Investigating oriented cell divisions from a molecular to a systems-level using optogenetics and micropatterning – LIVESPIN

Oriented cell divisions (OCDs) are essential to control tissue development, growth, and homeostasis. Defective OCD could be involved in a number of human diseases, such as microcephalies and polycystic kidney disease. Precise spindle orientation is important for the resolution of asymmetric cell fates, suggesting that OCDs control stem cell numbers and protect tissues against excessive proliferation: defects in mitotic spindle orientation have been associated with precancerous stages in gut stem cells. Understanding the mechanisms which govern this fundamental cellular event is of particular relevance in the context of homeostasis and pathogenesis in adult tissues, and may help devise preventive and curative strategies for human health.
Studies in models of oriented cell divisions have identified a conserved molecular cascade (the “LGN complex” and the dynein motor complex) as the major effector of spindle orientation, translating polarity cues from the cell cortex into localized pulling forces exerted on astral microtubules. This system is polarized and functional in a cell-cycle dependent manner, indicating that modulators, yet to be identified, play essential roles in fine tuning the localization, strength and duration of forces. It is important to understand how they orchestrate in space and time to constitute a molecular circuit able to detect, process and propagate the information leading to oriented division: what is the minimal level of asymmetry necessary to specify spindle orientation? Are there feedback loops that reinforce an initial polarization signal? Once polarity is initiated, is it robust to perturbations?
Genetic studies (loss and gain of function) in in vivo models of asymmetric division have been instrumental in characterizing molecules involved in spindle orientation, but they often fall short of addressing the systems-level properties discussed above, because they probe the molecule’s function in a global rather than local fashion. We think it is essential to develop complementary approaches in which fine spatial and temporal patterns of protein activity can be manipulated at will in simplified paradigms of spindle orientation.
The goal of this project is to address in single-cell experiments the mechanisms underlying mitotic spindle orientation in OCDs by combining advanced techniques in micropatterning, optogenetics and live cell imaging. Microadhesive patterns of defined size and shape offer the possibility to standardize and manipulate cell morphology in large numbers of cells, opening the way to systematic approaches and advanced screens. Optogenetic approaches use a light stimulus to control at a subcellular scale the activation of a molecular pathway, and therefore to measure how the cell responds to a local modification of its intracellular molecular context.
With these tools, we have two objectives: i) characterize at the systems-level how the known actors are orchestrated within spatio-temporally controlled circuits that process the flow of intracellular information leading to oriented division. In particular, we will explore the conditions required for an induced perturbation to be either filtered out or amplified and propagated to the whole cell scale by the endogenous machinery underlying cell polarity ii) identify new molecular actors/complexes governing the spindle orientation We will perform an RNAi screen for modulators of spindle orientation in a standardized orientation assay on micropatterns, and use optogenetics to assess and validate the molecular functions of candidate regulators.
This project constitutes an innovative approach to the study of OCD. The combined application of micropatterning and optogenetic tools in single cells paradigms of spindle orientation will give access to a wealth of quantitative data and allow a more refined analysis of mitotic spindle orientation, from a molecular to a systems-level.