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.

Wnt/β-catenin signaling and asymmetric cell division are essential to development and homeostasis in metazoans; these two mechanisms join into one in the Wnt/β-catenin Asymmetry (WβA) pathway in the nematode C. elegans. In WβA, nuclear asymmetry of two β-catenins, SYS-1 and WRM-1, is achieved by two parallel pathways that reduce SYS-1 and WRM-1 levels in the anterior daughter and increase their levels in the posterior daughter. While it is known that many conserved regulators of Wnt signaling are involved in WβA, how these components interact to achieve SYS-1 and WRM-1 asymmetry is not well understood. In this thesis, genetics, transgenics, and live-imaging are used to demonstrate how WβA regulates it’s multiple outputs. It is shown that APR-1/APC and PRY-1/Axin control asymmetric localization of both SYS-1 and WRM-1, and that Wnt signaling explicitly controls APR-1 regulation of either β-catenin via the kinase KIN-19/CKIα. Additionally, it is demonstrated that the Dishevelled proteins DSH-2 and MIG-5 are positive regulators of SYS-1, but negative regulators of WRM-1. Additionally, data from a screen designed to identify novel kinase regulators of Wnt signaling/asymmetric cell division is presented. Overall, this thesis takes current knowledge of conserved Wnt signaling component function and provides a compelling model of how those components are adapted to asymmetric cell division.

xiv, 81 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Protein-protein interaction networks translate environmental inputs into specific physiological outputs. The signaling proteins in these networks require regulatory mechanisms to ensure proper molecular function. Two common regulatory features of signaling proteins are autoinhibition and ultrasensitivity. Autoinhibition locks the protein in an inactive state through cis interactions with a regulatory module until it is activated by a specific input signal. Ultrasensitivity, defined as steep activation after a threshold, allows cells to convert graded inputs into more switch-like outputs and can lead to complex decision making behaviors such as bistability. Although these mechanisms are common features of signaling proteins, their molecular origins are poorly understood. I used the Drosophila Pins protein, a regulator of spindle positioning in neuroblast cells, as a model to study the molecular origin and function of autoinhibition and ultrasensitivity. Pins and its binding partners. Gαi and Mud, form a signaling pathway required for coordinating spindle positioning with cellular polarity in Drosophila neuroblasts. I found Pins switches from an autoinhibited to an activate state by modular allostery. Gαi binding to the third of three GoLoco (GL) domains allows Pins to interact with the microtubule binding protein Mud. The GL3 region is required for autoinhibitoon, as amino acids upstream and within GL3 constitute this regulatory behavior. This autoinhibitory module is conserved in LGN, the mammalian Pins orthologue. I also demonstrated that Gαi activation of Pins is ultrasensitive. A Pins protein containing inactivating point mutations to GLs l and 2 exhibits non-ultrasensitive (graded) activation. Ultrasensitivity is required for Pins function in vivo as the graded Pins mutant fails to robustly orient the mitotic spindle. I considered two models for the source of ultrasensitivity in this pathway: cooperative or "decoy" Gai binding. I found ultrasensitivity arises from a decoy mechanism in which GLs 1 and 2 compete with the activating GL3 for the input, Gai. These findings suggest that molecular ultrasensitivity can be generated without cooperativity. This decoy mechanism is relatively simple, suggesting ultrasensitive responses can be evolved by the inclusion of domain repeats, a common feature observed in signaling proteins. This dissertation includes previously published and unpublished co-authored material. / Committee in charge: Tom Stevens, Chairperson, Chemistry; Kenneth Prehoda, Member, Chemistry; Christopher Doe, Member, Biology; Peter von Hippel, Member, Chemistry; Karen Guillemin, Outside Member, Biology

