Browsing by Subject "Cilia"

The means by which cells, tissues, and organisms undergo morphogenesis are variable and highly regulated, and the mechanisms that govern cellular changes in response to signaling cues are poorly understood. This study seeks to address the role of a newly characterized protein in zebrafish in translating signaling cues into physical changes within a cell.

The Cordon–bleu (Cobl) gene is widely conserved in vertebrates, with developmentally regulated axial and epithelial expression in mouse and chick embryos. In vitro, Cobl can bind monomeric actin and nucleate formation of unbranched actin filaments, while in cultured cells it can modulate the actin cytoskeleton. However, an essential role for Cobl in vivo has yet to be determined. We have identified the zebrafish cobl ortholog and have used zebrafish as a model to assess the requirements for Cobl in embryogenesis. We find that cobl shows enriched expression in ciliated epithelial tissues during zebrafish organogenesis. The utilization of antibodies developed against Cobl shows that the protein is concentrated along the apical domain of ciliated cells, in close proximity to the apical actin cap.

Reduction of cobl by antisense morpholinos reveals an essential role in embryonic morphogenesis and organ development. A requirement for Cobl was shown for the proper function of various and ciliated epithelial organs. Cobl appears to direct the elongation of motile cilia in organs such as Kupffer’s vesicle and the pronephros. In Kupffer’s vesicle, the reduction in Cobl coincides with a reduction in the amount of apical F-actin. Additionally, Cobl may play a role during gastrulation cell movements and convergence and extension morphogenesis during early embryonic development. Thus, Cobl may represent a molecular activity that couples developmental patterning signals with local intracellular cytoskeletal dynamics to support elongation of motile cilia and tissue morphogenesis.

Cilia are hair-like structures found on a number of cells serving a variety of different functions. The common denominator between cilia is that they are made up of a microtubule-based projection called the axoneme and nucleated by a modified mother centriole termed the basal body. While there has been an extensive amount of work interrogating ciliogenesis, the requirements for cilia maintenance has less been appreciated. Recent work is also starting to reveal that the cilia biogenesis pathways for these different cilia are different, suggesting that the requirements for their maintenance may also be different.

TTBK2 is a key regulator of primary cilia assembly. In the following study, we identify novel pathways that regulate and are regulated by TTBK2 using BioID. Using tamoxifen-inducible Cre-recombinase cell and mouse systems, we are able to allow these systems to build cilia, deplete TTBK2 levels after cilia formation by tamoxifen induction, assess changes to different types of cilia over time, and validate some of the protein interactions we identified by BioID.

The actin cytoskeleton is a fundamental component of the cell and is involved in many processes, including cell division, cell migration, vesicle trafficking and cell polarity. The actin cytoskeleton has a very important role in embryogenesis as the cells within developing tissues proliferate, migrate, interpret extracellular cues, and shape complex tissues. The molecules that help the cell to interpret their environment and turn those cues into morphological changes are of great interest. One protein which may be involved in this manner is Cordon-bleu (Cobl).

In mouse embryos, Cobl's expression pattern resembles that of important developmental genes, is restricted to distinct domains, and changes dynamically throughout development as tissues are formed. While it is known that Cobl expression is regulated by developmental signaling pathways such as Shh and BMP, its molecular function at the cellular level remains elusive. In this study, we have identified molecular functions of Cobl. Cobl has C-terminal Wasp Homology-2 (WH2) domains which bind actin and nucleate new actin filaments in in vitro polymerization assays. Using cultured cells, we have determined that Cobl is involved in cytoskeletal remodeling during neurite branching and epithelial cell migration. We also demonstrate that Cobl interacts with the Syndapin family of adaptor proteins that link endocytosis and vesicle trafficking. Cobl colocalizes with Sdp2 in cultured epithelial cells and similarly localizes with Sdp1 and Sdp2 in developing mouse embryos. The localization of Cobl or Sdp2 in cultured epithelial cells is dependent on the other, as demonstrated using shRNA knockdown.

