Non-claret disjunctional (Ncd) is a kinesin-14 microtubule motor protein involved in the assembly and stability of meiotic and mitotic spindles in Drosophila oocytes and early embryos, respectively. Ncd functions by cross-linking microtubules through the tail and motor domains. It was originally believed that the role of the Ncd tail domain was to only statically bind microtubules. However, the Ncd tail domain has recently been shown to have properties that stabilize and bundle microtubules, and contribute to the overall motility of the Ncd protein. Continued characterization of the Ncd tail domain is essential to understanding the complete role of Ncd in cell division. This work explored the regulatory function and microtubule binding properties of the Ncd tail domain. Ncd activity is regulated during interphase by nuclear sequestration. GFP-Ncd fusion proteins, containing full length Ncd, individual Ncd domains, or combinations of Ncd domains, were used to identify the presence of a nuclear localization signal (NLS) in the Ncd polypeptide. The nuclear localization of only the GFP fusion proteins containing the Ncd tail sequence indicates that the NLS is contained within the tail domain. Subsequent, experiments performed with GFP fusion proteins containing segments of the tail domain indicate that essential NLS amino acid segments may span the length of the tail domain. Attempts to characterize the microtubule binding properties of the Ncd tail domain, using bacterially expressed MBP-Ncd tail-stalk, were unsuccessful. MBP-Ncd tail-stalk proteins aggregated under binding assay conditions, preventing an accurate determination of the stoichiometric binding relationship between Ncd and the tubulin dimer. / Master of Science

Oomycetes are fungi-like eukaryotic microorganisms, which are actually phylogenetic relatives of diatoms and brown algae, within the kingdom Stramenopila. Many oomycete species, mainly in the genera Phytophthora, Pythium and downy mildews, are devastating plant pathogens that cause multibillion-dollar losses to agriculture annually in the world. Some oomycetes are also animal pathogens, causing severe losses in aquaculture and fisheries, and occasionally causing dangerous infections of humans. Phytophthora species, represented by the Irish Potato Famine pathogen P. infestans and the soybean pathogen P. sojae, are arguably the most destructive pathogens of dicotyledonous plants among the oomycete species and thus have been extensively studied. This dissertation focuses on the model oomycete pathogen P. sojae to investigate specific aspects of its molecular biology and establish an efficient genetic manipulation tool. Specifically, in Chapter 1, I briefly introduce the basic concepts of oomycete biology and pathology, and summarize the experimental techniques used for studies of oomycete genetics over the past two decades. Because the approach to studying fungi and oomycetes are similar (indeed they were incorrectly placed in the same taxonomic group until recently), a special section reviews the emerging genome editing technology CRISPR/Cas system in these organisms together. Chapter 2 and Chapter 3 focus on one of the most important intracellular activities, nuclear localization of proteins, and describe the characterization of nuclear localization signals (NLSs) in P. sojae. This focus stemmed from my early work on genome editing in P. sojae, when I discovered that conventional NLS signals from SV40 used to target the TAL effector nuclease (TALEN) to the nucleus worked poorly in P. sojae. In the first part of this work (Chapter 2), I used confocal microscopy to identify features of nuclear localization in oomycetes that differ from animals, plants and fungi, based on characterization of two classes of nuclear localization signals, cNLS and PY-NLS, and on characterization of several conserved nuclear proteins. In the second part (Chapter 3), I determined that the nuclear localization of the P. sojae bZIP1 transcription factor is mediated by multiple weak nuclear targeting motifs acting together. In Chapter 4 and Chapter 5, I describe my implementation of nuclease-based technology for genetic modification and control of P. sojae. In Chapter 4, I describe the first use of the CRISPR system in an oomycete, including its use to validate the function of a host specificity gene. This is of particular importance because molecular techniques such as gene knockouts and gene replacements, widely used in other organisms, were not previously possible in oomycetes. The successful implementation of CRISPR provides a major new research capability to the oomycete community. Following up on the studies described in Chapter 4, in Chapter 5, I describe the generalization and simplification of the CRISPR/Cas9 expression strategy in P. sojae as well as methods for mutant screening. I also describe several optimized methodologies for P. sojae manipulation based on my 5 years of experience with P. sojae. / Ph. D.

