Cytoskeletal dynamics and function in oocytes
Using starfish as a model organism, the Lénárt group combines biochemistry with imaging assays to investigate how the fertilisable egg cell develops from the oocyte.
Figure 1: The actin filament network (gray) embedding the chromosomes (red).
Figure 2: Transparent starfish oocytes are uniquely suited for imaging meiotic divisions.
Previous and current research
All animal life begins with the fusion of sperm and egg. Our research is focused on the egg cell, specifically investigating how the fertilisable egg develops from the oocyte through meiotic divisions. Oocytes are exceptionally large cells, with diameters up to millimetres in size, because they store large amounts of nutrients to support embryonic development. Therefore, in oocytes and eggs, the cytoskeleton has to transport organelles, separate chromosomes, and organise cellular architecture in a very large cytoplasm. How the cytoskeleton adapts to this unusual size, and how these mechanisms differ from those in small somatic cells, is largely unknown.
We use starfish oocytes as a model system because they are easy to handle, complete meiosis rapidly, develop simply in seawater at room temperature, and are transparent – ideal for high-resolution imaging of cytoskeletal dynamics in live cells. We use confocal microscopy to image live oocytes and employ computational image analysis tools to extract quantitative parameters from these 3D time-lapse datasets. Parameters such as local concentrations or velocities of cellular components provide a quantitative assay for the biological process and, at the same time, serve as inputs for computational models of cytoskeletal dynamics. Model predictions are then tested in perturbation experiments using physical (e.g. laser microsurgery) or molecular (e.g. RNAi knock-down) manipulations. Biochemistry, in combination with the imaging assays, is used to identify the key molecular components in the process.
We have recently shown that meiotic chromosomes scattered in the large oocyte nucleus are collected by an actin meshwork and transported to the spindle, whose short microtubules cannot reach the chromosomes directly, as they do in somatic cells. This novel actin-based chromosome transport system forms as the nuclear envelope breaks down and fills the nuclear space with an actin meshwork, physically entrapping chromosomes. We were able to show that the actin meshwork contracts homogeneously; however, because it is mechanically anchored to the cell cortex, this homogeneous contraction is translated into directional transport towards the cortex where the spindle forms. By understanding the mechanism of chromosome transport essential to oocyte division and fertility, our studies revealed a novel design principle for a cytoskeletal ‘transport machine’ that is very different from previously known mechanisms of actin-driven intracellular transport.
Future projects and goals
Immediate goals include determining the detailed structure of the F-actin meshwork, understanding the molecular mechanisms of meshwork contraction, and identifying the mechanisms by which chromosomes attach to the meshwork. We will employ high-resolution imaging methods, including electron tomography and super-resolution light microscopy, to resolve single actin-filaments and to identify, localise and perturb molecules regulating actin filament dynamics that, combined with high-resolution imaging, will allow us to address the underlying molecular mechanisms. Longer term, we are interested in related cytoskeletal processes that occur in oocytes, eggs and early embryos, with the overall goal to mechanistically understand the organisational principles of the actin and microtubule cytoskeleton.