Kinetic control of eukaryotic chromatin structure by recursive topological restraints

Chromatin structure undergoes many changes during the cell cycle and in response to regulatory events. A basic unit of chromatin organization is the nucleosome core particle. However, very little is known about how nucleosomes are arranged into higher-order structures in vivo, even though the efficiency and precision of cell division imply high levels of structural organization. We propose abandoning the current paradigm of chromatin organization based on thermodynamics of the lowest energy state and replace it with the idea of a topologically restrained, high-energy structure. We propose that DNA is subject to a recursive topological restraint, and is anchored by hemicatenates that are part of the chromosomal scaffold. Long-distance _cis_-regulation of transcription is a natural consequence of recursive topological restraint. This new theory of chromatin structure has a multitude of consequences for key aspects of cellular biology

Single-cell approaches for understanding morphogenesis using Computational Morphodynamics

In multicellular organisms cells grow, divide and adopt different fates, resulting in tissues and organs with specific functions. In recent years, a number of studies have brought quantitative knowledge about how these processes are orchestrated, shed-ding new light on cells as active and central players in morphogenesis. We explore recent advances in understanding plant morphogenesis from a quantitative perspective, defining the re-search field of Computational Morphodynamics. The focus is on studies combining theoretical and experimental approaches integrating hypotheses of how molecular and mechanical regulation at the cellular level lead to tissue behaviour. Finally, we dis-cuss some of the main challenges for future work

Microtubule and chromosome dynamics during mitosis in budding yeast

As a cell divides, DNA must be replicated and faithfully segregated between the mother and daughter cells. This segregation is facilitated by the mitotic spindle, assembled to pull sister chromatids apart as the cell divides. In budding yeast, spindle pole bodies nucleate microtubules that make up the mitotic spindle, position it at the site of division, and physically link chromosomes to opposing poles via the kinetochores. The chromosomes are held together by cohesin, which is also involved in the architecture of chromatin. In this thesis, I have explored mechanisms controlling microtubule dynamics, kinetochore positioning and chromosome dynamics during mitotic cell division in budding yeast. Bik1 is a microtubule-associated protein shown to play a role in the cytosol to position the spindle before anaphase. In paper I, we have characterized the nuclear function of Bik1 and identified a novel role in clustering kinetochores prior to spindle elongation. Cells lacking nuclear Bik1 have a delayed cell cycle progression, with prolonged metaphase, and fail to cluster kinetochores. We also connect this function to the nuclear kinesin Cin8, which has previously been described to regulate kinetochore microtubule dynamics in metaphase. The spindle pole body anchors microtubule nucleating γ-tubulin complexes using two different receptors, Spc72 in the cytosol and Spc110 in the nucleus. In paper II, we have isolated ‘old’ Spc110, originating from the previous cell cycle, and mapped its phosphorylation sites. These analyses revealed that old Spc110 is phosphorylated at serine 36 and at a novel site, serine 11. Non-phosphorylatable mutant strains revealed that these sites influence microtubule dynamics and cell cycle progression. The Spc110S11A mutant strain frequently had brighter spindle microtubules with asymmetric distribution of α-tubulin. Furthermore, Spc110S11A S36A cells had slightly delayed cell cycle progression and spindle disassembly. The cohesin complex has been shown to shape the chromosomes into loops in budding yeast through a mechanism known as loop extrusion. This phenomenon has primarily been studied using genome-wide sequencing techniques, which report detailed population averages of contact frequencies throughout the genome. How chromosomes of individual cells are affected, and whether this looping affects physical compaction remains poorly understood. In paper III we have generated a microscopy-based system to study chromosome dynamics in single yeast cells by fluorescently tagging specific chromosomal loci. We then used this system to investigate how physical distances between the fluorescently marked loci change after inhibiting loop extruding cohesin. This study revealed that loop extrusion does not significantly affect physical distances but may limit the dynamic movement of chromosomes. In conclusion, these studies reveal novel mechanisms controlling spindle and chromosome dynamics during mitotic cell division: 1) We have uncovered a new role of Bik1 at the spindle. 2) We have mapped phosphorylation sites in old Spc110 and characterized a novel site. 3) We have created a system to study chromosome dynamics in single cells and found that loop extrusion does not significantly compact mitotic yeast chromosomes

TORC1 and PKA activity towards ribosome biogenesis oscillates in synchrony with the budding yeast cell cycle

Recent studies have revealed that the growth rate of budding yeast and mammalian cells varies during the cell cycle. By linking a multitude of signals to cell growth, the highly conserved Target of Rapamycin Complex 1 (TORC1) and Protein Kinase A (PKA) pathways are prime candidates for mediating the dynamic coupling between growth and division. However, measurements of TORC1 and PKA activity during the cell cycle are still lacking. Following the localization dynamics of two TORC1 and PKA targets via time-lapse microscopy in hundreds of yeast cells, we found that the activity of these pathways towards ribosome biogenesis fluctuates in synchrony with the cell cycle even under constant external conditions. Mutations of upstream TORC1 and PKA regulators suggested that internal metabolic signals partially mediate these activity changes. Our study reveals a new aspect of TORC1 and PKA signaling, which will be important for understanding growth regulation during the cell cycle

Investigating Cell Cycle Re-entry in the Drosophila brain: From the Pupa to the Adult

