The major objective of this project is to develop new enabling methods to visualize and control early stages of primary cilia formation. Our long-term goal is to develop quantitative image-based high content screens for therapeutics to human diseases resulting from primary cilium dysfunction. Most cells possess a primary cilium, an organelle that extends as a single long slender protrusion from the surface. Long ignored, in the last decade the primary cilium has been recognized as an essential cellular antenna that responds to light, sound, odors, chemicals and mechanical stimuli. The inability to correctly form or maintain a primary cilium causes numerous human diseases including polycystic kidney disease, retinal degeneration, a gamut of human syndromes (e.g. Bardet- Biedl, Meckel, Alstrom, MORM and Joubert) and has recently been correlated with several aggressive cancers. Current methods to study ciliogenesis have poor spatial-temporal resolution and afford little control over the process of cilia formation. Per consequence, standard methods are not robust and, as shown in our preliminary data, fail to discriminate between extremely important stages of cilia formation - most notably if the cilium is inside the cell or on the surface, as the location of the cilia will greatly affect its ability to sense the extracellular environment. Indee, the understanding of the dynamic process of ciliogenesis and its remodeling by vesicular traffic is still rudimentary and despite the importance and occurrence of primary cilia in most mammalian cells there is practically no visualization of these key steps. These major roadblocks hamper the ability to understand the cellular and molecular mechanisms of ciliogenesis and identify drugs that influence it. To overcome this longstanding barrier we will leverage our expertise in imaging of membrane traffic in two highly innovative Specific Aims. Firstly, we will develop a new image-based screen of early ciliogenesis using novel probes compatible with super-resolution imaging. Secondly, we will implement patterned microarrays and optogenetics approaches to control where and when ciliogenesis occurs. We will validate the methods and apply them to test hypotheses about roles of the exocyst and phosphoinositides in the emergence of the primary cilium. The latter is highly relevant to disease as MORM and Joubert syndromes are due to dysfunction of a phosphoinositides 5-phosphatase and optogenetics will enable directly testing of how phosphoinositides metabolism affects ciliogenesis. This toolkit will establish a new foundation to study early ciliogenesis and help set the stage for future screening of therapeutics to ciliary diseases.
Most cells have a fine hair-like antenna called a primary cilium that serves as an environmental sensor. Primary cilium dysfunction affects millions of people worldwide causing blindness, deafness, obesity, diabetes, heart and kidney disease, and is recently linked with cancer tumorigenesis. We will develop an innovative image-based toolkit to visualize and control ciliogenesis and enable new screens for therapeutics to ciliary diseases.
|Erdmann, Roman S; Takakura, Hideo; Thompson, Alexander D et al. (2014) Super-resolution imaging of the Golgi in live cells with a bioorthogonal ceramide probe. Angew Chem Int Ed Engl 53:10242-6|