Both an intuitive observation and maybe the most mysterious process of biology, aging describes the progressive deterioration of cellular functions with time. Asymmetric cell divisions stand at the center of ability to reset age in offspring and for stem cells to self-renew. This requires the asymmetric segregation of age-determinants, many of which have been identified in the budding yeast Saccharomyces cerevisiae. We here use budding yeast to explore fundamental aspects underlying the asymmetric inheritance of mitochondria and the concurrent rejuvenation of daughter cells. We show that in addition to the preferential inheritance of high-functioning mitochondria to daughter cells, a distinct population of high-quality organelles must also be retained within the mother cell. We find that both physical retention and qualitative maintenance of a distinct mitochondrial population at the mother cell tip depends on Mitochondrial F-box protein (Mfb1p) and that MFB1-deletion leads to premature aging. Our findings outline a critical balance between the need for daughter cell rejuvenation and the requirement to conserve replicative potential within the mother cell. The particular mechanism by which Mfb1p functions further lead us to uncover a critical role of globally maintained cellular polarity in form of an axial budding pattern in lifespan regulation, the functional significance of which thus far remained essentially unexplored. We also find that the asymmetric localization of Mfb1p depends on potentially novel structures of the actin cytoskeleton and the loss of Mfb1p-polarization with age may accurately predict remaining cellular lifespan.

During their lifecycle, cells must produce forces which play important roles in several subcellular processes. Force-producing components are organized into macromolecular assemblies of proteins that are often dynamic, and are constructed or disassembled in response to various signals. The forces themselves may directly be involved in subcellular mechanics, or they may influence mechanosensing proteins either within or outside these structures. These proteins play different roles: they may ensure the stability of the force-producing structure, or they may send signals to a coupled process. The generation and sensing of subcellular forces is an active research topic, and this thesis focusses on the roles of these forces in two key areas: cell division and neurotransmitter release. The first part of the thesis deals with the effect of force on cell wall growth regulation during division in the fission yeast Schizosaccharomyces pombe, a cigar-shaped, unicellular organism. During cytokinesis, the last stage of cell division in which the cell physically divides into two, a tense cytokinetic ring anchored to the cellular membrane assembles and constricts, accompanied by the inward centripetal growth of new cell wall, called septum, in the wake of the inward-moving membrane. The contour of the septum hole maintains its circularity as it reduces in size—an indication of regulated growth. To characterize the cell wall growth process, we performed image analysis on contours of the leading edge of the septum obtained via fluorescence microscopy in the labs of our collaborators. We quantified the deviations from circularity using the edge roughness. The roughness was spatially correlated, suggestive of regulated growth. We hypothesized that the cell wall growers are mechanosensitive and respond to the force exerted by the ring. A mathematical model based on this hypothesis then showed that this leads to corrections of roughness in a curvature-dependent fashion. Thus, one of the roles of ring tension is to communicate with the mechanosensitive septum growth processes and coordinate growth to ensure the daughter cells have a functional cell wall. The second part of the thesis deals with how ring tension is produced and sustained, using experimentally measured ultrastructure of the cytokinetic ring itself. Recent super-resolution experiments have revealed that several key proteins of the fission yeast constricting ring are organized into membrane-anchored complexes called nodes. The force producing protein myosin-II in these nodes exerts pulling forces on polymeric actin filaments that are synthesized from polymerizers residing in the nodes. How these forces are marshalled to generate ring tension, and how such an organization maintains its stability is unclear. Using a mathematical model with coarse-grained representations of actin and myosin, we showed that such a node-based organization reproduces previously measured ring tension values. The model explains the origin of experimentally observed bidirectional motion of the nodes in the ring, and showed that turnover of the nodes rescues the ring from inherent contractile instabilities that would be expected when a force-producing structure is made up of small object that effectively attract one another. Finally, the third part of the thesis deals with the role of forces produced by SNARE proteins at synapses between two neurons during neurotransmission. A key step here is synaptic release, where inside a neuron, membrane-bound compartments called vesicles filled with neurotransmitter fuse with the membrane of the neuron forming a transient fusion pore, and release their contents to the outside of the cell. These neurotransmitter molecules are sensed by another neuron that is physically separate from the neuron in question and this neuron propagates the signal henceforth. Thus, regulation of neurotransmitter release is a key step in neurotransmission. A fusion machinery consisting of several proteins facilitates membrane fusion, and pore nucleation requires the formation of a SNARE protein complex in this machinery, whose role during pore dilation is unclear. Using electrophysiological measurements, our collaborators experimentally measured the statistics of the size of single fusion pores in vitro, and observed that average pore sizes increased with the number of SNARE proteins. Using mathematical modeling, we showed that this effect was due to an entropic crowding force that expands the pore and increases with the number of SNAREs, and counteracts the energy barrier to fusion pore expansion.

