Marketa Schmidt-Cernohorska

Investigating The Biophysical Properties Of Centrosomes

Centrosomes are non-membrane bound cellular organelles whose main function is to nucleate and organize microtubules. As such, they coordinate the roles of the cytoskeleton in organelle positioning, intracellular transport, cell motility and establishment of polarity. During cell division, centrosomes contribute to spindle assembly and position the cleavage plane during cytokinesis. Centrosome abnormalities, both numerical and structural, are commonly observed in human cancers and are thought to be a driving force in tumorigenesis. While centrosome numerical abnormalities have been extensively studied, much less is known about structural abnormalities and indeed about the biophysical (material) properties of centrosomes in general. Centrosomes are composed of a pair of centrioles surrounded by the so-called pericentriolar material (PCM), within which microtubule nucleation and anchorage occurs. Centrioles assemble in a series of steps that have been extensively characterized at the molecular and structural level. In contrast, the surrounding PCM remains poorly defined. Classical experiments found the mitotic PCM to contain an underlying filamentous matrix scaffold that acts as a docking platform for other ‘client’ proteins that nucleate and organize microtubules. The molecular identity of this scaffold in vertebrates remains unclear. However, recent in vitro reconstitution experiments suggest that Centrosomin (Cnn) in Drosophila and Spindle-defective protein 5 (SPD-5) in C. elegans oligomerize to form a dynamic scaffold capable of recruiting other proteins. Those same in vitro experiments also showed scaffold polymers to initially behave as liquid droplets that are phase-separated from the cytoplasm, only hardening progressively over time. It is hard to reconcile this in vitro behavior with the ability of centrosomes in vivo to withstand pulling forces exerted on them via the microtubules they nucleate. In an attempt to resolve these conflicting findings, I plan to employ advanced microscopy-based techniques to for the first time examine the viscoelastic properties of centrosomes in vitro and in vivo. My proposal is divided into three aims.

In Aim 1, I will probe the mechanical properties of C. elegans centrosomes in vitro by atomic force microscopy (AFM), a method that relies on a fine cantilever to probe the stiffness of surface-accessible material by nanoindentation. I will also assess PCM scaffold stiffness in isolation after extracting centrioles and loosely bound PCM proteins with chaotropic agents and compare its properties with that of pure SPD-5 polymer to examine to what extent the mechanical properties of centrosomes are explained by those of their main structural component. Having established benchmarks for centrosomes in vitro, in Aim 2 I will compare these values with those obtained by Brillouin light scattering microscopy (BLSM), an optical method which assesses the viscoelastic properties of structures within a confocal volume. Critically, unlike AFM, Brillouin microscopy is a non-invasive technique, allowing me to monitor the behavior of centrosomes in vivo in the C. elegans embryo. This will open up questions such as how the mechanical properties of centrosomes change during their growth in mitotic prophase (centrosome maturation) and subsequent disassembly at mitotic exit. Finally, in Aim 3 I will turn to centrosomes in vertebrate cultured cells, using Brillouin microscopy to examine whether the structural abnormalities observed in tumor cells reflect alterations in the viscoelastic properties of the PCM, potentially resulting in premature centriole separation and overduplication and impacting their ability to function in cell division.

Together, the experiments in this proposal should result in a clearer picture of the biophysical principles that underlie functionality of the centrosome and the potential impact of structural abnormalities on centrosome function in cancer cells.