Genetically Encoded Calcium Indicator Illuminates Calcium Dynamics In Primary Cilia

Primary Cilia;Calcium;Genetically Encoded Calcium Indicator;ATP;Shear Force;Biomedical Engineering

Su, Steven HankongSpector, Alexander A. ;

Johns Hopkins University

关键词: Primary Cilia;    Calcium;    Genetically Encoded Calcium Indicator;    ATP;    Shear Force;    Biomedical Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/37095/SU-THESIS-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The primary cilium is an extraordinary, hair-like structure that extends outward from the plasma membrane of cells. Ubiquitous to almost every cell type in the human body, this singular, immotile structure, serves as an antenna for a cell, capable of transducing extracellular chemical and mechanical stimuli into intracellular signals. Primary cilia can facilitate signal transduction through an assortment of signaling pathways including Hedgehog, Wnt, and Calcium signaling, depending on the cell type. Despite the primary cilium’s role as a central signaling hub, direct visualization of real-time signaling within this organelle has never been achieved mainly due to the organelle’s minute physical dimensions and the inability to segregate primary cilia-specific signals from that of the vast cell body. Here, we directly address this challenge by developing a primary cilia-specific, genetically encoded calcium indicator, with high specificity, sensitivity, and dynamic range. Using our novel calcium indicator we visualized previously unobservable ATP-generated ciliary calcium spikes in mouse fibroblasts and shear-force-generated ciliary calcium fluxes in mouse kidney cells. The methodology employed in this study is generalizable and can be easily adapted to study small molecules of other primary cilia signaling pathways, and as such this work represents an important first step towards the elucidation and dissection of the intricacies in primary cilia signal transduction.