Zellwachstum und Zellteilung stellen zwei miteinander verknüpfte Prozesse dar, die dennoch grundsätzlich voneinander zu unterscheiden sind. Die Wiederaufnahme der Proliferation von neuralen Vorläuferzellen (Neuroblasten) im Zentralhirn von Drosophila nach der spät-embryonalen Ruhephase erfordert zunächst Zellwachstum. Der Erhalt der regulären Zellgröße ist eine wichtige Voraussetzung für die kontinuierliche Proliferation der Neuroblasten über die gesamte larvale Entwicklungsphase. Neben extrinsischen Ernährungssignalen ist für das Zellwachstum eine kontinuierliche Versorgung mit funktionellen Ribosomen notwendig, damit die Proteinsynthese aufrechterhalten werden kann. Mutationen im mushroom body miniature (mbm) Gen wurden über einen genetischen Screen nach strukturellen Gehirnmutanten identifiziert. Der Schwerpunkt dieser Arbeit lag in der funktionellen Charakterisierung des Mbm Proteins als neues nukleoläres Protein und damit seiner möglichen Beteiligung in der Ribosomenbiogenese. Der Vergleich der relativen Expressionslevel von Mbm und anderen nuklearen Proteinen in verschiedenen Zelltypen zeigte eine verstärkte Expression von Mbm in der fibrillären Komponente des Nukleolus von Neuroblasten. Diese Beobachtung legte die Vermutung nahe, dass in Neuroblasten neben generell benötigten Faktoren der Ribosomenbiogenese auch Zelltyp-spezifische Faktoren existieren. Mutationen in mbm verursachen Proliferationsdefekte von Neuroblasten, wirken sich jedoch nicht auf deren Zellpolarität, die Orientierung der mitotischen Spindel oder die Asymmetrie der Zellteilung aus. Stattdessen wurde eine Reduktion der Zellgröße beobachtet, was im Einklang mit einer Beeinträchtigung der Ribosomenbiogenese steht. Insbesondere führt der Verlust der Mbm Funktion zu einer Retention der kleinen ribosomalen Untereinheit im Nukleolus, was eine verminderte Proteinsynthese zur Folge hat. Interessanterweise wurden Störungen der Ribosomenbiogenese nur in den Neuroblasten beobachtet. Zudem ist Mbm offensichtlich nicht erforderlich, um Wachstum oder die Proliferation von Zellen der Flügelimginalscheibe und S2-Zellen zu steuern, was wiederum dafür spricht, dass Mbm eine Neuroblasten-spezifische Funktion erfüllt. Darüber hinaus wurden die transkriptionelle Regulation des mbm-Gens und die funktionelle Bedeutung von posttranslationalen Modifikationen analysiert. Mbm Transkription wird von dMyc reguliert. Ein gemeinsames Merkmal von dMyc Zielgenen ist das Vorhandensein einer konservierten „E-Box“-Sequenz in deren Promotorregionen. In der Umgebung der mbm-Transkriptionsstartstelle befinden sich zwei „E-Box“-Motive. Mit Hilfe von Genreporteranalysen konnte nachgewiesen werden, dass nur eine von ihnen die dMyc-abhängige Transkription vermittelt. Die dMyc-abhängige Expression von Mbm konnte auch in Neuroblasten verifiziert werden. Auf posttranslationaler Ebene wird Mbm durch die Proteinkinase CK2 phosphoryliert. In der C-terminalen Hälfte des Mbm Proteins wurden in zwei Clustern mit einer Abfolge von sauren Aminosäuren sechs Serin- und Threoninreste als CK2- Phosphorylierungsstellen identifiziert. Eine Mutationsanalyse dieser Stellen bestätigte deren Bedeutung für die Mbm Funktion in vivo. Weiterhin ergaben sich Evidenzen, dass die Mbm-Lokalisierung durch die CK2-vermittelte Phosphorylierung gesteuert wird. Obwohl die genaue molekulare Funktion von Mbm in der Ribosomenbiogenese noch im Unklaren ist, unterstreichen die Ergebnisse dieser Studie die besondere Rolle von Mbm in der Ribosomenbiogenese von Neuroblasten um Zellwachstum und Proliferation zu regulieren. / Cell growth and cell division are two interconnected yet distinct processes. Initiation of proliferation of central brain progenitor cells (neuroblasts) after the late embryonic quiescence stage requires cell growth, and maintenance of proper cell size is an important prerequisite for continuous larval neuroblast proliferation. Beside extrinsic nutrition signals, cell growth requires constant supply with functional ribosomes to maintain protein synthesis. Mutations in the mushroom body miniature (mbm) gene were previously identified in a screen for structural brain mutants. This study focused on the function of the Mbm protein as a new nucleolar protein, which is the site of ribosome biogenesis. The comparison of the relative expression levels of Mbm and other nucleolar proteins in different cell types showed a pronounced expression of Mbm in neuroblasts, particularly