Reconstructing the genomic network that coordinates cell polarity and the cell cycle, using microscopy-based functional genomics

An extraordinary capacity of all cells - from the simpler yeast cells to the most complex nerve cells in our bodies - is that to control space and time. In space, cells become asymmetrically polarized and adopt specialized shapes and growth modes both tailored and essential for the functions they need to perform; through time, cells make continually decisions as to when to grow or when to divide. Not surprisingly, when cells lose control over space or time they begin to malfunction, as is the case of metastatic cancerous cells that erratically conquer, grow and divide in wrong parts of the body at the wrong time.

Three decades of outstanding biomedical research have identified the molecular gearboxes that control cellular polarity ('space') and the cell division cycle ('time'), yet how those two fundamental processes are coordinated is virtually unknown. A better knowledge of that coordination could significantly advance our understanding of how cells and organisms live healthily and open the way to novel strategies for combatting a battery of diseases. What genes and proteins secure the coordination of polarity and the cell cycle, to secure for example that cells do not divide while they grow and vice-versa? Do they all constitute one single molecular machinery that acts together to secure cell polarity and cell cycle coordination, or do various submachineries exist that are exploited by cells in different situations? And what insights can those machineries give as to the origin of diseases like cancer and how to combat them?

These are questions that we aim to address with this project, where we will combine cutting-edge genetics, microscopy and computational functional genomics methods to investigate the coordination of cell polarity and the cell cycle in unprecedented detail.
The evolutionarily conserved molecular gearboxes that regulate cell polarity and the cell cycle were both largely discovered using the yeasts as experimental organisms, as they can be easily genetically manipulated and studied under the microscope. Therefore, in this project we will build upon that work and exploit the power of the yeast system and of state-of-the-art technologies to systematically explore the yeast genome and enquire which genes coordinate polarity and the cell cycle, using a commercial collection of 3700 yeast lines lacking each gene in the genome readily available in our laboratory. We have developed a genetic tool that allows us to monitor precisely the polarity status of each cell and simultaneously its cell cycle state. We will introduce that so-called 'cell polarity/cell cycle (CP/CC) biosensor' in the 3700 yeast lines, and image them using a robotized high-resolution OperaLX microscope - the only one in the market with the required throughput and resolution - and analyze all the images generated using automated image analysis computer programmes. These are protocols that our laboratory has pioneered, putting us in a unique position to carry out this project and address these questions for the very first time.

Lastly, we will identify which of the genes found to coordinate cell polarity and cell cycle in yeast also function analogously in cultured human cells and whether mutations in those genes are found to be enriched in cancer/diseases databases, indicating they may be used as leads for the design of future therapeutics.

Cell polarity and the cell cycle are two fundamental biological processes whose deregulation leads to countless pathologies, including cancer. Decades of research - notably in yeast - have discovered the molecular gearboxes controlling each of those processes, yet it is virtually unknown how they are molecularly coordinated. This project aims to comprehensively identify for the first time genes and proteins that coordinate cell polarity and the cell cycle using an integrated Systems Biology approach - combining genetics, systematic gene knockouts, high-throughput/high-content microscopy and computational/bioinformatics methods - and the fission yeast (Schizosaccharomyces pombe) as model organism.

In S. pombe, cell polarity and cell cycle progression are tightly correlated. We developed a 'biosensor' - CRIB-mCherry (a protein that interacts with active Cdc42) co-expressed with Cdc13-GFP (Cyclin B) - that allows us to simultaneously assess under the microscope the cell polarity and cell cycle (CP/CC) state of S. pombe cells and cell populations. We will generate a genome-wide collection of knockout yeast strains expressing the 'CP/CC biosensor', image the collection using high-throughput confocal microscopy and use custom-developed image analysis/Machine Learning tools to quantitate the biosensor signal from all knockouts and identify those with abnormal polarity, abnormal cell cycle, or both.

Using bioinformatics, we will then seek to clarify how the genes (corresponding to the knockouts identified) regulate CP/CC coordination by linking them to existing pathways of cell polarity and the cell cycle, and investigate their relevance for diseases in particular cancer. We will then experimentally validate and characterize the identified CP/CC coordinating genes in yeast. Finally, we will test whether their orthologues also play a similar role in human cultured epithelial cells, providing the first examples of such genes for humans.

The proposed research will lead to advances that could impact not only the scientific community but also a number of non-academic beneficiaries:

POTENTIAL IMPACT FOR HEALTH AND THE GENERAL PUBLIC.
- Through this project, new and deep knowledge will be gained with regards to the fine coordination of cell polarity and cell division - two fundamental biological processes of medical relevance - by identifying for the first time families of genes and proteins coordinating those processes and clarifying how those genes/proteins participate in cancer or disease pathways. This will generate a wealth of information that could in the longer term inspire novel therapeutics for the long-term benefit human health and the general public.

IMPACT FOR THE BIOTECHNOLOGY AND PHARMACEUTICAL INDUSTRY
Many of the technological tools developed in this project could have potential uses for the biotech and pharmaceutical industries.
- Due to the conserved molecular mechanisms that govern cell cycle and cell polarity in yeast and humans, our yeast strain expressing the cell polarity/cell cycle (CP/CC) biosensor could be directly exploited as a tool for chemical screening to identify molecules able to interfere with cell polarity, cell cycle progression or the coordination of both, helping to select for potential anti-cancer drugs.
- Moreover, an analogous CP/CC biosensor could also be developed for human cells and CP/CC biosensor-expressing human cell lines established. Building upon our validation work with human cells (section 4.6), this could enable partial or genome-wide RNAi screening for human cell-specific CP/CC genes, as well as screening libraries of drugs and chemical compounds capable of interfering with the machineries that coordinate cell polarity/cell cycle or conversely capable of reverting the lack of their coordination in selected cancer/disease cell lines. Both these could be of interest to the drug industry.
- Some of the computational tools and algorithms that we will develop - for multi-dimensional phenotypic (morphology/fluorescence) profiling; bioinformatics and statistical network inference; etc - could be adapted for the design of research or medical diagnostic tools. For example, based on our validation work with human cells (section 4.6) we could adapt our custom-made algorithms to quickly recognize aberrant cell polarity/cell cycle signatures of cell populations, to quickly analyze or compare pathological tissue samples, suggest possible molecular pathways involved in the pathologies, etc.
- Advances in software for image and data management, analysis or integration with data analysis frameworks and with online databases could similarly be of interest in a wide range of applications for biomedical/biotechnology companies, for high-throughput/high-content screening, medical imaging and diagnostics, and others.

BENEFITS TO SOCIETY/ETHICS
- Finally, today there is a strong demand to replace animal experiments with invertebrate model systems and cell culture assays because of the societal and ethical problems that animal experimentation raises. By using yeast and human cells to molecularly dissect the machineries that coordinate cell polarity and the cell cycle - two fundamental biological processes whose deregulation leads to many pathologies including cancer - our work will contribute to the advancement of this important area of research, while addressing Replacement and related pressing societal problems.