G0 associated with terminal differentiation represents the most common cellular state in adult multicellular organisms, yet it is poorly understood. In past years, various tissues of the fruit fly Drosophila melanogaster have served as a great model system to understand how cells establish and maintain their non-dividing state. While the Drosophila brain has been extensively studied in the context of neurodevelopment, relatively little is known about how the flexibility of cell cycle exit in terminally differentiated neurons and glia. In Chapter 2 of my dissertation, I show that postmitotic neurons and glia in the developing Drosophila pupa brain can be forced to re-enter the cell cycle and undergo mitosis after they have exited the cell cycle. Neurons can re-enter the cell cycle up to 24 hours after they have exited the cell cycle whereas glia exhibit greater flexibility and can undergo cell division up to over 48h after they exit the cell cycle. Forcing re-entry in neurons results in cell death, while glial cell division can result in tumor-like growths. Neurons and glia are some of the longest lived cells in metazoans. How these cells deal with ageing-related damage is poorly understood. My work summarised in Chapter 3 shows that polyploid cells accumulate in the adult fly brain and that polyploidy protects against DNA damage-induced cell death. Multiple types of neurons and glia that are diploid at eclosion, become polyploid in the adult Drosophila brain. The optic lobes exhibit the highest levels of polyploidy, associated with an elevated DNA damage response in this brain region. Inducing oxidative stress or exogenous DNA damage leads to an earlier onset of polyploidy, and polyploid cells in the adult brain are more resistant to DNA damage-induced cell death than diploid cells. Our results suggest polyploidy may serve a protective role for neurons and glia in adult Drosophila melanogaster brains.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163282/1/shyama_1.pd

Development of model systems to reconstruct the unicellular prehistory of animals : an emphasis on the cell cycle

El origen de la multicelularidad animal tiene sus raíces en el proceso de división celular. Conocer las bases moleculares del control de la divisón celular en animales y en sus parientes unicelulares tiene el potencial de permitirnos comprender qué cambios ocurrieron para permitir el origen de la multicelularidad. Sin embargo, nuestra capacidad experimental en los parientes unicelulares de los animales está bastante limitada. En esta tesis se ha contribuido al desarrollo de la especie Capsaspora owczarzaki como un organismo modelo, al desarrollar herramientas genéticas de transfección y herramientas de sincronización de ciclo celular. La caracterización del ciclo celular de Capsaspora ha permitido saber que muchos genes importantes en el ciclo celular de animales también poseen actividad transcripcional en Capsaspora, incluyendo los ortólogos principales de las ciclinas y CDKs de animales. Asimismo, el desarrollo de herramientas de transfección abre la puerta a nuevos estudios funcionales a nivel molecular en esta especie, lo cual podrá permitir conocer las funciones de muchos genes relacionados con la multicelularidad animal en el contexto de una especie unicelular.The origin of animal multicellularity has its roots in the process of cell division. Understanding the molecular basis of cell division in animals and their unicellular relatives has the potential to elucidate what changes in the control of cell division played a role, if any, in the transition to multicellularity. However, the experimental amenability of the closest relatives of animals is yet very limited. This thesis contributes to the development of Capsaspora owczarzaki, a close unicellular relative of animals, as a model organism, by developing genetic tools for DNA transfection and culture synchronization tools to study the cell cycle. Our characterization of the Capsaspora cell cycle revealed that many genes important in the cell cycle of animal cells are also transcriptionally regulated in Capsaspora, including the main orthologs of animal cyclins and CDKs present in Capsaspora. Likewise, the development of genetic tools opens the door to new functional studies in this species, which will allow to understand the role of many genes related to multicellularity under the context of a unicellular species

Circadian rhythm and cell cycle:two synchronized processes

Circadian rhythms are biological processes found in most living organisms, displaying a roughly 24-hour period, responding primarily to light darkness cycles in an organism's environment. At the cellular level, the circa-24h rhythmicity is generated by a molecular clock based on a transcription-translation feedback network and consists of a cell-autonomous and self-sustained oscillator. In conditions where cells proliferate, the cell division cycle can be also considered as an oscillator. Since both processes run with similar periods in several mammalian cells, it is reasonable to expect that interactions between these two cycles may cause synchronization. Many studies reported evidences of interactions between the circadian and the cell cycles in different organisms. In particular, it appeared that in several systems, specific cell cycle phases occur in distinct temporal windows rather than being randomly distributed in time. These findings led to the concept of circadian gating of the cell cycle, through which the circadian clock can favor or forbid cell cycle transitions at specific circadian phases. However, it was also reported the converse, namely an effect of cell division on the circadian oscillator. Even though interactions between the circadian clock and the cell cycle have been identified in both directions, the dynamical consequences and the directionality of the coupling at the single-cell level were not extensively investigated. In order to better characterize the potential synchronization in mammalian cells, we estimated the mutual interactions between circadian clock and cell cycle in NIH3T3 mouse fibroblasts by the use of time-lapse fluorescent microscopy in combination with statistical analysis and mathematical modeling. NIH3T3 cells, harboring a fluorescent reporter under the control of the circadian RevErb-a gene promoter, were imaged for several days allowing the simultaneous detection of circadian oscillations and time of divisions. The analysis of thousands of circadian cycles in dividing cells indicated that both oscillators are synchronized, with cell divisions occurring about 5 h before the peak of the circadian RevErb-a reporter. We tested several perturbations such as different serum concentrations, different temperatures, treatment with pharmacological compounds and shRNA-mediated knockdown of circadian regulators. Surprisingly, this showed that circadian rhythm and cell cycle remain synchronized over the wide range of conditions probed. Our data showed that this synchronization state reflects an unexpected predominant influence of the cell cycle on the circadian oscillator, and did not support the leading hypothesis about a circadian gating of the cell cycle. The stochastic modeling of two interacting phase oscillators allowed us to identify the parameters of the coupling functions, revealing an acceleration of circadian phase after the division. The work presented in this thesis sheds light on the interaction between two fundamentally recurrent cellular processes in mammalian cells and provides a deeper understanding of the role of the circadian clock in proliferating cells and tissues. These findings might have significant implications for chronobiology and chronotherapeutics