in the fibrillar component of the nucleolus, suggesting that in addition to nucleolar components generally required for ribosome biogenesis, more neuroblast specific nucleolar factors exist. Mutations in mbm cause neuroblast proliferation defects but do not interfere with cell polarity, spindle orientation or asymmetry of cell division of neuroblasts. Instead a reduction in cell size was observed, which correlates with an impairment of ribosome biogenesis. In particular, loss of Mbm leads to the retention of the small ribosomal subunit in the nucleolus resulting in decreased protein synthesis. Interestingly, the defect in ribosome biogenesis was only observed in neuroblasts. Moreover, Mbm is apparently not required for cell size and proliferation control in wing imaginal disc and S2 cells supporting the idea of a neuroblast-specific function of Mbm. Furthermore, the transcriptional regulation of the mbm gene and the functional relevance of posttranslational modifications were analyzed. Mbm is a transcriptional target of dMyc. A common feature of dMyc target genes is the presence of a conserved E-box sequence in their promoter regions. Two E-box motifs are found in the vicinity of the transcriptional start site of mbm. Gene reporter assays verified that only one of them mediates dMyc-dependent transcription. Complementary studies in flies showed that removal of dMyc function in neuroblasts resulted in reduced Mbm expression levels. At the posttranslational level, Mbm becomes phosphorylated by protein kinase CK2. Six serine and threonine residues located in two acidic amino acid rich clusters in the C-terminal half of the Mbm protein were identified as CK2 phosphorylation sites. Mutational analysis of these sites verified their importance for Mbm function in vivo and indicated that Mbm localization is controlled by CK2-mediated phosphorylation. Although the molecular function of Mbm in ribosome biogenesis remains to be determined, the results of this study emphasize the specific role of Mbm in neuroblast ribosome biogenesis to control cell growth and proliferation.

Cells divide to create two daughter cells through cytokinesis. Daughter cells of different sizes are created by shifting the position of the cleavage furrow. The cleavage furrow forms at the position of the metaphase plate so in asymmetric cytokinesis the spindle is shifted towards one pole. Unlike most systems, Drosophila neuroblasts have a centrally localized metaphase plate but divide asymmetrically. Drosophila neuroblasts divide asymmetrically due to the presence of a polarized myosin domain at the basal pole during mitosis. I investigated the mechanism by which the basal myosin domain produces asymmetric cytokinesis and the pathway regulating this domain. We tested several mechanisms by which the basal myosin domain could lead to asymmetric cytokinesis. Based on surface area and volume measurements, I demonstrated that asymmetric addition of new membrane is not involved. I determined that neuroblasts exhibit asymmetric cortical extension during anaphase with the apical pole extending 2-3 times more than the basal pole. Mutants that lose basal myosin extend equally at both poles supporting this model. Mutants that retain apical myosin exhibited symmetric cortical extension but still divided asymmetrically, demonstrating that asymmetric cortical extension is not required for asymmetric cytokinesis. Observations of the mitotic spindle show that the cleavage furrow forms at a position biased towards the basal pole when compared to the position of the metaphase plate even though this position is still equidistant between the centrosomes. I observed that midzone components shift basally in a basal domain dependent manner suggesting that contraction of the basal domain leads to new microtubule-cortex interactions at a position away from the spindle midzone. I demonstrated that the basal domain is regulated by the heterotrimeric G protein, Gβ13F, which is activated by Pins. In Gβ mutants, the localization of all basal components (myosin, anillin, and pavarotti) is lost and the cells divide symmetrically. Although the basal domain is contiguous with equatorial myosin, it is not regulated by the same pathway and photobleaching experiments indicate that they exhibit different behaviors during anaphase suggesting a difference in temporal regulation. This dissertation includes previously published coauthored material.

