Total internal reflection fluorescence microscope optics.
Quantitative behavioural phenotyping of C. elegans mutants
The goal of reverse genetics is to prevent a gene from performing its normal function and to determine what effect this has on an organism. The technology for perturbing genes in this way has advanced rapidly, in principle giving us an entry point to study the function of every gene. The problem is that we often cannot go any further because loss of the gene does not lead to an effect on an organism that is observable by eye, even to trained experts. This is still true for the nematode worm C. elegans, which is arguably the best-described animal on the planet: we know know the wiring diagram of all its neurons and the developmental lineage of all its cells from the single-celled embryo to the adult. However, we lack a correspondingly comprehensive view of its behaviour. One my goals is to change this using automated imaging of freely crawling worms and quantitative analysis to understand their behaviour.
I am also interested in behaviour more broadly. There are still fundamental questions that don't have satisfactory answers. What are the limits of behavioral quantification? Can we develop a language to describe behavior that is both precise and universal? Is it possible to describe behavior completely, analogously to the complete wiring diagram of C. elegans neurons?
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?
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.