Total internal reflection fluorescence microscope optics.
Current Research
Quantitative behavioural phenotyping of C. elegans mutants
An important idea in C. elegans behavioural research is that if a gene is involved in a visible behaviour, then mutations that break that gene might lead to detectable behavioural changes. In that case, worms that carry the mutation can be tracked in a population and studied using the tools of genetics and molecular biology. However, many mutants have subtle phenotypes that are difficult or impossible to see by eye. To broaden the applicability of this powerful approach to genetics, we are using an automated system to record high-resolution video of freely behaving worms and a computer vision system to identify and classify worms based on features of their movement, morphology, and posture yielding a rich phenotypic “fingerprint”. You can find more information about the tracking and analysis project at the Worm Tracker website. We hope to provide a major update by the end of 2011.
Having a lot of quantitative behavioural data gives us the chance to discover new things about normal worm behaviour but also about how mutations can modulate the behaviour. If mutations in two genes lead to very similar behavioural defects, this is a hint that the affected genes may have related functions. Therefore, phenotypic clustering leads to new hypotheses for gene function.
Predatory behaviour of the nematode Pristionchus pacificus
C. elegans feeds on bacteria, in the lab this usually means E. coli, but there are other nematodes like Pristionchus that also feed on other nematodes. We're using the same tracking and analysis technology that we use for studying C. elegans mutants to study Pristionchus pacificus. In the first place we're interested in how Pristionchus behaviour relates to C. elegans behaviour on and off bacterial food but it will also be interesting to try to understand their predatory behaviour. Do Pristionchus worms have a hunting strategy that we can identify and understand? Do they have a particular strike behaviour? Ultimately, can we identify the neurons and genes that regulate predation?
Previous Research
Blood clot mechanics
Blood clots are fascinating biological materials with important roles in physiology and disease: they stop bleeding when you cut yourself and help with wound healing but also block blood vessels in heart attacks and strokes. Motivated in part by our results on the mechanics of single fibrinogen molecules (see below) we wanted to understand their properties at higher levels. Blood clots can be extended to over three times their relaxed length before breaking. This large extensibility is accompanied by a large volume decrease, corresponding to a negative compressibility. We used electron microscopy to confirm that stretch leads to significant network ordering and fiber bundling. One possible explanation for the remarkable properties of fibrin clots is the unfolding of fibrinogen's mechanically labile alpha-helical coiled-coils, which we observed using single molecule atomic force microscopy. To bridge the macroscopic and molecular scales, we used small angle X-ray scattering to observe a disordering transition consistent with two-state molecular unfolding in stretched fibrin clots.
Hybrid TIRF/AFM Combines Molecular Specificity with High Resolution
New insights in biology often depend on new technologies that open windows on
nature on ever finer scales. To this end, I worked on a hybrid total internal reflection fluorescence and atomic force
microscope (TIRF/AFM) that can both image and manipulate biological samples in their native aqueous environment with
nanometer precision.
A cartoon of the experimental geometry of our TIRF/AFM instrument is shown at the right. The flexible AFM cantilever is scanned over the surface to produce an image while a laser is used to excite sample fluorescence through the objective of an inverted optical microscope. In this way, a combined fluorescence (a) and topographic (b) image can be generated. Using specific fluorescent antibodies, TIRF imaging yields spatial and chemical information, in this case revealing the presence of microtubules in a chick axon. In contrast, AFM reveals finer structural details at the growth cone tip and also quantitative height information that can be used to determine the arrangement of microtubule bundles in the axon (white rectangle) that is not clear from the TIRF image alone.
Single Molecule Mechanics
AFM can also be used in force mode to investigate the mechanics of biological
molecules one at a time. In this experiment, a flexible cantilever with a sharp tip is repeatedly brought into contact with
a surface covered in a molecule of interest and retracted. When the tip is retracted, sometimes a non-specifically adsorbed
molecule is extended from the surface. By monitoring the deflection of the cantilever, we can generate a force-extension
curve that contains information about the mechanics of single molecules. Molecules of current interest include cytoskeletal
proteins like spectrin and filamin and fibrinogen, the protein that forms the scaffold of blood clots.
Microtubule Mechanics and Intracellular Transport
Microtubules are stiff biological polymers that form a critical
part of the cellular cytoskeleton. Microtubules are often thought of as relatively static highways used by molecular motors
like kinesin and dynein to transport cargoes from one end of the cell to another. In some cases, this can be a good
approximation, but we have found that microtubules writhe wildly inside of many cell types. These fluctuations are not
thermal, but are in fact driven by the action of molecular motors. The image on the left shows the standard deviation of
intensity at each pixel over the course of a fluorescent movie of microtubules in a drosophila S2 cell. Regions with
significant motion appear brighter giving the cell interior the appearance of an intense flame. Interestingly, these
fluctuations can be harnessed by cellular cargoes to speed their transport so in some cases a better analogy for
microtubules than a system of highways is a chaotic molecular stir-bar to enhance diffusion!
Disclaimer: These pages are my personal pages. The opinions expressed here are not necessarily those of the Laboratory of Molecular Biology or the Medical Research Council.