Title:
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Combining scanning probe, confocal microscopy and electrophysiology : a new approach to studying the primary cilium
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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.
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