Browsing by Subject "cilia"

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Identification and characterization of novel ciliogenic machinery
(2017-05) Huizar, Ryan; Wallingford, John
Cilia are microtubule-based structures that project from almost every cell in the vertebrate body. In humans, there are two types of cilia, motile, which generate fluid flow across tissues of the ventricles, airway, and oviduct, as well as in propulsion in single cells, and primary, which are responsible for transducing many signaling pathways. Primary and motile cilia are dependent on a bidirectional trafficking process called intraflagellar transport (IFT) in order to bring material into the cilium, which governs their growth, maintenance, and signaling. IFT is mediated by two distinct protein complexes called IFT-A and IFT-B, which function in anterograde and retrograde transport, respectively. In motile cilia, an organization of multiple large protein complexes within the axoneme allow for wave-like motion to be produced. Instrumental to this motility are axonemal dynein arms, large motor protein complexes that slide along microtubule doublets in a coordinated manner to generate bending. Here, I describe two studies regarding ciliogenesis in multiciliated cells, a highly-specialized cell type decorated with dozens of motile cilia. First, I identify ANKRD55 as an IFT-B interactor. I demonstrate that this protein traffics through multiciliated cell axonemes and results in severe developmental defects in its absence. In addition, I describe early insights into the potential role this gene plays in cilia-related human disease. Together, these data suggest that ANKRD55 is a novel member of IFT-B. Second, I characterize the processes that underlie the cytoplasmic assembly of axonemal dynein arms, wherein various chaperones and cytoplasmic factors work in unison to fold and complex dynein arm subunits prior to ciliary transport. Using various imaging methods, I show that the factors responsible for dynein arm assembly localize to non-membrane bound cytoplasmic phase- separations in multiciliated cells, which we term DynAPs (Dynein Assembly Particles). I then demonstrate that machinery involved in phase separation of stress granules is required for formation of DynAPs and recruitment of dynein to axonemes.
The role of the PCP effector protein Fritz in convergent extension, ciliogenesis and hedgehog signaling
(2009) Ghosh, Srimoyee; John Wallingford
Fritz is one of the downstream players in the Planar Cell Polarity (PCP) pathway. PCP signaling was first identified in Drosophila, and many vertebrate homologues of PCP genes have been shown to be central to neural tube development and closure in vertebrate animals, including humans. Recently, it has also been shown that some PCP genes are involved in the formation of cilia. A critical function for cilia during embryonic development involves transducing Hedgehog signaling, which is essential for neural tube closure. However, the role of Fritz in vertebrate neural tube formation has not yet been studied. The expression patterns of Fritz during the early development of Xenopus laevis embryos have been determined. Also, phenotypic changes in Xenopus laevis embryos have been observed when the Fritz protein is knocked down by antisense morpholino oligonucleotide injection— embryos failed to close their neural tube properly, indicating that Fritz is involved in neural tube closure as well as convergent extension. GFP fusion to Fritz and immunostaining reveal that Fritz protein is localized at the base of cilia. Morpholino injection resulted in defective cilia inside the neural tube and in the epidermis of these morphant embryos. These results indicated that Fritz is an essential gene for cilia formation. Furthermore, the expression level of Hedgehog target genes was greatly reduced within Fritz morphants. Fritz morphants also exhibit characteristic craniofacial defects associated with defective Hedgehog signaling. Though much work is left to determine the exact role of Fritz, these experiments illustrate that the Fritz gene is involved in cilia formation, and consequently Hedgehog signaling, which is critical for neural tube closure and development in vertebrate animals. Neural tube closure and Hedgehog signaling are two processes that are essential for proper vertebrate development. The neural tube later gives rise to the brain and spinal cord, while Hedgehog signaling is necessary for brain development, limb development, and establishment of the midline. Considering that Fritz plays a role in both of these processes, a better understanding of Fritz can provide vital information about early vertebrate development. More importantly, because a lack of Fritz disrupts these processes, one can gain insight into why certain developmental defects occur. These include Holoprosencephaly, which results in severe defects in brain formation, and Spina Bifida, a spinal cord formation defect that is one of the most common birth defects worldwide. It is my hope that my work on Fritz has contributed to the body of knowledge regarding these types of defects, and that my work may be someday used to help prevent or treat these defects, and save the lives of children worldwide.