Microtubule-based mitochondrial transport into dendrites and axons is vital for sustaining neuronal function. Transport along microtubules proceeds in a series of plus- and minus-end directed movements facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective long distance transport remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites. To directly examine Miro's significance for kinesin- and/or dynein-mediated mitochondrial motility, we live imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of either antero- or retrograde mitochondrial transport by selectively impairing kinesin- or dynein-mediated movements, depending on the direction of net transport. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We concluded that dMiro promotes effective antero- and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport. To determine how Miro achieves this control mechanistically, we introduced point mutations that render each GTPase either constitutively active or inactive. Expression of either first GTPase mutant impaired antero- (inactive) or retrograde motor movements (active) in a direction dependent manner. The active state of the second GTPase domain up-regulated the number of consecutive kinesin motions during anterograde transport but impaired kinesin transport biases while the inactive second GTPase state impaired transport in either direction. Together, these data suggest that Miro's first GTPase domain is major factor that controls the execution of either the antero- or retrograde directional program while Miro's second GTPase may provide a signal that supports or disfavors transport. In addition, the active state of the first and the second GTPase domain increased the length of stationary mitochondria but only the first GTPase domain modified motile mitochondrial lengths. Overexpression of these mutations generated opposing effects. We conclude that both domains control antero- and retrograde transport in a switch-like manner.

Les kinases dépendantes des cyclines (CDKs) sont les régulateurs essentiels du cycle cellulaire. Au sein de la famille des cyclines, les fonctions de la cycline M (CycM), produit du gène FAM58A dont des mutations sont à l’origine du syndrome STAR, sont toujours inconnues. Dans une première partie, mon travail de thèse dévoile que CycM active CDK10, une CDK mystérieuse et toujours orpheline. CDK10/CycM phosphoryle le facteur de transcription ETS2 pour induire sa dégradation par le protéasome. Dans des cellules issues d’une patiente atteinte du syndrome STAR, les niveaux d’expression d’ETS2 sont plus élevés, et cette augmentation est liée à une diminution du niveau d’expression de CycM. ETS2 joue des rôles importants au cours du développement et dans la survenue de certains cancers. Ces travaux dévoilent l’existence d’un nouveau mécanisme de régulation de ETS2 et ils offrent le premier éclairage sur les mécanismes moléculaires à l’origine du syndrome STAR. Une deuxième partie de mon travail démontre que CDK10 et CycM sont des protéines localisées en zone centriolaire, en mitose et en quiescence. CDK10 et CycM régulent négativement la ciliogénèse et la longueur du cil primaire. Il dévoile un nouveau partenaire d’interaction et substrat de phosphorylation de CDK10/CycM, qui pourrait participer à cette régulation négative. Des anomalies de la ciliogénèse causent des défauts de développement caractéristiques de maladies génétiques nommées ciliopathies. Certains défauts de développement du syndrome STAR pourraient être liés à des anomalies de la ciliogénèse. / Cyclin-dependent kinases (CDKs) are activated by their cyclin subunits to drive the cell cycle. Among the cyclin family, the functions of the FAM58A gene product, cyclin M (CycM) remain unknown. However, FAM58A mutations lead to severe developmental defects which are reported as STAR syndrome. In the first part of my work, I show that CycM activates CDK10, an orphan and mysterious CDK. The novel protein kinase CDK10/CycM phosphorylates ETS2 to induce its degradation via the proteasome. ETS2 protein levels are increased in cells derived from a STAR patient, and this increase is attributable to decreased CycM levels. Altogether, these results reveal a new regulatory mechanism for ETS2, which plays key roles in cancer and development. They shed light on the molecular mechanisms underlying STAR syndrome. In a second part of my work, I show that CDK10 and CycM localize in a centriolar zone in mitosis and in quiescence. I show that CDK10 and CycM negatively regulate ciliogenesis and primary cilium length. Finally, I uncover a novel interactor and phosphorylation substrate of CDK10/CycM that may participate in this negative regulation. Ciliogenesis defects lead to severe developmental abnormalities called ciliopathies. Some STAR syndrome developmental anomalies could thus be linked to ciliogenesis defects.

Receptor-like kinases (RLKs) are encoded for by one of the largest gene families in Arabidopsis and represent the predominant form of cell surface receptors in plants. RLKs mediate signal transduction in diverse processes including steroid-mediated growth pathways, pathogen-triggered innate immune responses. Here I present characterization of mutant phenotypes, expression patterns, and genetic interactions for the BAK1 INTERACTING RECEPTOR (BIR) family of Leucine-rich Repeat-RLKs, three members of which have had no previous characterization. Furthermore, I show that cell death, aerial growth, and lateral root development defects in bir1-1 are suppressed by mutations of the LRR-RLK co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1); I identify a novel primary root growth phenotype in bir1-1 mutants, as well as a lateral root development phenotype for bir3 mutants; and primary root growth and aerial defects in bir3.bir4;bak1 triple mutants. Using an allelic series of bak1 mutations I show that bir phenotypes are dependent upon particular functions of BAK1, and propose that the BIR family exhibits a novel function, previously undescribed for LRR-RLKs, as regulators of co-receptor/ligand-binding receptor complex specificity.