The Drosophila neuroblast undergoes repeated asymmetric cell divisions that produce one daughter cell that assumes a neuronal fate and another that remains a neuroblast. During mitosis, the neuroblast polarizes the conserved Par polarity complex to the apical cortex, which is responsible for segregating fate determinants to the basal cell cortex. Polarity is accompanied by orientation of the mitotic spindle through the proteins Pins, Mud, and Dlg to ensure that the cleavage furrow properly segregates the fate determinants. The adaptor protein Inscuteable coordinates these two pathways. In my work, I have addressed how asymmetrically dividing cells are dynamically polarized during the cell cycle and how the resulting polarity is coupled to spindle position. To address how neuroblast polarity is dynamically controlled, I identified the protein Inscuteable as a continuously polarized cue for Par complex localization during mitosis. Inscuteable and Bazooka, a member of the Par complex, interact directly and form a complex that is regulated by the mitotic kinase Aurora A. Regulating this interaction allows for cell-cycle dependent establishment of polarity and for the subsequent loss of polarity after the cell divides. To investigate how Par complex directed polarity is connected to spindle position, I investigated the effect of Inscuteable binding on the spindle orientation ability of the protein Pins. When bound to Inscuteable, Pins' spindle orientation activity becomes repressed. Inscuteable competes with Mud for Pins binding and represses the Gai-Pins-Mud signaling pathway. Function of the parallel Pins-Dlg pathway remains unaffected. This repression behavior may allow differential timing of spindle attachment (through Dlg) and spindle shortening (through Mud) pathways that ensures correct alignment of the mitotic spindle. I was able to model the spindle orientation behavior of Pins using a synthetic protein containing activation sites that have different affinities for the activator. Changing the number and affinities of these activation sites leads to different response profiles that mimic the ultrasensitive behavior of Pins using a non-cooperative mechanism. Together, these regulatory mechanisms cooperate to allow for spatial and temporal control of polarity and for physical connection of polarity to the mitotic spindle. This dissertation includes previously published and unpublished co-authored material.

The large numbers of neurons that comprise the adult brain display an immense diversity. Repeated divisions of a relatively small pool of neural stem cells generate this neuronal diversity during development. To increase progress towards medical treatments for neurodegenerative diseases, it is of interest to understand both how neural stem cells generate the assortment of neurons and how these neurons come together to form a functional brain. Brain assembly occurs sequentially across time with early events laying the foundation for later events. Drosophila neural stem cells, neuroblasts (NBs), are an excellent model for investigating how neural diversity is generated and what roles early and late born neurons have in shaping the stereotypical adult brain structure. Generation of neural diversity, begins with specifying the diverse population of stem cells, called spatial patterning, and continues with diversifying neurons made from the diverse stem cells, called temporal patterning. Drosophila NBs exhibit both spatial and temporal patterning. Drosophila NBs have three types of division modes: type 0, type I and type II. Type II NBs expand the number of neurons made with progeny that exhibit a transit-amplifying division pattern, similar to that of mammalian outer subventricular zone (OSVZ) progenitors. Additionally, type II NBs exhibit temporal patterning across both the NB and their progeny to generate a large diversity of neurons that populate a conserved region of the brain responsible for many sensory and motor functions, called the central complex. Type II NBs have only been identified and studied during later stages in development, with nothing known about their origin or early divisions. In this dissertation, I describe the early lineages of the type II NBs within the Drosophila embryo. I show that type II NBs and lineages originate early in development, exhibit temporal patterning across both the NB and transit-amplifying progeny, and produce neurons that survive into the adult brain to innervate and potentially serve as a foundation within the adult central complex. Additionally, I explain how live imaging of the developing Drosophila brain can answer questions not easily addressed through other methods.

The Par complex protein, atypical protein kinase C (aPKC), plays an instrumental role in diverse cell polarities. aPKC is able to restrict substrate localization through a phosphorylation-induced cortical exclusion mechanism, allowing for the generation of molecularly distinct cortical domains. Thus, controlling the localization of aPKC is central to Par-mediated polarity but the mechanism by which aPKC is polarized remains poorly understood. In this dissertation I investigated the restriction of aPKC to the apical cortex of Drosophila neural stem cells, neuroblasts, as these cells dynamically polarize aPKC through repeated asymmetric cell divisions. The polarity created through aPKC phosphorylation must be tightly regulated in order to ensure proper balance between self-renewal and differentiation. To begin, I investigated whether or not aPKC’s so called ‘maturation’ by PDK1 phosphorylation is required for aPKC activity and localization. We found that aPKC’s phosphorylation by PDK1 is required for both polarity and full activity. An aPKC containing an unphosphorylatable activation loop mutation localizes symmetrically around the cortex in a manner independent of its binding partner, Par-6, suggesting that aPKC could interact with the cortex by an unknown mechanism. To investigate how aPKC is able to localize to the cortex independent of Par-6, I used an in vivo structure function analysis of domains within aPKC, accompanied by biochemical approaches. I identified a necessity for the aPKC C1 domain for binding to the neuroblast cortex. This interaction is mediated by negatively charged phospholipids. Neither aPKC interaction, with phospholipids or Par-6, is sufficient to restrict aPKC to the apical cortex. Thus, aPKC polarization utilizes a dual interaction mechanism that takes advantage of both protein-lipid and protein-protein interactions, and proper control of each of these signals is required to prevent neuroblast division defects. One interaction, mediated by the C1, is a general cortical targeting mechanism, whereas the other specifies polarization mediated by Par complex interactions. We conclude that a conformational change induced by these interactions activates aPKC’s catalytic activity, thereby coupling localization and activity. This dissertation includes unpublished co-authored material.