The immune system relies on the collaboration of heterogeneous cell types to respond to infection, develop immunological memory, and to maintain immunological tolerance. In response to infection, naïve lymphocytes must divide and give rise to differentiated effector cells while also regenerating a population of memory cells that may respond more efficiently to future infection. It has been demonstrated in B cells and T cells that the generation of these cell types may be accomplished simultaneously through asymmetric cell division. The second chapter of this thesis focuses on what factors may drive the divergence of cell fates in asymmetric cell division of CD8+ T cells. We demonstrate unequal expression of transcription factor TCF1 between cytokinetic sibling cells, which may be driven by unequal transduction of nutrient-sensitive PI3K/AKT/mTOR signaling. In chapter three, we extend our interrogation of asymmetric cell division in lymphocytes to the development of regulatory T cells, which are important for the maintenance of immunological self-tolerance. It has been shown that there is some overlap in the T cell receptor repertoires of Tregs and conventional CD4+ T cells. We propose that this overlap may be a result of an asymmetric cell division, giving rise to one Treg and one conventional CD4+ T cell. We demonstrate asymmetric Foxp3 expression between cytokinetic sibling cells found in the thymus as well as from an in vitro Treg induction model. We also show that in vitro upregulation of Foxp3, the major Treg-associated transcription factor, is inhibited by cell cycle inhibitors, further linking the act of cell fate divergence to a divisional event.

The asymmetric segregation of fate determinants is a conserved process by which differential cell fate can be acquired upon cell division. In this thesis we investigate how the asymmetric localisation of fate determinants is achieved in Drosophila neuroblasts (NBs, Neural Stem Cells). In particular we focus on the localisation of the fate determinant Miranda, which is segregated to the basal pole of the NB cell cortex in mitosis and carries a series of signalling molecules into one of the two daughter cells, promoting differentiation. The most widely accepted model for how Miranda becomes polarised at mitosis is based on its phosphorylation by the apically localised kinase, aPKC (atypical protein kinase C). This model proposes that aPKC localises to the apical cortex and phosphorylates Miranda, excluding it from the apical domain by phosphorylation of Miranda’s membrane binding motif. However, earlier work demonstrated that the acto-myosin cell cortex is essential for asymmetric Miranda localisation. Thus far these two models have not been successfully integrated. In this thesis we generated flies carrying fluorescent reporters for apical and basal polarity proteins and imaged their localisation live. We reveal that localisation appears to happen in two stages. Firstly, Miranda is localised uniformly to the plasma membrane, from where it is cleared by aPKC at the onset of prophase in an actin independent manner. After NEB, Miranda returns to the cell cortex, localising to a basal crescent in an acto-myosin dependent manner. Furthermore, the size of the basal domain to which Miranda localises appears to be under the control of Rho kinase, and linked to cell size asymmetry. Together these data suggest that in mitosis, Miranda localisation is under structural control. Therefore, we reveal that aPKC and Actin-myosin activity contribute to Miranda localisation at distinct time points in the cell cycle.