During Arabidopsis embryogenesis, cell-cell signaling plays an essential role in establishing an organized body plan centered around two major axes of development: apical-basal and radial. Two topics of great interest are how the layered structure is initiated and maintained during and after embryogenesis and how communication between layers is achieved to allow for coordinated development. Recent research involving Receptor-Like Kinases (RLKs) in plants suggests that their roles in integrating various signals are important in many aspects of development, including embryonic and post-embryonic patterning. The research presented here describes the roles of two pairs of RLKs with independent roles in two different signaling environments. The first RLK pair, GSO1 and GSO2, function in root development at the transition to photoautotrophic nutrition to integrate sugar signals and regulate root growth. GSO1 and GSO2 regulate root epidermal cell identity by controlling the pattern of cell division of stem cells. The second pair of RLKs, RPK1 and TOAD2, function to control root development by regulation of meristem proliferation and a coordinated response to signaling molecules of the CLE family. The response of wild-type roots to treatment with CLE peptides (A-type) is meristem growth arrest, resulting in short roots. toad2 mutants are insensitive to the effect of CLE peptides in reducing meristem size and TOAD2 also regulates RPK1 upon CLE stimulation. Although responding to different signals, the two pairs of RLK share a common output of regulating cell proliferation in and around the root meristem, especially in the epidermis of the root.

Viruses represent the most abundant biological entities on earth where, they are able to interact with all kingdoms of life. Yet their diversity, ecology and evolutionary aspects are only beginning to be fully elucidated, mainly due to technical limitations. The vast majority of the microbial world remains elusive to culture; more than 90% of genome sequenced viral isolates infect only 5 of the 54 prokaryotic phyla that are currently recognized. In contrast, viral metagenomics bypasses the need for cultures by directly sequencing fragmented genetic material of environmental viral communities. This dissertation uses viral metagenomics by applying well-tested bioinformatic protocols and expanding them to compare and contrast patterns of diversity, richness and specialization of large viral metagenomic datasets, in both local and global scales. First I demonstrate the utility of a functional-based perspective by adopting the protein cluster environment to estimate global viral diversity. Then, I use this PC approach to analyze metagenomes from two ecologically different environments, which by uncovering local gene specialization showcases the adequacy of a gene-centered workflow. Then I continue to expand upon this PC framework to study the Tara Oceans virome analyses of these data reveal patters of diversity that support a seed bank model. Finally, in search of a more meaningful ecological unit, I move from a gene-centered standpoint towards a population-based frame. We adopted a novel metagenomic technique that allowed me to uncover the discontinuity in the genomic sequence space, thus empirically defining a population. This final contribution will allow to sort and count viral communities, the first step to applying ecological and evolutionary theory.

Hypertrophic cardiomyopathy (HCM) is a primary disease of the myocardium. 4-11% of HCM is caused by mutations in cardiac troponin T (cTnT) and 65% of them are within the tropomyosin (TM)-binding TNT1 domain. Two of the known mutational hotspots within TNT1 are in the N and C-terminal domains. Unlike the N-terminal domain; no high-resolution structure exists for the highly conserved C-terminal domain limiting both our ability to understand the functional role of this extended domain in myofilament activation and molecular mechanism(s) of HCM. The Δ160E mutation is an in-frame deletion of a glutamic acid residue at position 160 of cTnT. This TNT1 C-terminal mutation is associated with an especially poor prognosis. The Δ160E mutation is located in a putative "hinge region" immediately adjacent to the unstructured flexible linker connecting the TM-binding TNT1 domain to the Ca²⁺-sensitive TNT2 domain. Unwinding of this α-helical hinge may provide the flexibility necessary for thin filament function. Previous regulated in vitro motility assay (R-IVM) data showed mutation-induced impairment of weak actomyosin binding. Thus, we hypothesized that the Δ160E mutation repositions the flexible linker which impairs weak electrostatic binding and ultimately leads to severe cardiac remodeling. The goal of our studies is two-fold: 1) to gain high-resolution insight into the position of the cTnT linker with respect to the C-terminus of TM, and 2) to identify Δ160E-induced positional changes using Fluorescence Resonance Energy Transfer (FRET) in a fully reconstituted thin filament. To this end, residues in the middle and distal regions of the cTnT linker were sequentially cysteine-substituted (A168C, A177C, A192C and S198C) and labeled with the energy donor IAEDANS. The energy acceptor, DABMI was attached to cysteine 190 (C190) in the C-terminal region of TM and FRET measurements were obtained in the presence and absence of Ca²⁺ and myosin subfragment 1 (S1). An all-atom thin filament model in the Ca²⁺–on state was employed to predict the pathogenic effects of the Δ160E mutation on the structure and the dynamics of the cTnT linker region. Our data suggest that the linker domain runs alongside the C-terminus of TM and is differentially repositioned by calcium, myosin and the Δ160E mutation. The Δ160E mutation moves the linker closer to the C-terminus of TM. The in silico model supported this finding and demonstrated a mutation-induced decrease in linker flexibility. Moreover, the model predicted a pathogenic change in the orientation of the middle region of the linker and in the position of the Ca²⁺-sensitive TNT2 domain and the TM-binding TNT1 domain in response to Δ160E mutation. Collectively, our findings suggest that the Δ160E mutation-induced changes in the structure, position and dynamics of the linker region cause steric blocking of weak myosin binding sites on actin and subsequent impairment of contraction and disruption of sarcomeric integrity. These studies, for the first time, provided information regarding the role of the extended linker in both myofilament activation and disease.