Phenotypic heterogeneity in terms of cell size, morphology, and metabolic status, which are believed to help the population survive under stress conditions, is known in mycobacterial populations. Such population heterogeneity had been observed in in vitro cultures, TB patients, and in animal models. Our laboratory had earlier shown that about 20-30% of the 15% septating cells of Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium xenopi mid-log phase cultures divide by highly deviated asymmetric cell division (ACD), generating subpopulations of short cells and normal-sized/long cells. The remaining 70-80% of the septating cells divide by symmetric cell division (SCD) with 5-10% deviation of the division site constriction from the median. The proportion of short cells amounted to about 3-5% of the total population, while the remaining 97-98% of the population was constituted by normal-sized/long cells. This proportion of short cells has been found to be consistent and reproducible irrespective of culture media. Comparable proportion of short cells of tubercle bacilli has been found in the freshly diagnosed pulmonary tuberculosis patients’ sputum also. It indicated that such processes must be occurring in the tubercle bacillary population in the TB patients too and that the presence of short cells has some physiological relevance. Thus, ACD has been found to be one of the mechanisms that mycobacteria use to generate cell size heterogeneity in the population. However, there has not been any study on the mechanism that generate such subpopulation of short cells through ACD. Therefore, in the present study, we investigated the mechanism behind the ACD that generates short cells in the Mycobacterium smegmatis and Mycobacterium tuberculosis populations. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on all the different areas of research in bacterial physiology that are linked by the present study. These areas of research include bacterial cell division process per se, the proteins involved in bacterial cell division, cell division in general in mycobacteria, different modes of cell division in mycobacteria, generation of cell size heterogeneity through symmetric and asymmetric cell division in mycobacteria, cell-cell communication in bacterial systems, and the characteristics and physiological significance of diadenosine polyphosphates from bacteria to humans. The complete account of the research in these areas that are linked in the present study thus justifies the introduction to the results that are given in the ensuing chapters. The Chapter 2 forms the Materials and Methods used in the present study. Here a detailed description of the methods used for the cell division bioassay used to score for the proportion of cells undergoing symmetric and asymmetric divisions, the biochemical assays performed to find out the biochemical identity of the molecule, the isolation and fractionation methods for the ACD-IM from the concentrated culture supernatants (CS), and finally the mass spectrometric methods used for the elucidation of the structure of the molecule are given The Chapter 3 forms the first data chapter that presents results on the detection of the presence of the asymmetric cell division inducing molecule (ACD-IM) from the concentrated culture supernatant (CS) of Mycobacterium smegmatis and Mycobacterium tuberculosis, as inducing ACD in higher proportions of cells. Further, it shows that the levels of ACD-IM increase at late growth phases of 0.8 and 1.0 OD600 nm. The chapter is concluded with a discussion of the results. The Chapter 4 describes the biochemical analyses of the CS to find out the chemical nature of the ACD-IM. DNase I, snake venom phosphodiesterase (SVP), RNase A, lipase, and proteinase K were used to find whether exposure of CS of M. smegmatis and M. tuberculosis cells to these enzymes could abolish the ACD inducing activity of the molecule in the CS on the respective cells. These experiments showed that the ACD-IM was susceptible to DNase I and SVP, but not to RNase A, lipase, or proteinase K. However, proteinase K showed direct effect on the cells by decreasing the proportion of cells dividing by ACD, indicating the probable presence of the receptor to the molecule on the external cell surface. The data also shows the conservation of the molecule in M. smegmatis and M. tuberculosis by demonstrating that the CS of M. smegmatis could induce ACD in higher proportions of M. tuberculosis cells and vice versa. The structural identification of ACD-IM present in the concentrated CS of M. smegmatis and M. tuberculosis using LC-ESI-MS and MS-MS analyses as diadenosine hexaphosphate (Ap6A). The structure of the natural Ap6A molecule was confirmed using synthetic Ap6A molecule subjected to LC-ESI-MS and MS-MS analyses. Further, the ACD inducing activity of the synthetic Ap6A molecule, its susceptibility to DNase I and SVP, but not to RNase A, lipase and proteinase K were verified to establish that the structural, biochemical, and functional properties of the naturally occurring Ap6A in the CS of M. smegmatis and M. tuberculosis were identical to those of synthetic Ap6A. The chapter is concluded with a discussion of the results. The Chapter 5 presents the data on the genes involved in the degradation and synthesis of Ap6A in M. smegmatis and M. tuberculosis. The knockout of MSMEG_2936 gene of M. smegmatis resulted with significant increase in the ACD proportion. The quantitation of Ap6A in the CS obtained from this strain found significantly higher concentration than wild type. These results showed that MSMEG_2936 protein might catalyse the degradation of Ap6A in mycobacteria. The knockout of MSMEG_2932 gene of M. smegmatis resulted with significant reduction in the ACD proportion. The quantitation of Ap6A in the CS obtained from this strain found comparable to wild type. These results showed that MSMEG_2932 protein might not involve in the synthesis of Ap6A in mycobacteria. The chapter is concluded with a discussion of the results. Thus, the present study, for the first time, establishes the identity, structure, and function of Ap6A as the molecule that induces asymmetric cell division in mycobacteria.

