt signaling and reveal a Fuz-dependent negative feedback loop controlling Wnt/β-catenin signaling.
Investigation of the role of Fritz and its associated factors, septin and CCT in ciliogenesis of Xenopus laevis epidermis
(2014-05) Kim, Su Kyoung; Wallingford, John B.; De Lozanne, Arturo; Fischer, Janice; Gross, Jeffrey; Stevens, Scott
Cilia are evolutionarily conserved microtubule-based organelles projecting from nearly all vertebrate cells, and ciliary defects result in a variety of human disorders known as ciliopathies. Recent studies have shown that several planar cell polarity (PCP) proteins are essential for cilia functions. Here, we focused on Fritz, known as a novel PCP effector protein in Drosophila, in multi-ciliated cells in the epidermis of Xenopus laevis embryos. To investigate the role of Fritz, using confocal and scanning electron microscopy, we discovered that Fritz localizes along the ciliary axonemes and that knockdown of Fritz causes severe reductions in both axoneme length and number. Then, using pull-downs and mass-spectrometry, we identified Chaperonin Containing T-complex polypeptide 1 (CCT) and septin as interacting partners of Fritz. CCT is the key chaperonin interacting with septins, and both have been implicated in ciliogenesis. Using tagged CCT subunit constructs, we found that the tagged CCTα and CCTε co-localize with Fritz along the ciliary axonemes of multi-ciliated cells. Knockdown of Fritz resulted in the accumulation of CCT at the apical cytoplasm of multi-ciliated cells; however, it was confirmed that Fritz does not affect the CCT holoenzyme assembly. Septins, another interacting partner of Fritz, are novel cytoskeletal elements. Using septin antibodies, we found that endogenous septins also localize along the ciliary axonemes and accumulate in the apical cytoplasm of multi-ciliated cells in Fritz morphants. Similar ciliary defects were observed in septin morphants. Our results reveal that Fritz is essential for ciliogenesis, and that CCT and septin interact with Fritz to control ciliogenesis in Xenopus multi-ciliated cells. Additionally, tubulin acetylation is markedly reduced by Fritz knockdown, suggesting that Fritz affects tubulin acetylation.
Protein trafficking within ciliated cells
(2021-08-13) Hibbard, Jaime; Wallingford, John B.; Dickinson, Daniel; Marcotte, Edward; Finkelstein, Ilya; Kim, Jonghwan
Cilia are microtubule-based organelles that are crucial for embryonic development and tissue homeostasis post-development. Cilia are composed of thousands of proteins that must be trafficked from their site of synthesis in the cell body. Active transport of protein cargoes within cilia is mediated by the intraflagellar transport complex (IFT). IFT trafficking within cilia is well studied, but the role of IFT proteins in the cytoplasm is less understood. In my graduate work, I investigated modes of protein trafficking within ciliated cells. In Chapter 1, I introduce the routes by which ciliary proteins can traffic from their site of synthesis to the basal body. In Chapter 2, I describe my work studying the mechanism of IFT trafficking from the cytoplasm to the basal body. Finally, in Chapter 3, I introduce a pipeline to identify protein cargoes of IFT. This work provides new insights into protein trafficking within ciliated cells, which is crucial for ciliary signaling.
The Small GTPase Rsg1 is important for the cytoplasmic localization and axonemal dynamics of intraflagellar transport proteins
(Cilia, 2013-10-07) Brooks, Eric R.; Wallingford, John B.
Background:Cilia are small, microtubule-based protrusions important for development and homeostasis. We recently demonstrated that the planar cell polarity effector protein Fuz is a critical regulator of axonemal intraflagellar transport dynamics and localization. Here, we report our findings on the role of the small GTPase Rsg1, a known binding partner of Fuz, and its role in the dynamics and cytoplasmic localization of intraflagellar transport proteins. Results: We find that Rsg1 loss of function leads to impaired axonemal IFT dynamics in multiciliated cells. We further show that Rsg1 is required for appropriate cytoplasmic localization of the retrograde IFT-A protein IFT43. Finally, we show that Rsg1 governs the apical localization of basal bodies, the anchoring structures of cilia. Conclusions: Our data suggest that Rsg1 is a regulator of multiple aspects of ciliogenesis, including apical trafficking of basal bodies and the localization and dynamics intraflagellar transport proteins.