Development of the central nervous system in Drosophila melanogaster relies on neural stem cells called neuroblasts. Neuroblasts divide asymmetrically to give rise to a new neuroblast as well as a small daughter cell which eventually generates neurons or glia cells. Between each division, neuroblasts have to re-grow to be able to divide again. In previous studies, it was shown that neuroblast proliferation, cell size and the number of progeny cells is negatively affected in larvae carrying a P-element induced disruption of the gene mushroom body miniature (mbm). This mbm null mutation called mbmSH1819 is homozygously lethal during pupation. It was furthermore shown that the nucleolar protein Mbm plays a role in the processing of ribosomal RNA (rRNA) as well as the translocation of ribosomal protein S6 (RpS6) in neuroblasts and that it is a transcriptional target of Myc. Therefore, it was suggested that Mbm might regulate neuroblast proliferation through a role in ribosome biogenesis. In the present study, it was attempted to further elucidate these proposed roles of Mbm and to identify the protein domains that are important for those functions. Mbm contains an arginine/glycine rich region in which a di-RG as well as a di-RGG motif could be found. Together, these two motifs were defined as Mbm’s RGG-box. RGG-boxes can be found in many proteins of different families and they can either promote or inhibit protein-RNA as well as protein-protein interactions. Therefore, Mbm’s RGG-box is a likely candidate for a domain involved in rRNA binding and RpS6 translocation. It could be shown by deletion of the RGG-box, that MbmdRGG is unable to fully rescue survivability and neuroblast cell size defects of the null mutation mbmSH1819. Furthermore, Mbm does indeed rely on its RGG-box for the binding of rRNA in vitro and in mbmdRGG as well as mbmSH1819 mutants RpS6 is partially delocalized. Mbm itself also seems to depend on the RGG-box for correct localization since MbmdRGG is partially delocalized to the nucleus. Interestingly, protein synthesis rates are increased in mbmdRGG mutants, possibly induced by an increase in TOR expression. Therefore, Mbm might possess a promoting function in TOR signaling in certain conditions, which is regulated by its RGG-box. Moreover, RGG-boxes often rely on methylation by protein arginine methyltransferases (in Drosophila: Darts – Drosophila arginine methyltransferases) to fulfill their functions. Mbm might be symmetrically dimethylated within its RGG-box, but the results are very equivocal. In any case, Dart1 and Dart5 do not seem to be capable of Mbm methylation. Additionally, Mbm contains two C2HC type zinc-finger motifs, which could be involved in rRNA binding. In an earlier study, it was shown that the mutation of the zinc-fingers, mbmZnF, does not lead to changes in neuroblast cell size, but that MbmZnF is delocalized to the cytoplasm. In the present study, mbmZnF mutants were included in most experiments. The results, however, are puzzling since mbmZnF mutant larvae exhibit an even lower viability than the mbm null mutants and MbmZnF shows stronger binding to rRNA than wild-type Mbm. This suggests an unspecific interaction of MbmZnF with either another protein, DNA or RNA, possibly leading to a dominant negative effect by disturbing other interaction partners. Therefore, it is difficult to draw conclusions about the zinc-fingers’ functions. In summary, this study provides further evidence that Mbm is involved in neuroblast proliferation as well as the regulation of ribosome biogenesis and that Mbm relies on its RGG-box to fulfill its functions. / Die Entwicklung des zentralen Nervensystems von Drosophila melanogaster beruht auf neuronalen Stammzellen genannt Neuroblasten. Neuroblasten teilen sich asymmetrisch und bringen dabei sowohl einen neuen Neuroblasten als auch eine kleinere Tochterzelle hervor, die wiederum letztlich Neuronen oder Gliazellen generiert. Zwischen jeder Zellteilung