Controlling fates of stem and progenitor cells is one of the central goals of regenerative medicine. However, conventional cell enumeration methods are unable to distinguish between the effects of cell death, proliferation, and differentiation through molecular interventions on the output of specific cell types. We have devised a strategy to simultaneously obtain proliferation and death rates in cultures of highly purified erythroid progenitors. The approach is based on combining cell-surface marker analysis, cell-division tracking and 7-amino-actinomycin-D staining to monitor cell death. A compartment model of cell proliferation was developed to evaluate cell generation-specific length of cell-division, rates of entry into division, and cell death, from the experimental cell-division tracking data obtained following stimulation with erythropoietin (EPO) and Stem cell factor (SCF). The results indicated that EPO and SCF, either as single factor or in combination, differentially affect the rates of differentiation, length of cell-division and rates of death.

The success of Mycobacterium tuberculosis as a pathogen is due to its remarkable ability to: (i). adapt to and survive inside activated macrophages under nonproliferating condition, (ii). put up drug resistance and (iii). enter into hypoxia-induced dormancy and remain in nonproliferating condition, be resistant to drugs, and get reactivated into proliferation when favourable conditions arise. Thus, regulation of cell division (arrest and resumption) is an obligatory event that is critical to the pathogen for the establishment of successful infection, latency and reactivation process in human host. Therefore, in order to understand and combat the successful survival strategy of the bacterium inside the host macrophages or in granuloma, a basic knowledge of the regulation of cell division in tubercle bacillus is essential. Bacterial cytokinetic protein FtsZ (a tubulin homologue) is the key regulatory molecule for cell division and its intracellular level is critical for initiation of cell division in bacteria. Therefore, in order to understand the regulation cell division by expression and maintenance of ftsZ mRNA and protein, we initiated studies on the transcriptional regulation of ftsZ gene in the slow growing pathogen, M. tuberculosis, and in the fast-growing saprophyte M. smegmatis. Identification of regions containing ftsZMt promoter activity In order to identify promoter activity-containing regions of ftsZ gene of M. tuberculosis H37Rv (ftsZMt) in vivo, different regions upstream of ftsZMt namely, the ftsQ-ftsZ intergenic region, the ftsQ open reading frame (ORF), and different regions of ftsQ ORF, were cloned in a gfp reporter plasmid and analyzed for gfp expression in M. smegmatis mc2155 cells. Flow cytometric analysis of exponentially grown M. smegmatis mc2155 cells containing these transcription fusion constructs revealed GFP expression in the cells harbouring ftsQ-ftsZ intergenic region (172 bp), the entire ftsQ ORF (945 bp), and 5’ 467 bp and 3’ 217 bp regions of ftsQ ORF. RT-PCR analyses on RNA from M. smegmatis mc2155 cell transformants carrying the entire ftsQ ORF-ftsQ-ftsZ intergenic region containing construct, as well as on total RNA from M. tuberculosis confirmed that the regions identified indeed elicit promoter activity. RT-PCR analysis on M. tuberculosis RNA as well as semi-quantitative RT-PCR analyses of gfp transcripts driven by cloned MtftsZ promoter regions in M. smegmatis cells showed that about 70% of the total promoter activity comes from ftsQ ORF and there is co-transcription of ftsQ-ftsZ genes. Multiple transcripts code for ftsZMt Primer extension analysis, using primers annealing at different positions in the ftsQ-ftsZ chromosomal region, on RNA from M. tuberculosis as well as from M. smegmatis transformants containing 1.117 kb ftsZMtpromoter region in a promoter probe vector, identified origin of six different transcripts (T1-T6) for the gene. Among them, five transcripts (T1, T2, T3, T4, and T6) were detected in M. tuberculosis cells at exponential phase of growth. T5 could be detected only in M. smegmatis transformants containing 1.117 kb ftsZMt promoter upstream of mycgfp2+ reporter gene. Transcript T1 and T2 originate in the ftsQ-ftsZ intergenic region, while T3, T4, and T6 start in the ftsQ ORF. Analysis of sequence in the –10 and –35 regions of the corresponding promoters for the individual transcripts identified high GC content of the regions, which is characteristic of promoters of M. tuberculosis. All of the individual promoter sequences were independently cloned in a promoter probe vector and confirmed that they are true promoters, active in M. smegmatis cells, and that the T1-T6 transcripts were not products of RNA processing. Differential expression from the multiple ftsZMt promoters In order to study the activity and regulation of ftsZMt promoters in M. tuberculosis cells, which is a slow grower and also asymptomatically survives as