<p>Robust regulation of spindle orientation is essential for driving asymmetric cell divisions (ACDs), which generate cellular diversity within a tissue. During the development of the multilayered mammalian epidermis, mitotic spindle orientation in the proliferative basal cells is crucial not only for dictating daughter cell fate but also for initiating stratification of the entire tissue. A conserved protein complex, including LGN, Nuclear mitotic apparatus (NuMA) and dynein/dynactin, plays a key role in establishing proper spindle orientation during ACDs. Two of these proteins, NuMA and dynein, interact directly with astral microtubules (MTs) that emanate from the mitotic spindle. While the contribution of these MT-binding interactions to spindle orientation remains unclear, these implicate apical NuMA and dynein as strong candidates for the machinery required to transduce pulling forces onto the spindle to drive perpendicular spindle orientation. </p><p> In my work, I first investigated the requirements for the cortical recruitment of NuMA and dynein, which had never been thoroughly addressed. I revealed that NuMA is required to recruit the dynein/dynactin complex to the cell cortex of cultured epidermal cells. In addition, I found that interaction with LGN is necessary but not sufficient for cortical NuMA recruitment. This led me to examine the role of additional NuMA-interacting proteins in spindle orientation. Notably, I identified a role for the 4.1 protein family in stabilizing NuMA's association with the cell cortex using a FRAP (fluorescence recovery after photobleaching)-based approach. I also showed that NuMA's spindle orientation activity is perturbed in the absence of 4.1 interactions. This effect was demonstrated in culture using both a cortical NuMA/spindle alignment assay as well as a cell stretch assay. Interestingly, I also noted a significant increase in cortical NuMA localization as cells enter anaphase. I found that inhibition of Cdk1 or mutation of a single residue on NuMA mimics this effect. I also revealed that this anaphase localization is independent of LGN and 4.1 interactions, thus revealing two independent mechanisms responsible for NuMA cortical recruitment at different stages of mitosis. </p><p> After gaining a deeper understanding of how NuMA is recruited and stabilized at the cell cortex, I then sought to investigate how cortical NuMA functions during spindle orientation. NuMA contains binding domains in its N- and C-termini that facilitate its interactions with the molecular motor dynein and MTs, respectively. In addition to its known role in recruiting dynein, I was interested in determining whether NuMA's ability to interact directly with MTs was critical for its function in spindle orientation. Surprisingly, I revealed that direct interactions between NuMA and MTs are required for spindle orientation in cultured keratinocytes. I also discovered that NuMA can specifically interact with MT ends and remain attached to depolymerizing MTs. To test the role of NuMA/MT interactions in vivo, I generated mice with an epidermal-specific in-frame deletion of the NuMA MT-binding domain. I determined that this deletion causes randomization of spindle orientation in vivo, resulting in defective epidermal differentiation and barrier formation, as well as neonatal lethality. In addition, conditional deletion of the NuMA MT-binding domain in adult mice results in severe hair growth defects. I found that NuMA is required for proper spindle positioning in hair follicle matrix cells and that differentiation of matrix-derived progeny is disrupted when NuMA is mutated, thus revealing an essential role for spindle orientation in hair morphogenesis. Finally, I discovered hyperproliferative regions in the interfollicular epidermis of these adult mutant mice, which is consistent with a loss of ACDs and perturbed differentiation. Based on these data, I propose a novel mechanism for force generation during spindle positioning whereby cortically-tethered NuMA plays a critical dynein-independent role in coupling MT depolymerization energy with cortical tethering to promote robust spindle orientation accuracy. </p><p> Taken together, my work highlights the complexity of NuMA localization and demonstrates the importance of NuMA cortical stability for productive force generation during spindle orientation. In addition, my findings validate the direct role of NuMA in spindle positioning and reveal that spindle orientation is used reiteratively in multiple distinct cell populations during epidermal morphogenesis and homeostasis.</p> / Dissertation

