The question of cell size regulation

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Table of contents

Acknowledgments
Abstract
List of abbreviations
1 The question of cell size regulation
1.1 Dening cell size
1.1.1 Cell mass and cell volume
1.1.2 Cell size in proliferating cells: coordination between growth and cell cycle progression
1.2 Cell size homeostasis in metazoan cells
1.2.1 Cell size vs. Organ size
1.2.2 Intrinsic vs. extrinsic regulation of cell size
1.2.2.1 Flexibility upon environmental changes
1.2.2.2 Coupling of cell growth and cell cycle progression?
1.2.3 Proliferating cells in multicellular organisms
1.2.4 Evolutionary point of view on the requirement of growth regulation in unicellular and multicellular organisms
1.3 Conclusion
2 Size homeostasis study: question, challenges and concepts step by step
2.1 Sizer, timer & adder
2.2 Coordination of cell cycle progression to growth
2.3 Models of size-sensing mechanism
2.3.1 Geometric measurement of absolute length
2.3.2 Volume measurement through titration-based mechanism
2.3.3 Surface area measurement
2.3.4 Conclusion: the importance of cell volume
2.4 An emergent role for growth regulation in size control
2.4.1 The regulation of growth
2.4.1.1 Growth in volume
2.4.1.2 Growth in mass
2.4.1.3 Growth and metabolism
2.4.1.4 Density throughout the cell cycle
2.4.1.5 Conclusion
2.4.2 Measuring cell growth at the single-cell level
2.4.2.1 New techniques enabling single-cell measurement
2.4.2.2 From population-level studies to single-cell measurement
2.5 Combination of processes?
2.5.1 Independent control in each cell cycle phase
2.5.2 One overarching mechanism
2.5.3 Parallel and concurrent processes
2.6 Robustness and exibility of size control
2.6.1 Apparent exibility of size thresholds
2.6.2 What we can learn from studying cells in dierent growth environments
2.7 Conclusion
3 Current understanding of size homeostasis in yeast and bacteria
3.1 Cell size homeostasis in bacteria
3.1.1 Brief description of the cell cycle of bacteria
3.1.2 Phenomenological description and recent emergence of the adder model
3.1.3 Beyond the adder observation, current mechanisms debated
3.1.3.1 Control in each sub-periods
3.1.3.2 Molecular players
3.1.4 Trying to unify the dierent ndings in the eld of bacteria
3.1.4.1 How do these controls combine to generate an adder?
3.1.4.2 Lessons from model-free approaches
3.2 Cell size homeostasis in yeast
3.2.1 Size control in S. pombe
3.2.1.1 S. pombe, cell cycle and size-checkpoints
3.2.1.2 S. pombe is the classical example of a sizer in wild-type cells
3.2.1.3 S. pombe. grows in a bilinear fashion
3.2.1.4 In search for the mechanism generating geometric sensing of size
3.2.2 Size control in S. cerevisiae
3.2.2.1 S. cerevisiae, cell cycle and checkpoints
3.2.2.2 Lack of consensus about the growth behaviour of S. cerevisiae
3.2.2.3 Daughter cell of S. cerevisiae behave in an adder-like manner
3.2.2.4 Whi5 and the inhibitor-dilution size sensor model for G1/S transition
3.3 Lessons from yeast and bacteria
4 Cell size homeostasis in metazoan cells
4.1 Growth and cell cycle pathways in mammalian cells
4.1.1 Pathways regulating the growth rate
4.1.1.1 The mTOR pathway
4.1.1.2 Other pathways
4.1.1.3 Conclusion
4.1.2 Cell cycle regulation
4.1.2.1 Brief overview of the mammalian cell cycle
4.1.2.2 Commitment to a new the cell cycle: checkpoint(s) for growth during G0 and G1
4.2 Population-level studies and evidence of size control in early work
4.2.1 Evidence of size-sensing in G1
4.2.2 Results challenging the idea of size control in metazoan cells
4.3 The challenging problem of characterizing cell growth in metazoan cells
4.4 Current views on the homeostatic process in metazoan cells
4.4.1 Cells grow exponentially or super-linearly
4.4.2 No clear role for time modulation, possible existence of a growth-rate modulation