dormant bacteria for decades in human granuloma, a stably genome-integrated plasmid was required where activity of the promoters can be studied by means of stable and enhanced gfp expression. For that purpose, an L5-mycobacteriophage attP (attachment site)-specific integration proficient promoter probe vector, which contains a stable gfp gene (mycgfp2+) whose codon has been optimized for mycobacterial expression, was generated. Using the vector, all the six promoter regions (P1-P6) were studied in M. smegmatis and M. tuberculosis cells. Flow cytometric and semi-quantitative RT-PCR analyses showed that promoter P5 is unable to elicit activity in M. tuberculosis cells, unlike in M. smegmatis transformants. Semi-quantitative RT-PCR analyses showed that expression of P3 is only 10-20% of the total promoter activity. Promoters P1, P2, P4 and P6 showed 50-80% activity of the total promoter activity and their activity were comparable in M. smegmatis and M. tuberculosis. The presence of multiple promoters reflects the requirement to maintain high basal level of, or to differentially regulate a critical level of, FtsZ expression during different pathogenic stages of tubercle bacilli. In order to investigate the role of multiple promoters, we verified the levels of expression of the five transcripts from the five ftsZ promoters in M. tuberculosis cells under conditions of growth inside mouse macrophage cell line and also under various stress conditions mimicking those that exist in the granuloma environment, like conditions of nonreplicating persistence, gradual nutrient depletion stress, oxidative stress, surface tension stress, acidic stress, heat shock, DNA damaging conditions and osmotic stress. For this purpose, individual promoter regions were cloned into a stably inheritable gfp reporter plasmid vector, and into an L5 mycobacteriophage attP (attachment site)-specific integration-proficient variant of the same vector, for the expression of the promoters from the chromosomal locus in M. smegmatis and M. tuberculosis cells. Quantitative primer extension analyses, semiquantitative RT-PCR analyses on RNA from M. tuberculosis cells grown under these different conditions, and quantitative GFP fluorescence analyses in these cells showed differential activation of the five promoters under different conditions of growth. Under hypoxic and nutrient-depleted stationary phase of growth, two new promoters, Tdor and Ts, in the ftsQ ORF were identified, and these promoters showed maximal activity only under those specific conditions of growth. None of the ftsZ promoters were found to be responsive to stringent response mediated by overexpression of M. tuberculosis RelA. None of the promoters were also found to be responsive of overexpression of heat-shock sigma factor SigH in M. tuberculosis, implicating new pathway of regulation of ftsZ promoters. Multiple promoters driving expression of ftsZ gene of M. smegmatis Similar studies, which were carried out on the identification, structural and functional characterization, regulation of the promoters of cell division gene ftsZ in the fast growing saprophyte M. smegmatis cells, showed the presence of four ftsZ promoters, three of which originates from the 249 bp ftsQ-ftsZ intergenic region and one from the ftsQ ORF. RT-PCR analysis showed that both ftsQ and ftsZ are co-transcribed. Cloning and expression analysis of the individual promoters mapped by primer extension in a GFP based reporter plasmid showed that all the four putative regions are true promoters. Quantitative primer extension on RNA from a synchronously grown culture identified P2 promoter to be responsive to either initiation of cell division or stress, although expression of P1, P3, and P4 did not vary with respect to synchronous division. Quantitative primer extension analysis and semi-quantitative RT-PCR analysis on the RNA from M. smegmatis cells showed that under various stress conditions, P2 activity goes down significantly. Under nutrient depleted stationary phase and hypoxic nonreplicating persistence stage-2, the levels of P2 and P3 activity could hardly be detected, whereas, expression from P1 and P4 goes down only in hypoxia. Level of total ftsZ mRNA remains almost the same under various stress conditions, although upon hypoxia and stationary phase the level goes down almost two fold. Thus, in fast growing M. smegmatis too, multiple ftsZ promoters are differentially regulated under various stress conditions and a critical level of ftsZ mRNA is maintained. Taken together, the study of ftsZ promoters of a slow-growing pathogenic mycobacterium and a fast growing non-pathogenic mycobacterium indicate that differential expression of the multiple promoters, along with conditional activation of stage specific promoters like Pdor or Ps, is one of the mechanisms through which the bacilli probably maintain required levels of FtsZ protein that are crucial for the cell survival, probably through cytoskeletal maintenance, and cell division.