Doctor of Philosophy / Department of Biology / Michael A. Herman / All multicellular organisms consist of a variety of cell types. One of the mechanisms to generate this cellular diversity is the asymmetric cell division, which requires the establishment of cell polarity. In Caenorhabditis elegans hermaphrodites, 807 of 949 somatic cell divisions are asymmetric. The centrosome and the Wnt signaling pathway both have been shown to regulate cell polarity and subsequently asymmetric divisions in many model organisms. However, it is not clear whether the Wnt signaling pathway manipulates the cell polarity through specific cellular organelles, such as the centrosome. To address this question, we examined a centrosomal component, SPD-5, to see whether it cooperates with the Wnt signaling pathway to regulate certain asymmetric cell divisions. We showed that SPD-5, which was originally found to be critical for the embryonic development, also played a role during certain post-embryonic cell divisions in C. elegans. Specifically the asymmetric divisions of seam cells that required SPD-5 function were also known to be regulated by the Wnt signaling pathway. Thus the stem-cell like seam cell divisions could be an intriguing system to study the interaction of centrosomes and the Wnt pathway. We found that SPD-5 was required for a successful cell division, similar to other centrosomal components. This suggests that SPD-5 still functions as a centrosomal component during C. elegans post-embryonic development. It has been shown that establishment of seam cell polarity relies on the asymmetric localization of certain Wnt pathway components. Interestingly, we found that SPD-5 was required for the proper localization of several Wnt components in a way that was independent of a key MTOC (microtubule-organizing center) member γ-tubulin. In addition, SPD-5 genetically interacted with the Wnt pathway components APR-1/APC and POP-1/Tcf to regulate asymmetric divisions of seam cells. These data suggest that SPD-5 interacts with the Wnt signaling pathway in controlling the polarity of seam cells. Overall, our results suggest a novel role of SPD-5 in cooperating with the Wnt signaling pathway to regulate cell polarity and asymmetric cell division, in addition to its function as a centrosomal component.

Human β-catenin is a dual-functioned protein responsible for regulating cell-cell adhesion and gene transcription. To activate gene transcription, β-catenin must be shuttled into the nucleus where it interacts with various co-activators to activates gene transcription. Various studies have identified proteins that bind to specific amino acid sequences in β-catenin for proper gene transcription regulation. Compared to the single beta-catenin in most animals, C. elegans surprisingly contains four β-catenins. Though structurally similar, these beta-catenins became distinct during nematode evolution, resulting in four β-catenins that differ in functions. SYS-1 is one such β-catenin that loses its adhesion ability and is specialized in activating transcription of genes in the nucleus. Across different animals, β-catenin shares similar amino acid sequences and structure. SYS-1, while it shares the similar structure to other β-catenins, is the most divergent C. elegans beta-catenin when comparing amino acid sequences. In addition, while SYS-1 interacts with homologs of proteins that bind to and regulate human β-catenin, the binding sites of those proteins to SYS-1 is unknown. Here, we identify novel sites for beta-catenin’s gene transcription role within SYS-1 that greatly differed from human β-catenin. We also identify a novel mechanism for beta-catenin nuclear import, which is still largely unknown in any system, by identifying a candidate importer that associates with SYS-1 is required for SYS-1 dependent cell fate. In summary, though SYS-1 has a well-conserved function dictating cell fate in response to developmental signals, it has evolved novel regulatory, functional and localization mechanisms and therefore serves as a model for the plasticity nuclear importer that helps shuttle SYS-1 into the nucleus identified specific regions in SYS-1 that is involved in activating transcription which will result in cell fate changes.

<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

<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

Animal cells, as distinct as epithelia and migratory cells, have cell polarity that is defined by a common set of molecules. The Par complex polarizes the cortex of animal cells through the activity of atypical protein kinase C (aPKC). In this work, I aimed to determine the mechanism of aPKC substrate polarization and identify common characteristics of aPKC substrates that are polarized by phosphorylation. I found that several diverse Par-polarized proteins contain short highly basic and hydrophobic motifs that overlap with their aPKC phosphorylation sites. These Phospho-Regulated Basic and Hydrophobic (PRBH) motifs mediate plasma membrane localization by electrostatics-based phospholipid binding when unphosphorylated but are displaced into the cytoplasm when phosphorylated. To assess whether the Par complex polarizes other proteins by this mechanism, I developed an algorithm to identify potential PRBH motifs and score these linear motifs for basic and hydrophobic character, as well as the quality and number of aPKC phosphorylation sites. Using this algorithm, I identified numerous putative PRBH candidates in the fruit fly proteome and performed two screens of these candidates for Par-polarized proteins. The first screen focused on determining whether aPKC regulates cortical targeting of proteins that are reported to be polarized. This screen identified the Rho GAP crossveinless-c (cv-c) to be a novel aPKC substrate and found that aPKC is sufficient to polarize cv-c in a reconstituted polarity assay. The second screen characterized the localization of putative PRBH motif-containing proteins in vivo. This screen identified a previously uncharacterized protein, CG6454, to be basolateral in epithelia; however, ex vivo experiments found it to have a Ca2+-dependent and aPKC-independent membrane targeting mechanism. Overall this work identified a common mechanism for Par substrate polarization and used knowledge of this mechanism to identify a novel Par effector. This dissertation contains previously published coauthored materials as well as unpublished materials. / 2019-05-08