müssen die Neuroblasten wieder auf ihre ursprüngliche Größe wachsen, sodass sie zur erneuten Teilung in der Lage sind. In vorhergehenden Studien konnte gezeigt werden, dass sowohl die Proliferation der Neuroblasten, deren Zellgröße als auch die Anzahl ihrer Tocherzellen reduziert ist in Larven, die eine P-Element-induzierte Unterbrechung des Gens mushroom body miniature (mbm) tragen. Diese mbm-Nullmutation, genannt mbmSH1819, ist homozygot letal während des Puppenstadiums. Es konnte außerdem gezeigt werden, dass das nucleoläre Protein Mbm eine Rolle in der Prozessierung ribosomaler RNA (rRNA), sowie der Translokation des ribosomalen Proteins S6 (RpS6) in Neuroblasten erfüllt und dass seine Transkription durch Myc reguliert wird. Daher wurde geschlussfolgert, dass Mbm die Proliferation von Neuroblasten durch eine Funktion in der Ribosomenbiogenese regulieren könnte. In der vorliegenden Studie wurde das Ziel verfolgt, weitere Hinweise auf diese möglichen Funktionen von Mbm zu finden und die Proteindomänen zu identifizieren, die dafür benötigt werden. Mbm beinhaltet einen Arginin/Glycin-reichen Abschnitt, der ein di-RG sowie ein di-RGG Motiv enthält. Diese beiden Motive wurden zusammen zu Mbms RGG-Box definiert. RGG-Boxen finden sich in vielen Proteinen verschiedener Familien und sie können sich sowohl verstärkend als auch inhibierend auf Protein-RNA- sowie Protein-Protein-Interaktionen auswirken. Somit stellt Mbms RGG-Box einen vielversprechenden Kandidaten dar für eine Proteindomäne, die in die rRNA-Bindung sowie die Translokation von RpS6 involviert ist. Es konnte gezeigt werden, dass Mbm mit deletierter RGG-Box (MbmdRGG) nicht in der Lage ist, die Überlebensfähigkeit und die Neuroblastengröße der Nullmutation mbmSH1819 vollständig zu retten. Des Weiteren benötigt Mbm die RGG-Box, um rRNA in vitro zu binden und in mbmdRGG sowie mbmSH1819 Mutanten konnte eine partielle Delokalisation von RpS6 beobachtet werden. Die korrekte Lokalisation von Mbm selbst scheint auch von der RGG-Box abzuhängen, da MbmdRGG teilweise in den Nukleus delokalisiert ist. Interessanterweise ist außerdem die Proteinsyntheserate in mbmdRGG Mutanten erhöht, was möglicherweise in einer Erhöhung der TOR-Expression begründet ist. Somit könnte Mbm unter bestimmten Bedingungen eine verstärkende Funktion im TOR-Signalweg erfüllen, die durch seine eigene RGG-Box reguliert wird. Des Weiteren sind RGG-Boxen hinsichtlich ihrer Funktion häufig von der Methylierung durch Protein-Arginin-Methyltransferasen (in Drosophila: Darts – Drosophila arginine methyltransferases) abhängig. Mbm könnte innerhalb seiner RGG-Box symmetrisch dimethyliert sein, allerdings sind die Ergebnisse in dieser Hinsicht sehr zweifelhaft. Jedenfalls scheinen Dart1 und Dart5 nicht imstande zu sein, Mbm zu methylieren. Außerdem beinhaltet Mbm zwei Zink-Finger-Motive des C2HC-Typs, die in die Bindung von rRNA involviert sein könnten. Eine vorhergehende Studie konnte zeigen, dass die Mutation der Zink-Finger, mbmZnF, zwar nicht zu einer Veränderung der Neuroblastengröße führt, allerdings, dass MbmZnF ins Zytoplasma delokalisiert vorliegt. In der vorliegenden Studie wurden die mbmZnF Mutanten in die meisten Experimente mit einbezogen. Allerdings sind die Ergebnisse rätselhaft, da mbmZnF-mutierte Larven sogar eine geringere Überlebensrate zeigen als die mbm Nullmutanten und da MbmZnF eine stärkere Bindungsaffinität zu rRNA zeigt als wildtypisches Mbm. Dies weist auf eine unspezifische Interaktion zwischen MbmZnF und einem anderen Protein, RNA oder DNA hin, was einen dominant-negativen Effekt auslösen könnte, indem andere Interaktionspartner gestört werden. Somit gestaltet es sich schwierig, Schlussfolgerungen zur Funktion der Zink-Finger zu ziehen. Zusammengefasst liefert die vorliegende Studie weitere Anhaltspunkte, dass Mbm in der Neuroblastenproliferation sowie der Regulation der Ribosomenbiogenese involviert ist und dass Mbm seine RGG-Box benötigt, um seine Funktionen zu erfüllen.

