| dc.identifier.uri | http://hdl.handle.net/11401/76511 | |
| dc.description.sponsorship | This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. | en_US |
| dc.format | Monograph | |
| dc.format.medium | Electronic Resource | en_US |
| dc.language.iso | en_US | |
| dc.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dc.type | Dissertation | |
| dcterms.abstract | Cilia are small hair-like projections extending from nearly all eukaryotic cell surfaces. Cilia are associated with critical cellular functions such as cellular motility as well as acting as the cellular antennae for signaling pathways and sensory functions. In recognition of this, there has been intense research to elucidate the molecular mechanism of ciliary formation and maintenance in order to understand the many and heterogeneous diseases (cilia-related diseases termed the ciliopathies) resulting from ciliary defects. Chibby (Cby) is an evolutionarily conserved coiled-coil protein that was initially isolated as an antagonist of the canonical Wnt/β-catenin signaling pathway. Generation of CbyKO mice revealed phenotypes characteristic of ciliopathic diseases. Subsequently, it was revealed that Cby is a critical mediator of cilia formation through its ability to recruit membranous vesicles to the ciliary base. However, the molecular mechanism as to how exactly Cby facilitates recruitment and subsequent fusion of small vesicles to centrioles remains unclear. In order to gain insight into the role of Cby in vesicle recruitment, I first examined Cby dynamics at the bases of cilia in order to gain insight into the role Cby plays in vesicle recruitment. The fluorescence recovery after photobleaching (FRAP) data indicates that Cby is localized to a region of the cilia that is not readily accessible thru free diffusion. Second, I compiled a comprehensive list of potential Cby interacting partners, with a focus on basal body proteins. The tandem affinity purification (TAP) technology was used to identify the novel Cby interacting partners FAM92A and FAM92B. Beyond the few studies that suggest that FAM92A plays a role in embryogenesis, little else is known about the function of the FAM92 family of proteins. Members of this family contain a putative BAR-domain. BAR-domains are a highly conserved domain that forms a crescent-shaped homodimer and is found in many proteins involved in membrane dynamics. I characterized FAM92A and FAM92B as novel Cby interacting partners that localized to the base of cilia. The BAR-domain of FAM92A and FAM92B was sufficient for this interaction as well as the FAM92 proteins ability to homodimerize. In the absence of Cby, FAM92A and FAM92B localization to the base of cilia was disrupted. This suggests that Cby acts upstream of the FAM92 proteins, possibly recruiting the FAM92 proteins to the basal body. Additionally, siRNA mediated knockdown of FAM92A decreased cilia formation, which implicates the FAM92 proteins involvement in cilia formation. Furthermore, ectopic expression of FAM92 and Cby induced membrane tubule-like structures. Overall, I have identified and characterized the BAR-domain containing proteins, FAM92A and FAM292B, as novel Cby interacting partners. The BAR-domain properties of FAM92 proteins could provide the crucial link between Cby’s ability to recruit vesicles and the subsequent membrane fusion necessary for cilia formation. | |
| dcterms.abstract | Cilia are small hair-like projections extending from nearly all eukaryotic cell surfaces. Cilia are associated with critical cellular functions such as cellular motility as well as acting as the cellular antennae for signaling pathways and sensory functions. In recognition of this, there has been intense research to elucidate the molecular mechanism of ciliary formation and maintenance in order to understand the many and heterogeneous diseases (cilia-related diseases termed the ciliopathies) resulting from ciliary defects. Chibby (Cby) is an evolutionarily conserved coiled-coil protein that was initially isolated as an antagonist of the canonical Wnt/β-catenin signaling pathway. Generation of CbyKO mice revealed phenotypes characteristic of ciliopathic diseases. Subsequently, it was revealed that Cby is a critical mediator of cilia formation through its ability to recruit membranous vesicles to the ciliary base. However, the molecular mechanism as to how exactly Cby facilitates recruitment and subsequent fusion of small vesicles to centrioles remains unclear. In order to gain insight into the role of Cby in vesicle recruitment, I first examined Cby dynamics at the bases of cilia in order to gain insight into the role Cby plays in vesicle recruitment. The fluorescence recovery after photobleaching (FRAP) data indicates that Cby is localized to a region of the cilia that is not readily accessible thru free diffusion. Second, I compiled a comprehensive list of potential Cby interacting partners, with a focus on basal body proteins. The tandem affinity purification (TAP) technology was used to identify the novel Cby interacting partners FAM92A and FAM92B. Beyond the few studies that suggest that FAM92A plays a role in embryogenesis, little else is known about the function of the FAM92 family of proteins. Members of this family contain a putative BAR-domain. BAR-domains are a highly conserved domain that forms a crescent-shaped homodimer and is found in many proteins involved in membrane dynamics. I characterized FAM92A and FAM92B as novel Cby interacting partners that localized to the base of cilia. The BAR-domain of FAM92A and FAM92B was sufficient for this interaction as well as the FAM92 proteins ability to homodimerize. In the absence of Cby, FAM92A and FAM92B localization to the base of cilia was disrupted. This suggests that Cby acts upstream of the FAM92 proteins, possibly recruiting the FAM92 proteins to the basal body. Additionally, siRNA mediated knockdown of FAM92A decreased cilia formation, which implicates the FAM92 proteins involvement in cilia formation. Furthermore, ectopic expression of FAM92 and Cby induced membrane tubule-like structures. Overall, I have identified and characterized the BAR-domain containing proteins, FAM92A and FAM292B, as novel Cby interacting partners. The BAR-domain properties of FAM92 proteins could provide the crucial link between Cby’s ability to recruit vesicles and the subsequent membrane fusion necessary for cilia formation. | |
| dcterms.available | 2017-09-20T16:50:30Z | |
| dcterms.contributor | Takemaru, Ken-Ichi | en_US |
| dcterms.contributor | Talmage, David | en_US |
| dcterms.contributor | Garcia-Diaz, Miguel | en_US |
| dcterms.contributor | Kernan, Maurice. | en_US |
| dcterms.creator | Chen, Xingwang | |
| dcterms.dateAccepted | 2017-09-20T16:50:30Z | |
| dcterms.dateSubmitted | 2017-09-20T16:50:30Z | |
| dcterms.description | Department of Molecular and Cellular Pharmacology. | en_US |
| dcterms.extent | 122 pg. | en_US |
| dcterms.format | Monograph | |
| dcterms.format | Application/PDF | en_US |
| dcterms.identifier | http://hdl.handle.net/11401/76511 | |
| dcterms.issued | 2015-05-01 | |
| dcterms.language | en_US | |
| dcterms.provenance | Made available in DSpace on 2017-09-20T16:50:30Z (GMT). No. of bitstreams: 1
Chen_grad.sunysb_0771E_12517.pdf: 3068759 bytes, checksum: 3f20f6b9df7baad021cbc3bf552c68a6 (MD5)
Previous issue date: 2015 | en |
| dcterms.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dcterms.subject | Developmental biology | |
| dcterms.subject | BAR-domain, Chibby, ciliogenesis, ciliopathy, FAM92A, FAM92B | |
| dcterms.title | Identification and characterization of the novel BAR-domain containing proteins, FAM92A and FAM92B, as interacting partners of the ciliary protein Chibby | |
| dcterms.type | Dissertation | |
