| dc.description.abstracteng | I begin with a brief account of the topic of mechanosensation, followed by an outline of how the project evolved. The experimental techniques used and their rationale are described. The section ends with a short description of the thesis structure.
Out of all sensory modalities, mechanosensation is of special interest to biophysicists, since its transduction mechanism in most cases involves a direct mechanical gating of ion channels, as opposed to gating by a chemical messenger. One important class of mechanosensory organs are the chordotonal organs. These perform proprioceptive and other mechanosensory functions in insects and crustaceans. There is a wide diversity of chordotonal organs across species and also within a single species, but there is a great deal of structural overlap. This makes them rather interesting to study, because results obtained by studying the mechanics of one type of chordotonal organ can in principle be applied to others. The mechanical properties of these organs are thought to be correlated to the sensory functions. Mutant studies, laser Doppler vibrometry and other techniques have given us some information on the functioning of these organs, but direct mechanical probing of their components had hitherto not been carried out. This was the motivation for my project, in which I measured mechanical properties of a particular chordotonal organ – the lateral pentascolopidial (lch5) organ – that plays a key role in proprioceptive locomotion control in Drosophila larvae.
In the early stages of the project, the mechanical properties of Johnston’s organ – the antennal hearing organ in the adult fruit fly Drosophila melanogaster – appeared attractive, because it functions using an active process very similar to that operating in the vertebrate ear. The idea was to measure active fluctuations from the sensory cilia of the organs, which are believed to be the main transducing element. This would involve initially measuring forces at the arista, which is the external sound receiver of the antenna, and then moving inwards. Attempts to measure forces at the arista using optical tweezers were unsuccessful owing to thermal damage. There were also other difficulties, mainly that optical trapping requires a water sample and the fly does not survive under water. We also understood that probing the internal structures of Johnston’s organ would not yield conclusive results, since this would require perforating the cuticle and would impact the mechanics. We then shifted our attention to the lch5 organ since it is also a chordotonal organ, albeit simpler in structure than Johnston’s organ. We first decided to repeat the bead-trapping experiment on the lch5 organ. For these experiments I used a dissected fillet preparation of the larva. Here the muscles presented an obstacle to bringing the bead in contact with the organ. This was overcome by digesting the muscles with collagenase. However, once the bead was stuck to the organ it could not be trapped, and no fluctuations could be measured. Since the ciliary mechanics appeared inaccessible, we shifted to whole-organ mechanics.
Since the lch5 organ is believed to regulate the crawling mechanism, we decided to focus on this aspect. We captured detailed videos of the deformation of the cuticle as the larva crawls. Also, I obtained high-resolution images of the lch5 organ using DIC microscopy and a self-designed preparation. This involved flattening the larva between a slide and a coverslip, such that the gut was squeezed out and the sample was rendered transparent. This “squished prep” proved highly useful to us in the laser ablation experiments that we performed in the later stages of the project.
The next set of experiments was to measure the mechanics of the lch5 organ in a fillet prep using a tungsten needle. We applied tension to the whole organ in situ by transverse deflection. Upon release of force, the organ displayed overdamped relaxation with two widely separated time constants, tens of milliseconds and seconds respectively. When the muscles covering the lch5 organ were excised, the slow relaxation was absent and the fast relaxation became faster. We also observed the change in shape of the organ as it was deformed by the needle. A cusp-like shape was seen. The ends of the organ were fixed during the entire process, which meant that the length of the organ increased, and once the needle was released, relaxed back to its original value. Interestingly, most of the strain in the stretched organ is localized in the cap cells, which account for two-thirds of the length of the entire organ, and could be stretched to nearly a 10% increase in length without apparent damage.
Next, laser ablation experiments on the lch5 organ were carried out. For this, the earlier mentioned “squished prep” was employed. Using a UV laser, the organ was then severed at different points, and its retraction was observed. It was found that cap cells retracted by over 100 μm after being severed from the neurons, indicating considerable steady state stress and strain in these cells. Also, in a myosin knockdown mutant, a much smaller retraction in comparison to the control was seen. Given the fact that actin as well as myosin motors are abundant in cap cells, the results point to a mechanical regulatory role of the cap cells in the lch5 organ, and a significant contribution of myosin motors to this process.
The final set of experiments was to develop a technique to measure forces in the lch5 organ using calibrated glass needles. We have optimized the method and made it suitable for future investigations. | de |