The pluripotent cells in an early mammalian embryo give rise to the multitudes of cell types in an adult organism. How they change their inner structure to take on the various identities of these many cell types remains elusive. The microtubule cytoskeleton mediates many key cellular processes such as intracellular transport and cell division, and takes on unique arrangements according to the changing needs of the cell. In this way, the microtubule cytoskeleton acts as a “fingerprint” in the cell, shaping its form and function. Thus, manipulation of microtubules is an operation in fine subcellular precision. Traditional pharmacological or genetic methods can have robust effects but are challenging to execute in a specific way. To understand the role microtubules play in determining the identity of a cell, we have developed a toolbox of light-inducible methods to modify their arrangement with subcellular resolution and sub-second response times.
Microtubules in the early mouse embryo emanate from a non-centrosomal microtubule-organising centre, termed the “interphase bridge”, which is anchored by the calmodulin-modulated spectrin-associated protein 3 (CAMSAP3). This unique arrangement is essential for the formation of the pluripotent inner cell mass, defining cells that will give rise to the embryo proper, a fundamental process in embryogenesis. Despite this, we know little about how the inner structure of a cell contributes to their identity during preimplantation development.
We have used genetic engineering to develop a light-inducible optogenetically-controlled CAMSAP3 variant, which disassociates from microtubules under blue-light illumination. To complement this approach, we are also using the light-switchable drugs, photostatins and SBTubs, which allow for ease of use and reversible inhibition of microtubule growth. These tools allowed us to destabilise the microtubule network with high precision and investigate microtubule growth hotspots in iPSCs and the living preimplantation embryo, which will be combined with a 4-dimensional map of microtubule growth rates during acquisition of cell identity.
The development of light-inducible tools to manipulate the microtubule network allows us to pinpoint these effects in a precise manner, giving us the power to implement this whenever and wherever we need to uncover the mysteries of the unique microtubule arrangement in pluripotent cells of the living preimplantation mammalian embryo and its link to cellular identity.