Previous studies demonstrated that a hypomorphic allele of Cobl interacts genetically with Looptail, in midbrain neurulation. Looptail mutants are deficient in the gene Vangl2, a member of the planar cell polarity pathway that coordinates the morphogenesis of a sheet of cells. To discover other roles for Cobl in the developing mouse, we have generated a conditionally null allele of Cobl. We find that outbred Cobl homozygous mutants are viable, but that they have inner ear defects. Together, our studies demonstrate that Cobl is a tissue-specific actin nucleator whose localization is regulated by its interaction with Syndapins and which functions in the development of sensory epithelia.

Human vision begins in the retina, where ~100 million photoreceptor neurons absorb light and respond to it, transferring the information to the brain where ultimately an image is created. Just like a camera’s sensor, an essential quality of photoreceptors for functional vision is their incredible sensitivity—our rod photoreceptors can detect the smallest unit of light possible, a single photon. To achieve this level of sensitivity, the photoreceptor evolved a primary cilium-derived light sensor organelle called the outer segment, which is a massive 30µm-long cylinder filled with a stack of ~1000 perfectly flattened disc membranes. The disc membrane houses the protein machinery necessary for generating light responses, including rhodopsin, the transmembrane photopigment protein responsible for absorbing light. By stacking 1000 discs, each with two rhodopsin containing membrane bilayers, the light absorbing membrane surface area of the retina is increased ~2000 fold, enabling the incredible sensitivity of the rod photoreceptor.

We completed a mass spectrometry project identifying the proteins which specifically reside in the photoreceptor disc. Except for one 6 kDa protein called PRCD, the proteins identified were previously known to reside in photoreceptor discs and their functions were well studied. PRCD is a recently discovered protein whose mutations are linked to retinal degeneration in canine and human patients, and had previously unknown localization in any cell type. Virtually nothing was known about this protein, so this dissertation sought to biochemically characterize the protein, understand how its mutation leads to blindness in dogs and humans, and elucidate its function in photoreceptor discs.

To biochemically characterize the protein, we generated an antibody to its C terminus, and used it to confirm its localization specific to discs by immunohistochemistry. By analyzing the multiple bands PRCD protein produces on Western blot, we discovered that the protein is post translationally modified by lipid acylation and phosphorylation. Furthermore, mutagenesis experiments determined that the lipid is attached to the single cysteine residue of PRCD, which is mutated in blind canine and human patients. This disease-causing mutation results in complete mislocalization of PRCD from the outer segment, and its degradation—effectively resulting in a null PRCD mutant allele.

Pull down experiments revealed PRCD specifically binding to rhodopsin, which was confirmed by reciprocal immunoprecipitation and co-chromatography experiments. Bolstering this result, we found that PRCD was nearly absent from rhodopsin knockout mouse retinas and without outer segment localization. This result contrasted a large cohort of other outer segment proteins; all of them except guanylate cyclase 1 were trafficked to the outer segment and expressed in relative abundance. Through reciprocal co-immunoprecipitations, we discovered that guanylate cyclase 1 is also a rhodopsin binding protein, and that this interaction is dependent on gentle detergent conditions, likely hindering its identification in the past. These results reveal that the bulk of disc specific proteins have their own, uncharacterized trafficking pathway(s), independent of rhodopsin.

To elucidate the function of PRCD in photoreceptor discs, we generated and characterized a PRCD knockout mouse, which develops a normally layered retina. The abundance and localization of disc proteins is normal in young animals, and so are their rod photoresponses. PRCD knockout mouse photoreceptors degenerate slowly, and by electron microscopy, outer segments from PRCD knockout mice are disorganized and display a phenotype similar to dogs containing C2Y mutation in PRCD. The studies presented in this dissertation are the first to lay the biochemical ground work for characterizing PRCD, and elucidate its function in photoreceptor disc membranes.