The development of multicellular organisms is dependent on the tight coordination between tissue growth and morphogenesis. The stereotypical orientation of cell divisions has been proposed to be a fundamental mechanism by which proliferating and growing tissues take shape. However, the actual contribution of stereotypical cell division orientation (SDO) to tissue morphogenesis is unclear. In zebrafish, cell divisions with stereotypical orientation have been implicated in both body axis elongation and neural rod formation, although there is little direct evidence for a critical function of SDO in either of these processes. Making use of extended time-lapse, multi-photon microscopy and a careful three-dimensional analysis of cell division orientation, we show that SDO is required for neural rod midline formation during neurulation, but dispensable for body axis elongation during gastrulation. Our data indicate that SDO during both gastrulation and neurulation is dependent on the non-canonical Wnt receptor Frizzled 7 (Fz7), and that interfering with cell division orientation leads to severe defects in neural rod midline formation, but not body axis elongation. These findings suggest a novel function for Fz7 controlled cell division orientation in neural rod midline formation during neurulation. They also shed new light on the field of cell division orientation by uncoupling it from tissue elongation.

The bacterial cell wall, composed of peptidoglycan (PG), is an essential component of the cell envelope. This macromolecular structure fortifies the cell membrane, determines cell shape, and helps prevent osmotic lysis. The synthesis and remodeling/recycling of this polymer is mediated by PG synthases and hydrolases, respectively. Proper control of the PG hydrolases is particularly important since misregulation of these enzymes can lead to lethal breaches in the cell wall. Surprisingly, however, the precise molecular mechanisms governing the activities of these enzymes remain poorly understood. To help understand how PG hydrolases are regulated, I examined how their activity is controlled during cytokinesis in Escherichia coli. One important class of PG hydrolases necessary for cell division is the LytC-type amidases (AmiA, AmiB and AmiC). These enzymes require activation by the LytM factors EnvC and NlpD. My work focused on elucidating the mechanism by which the LytM factors activate the amidases. Using a genetic enrichment strategy, I isolated amiB misregulation mutants. Interestingly, the mutations mapped to a region of AmiB found only in cell separation amidases. Structural analysis of an AmiB ortholog indicates that this region corresponds to an alpha-helical domain that appears to occlude the active site. Thus, activation of the amidases by the LytM factors likely occurs via a conformational change that displaces the regulatory helix from the active site. In addition to amidase regulation, I also investigated how the LytM activators are recruited to, and regulated at the site of division. Using genetic and biochemical approaches, I showed that EnvC is directly recruited to the division site by FtsEX, an ATP-binding transporter- like complex. Interestingly, ATPase-defective FtsEX derivatives can still recruit EnvC to the divisome, but fail to promote cytokinesis. These results support a model where conformational changes induced by the ATPase activity of FtsE are directly and specifically transmitted to the amidases via FtsX and EnvC. This model is attractive because it provides a mechanism for converting the potentially dangerous activity of septal PG splitting into a discrete process which can be cycled on and off in coordination with the division process.

Mathematical modeling has been important in the study of biology. Two main challenges with modeling biological problems are the lack of quantitative data and the complexity of biological problems. With the invention of new techniques, like single molecule transcript counting, very quantitative gene expression measurements at the level of single transcript in individual cells can now be obtained. Biological systems are very complex, involving many reactions and players with unknown reaction rates. To reduce the complexity, scientists have often proposed simplified phenomenological models that are tractable and capture the main essence of the biological systems. These simplified models allow scientists to describe the behavior of biological systems with a few meaningful parameters. In this thesis, by integrating quantitative single-cell measurements with phenomenological modeling, we study the (1) roles of Wnt ligands and receptors in sensing and amplification in Caenorhabditis elegans’ P cells and (2) regulation of rDNA transcription in Saccharomyces cerevisiae. The initiation of cell polarity consists of two sequential processes: an external gradient is first sensed and then the resulting signal is amplified by intracellular signaling. It is challenging to determine the role of proteins towards sensing and amplification as these two processes are intertwined. We integrated quantitative single-cell measurements with phenomenological modeling to determine the roles of Wnt ligands and receptors in sensing and amplification in the P cells of Caenorhabditis elegans. By systematically exploring how P cell polarity is altered in Wnt ligand and receptor mutants, we inferred that ligands predominantly affect sensing, whereas receptors are needed for both sensing and amplification. Most eukaryotes contain many tandem repeats of ribosomal RNA genes of which only a subset is transcribed at any given time. Current biochemical methods allow for the determination of the fraction of transcribing repeats (ON) versus nontranscribing repeats (OFF) but do not provide any dynamical information. By using the single molecule transcript counting technique complemented with theoretical modeling, we determine the rate of switching from OFF to ON (activation rate) and the average number of RNA molecules produced during each transcriptional burst (burst size). We explore how these two variables change in mutants and different growth conditions.