Php4 is a subunit of the CCAAT-binding protein complex that has a negative regulatory function during iron deprivation in the fission yeast Schizosaccharomyces pombe. Under low iron conditions, Php4 fosters the repression of genes encoding iron using proteins. In contrast, under iron-replete conditions, Php4 is inactivated at both transcriptional and post-transcriptional levels. Our group has already described that Php4 is a nucleo-cytoplasmic shuttling protein, which accumulates into the nucleus during iron deficiency. On the contrary, Php4 is exported from the nucleus to the cytoplasm in response to iron abundance. Php4 possesses a leucine-rich NES (93LLEQLEML100) that is necessary for its nuclear export by the exportin Crm1. Our current study aims at understanding the mechanism by which Php4 is imported in the nucleus during iron starvation. Through microscopic analyses using different mutant strains, we showed that the nuclear localization of Php4 is independent of the other subunits of the CCAAT-binding core complex namely Php2, Php3 and Php5. Deletion mapping analysis of Php4 identifies two putative nuclear localization sequences (NLSs) in Php4 (171KRIR174 and 234KSVKRVR240). Using chimeric proteins that consist of GFP fused to Php4, we engineered substitutions of the basic amino acid residues 171AAIA174 and 234ASVAAAA240 and analyzed the functionality of both NLSs. We observed that both monopartite NLSs play critical role for Php4 nuclear localization. We also observed that mutant strains of cut15+, imp1+ or sal3+ exhibited defects in nuclear targeting of Php4, revealing that nuclear accumulation of Php4 is dependent on two karyopherin α (Imp1 and Cut15) and one karyopherin β (Sal3) receptors. Consistently, the Php4-mediated repression activity is abolished in the absence of two functional NLSs. Moreover, loss of Imp1, Cut15 or Sal3 resulted in increased expression of isa1+, which is a target gene of Php4. Co-immunoprecipitation assay (Co-IP) reveals physical interaction of Php4 with Imp1, Cut15 and Sal3 in vitro. Collectively, our results demonstrate that Php4 has two distinct NLS regions responsible for its nuclear localization. Furthermore, karyopherin α and β receptors play a role in the nuclear import of Php4. Because Php4 is essential for growth under low iron conditions, the presence of two NLSs would ensure the protein to reach its nuclear destination when cells undergo a transition from iron-sufficient to iron-limiting conditions.

Tumor suppressor p53-binding protein 1 (53BP1) is an integral part of a sophisticated network of cellular pathways termed as the DNA damage response (DDR). These pathways are specialized in the maintenance of genome integrity. Recently, it was reported that nuclear import of 53BP1 depends on importin ß. Here, I used fluorescence microscopy and co-immunoprecipitation experiments to identify its nuclear localization signal (NLS). Clusters of basic amino acids 1667-KRK-1669 and 1681-KRGRK- 1685 were required for 53BP1 interaction with importin ß and for its nuclear localization. Short peptide containing these two clusters was sufficient for interaction with importin ß and targeting EGFP to the nucleus. Additionally, the effect of 53BP1 phosphorylation at S1678 on its nuclear import was examined. Mimicking the phosphorylation in the 53BP1-S1678D mutant decreased the binding to importin ß and resulted in a mild defect in 53BP1 nuclear import. However, 53BP1 entered the nucleus continuously during the cell cycle, suggesting that CDK-dependent phosphorylation of S1678 probably does not significantly contribute to the regulation of 53BP1 nuclear transport. Taken together, 53BP1 NLS meets the attributes of a classical bipartite NLS. Although no cell cycle-dependent regulation of its import was observed, the...