xiii, 92 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. / Cellular polarity is a biological mechanism that is conserved across metazoa and is used in many different biological processes, one of which is stem cell self-renewal and differentiation. Stem cells generate cellular diversity during development by polarizing molecular determinants responsible for directing one daughter cell to maintain stem cell-like qualities and the other daughter cell to initiate a specific cell fate. The stem cell self-renewal versus differentiation choice is critical to avoid overproliferation of stem cells and tumor formation or underdevelopment of tissues and early animal death. Drosophila neural stem cells (neuroblasts) undergo asymmetric cell division (ACD) to populate the fly central nervous system and provide an excellent model system to study processes involving cellular polarity, ACD, stem cell self-renewal, and differentiation. Neuroblasts divide unequally to produce a large, apical self-renewing neuroblast and a small, basal ganglion mother cell that goes on to divide and form two neurons or glia. In this way, a small population of neuroblasts can give rise to thousands of neurons and glia to generate a functional central nervous system. Atypical Protein Kinase C (aPKC) is critical to establish and maintain neuroblast polarity, ACD, stem cell self-renewal, and differentiation. aPKC is part of the evolutionarily conserved Par complex, whose other members include Bazooka and Par-6, and they localize to the neuroblast apical cortex and function to restrict cell-fate determinants into one daughter cell. How aPKC is asymmetrically localized and how its activity translates into cell-fate specification are of incredible importance as apkc mutants where localization is disrupted no longer segregate cell-fate determinants. This work will show that Cdc42 recruits the Par-6/aPKC complex to the neuroblast apical cortex independent of Bazooka. Once there, aPKC phosphorylates the cell-fate determinant Miranda to exclude it from the apical cortex and restrict it basally. Par-6 and Cdc42 regulate aPKC kinase activity though inter- and intramolecular interactions that allow high aPKC kinase activity at the apical cortex and suppressed activity elsewhere. Cdc42 also functions to keep aPKC asymmetrically localized by recruiting the PAK kinase Mushroom bodies tiny to regulate cortical actin and provide binding sites for cortical polarity determinants. This dissertation includes previously published co-authored material. / Adviser: Kenneth Prehoda

Universiẗat, Diss., 2005--Düsseldorf.

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

The adoption and subsequent retention of distinct cellular fates upon cell division is a critical phenomenon in the development of multicellular organisms. A well-studied example of this process is stem cell divisions; stem cells must possess the capacity to self-renew in order to maintain a stem cell population, as well as to generate differentiated daughters for tissue growth and repair. Drosophila neuroblasts are the neural stem cells of the central nervous system and have emerged as an important model for stem cell divisions and the genetic control of daughter cell identities. Neuroblasts divide asymmetrically to generate daughters with distinct fates; one retains a neuroblast identity and the other, a ganglion mother cell, divides only once more to generate differentiated neurons and glia. Perturbing the asymmetry of neuroblast divisions can result in the failure to self-renew and the loss of the neural stem cell population; alternatively, ectopic self-renewal can occur, resulting in excessive neuroblast proliferation and tumorigenesis. Several genetic lesions have been characterized which cause extensive ectopic self-renewal, resulting in brains composed of neuroblasts at the expense of differentiated cells. This contrasts with wild type brains, which are composed mostly of differentiated cells and only a small pool of neuroblasts. We made use of these mutants by performing a series of microarray experiments comparing mutant brains (consisting mostly of neuroblasts) to wild type brains (which are mostly neurons). Using this approach, we generated lists of over 1000 putatively neuroblast-expressed genes and over 1000 neuronal genes; in addition, we were able to compare the transcriptional output of different mutants to infer the neuroblast subtype specificity of some of the transcripts. Finally, we verified the self-renewal function of a subset of the neuroblast genes using an RNAi-based screen, resulting in the identification of 84 putative self-renewal regulators. We went on to show that one of these genes, midlife crisis (mammals: RNF113a), is a well-conserved RNA splicing regulator which is required in postmitotic neurons for the maintenance of their differentiated state. Our data suggest that the mammalian ortholog performs the same function, implicating RNF113a as an important regulator of neuronal differentiation in humans.