DNA is packaged into the nucleus of a mammalian cell as a nucleoprotein complex called chromatin. Changes in chromatin structure occur during processes that are critical to an understanding of mammalian cell biology such as cell division. Existing fixed-cell techniques have provided insight into chromatin organization in the mammalian nucleus. In addition, fluorescence microscopy techniques have allowed us to study changes in chromatin structure in living cells. However, most of these fluorescence techniques cannot be used for tissue imaging or long-term imaging due to photobleaching. In this thesis, we demonstrate that a label-free technique called Stimulated Raman Scattering (SRS) microscopy can be used to solve these problems and study chromatin structure in living mammalian cells both in culture and in tissue. SRS is a vibrational microscopy technique that takes advantage of intrinsic contrast arising from specific chemical bonds in a molecule. Nucleic acids have specifc phosphate and CH vibrations that can be used to determine their cellular distributions. Imaging at specific phosphate peaks using fingerprint SRS microscopy allows the detection of polytene chromosomes in Drosophila salivary gland cells and condensed chromatin in metaphase mammalian cells. In addition, we develop a technique called multicolor SRS microscopy, in which we image at several wavelengths across the CH vibrational band, and then use linear combination to simultaneously determine the nucleic acid, lipid and protein distributions in living mammalian cells. This technique achieves greater contrast than imaging at the phosphate vibrational peak due to the stronger SRS signal in the high wavenumber CH band and so allows us to determine chromatin structure in interphase mammalian cells. This technique also allows long-term imaging of living mammalian cells and the imaging of tissue such as mouse skin. The technique is used to monitor mammalian cell division in culture and paves the way for similar studies in living tissue. This technique will provide insight into cell division, differentiation and apoptosis during development and in disease models such as cancer.

Eukaryotic gene expression requires hundreds of proteins and several RNA factors to facilitate nuclear RNA processing. These RNA processing events include RNA transcription, pre-mRNA splicing, pre-ribosomal RNA (pre-rRNA) processing and trafficking of RNA to its proper location in the cell. As we learn more about the molecular details of the factors governing these highly coordinated processes it is becoming increasingly clear that a subset of factors participate in multiple RNA processing pathways to ensure faithful gene expression. Our work completes the characterization of the Abelson pre-mRNA splicing mutants. We have discovered that the prp27-1 splicing mutant is a severe loss of function allele of RAT1, an essential 5’→3’ exoribonuclease. Several alleles of RAT1 have been previously isolated with each conferring an array of phenotypes thus making the elucidation of its essential in vivo function difficult. We set out to determine how mutations within a specific region determines the RNA processing pathway in which Rat1p has been implicated to function within. In our analysis of Rat1p function we discovered the prp27-1 allele exhibits novel 3’ end processing defects never reported in previous rat1 mutants. We performed mutational analysis to examine the coupling of 5’ and 3’ exonucleolytic activities in nuclear RNA processing events. Through our study we have discovered a means by which the cell coordinately regulates the nuclear RNA degradation complexes to ensure efficient processing of pre-RNAs for the faithful execution of eukaryotic gene expression. Additionally, we offer evidence in support of role for Rat1p in promoting mitotic events in vivo. / text