4.5 Conclusion
5 General conclusion and aims of this study
5.1 General conclusion
5.2 Aims of this study
6 Methods
6.1 Long time-lapse acquisition in animal cells to characterize growth: state of the art and challenges
6.2 Fluorescence-exclusion based volume measurement
6.2.1 Volume measurement method: principle and validation
6.2.1.1 Method principle
6.2.1.2 Theoretical validation
6.2.1.3 Experimental validation
6.2.2 Image analysis optimization
6.2.3 Protocol optimization for long time-lapse acquisition
6.2.3.1 Improve nutrient access
6.2.3.2 Standardized cell culture protocol to try reduce variability
6.2.3.3 Analytical validation of the quality of growth in the experiments
6.2.3.4 Improving the uorescent probes
6.3 Cell growth analysis: clonal & single-cell curves, cell cycle transitions
6.3.1 Single-cell curves
6.3.1.1 Careful control of the sources of volume curves uctuations
6.3.1.2 Semi-automated analysis of hundreds of single-growth curves
6.3.2 Clonal growth curves
6.3.3 Cell cycle transitions keypoint analysis
6.4 Developing tools to induce asymmetrical divisions
6.4.1 Inducing asymmetrical divisions using micro-channels
6.4.1.1 Micro-channels induce asymmetrical cell divisions and allow volume measurement
6.4.1.2 Optimization of the micro-channels device to improve nutrient access
6.4.2 Asymmetrical patterns
6.4.3 Drug-induced asymmetrical distribution of organelles
6.5 Choice of cell types
6.5.1 Description of the cell types used in this study
6.5.2 Establishing new stable lineages
6.6 Statistical analysis
7 Results
7.1 Summary
7.2 Manuscript in preparation for submission
7.3 Concluding remarks
7.3.1 Analysis planed before the submission
7.3.2 Important experiments needed to complete the work
8 Discussion
8.1 Single cell growth measurement in mammalian cells
8.1.1 Single cell measurement of volume with the FXm
8.1.1.1 FXm allows measurement of both suspended and adherent cells
8.1.1.2 FXm allows measurement of cell volume
8.1.1.3 Results from direct measurement of single cell growth might precise some of the previous ndings
8.1.1.4 FXm measurements provide a robust set of data for the study of size homeostasis
8.1.2 Current limitations of the FXm
8.1.2.1 Limitation to cells which do not internalize the probe
8.1.2.2 FXm currently requires a device conning the cells
8.2 Growth and time modulation
8.2.1 Time modulation
8.2.1.1 G1 duration is correlated with volume in HT29 and HeLa cells and gated by a minimum duration
8.2.1.2 S and G2 phases
8.2.1.3 Conclusion on the role of time adaptation
8.2.2 Growth modulation
8.2.2.1 Experimental evidence
8.2.2.2 Growth regulation and cellular homeostasis
8.2.2.3 Conclusions on the role of growth modulation
8.2.3 Conclusion on the respective contribution of time and growth modulation in size control
8.2.3.1 Classication of the results into three types of combinations of growth and time modulation
8.2.3.2 Identication of three distinct rate-limiting processes for cell cycle and cell growth
8.3 Is there a unique mechanism or several processes resulting in the adder?
8.3.1 Generality of the adder, from bacteria to mammalian cells
8.3.1.1 Generality of the adder in our results
8.3.1.2 Generality of the adder in the litterature
8.3.2 Is there enough evidence to conclude that size homeostasis is an adder in mammalian cells?
8.3.2.1 Statistical resolution is currently lacking to conrm the adder in any organism
8.3.2.2 Testing the adder
8.3.3 Size homeostasis is a exible process
8.3.3.1 Bacteria and S. cerevisiae: not always an adder
8.3.3.2 Why exibility is important when trying to build a model for size homeostasis
8.3.3.3 Conclusion: phenomenological adder and molecular adder
8.3.4 Several scenarios can explain the apparent adder in mammalian cells
8.4 Perspectives: a combination of processes, both single and tissue-level determined?
9 Conclusion
Bibliography