Targeting the primary cilium 

The primary cilium is an incredibly fine process that protrudes from the apical surface of cells.  Most cells have a primary cilium, though it temporarily vanishes during mitosis.  For many years the primary cilia were largely neglected, just a matter of curiosity to most.   Indeed this process is so fine that it is largely impossible to see without specific markers or electron microscopy.   However a variety of devastating inherited mutations – known as ciliopathies – were discovered that affect transport to the primary cilia (Guemez-Gamboa et al., 2014).  This has led to an increasing recognition of the importance of this fine cellular appendage. The molecular characterization of the proteins involved in ciliogenesis and function has made it possible to selectively target fluorescent proteins to light up cilia quite specifically (Follit et al., 2010).

In a tour de force of live cell imaging, Sherpa and colleagues in the Nauli lab at Chapman University showed that cilioplasmic and cytosolic cAMP pools are differentially regulated. Tolvaptam, a competitive antagonist of the vasopressin Receptor (V2) and mechanical shear both cause changes in the cAMP levels of the cilioplasm (Sherpa et al., 2019).  This recent success provides a great example of how Montana Molecular’s targeted cADDis sensors enable studies of signaling kinetics in this fascinating, if small, hub of cellular communication.  

Many thanks to Rinzhin T. Sherpa in the School of Pharmacy of Chapman University for sharing these amazing video images of cAMP dynamics in the primary cilium of renal epithelial cells!

It is now well appreciated that crucial signaling pathways are located in the cilia.  Hedgehog, Wnt, and Notch signaling all occurs in the cilia (Wheway et al., 2018).  Importantly, there is specialized G-protein receptor signaling in the cilia.  For example, the somatostatin receptor activates adenylyl cyclase 3, which is localized to the cilum.  Intriguingly, knocking out the AC3 expressed in neurons of the brain leads to memory deficits in mice (Wang et al., 2011).  The past decade of signaling research has led us to the surprising realization that the cilium, while slight in stature, is a crucial hub of signaling.  

The restricted localization of receptors and signaling proteins to the cilium suggests that there might be very localized signaling circuits confined to just the small space within the cilium.  But what about the freely diffusible second messengers like Ca2+ and cAMP?  The Clapham lab targeted  a genetically encoded green fluorescent Ca2+ sensor (GCaMP3) to the cilia and found that Ca2+  entry into the Cilia, through TRP channels, was largely confined to the cilia and had little effect on the cytosolic Ca2+ levels.  More recently the Mirshahi lab targeted the cADDis sensors to study cAMP levels in the cilia to reveal a fascinating regulation of cAMP levels that was quite different from the adjacent cytosol (Moore et al., 2016). 

References

K. Hilgendorf, et al. Omega-3 Fatty Acids Activate Ciliary FFAR4 to Control Adipogenesis Cell November, 2019

Follit, J.A., Li, L., Vucica, Y., and Pazour, G.J. (2010). The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence. J. Cell Biol. 188, 21–28. 

Guemez-Gamboa, A., Coufal, N.G., and Gleeson, J.G. (2014). Primary Cilia in the Developing and Mature Brain. Neuron 82, 511–521. 

Moore, B.S., Stepanchick, A.N., Tewson, P.H., Hartle, C.M., Zhang, J., Quinn, A.M., Hughes, T.E., and Mirshahi, T. (2016). Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics. Proceedings of the National Academy of Sciences 113, 13069–13074. 

Sherpa, R.T., Mohieldin, A.M., Pala, R., Wachten, D., Ostrom, R.S., and Nauli, S.M. (2019). Sensory primary cilium is a responsive cAMP microdomain in renal epithelia. Sci. Rep. 9, 6523. 

Wang, Z., Phan, T., and Storm, D.R. (2011). The type 3 adenylyl cyclase is required for novel object learning and extinction of contextual memory: role of cAMP signaling in primary cilia. Journal of Neuroscience 31, 5557–5561. 

Wheway, G., Nazlamova, L., and Hancock, J.T. (2018). Signaling through the Primary Cilium. Front Cell Dev Biol 6, 8.