<p>Stem cells are defined by their ability to make more stem cells, a property known as self-renewal and their ability to generate cells that enter differentiation. One mechanism by which fate decisions can be effectively controlled in stem cells is through asymmetric division and the correct partitioning and inheritance of cell fate determinants. While hematopoietic stem cells have the capacity to divide through asymmetric division, the molecular machinery that regulates this process is unknown and whether its activity is required in vivo remains unclear. Here we show that Lis1, a dynein-binding protein and regulator of asymmetric division, is critically required for blood development and for hematopoietic stem cell renewal in fetal and adult life. In particular, conditional deletion of Lis1 led to a severe bloodless phenotype and embryonic lethality in vivo. In both fetal and adult mice, loss of Lis1 led to a failure of normal self-renewal, which included impaired colony-forming ability in vitro and defects in long-term reconstitution ability following transplantation. As a possible mechanism, we find that the absence of Lis1 in hematopoietic cells, in part, accelerates differentiation linked to the incorrect inheritance of cell fate determinants. Furthermore, using a live cell imaging strategy, we find that the incorrect inheritance of cell fate determinants observed following the loss of Lis1 is due defects in spindle positioning and orientation. Finally, using two animal models of undifferentiated myeloid leukemia, we show that Lis1 is critical for the aberrant cell growth that occurs in cancer. Deletion of Lis1 both at the early and late stages of myeloid leukemia blocked its propagation in vivo and led to a marked improvement in survival. Together, these data identify Lis1 and the directed control of asymmetric division as key regulators of normal and malignant hematopoietic development.</p> / Dissertation

No description available.

<p>During development, oriented cell divisions are crucial for correctly organizing and shaping a tissue. Mitotic spindle orientation can be coupled with cell fate decisions to provide cellular diversity through asymmetric cell divisions (ACDs), in which the division of a progenitor cell results in two daughters with different cell fates. Proper tissue morphogenesis relies on the coupling of these two phenomena being highly regulated. The development of the mouse epidermis provides a powerful system in which to study the many levels that regulate ACDs. Within the basal layer of the epidermis, both symmetric and asymmetric cell divisions occur. While symmetric divisions allow for an increase in surface area and progenitor cell number, asymmetric divisions drive the stratification of the epidermis, directly contributing additional cell layers (Lechler and Fuchs 2005; Poulson and Lechler 2010; Williams, Beronja et al. 2011). </p><p>Utilizing genetic lineage tracing to label individual basal cells I show that individual basal cells can undergo both symmetric and asymmetric divisions. Therefore, the balance of symmetric:asymmetric divisions is provided by the sum of individual cells' choices. In addition, I define two control points for determining a cell's mode of division. First is the expression of the mInscuteable gene, which is sufficient to drive ACDs. However, there is robust control of division orientation as excessive ACDs are prevented by a change in the localization of NuMA, an effector of spindle orientation. Finally, I show that p63, a transcriptional regulator of stratification, does not control either of these processes, rather it controls ACD indirectly by promoting cell polarity. </p><p>Given the robust control on NuMA localization to prevent excess ACDs, I sought to determine how targeting of NuMA to the cortex is regulated. First, I determined which regions within the protein were necessary and sufficient for cortical localization. NuMA is a large coiled- coil protein that binds many factors important for ACDs, which include but are not limited to: microtubules, 4.1, and LGN. Interestingly, while the LGN binding domain was necessary, it was not sufficient for proper NuMA localization at the cortex. However, a fragment of NuMA containing both the 4.1 and LGN binding domains was able to localize to the cortex. Additionally, the NuMA-binding domain of 4.1 was able to specifically disrupt NuMA localization at the cortex. These data suggested an important role for a NuMA-4.1 interaction at the cortex. While the 4.1 binding domain was not necessary for the cortical localization of NuMA, it was important for the overall stability of NuMA at the cortex. I hypothesize that 4.1 acts to anchor/stabilize NuMA at the cortex to provide resistance against pulling forces on the mitotic spindle to ensure proper spindle orientation.</p><p>Finally, to determine if post-translational modifications of NuMA could regulate its localization I tested the importance of a conserved Cdk-1 phosphorylation site. Interestingly, a non-phosphorylatable form of NuMA localized predominately to the cortex while the phosphomimetic protein localized strongly to spindle poles. In agreement with these data, use of a CDK-1 inhibitor was able to enhance the cortical localization of NuMA. Unexpectedly, the non-phosphorylatable form of NuMA did not require LGN to localize to the cortex. Additionally, restoration of cortical localization of the phosphomimetic form of NuMA was accomplished by the overexpression of either LGN or 4.1. Thus, phosphorylation of NuMA may alter its overall affinity for the cortex. </p><p>Overall, my studies highlight two important regulatory mechanisms controlling asymmetric cell division in the epidermis. Additionally, I show a novel role for the interaction between NuMA and 4.1 in providing stability at the cortex. This will ultimately provide a framework for analysis of how external cues control the important choice between asymmetric and symmetric cell divisions.</p> / Dissertation