Studies on the Kinesin-9 family in motile cilia formation and function
(2021-11-22) Konjikusic, Mia Jasmina; Wallingford, John B.; Gray, Ryan S.; Vokes, Steven; Stein, David; Marcotte, Edward
Kinesins are microtubule-based motors that are an integral part of basic cell biological processes. From driving intracellular transport along microtubules, to orchestrating chromosome segregation during mitosis, and finally to building specialized organelles such as the cilium, the 45 mammalian kinesins have adopted specific functions in vivo during the development of the organism. In this work, we defined the roles of the Kinesin-9 family, Kif6 and Kif9, in vivo in motile cilia. Motile cilia are microtubule-based, whip-like projections on multiciliated cells that function in fluid flow and cell motility in single cells, such as sperm. We have performed extensive genetic analysis of KIF6 in human, mouse, and zebrafish. By modeling a KIF6 allele found in a human patient with intellectual disabilities, we revealed a role in ependymal cell ciliogenesis in the mouse. While ependymal cell cilia are reduced in the Kif6 mouse mutant, we did not observe similar defects in other multiciliated cell types. Analysis of kif6 mutant zebrafish further confirmed an evolutionarily conserved role in ependymal cell cilia formation. Together these data suggest Kif6 may be a tissue specific kinesin required for ependymal cell ciliogenesis. On the other hand, we have performed an extensive cell biological analysis of Kif9 in Xenopus laevis multiciliated cells to reveal a specific role in ciliary motility. Loss of Kif9 leads to a reduction in every known ciliary motility complex from the distal tip of motile cilia. Our in vitro analysis shows that Kif9 is a slowly processive motor along microtubules with a speed of ~8nm/second, while we observe no processivity in vivo in multiciliated cells. These data suggest that Kif9 is contributing to ciliary beating through a force generation mechanism, similar to other motor proteins in the cilium, while also contributing to the localization of other motility complexes in the cilium. Our work highlights the importance of in vivo analysis of kinesins, and further defines the functions of the Kinesin-9 family.
Systematic analysis of Rfx2 target genes in vertebrate multiciliated cells
(2017-08-25) Tu, Fan; Wallingford, John B.; Marcotte, Edward M.; Miller, Kyle M.; O'Halloran, Theresa; Paull, Tanya T.
Multiciliated cells (MCCs) drive directional fluid flow in diverse tubular organs and are essential for development and homeostasis of the vertebrate central nervous system, airway, and reproductive tracts. These cells are characterized by dozens or hundreds of long, motile cilia that beat in a coordinated and polarized manner. In recent years, genomic studies have not only elucidated the transcriptional hierarchy for MCC specification, but also identified myriad new proteins that govern MCC ciliogenesis, cilia beating, or cilia polarization. Interestingly, this burst of genomic data has also highlighted the obvious importance of the “ignorome,” that large fraction of vertebrate genes that remain only poorly characterized. Understanding the function of novel proteins with little prior history of study presents a special challenge, especially when faced with large numbers of such proteins. Here, we explored the MCC ignorome by defining the subcellular localization of 260 poorly defined proteins in vertebrate MCCs in vivo. Based on this localization data, we selected some targets of MCC ignorome for further functional studies because they could possibly play key roles in the regulation of ciliogenesis. We characterized Myo5c as the motor for basal body apical migration, vi Arhgef18 as the RhoA signaling activator at the basal bodies, and Dennd2b as a regulator of actin network formation and ciliogenesis. All of these findings have deepened our understanding about molecular mechanisms of related cellular process. This study exemplifies the power of high content protein localization screening as the bridging step between large-scale omics data and functional study of specific proteins.
Transcriptional control of epithelial morphogenesis
(2013-05) Chung, Mei-I; Wallingford, John B.
How tissues and organs develop into their final shape during embryogenesis is a fascinating and long-standing question in developmental biology. Tissue morphogenesis is driven by a variety of events at the cellular level and individual cell shape change is one of the central morphogenetic engines. Thus, it is crucial to understand what signals specify the correct cell behavior in specific groups of cells during development. For my doctoral studies, I have focused on two cell shape change events, apical constriction and cilia assembly. First, we present data demonstrating that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut, where Shroom3 transcription requires the Pitx1 transcription factor. We identified a Pitx-responsive regulatory element in the genomic DNA upstream of Shroom3, and showed that Pitx proteins directly activated Shroom3 transcription in Xenopus. Moreover, we showed that ectopic expression of Pitx proteins was sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrated new breadth to the requirements for Shroom3 in morphogenesis, and also provided a cell-biological mechanism for Pitx transcription factors action during morphogenesis. Next, we focused on understanding the transcriptional regulation of ciliogenesis. We first showed that Rfx2 transcription factor broadly controlled ciliogenesis, and by RNA- and ChIP-sequencing, we showed that Rfx2 directly regulated a wide range of genes encoding diverse ciliogenic machinery. Finally, in addition to ciliogenesis regulation, a large number of non-ciliary genes in our Rfx2 dataset led us to identify a novel role of Rfx2 in controlling insertion of multi-ciliated cells into the overlying mucociliary epithelium. Moreover, we showed here that Slit2, a target of Rfx2, was involved in multi-ciliated cell movements, possibly through mediating cortical E-cadherin level. This work allowed us to begin building a genetic network controlling multi-ciliated cells in mucociliary epithelium. Together, we showed a transcriptional regulation of apical constriction driving gut morphogenesis and a comprehensive transcriptional network that governs multi-ciliated cell development.