Alpha Omega Alpha Honor Medical Society

2014 Research Abstract

The role of IFT88 on ciliogenesis of motile respiratory epithelium and the regulation of ciliary motility

Investigator: Grant Turner

Mentor: Joseph Sisson, MD

Cilia are essential components of multiple organ systems. Inherited defects in ciliogenesis, the formation of cilia, are often lethal in utero, indicating a critical role for cilia in growth and development. Intraflagellar transport proteins (IFTs) are a diverse group of proteins that are essential for moving proteins on and off of the axoneme and found to have multiple important functions in the structure and function of cilia. For example, defects in IFT88 cause severe defects in the sensing cilia in the kidneys, resulting in polycystic kidney formation, and in the lung, where bronchiectasis results. While the role of IFTs in ciliogenesis has been established, the specific role IFT88 plays in airway cilia structure and function is unknown. We hypothesized that: 1) IFT88 is required for de novo ciliogenesis in vitro; and 2) IFT88 is required to translocate the methacholine receptor, which is part of a cilia motility regulator complex, from the cilia basal body into the ciliary membrane. To test these hypotheses, we used airway cells derived from inducible IFT88 conditional knockout mice grown on air liquid interface (ALI) in the presence and absence of tamoxifen, which controls the expression of the IFT88 gene in our model. We designed experiments to measure the motile cilia formation to quantify ciliogenesis during airway cell differentiation as a function of time. We also measured changes in ciliary beat frequency (CBF) triggered by methacholine (MCH), a ciliary agonist that requires IFT88 placement compared to a β-agonist, procaterol, whose receptor resides in the non-ciliary membrane. Our data show that in the absence of IFT88, there is a significantly decreased number of motile points measured as a function of time, indicating impaired ciliogenesis, while the ciliary beat frequency (CBF) remains unchanged for those ciliated cells that remain. We also demonstrated a decreased CBF response to methacholine in the absence of IFT88, compared to cells containing IFT88, but found they both remained equally sensitive to procaterol stimulation. Through Western Blot and confocal imaging, we confirmed that this is not a decrease in total protein of the M3 acetylcholine receptor, but an absence of M3 on the ciliary membrane, leaving the cells deficient in the ability to respond to the methacholine stimulus. In summary, we found that IFT88 is required for motile ciliogenesis and methacholine responsiveness in mouse airway cells in ALI. Our work will help lead to the recognition of new phenotypes of genetic and acquired cilia disorders that are based on dysregulation of IFTs in ciliary motility and not just anatomical structural defects of the axoneme.

Supported by Sisson NIAAA 5-RO1 AA008769-21 and AOA Carolyn L. Kuckein Student Research Fellowship.

Last updated: 2/20/2014

Updated on February 20, 2015.

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