HIF Stabilization Weakens Primary Cilia

Abstract

Although solitary or sensory cilia are present in most cells of the body and their existence has been known since the sixties, very little is known about their functions. One suspected function is fluid flow sensing- physical bending of cilia produces an influx of Ca++, which can then result in a variety of activated signaling pathways. Defective cilia and ciliary-associated proteins have been shown to result in cystic diseases. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a progressive disease, typically appearing in the 5th decade of life and is one of the most common monogenetic inherited human diseases, affecting approximately 600,000 people in the United States. Because the mechanical properties of cilia impact their response to applied flow, we asked how the stiffness of cilia can be controlled pharmacologically. We performed an experiment subjecting cilia to Taxol (a microtubule stabilizer) and CoCl2 (a HIF stabilizer to model hypoxia). Madin-Darby Canine Kidney (MDCK) cells were selected as our model system. After incubation with a selected pharmacological agent, cilia were optically trapped and the bending modulus measured. We found that HIF stabilization significantly weakens cilia. These results illustrate a method to alter the mechanical properties of primary cilia and potentially alter the flow sensing properties of cilia.

Mechanical Properties of a Primary Cilium from the Stochastic Motions of the Cilium Tip

“…We have previously investigated pharmacological alterations of the ciliary bending modulus (20) and here we present new data ( Figure 3) showing similar effects. We examined the effects of two drugs, Taxol (paclitaxel) and CAY10585.…”

“…Our experimental protocols for the growth, maintainance, and pharmacological manipulations of ciliated epithelial cells and optical trapping of cilia have been published elsewhere (15,20,22), so here we only provide a brief summary. Conditionally immortalized epithelial cells originally microdissected from the cortical collecting duct of an Immortomouse are grown to confluence and allowed to differentiate for several days, during which time a cilium emerges.…”

“…Taxol is a microtubule stabilizer, yet prior studies applying Taxol to microtubules (31) and cilia (20) show that the flexural rigidity decreases, a counterintuitive result. Based on molecular dynamics simulations, it was proposed (32) that Taxol increases axonemal stability by allowing the increased flexibility to relieve internal stresses.…”

“…Furthermore, we determined EI e f f when either Taxol, a microtubule stabilizer, or the small molecule CAY10585 is added to the culture medium. Hypoxia-inducible factor HIF-1 is destabilized by CAY10585 (19) and preliminary results demonstrate a link between ischemia-hypoxia and cilium bending modulus (20).…”

“…Furthermore, we determined EI e f f when either Taxol, a microtubule stabilizer, or the small molecule CAY10585 is added to the culture medium. Hypoxia-inducible factor HIF-1 is destabilized by CAY10585 (19) and preliminary results demonstrate a link between ischemia-hypoxia and cilium bending modulus (20).After presenting the data, we provide several attempts to determine the source of this apparent length dependence by: 1) Use of different boundary conditions; 2) Careful accounting of viscous forces along the axoneme; 3) Allowing the bending modulus to vary with position; 4) allowing the basal body to undergo active fluctuation; and 5) modeling the cilium as a collection of elastic shells rather than a homogeneous beam. We will show that a combination of improved structural modeling (orthotropic shell model) and stochastic modeling of ATP-driven actin cortex fluctuations of the basal body (21) reconcile our experimental data.…”