The causes of Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease that results in skeletal muscle paralysis, remain unclear. However, a nuclear DNA and RNA binding protein called TAR DNA binding protein 43 (TDP-43) has emerged as a critical marker of ALS pathology. A previous drug screen conducted in the Zarnescu laboratory showed that anti-diabetic drugs can rescue lethality in a fruit fly model of ALS based on TDP-43. These results suggested that the insulin signaling pathway might be altered in motor neurons in a TDP-43 dependent manner. Therefore, we hypothesized that the insulin pathway is interacting with TDP-43 in vivo and may be contributing to TDP-43neurotoxicity. Using genetic interaction approaches in flies we found that TDP-43dependent locomotor defects are sensitive to the levels of insulin receptor activity. In addition, genetic interaction data suggest that Akt is hyperactivated in motor neurons expressing TDP-43, possibly as a compensatory mechanism to enable survival. Finally, upregulating protein synthesis through S6K and 4EBP appears to have beneficial effects. These findings support our hypothesis and provide insights into potential therapeutic strategies to help treat this devastating disease.

Experts in the field of molecular and cellular biology (MCB) use domain-specific reasoning strategies to navigate the unique complexities of the phenomena they study and creatively explore problems in their fields. One primary goal of instruction in undergraduate MCB is to foster the development of these domain-specific reasoning strategies among students. However, decades of evidence-based research and many national calls for undergraduate instructional reform have demonstrated that teaching and learning complex fields like MCB is difficult for instructors and learners alike. Therefore, how do students develop rich understandings of biological mechanisms? It is the aim of this dissertation work to explore features of expertise and knowledge building in undergraduate MCB by investigating knowledge organization and problem-solving strategies. Semi-structured clinical think-aloud interviews were conducted with introductory and upper-division students in MCB. Results suggest that students must sort ideas about molecular mechanism into appropriate mental categories, create connections using function-driven and mechanistic rather than associative reasoning, and create nested and overlapping ideas in order to build a nuanced network of biological ideas. Additionally, I characterize the observable components of generative multi-level mechanistic reasoning among undergraduate MCB students constructing explanations about in two novel problem-solving contexts. Results indicate that like MCB experts, students are functionally subdividing the overarching mechanism into functional modules, hypothesizing and instantiating plausible schema, and even flexibly consider the impact of mutations across ontological and biophysical levels. However "filling in" these more abstract schema with molecular mechanisms remains problematic for many students, with students instead employing a range of developing mechanistic strategies. Through this investigation of expertise and knowledge building, I characterize several of the ways in which knowledge integration and generative explanation building are productively constrained by domain-specific features, expand on several discovered barriers to productive knowledge organization and mechanistic explanation building, and suggest instructional implications for undergraduate learning.

Mayer-Rokitansky-Kuster-Hauser Syndrome is a congenital disorder of the female reproductive tract due to impaired Müllerian duct development. There are three known categorical presentations: isolated, atypical, and MURCS association. Several developmentally significant factors including inappropriate AMH/AMHR interaction, and mutations in the WNT gene family and HOXA7-13 cluster have been studied. There has also been investigation into an autosomal dominant pattern of inheritance in families with multiple cases of the syndrome. Due to the presence of multiple subsets of patients with similar genetic abnormalities, it seems unlikely that a single etiology will be discovered.

Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The orthologue of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, PHI-33, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppress cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.