Examination of Calcium’s Role in Mammalian Primary Intraciliary Protein Trafficking

Primary cilia;calcium signaling;calcium concentration;Intraflagellar Transport;mammalian;steady-state;transient

Adstamongkonkul, Pichet ; Clapham, David E.,Mooney, David J.,Habbal, Fawwaz,Needleman, Daniel J.

University:Havard University

Department:Engineering and Applied Sciences - Engineering Sciences

关键词: Primary cilia;    calcium signaling;    calcium concentration;    Intraflagellar Transport;    mammalian;    steady-state;    transient;   

Others  :  https://dash.harvard.edu/bitstream/handle/1/37365827/ADSTAMONGKONKUL-DISSERTATION-2020.pdf?sequence=1&isAllowed=n

美国|英语

来源: Digital Access to Scholarship at Harvard

【 摘 要 】

The primary cilium is an organelle found on nearly every cell in our body. It is a small organelle, averaging from 5-10 micrometers in length and about 500 nanometers in diameter. The loss or defect in proteins specific to primary cilia functions result in a class of diseases collectively called ciliopathies. There are many signaling pathways that localize to primary cilia, but how they work and are regulated are not fully known. Research has demonstrated that primary cilia possess ion channels and have an elevated resting calcium concentration. What role calcium plays in primary cilia function is unclear. Further, protein synthesis does not occur inside primary cilia. Proteins may enter cilia passively, by diffusion, or actively by a process that trafficks proteins in and out of cilia. This process is referred to as Intraflagellar Transport (IFT) and is completed by a multiprotein complex. The singular and small dimensions of a primary cilium pose technical challenges to answer these questions.The aim of this thesis is to understand IFT as it relates to calcium in primary cilia and study IFT dynamics at long and short timescales. An assay that involves tracking two IFT particles using a sparse labeling strategy coupled to advanced imaging modalities was created. Changes in calcium concentration were measured with genetically encoded calcium sensors, localized to either the cytoplasm or cilia. Trafficking rates of the IFT proteins (IFT88 and IFT25), with varying calcium concentrations and time courses were then quantified with a semi-automated analysis technique. Short-timescale studies employed a calcium caging compound loaded into cells and released calcium ions with a short-wavelength laser. Results from this study demonstrated that IFT proteins in mouse fibroblast primary cilia travel faster in the direction from the base to the tip of the cilia (anterograde) than in reverse direction (retrograde), an observation consistent with other published studies. However, perturbing calcium concentration did not elicit changes in the velocities of the IFT proteins at any timescale.Changes in calcium concentration did not alter the IFT velocities in primary cilia and so the role of elevated resting calcium concentration remains unclear. Future investigations will require extensive studies of the entire IFT complex and its interaction with calcium. Development of new assays to probe activities of calcium-binding proteins, membrane and microtubule states in primary cilia during changes of calcium concentration will also advance understanding in ciliary functions.