The centromere is a chromosomal locus required for accurate segregation of sister chromatids during cell division. They are maintained epigenetically in most eukaryotes, by incorporating the H3 variant CENP-A, and can, in rare instances, change location on the chromosome throughout generations. Centromeres are transcribed, and an active transcription chromatin signature is required for centromere maintenance. For this reason, insight into the nature of this so-called “centrochromatin” is essential for understanding a centromere’s place in the chromosome. The body of work contained in this thesis shows my efforts to understand the centromere in the context of chromatin, revealing interactions and new evidence for repressive chromatin domains with centromere activity, in two different vertebrate models: chicken DT40 cells and human HeLa cells. Centromeres are generally embedded within large domains of heterochromatic repetitive sequences in most eukaryotes, and mapping “centrochromatin” to high-resolution has proven difficult. However, chromosomes 5, 27 and Z of Gallus gallus are not located within repeat arrays, and are fully sequenced. CENP-A distribution on these centromeres has been mapped by ChIP-seq, and I have performed ChIP against selected histone modifications as part of a collaboration. While levels of heterochromatin are naturally quite low in these centromeres, I have shown that repressive polycomb chromatin instead is enriched in these non-repetitive centromeres, suggesting a replacement of one silenced chromatin state with another. Additional mapping of these centromeres showed a pattern of active chromatin marks distinct from that reported for human cells, which exhibited dynamic distribution throughout the cell cycle. Furthermore, conditionally generated neocentromeres in DT40 cells revealed that centrochromatin formation lowers, but does not eliminate, active transcription. To directly study the interaction of polycomb and heterochromatin with centrochromatin, I used a synthetic Human Artificial Chromosome (HAC), which allows for specific conditional targeting of chromatin modification enzymes, allowing manipulation of the underlying chromatin. Enrichment of the polycomb chromatin state on the HAC centromere, by EZH2 tethering, reduced its active transcriptional chromatin signature, but did not impair its actual transcription or mitotic activity. However, direct tethering of polycomb secondary silencing effector PRC1 caused centromere loss, and this effect was mimicked with homologous heterochromatin factors, indicating that centromeres can subsist within repressive chromatin domains, but are lost when direct repression is applied. To understand the contribution of the local repressive heterochromatin to centromere stability, I erased heterochromatin marks from the HAC centromere by tethering JMJD2D (an H3K9me3 demethylase): long-term (but not short-term) heterochromatin loss impaired CENP-A assembly, perturbed mitotic behaviour, and resulted in significant HAC mis-segregation. These results strongly suggest that local heterochromatin is essential to maintain normal CENP-A dynamics and centromere function. Together with previous observations, these data suggest that a repressive chromatin environment contributes to centromere stability, and that centromeres likely have natural mechanisms to maintain their transcriptional activity within such domains.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 140-160).

Thesis--Cornell University. / Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Preliminary studies have shown that manuka honey affects the cell cycle of MRSA by impeding cell division, but mode of action was unknown. Cell division depends on the formation of septa and cleavage of peptidoglycan at cytokinesis. This study investigated how manuka honey might alter the cell cycle of EMRSA-15. Physiological and chemical changes in the bacteria exposed to manuka honey were determined using time to kill studies, confocal and electron microscopy. Data indicated that honey had a bactericidal effect on MRSA, inhibiting the cell cycle cytokinesis. Increased septum formation was noted in honey treated cells by transmission electron microscopy. Cell division components including FtsZ and Endo-B-N-Acetylglucosaminidase were investigated using cell wall turbidity assays, zymography, immunofluorescence and immuno gold labelling. Manuka honey treated MRSA cells showed a marked reduction in hydrolase activity after 12 hours compared to untreated cells. The immunofluorescence indicated an initial increase in FtsZ production followed by a significant decrease by 24 hours. PCR of FtsZ showed a 10% increase in production after 1 and 4 hours. Localization by gold labelling gave inconclusive results. Immunofluorescence of Endo-B-N-Acetylglucosaminidase showed a decrease in the amount of enzyme over 24 hours and localization by gold labelling indicated altered distribution of this enzyme. PCR showed no significant difference in expression. 2-D electrophoresis showed a differing proteomic profile between control cells and those treated with honey, with a potential target protein being identified. Methylglyoxal (an antibacterial component of manuka honey) was investigated after a report named this as potentially the active component of manuka honey. Results showed it has an effect but is not wholly responsible for the effects induced by manuka honey. It was concluded that increased numbers of cells with septa were formed and alteration in production of proteins and enzymes resulted in MRSA cells exposed to bactericidal concentrations of manuka honey. The work was also carried out with artificial honey controls, indicating that effects seen were not due to sugar content within honey or methylglyoxal content.