The primary cilium, a solitary hair-like structure that extends out of the apical membrane of many cell types, has been investigated here. The current paradigm suggests that the cilium behaves as a cantilever-like object which bends in a continuous manner along the length of the cilium. This thesis aims to investigate whether this paradigm is appropriate. Here, primary cilia are imaged by atomic force microscopy and light microscopy in live and fixed states. Using atomic force microscopy to do force spectroscopy, it is shown by buckling the primary cilium that the cilium has a Young's modulus of 3.5 ± 1.3 MPa, a value which places it an order of magnitude stiffer than previously recognised. Calcium imaging has been used to identify the presence of calcium fluxes in response to fluid flow and actuation of the primary cilium with the atomic force microscope (AFM). A new technique, coined Vertical Deflection Mapping, has been developed, whereby a defined force is applied by the AFM at a known distance from the base of the cilium; it was observed that the cilium had a spring constant of (3.9 ± 2) X 10-5 Nm-1, approximately an order of magnitude more sensitive than previously recognised. It was found that the spring constant decreased towards the tip of the cilium as a function of the reciprocal of the length squared. This relationship is best represented by a rigid body bending from the base. These results were compared to results collected on a cantilever, which displayed results that tended towards the reciprocal of the length cubed. Preliminary work has been completed on combining light microscopy and AFM with a patch-clamp electrophysiology set-up allowing all three systems to be used simultaneously. This will allow a new method of delving into the functioning of mechanosensitive ion channels in the primary cilium and other structures.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:601100 |
| Date | January 2012 |
| Creators | Kocher, Stephen James |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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