Thesis advisor: Junona Moroianu / During the papillomavirus replication cycle, the L2 minor capsid protein enters the nucleus in the initial phase after uncoating of the incoming virions and in the productive phase when L2 together with L1 major capsid protein mediate the encapsidation of the newly replicated viral genome. L2 proteins of both high risk HPV16 L2 and low risk HPV11 L2 have two nuclear localization signals (NLSs): one at the N-terminus (nNLS) and one at the C terminus (cNLS). The purpose of these experiments is to determine the minimal mutations necessary to inhibit the function of the NLSs. In this study, subcellular localization of enhanced green fluorescent protein (EGFP) fusions with full length L2 and L2 mutants lacking either the cNLS (EGFP-L2ΔC), nNLS (EGFP-L2ΔN), or both NLSs (EGFP-L2ΔNΔC) was analyzed in HeLa cell transfection assays. Full length HPV16 L2 and HPV11 L2 proteins localize to the nucleus. For both HPV16 and 11 L2, each NLS could independently mediate nuclear import in vivo. EGFP fusions were also made with mutated nNLS (EGFP-L2ΔCSbN) or mutated cNLS (EGFP-L2ΔNSbC). Transfected HeLa cells were examined by fluorescence microscopy and quantitative studies were done. In both HPV16 and 11 L2 proteins, mutation of basic residues in either NLS inhibited its nuclear import ability. / Thesis (BS) — Boston College, 2008. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Bone morphogenetic protein 4 (Bmp4) is a well known growth factor that regulates gene expression through the SMAD signaling pathway. Bmp4 is involved in many developmental processes and has been identified as an important factor in several cancers, including melanoma, ovarian cancer, and colon cancer. Madoz-Gurpide et al. recently observed Bmp4 in the nuclei of a minor percentage of cells in colon cancer tissues. In addition, our lab has recently discovered a nuclear variant of Bmp2 (nBmp2), the TGF-β family member most closely related to Bmp4. These observations led us to hypothesize that a nuclear variant of Bmp4 (nBmp4) also exists. The results of chapter one report the existence of a nuclear variant of Bmp4. nBmp4 is translated from an alternative start codon downstream of the signal peptide sequence which allows a bipartite nuclear localization signal to direct translocation of nBmp4 to the nucleus. Chapter 2 and 3 further report that nBmp4 interacts with several subunits in the SCF E3 ubiquitin ligase, namely two Regulator of Cullins (ROC) proteins, five Cullin proteins, and two F-box proteins. Due to the known role of the SCF E3 ubiquitin ligase in regulating the cell cycle, the effect of nBmp4 on cell cycle progression was analyzed and the results show that nBmp4 affects the cell cycle by causing cells to accumulate in G0/G1. The association of nBmp4 and the SCF E3 ubiquitin ligase components and the affect that nBmp4 has on the cell cycle suggest that nBmp4 functions in the nucleus by inhibiting the SCF E3 ubiquitin ligase from ubiquitinating target proteins that are involved in regulating cell cycle progression. Finally, the initial stages in the generation of an nBmp4 over-expression mouse are described. The results of this research clearly change the traditional paradigm that Bmp4 performs all of its functions via extracellular signaling and introduce the existence of a nuclear variant that is involved in cell cycle regulation.

Non-viral gene delivery is plagued by low transfection levels compared to viral delivery. The nuclear envelope presents a significant obstacle for non-viral vectors. A peptide-based nuclear localizing sequence has been incorporated into non-viral vectors to traverse the nuclear envelope. Here, we selected a photo-chemical method for covalently labeling the peptide onto plasmid DNA. The hypothesis of this work was to incorporate a nuclear localizing sequence into a non-viral delivery vector, demonstrate increased nuclear uptake and show a subsequent increase in transgene expression both in vitro and in vivo. We focused on pursuing in vitro and in vivo methods by which to test non-viral vectors for increases in gene expression based on the nuclear localizing sequence. Hydrodynamic dosing and intramuscular dosing (followed by electroporation) are two efficient delivery routes for dosing DNA in vivo. Through preliminary experiments, we became confident that whole animal bioluminescent imaging was a reliable and quantitative method by which to detect luciferase expression by either delivery route. Moving forward, both hydrodynamic and intramuscular dosing would be used to test formulations for nuclear localizing ability in vivo. Nuclear localizing peptides containing a photo-activatable functionality were synthesized and characterized. We quantitatively explored the photo-labeling capabilities on plasmid DNA via a radioactive peptide. In vitro, tissue culture-based experiments were carried out to show increased nuclear uptake by confocal microscopy as well as increased transgene expression. Throughout the literature, achieving an increase in expression by incorporating a nuclear localizing sequence into a non-viral vector has been elusive. The complexity of achieving this goal is increased when considering an in vivo system for improving gene transfer efficiency. Several strategies have been explored to demonstrate an increase in reporter gene expression from this type of non-viral vector, and the methods developed herein can be applied to other nuclear localizing vectors.

Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF-hand motif characteristic of the classical calpains, calpain 1 and 2, but retains catalytic and Ca2+ binding non EF domains. Tra-3, an ortholog of CAPN5, is involved in necrotic cell death in C.elegans; although specific role of CAPN5 has not been investigated in the mammalian CNS. I compared relative mRNA levels of calpains in rat CNS, which revealed that CAPN5 is the second most highly expressed calpain. We examined relative levels of CAPN5 from late embryonic day 18 to postnatal day 90 and found lower mRNA but higher protein levels during CNS development. Using X –gal staining in Capn5 +/- mice, immunostaining of rat brain sections and SH-SY5Y cells, and subcellular fractionation of rat brain cortex, we found that CAPN5 is a non-cytoplasmic calpain localized in the nucleus and enriched in synaptic mitochondria. Proteinase K treatment of mitochondria and mitoplasts from B35 rat neuroblastoma cells and rat synaptic mitochondria revealed CAPN5 was localized on the inner mitochondrial membrane and released from mitochondria on membrane permeabilization with alamethicin. We used immunolabelling, confocal imaging, nuclear subfractionation and transient transfections to evaluate the subnuclear localization of CAPN5. CAPN5 was detected in punctate domains and associated with promyelocytic leukemia (PML) protein, a tumor suppressor protein. We further demonstrated that CAPN5 carries a nonconventional bipartite nuclear localization signal. Together, these findings demonstrate that CAPN5 is a non-cytosolic calpain, abundant in the CNS and localized to the mitochondria inner membrane and nuclear PML bodies.

Potato yellow dwarf virus (PYDV) is the type species of the genus Nucleorhabdovirus and, like all members of this genus, replication and morphogenesis occurs inside the nuclei of infected cells. Protein localization prediction algorithms failed to identify a nuclear localization signal (NLS) in PYDV nucleocapsid (N) protein, although PYDV-N has been shown to localize exclusively to the nucleus when expressed as a green fluorescent protein (GFP):N fusion in plant cells. Deletion analysis and alanine-scanning mutagenesis identified two amino acid motifs, 419QKR421 and 432KR433, that were shown to be essential for nuclear import and interaction with importin-α. Additional bimolecular fluorescence complementation showed that the PYDV-N-NLS mutants cannot be ferried into the nucleus via interaction with PYDV-P or-M. In contrast, interaction with N-NLS mutants appeared to retard the nuclear import of PYDV-P. Taken together, it was determined that PYDV-N contains the bipartite NLS 419QKRANEEAPPAAQKR433. Similarly, alanine-scanning mutagenesis was performed to determine the regions responsible for the nuclear import of PYDV-M and -P. A non-canonical NLS was identified in PYDV-P, consisting of three regions in the N-terminus of the protein required for nuclear import. PYDV-P does not interact with any Nicotiana benthamiana importins, but was found to interact with importin-α7 and -α9 of the non-host plant Arabidopsis thaliana. Two amino acids of PYDV-M, 225KR226, were found to be critical for nuclear import and interaction with importin-α. In addition, site-directed mutagenesis identified that amino acids 223LL224 of PYDV-M, which are adjacent to the two amino acids identified as responsible for nuclear import, are critical for inducing invaginations of the inner nuclear membrane. Bimolecular fluorescence complementation (BiFC) was then used to identify any differences in localization and interaction caused by the mutations introduced to PYDV-P and -M. The PYDV-P and -M proteins were still able to interact with other PYDV proteins, although the localization of the interaction differs between mutants.

Dictyostelium is a model eukaryote for the study of a multitude of fundamental cellular processes as well as several human diseases. Despite its extensive study relatively little is known about its nucleolus. Only three nucleolar proteins have been identified. The nucleolus in Dictyostelium is different than that of other eukaryotes since it is neither bipartite nor tripartite, possessing no visible subcompartments at the ultrastructural level. Moreover, it exists as two to four patches adjacent to the inner nuclear envelope instead of within the nucleoplasm. The aim of this study was thus to identify and characterize novel nucleolar proteins in Dictyostelium in order to better understand the structure and function of its nucleolus. Previous work had shown that NumA1, a protein linked to cell cycle in Dictyostelium, localizes to similar intranuclear patches suggesting it may be nucleolar. NumA1-binding partners Ca2+-binding protein (CBP) 4a and puromycin-sensitive aminopeptidase A may therefore also reside in the nucleolus. Based on the function of a potential NumA1 homologue in other organisms, BRG1-associated factor 60a homologue Snf12 and checkpoint kinase 2 (Rad53 in yeast) homologue forkhead-associated kinase (Fhk) A were chosen as potential nucleolar proteins in Dictyostelium that may also be involved in cell cycle events. Using a diversity of approaches, this study found that NumA1, CBP4a, Snf12, and FhkA are nucleolar proteins in Dictyostelium while puromycin-sensitive aminopeptidase A is nucleoplasmic. Several nuclear localization signals (NLSs) were identified in these proteins some of which also act as nucleolar localization signals (NoLSs). These NLS/NoLSs (within NumA1 and Snf12) represent the first NoLSs and first NLS/NoLSs identified in Dictyostelium. Treatment with the rDNA transcription inhibitor AM-D led to the budding of nucleolar CBP4a, Snf12, and FhkA from the nucleus to the cytoplasm, a phenomenon not previously observed in any organism. This study also examined for the first time the redistribution of nucleolar proteins during mitosis, a time when the nucleolus disassembles into its component parts. The nuclear envelope was also shown to become permeable at this time. Finally, multiple nucleolar subcompartments were identified suggesting compartmentalization of different functions in the Dictyostelium nucleolus.