474 research outputs found

    Development of variable and robust brain wiring patterns in the fly visual system

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    Precise generation of synapse-specific neuronal connections are crucial for establishing a robust and functional brain. Neuronal wiring patterns emerge from proper spatiotemporal regulation of axon branching and synapse formation during development. Several neuropsychiatric and neurodevelopmental disorders exhibit defects in neuronal wiring owing to synapse loss and/or dys-regulated axon branching. Despite decades of research, how the two inter-dependent cellular processes: axon branching and synaptogenesis are coupled locally in the presynaptic arborizations is still unclear. In my doctoral work, I investigated the possible role of EGF receptor (EGFR) activity in coregulating axon branching and synapse formation in a spatiotemporally restricted fashion, locally in the medulla innervating Dorsal Cluster Neuron (M- DCN)/LC14 axon terminals. In this work I have explored how genetically encoded EGFR randomly recycles in the axon branch terminals, thus creating an asymmetric, non-deterministic distribution pattern. Asymmetric EGFR activity in the branches acts as a permissive signal for axon branch pruning. I observed that the M-DCN branches which stochastically becomes EGFR ‘+’ during development are synaptogenic, which means they can recruit synaptic machineries like Syd1 and Bruchpilot (Brp). My work showed that EGFR activity has a dual role in establishing proper M-DCN wiring; first in regulating primary branch consolidation possibly via actin regulation prior to synaptogenesis. Later in maintaining/protecting the levels of late Active Zone (AZ) protein Brp in the presynaptic branches by suppressing basal autophagy level during synaptogenesis. When M-DCNs lack optimal EGFR activity, the basal autophagy level increases resulting in loss of Brp marked synapses which is causal to increased exploratory branches and post-synaptic target loss. Lack of EGFR activity affects the M-DCN wiring pattern that makes adult flies more active and behave like obsessive compulsive in object fixation assay. In the second part of my doctoral work, I have asked how non-genetic factors like developmental temperature affects adult brain wiring. To test that, I increased or decreased rearing temperature which is known to inversely affect pupal developmental rate. We asked if all the noisy cellular processes of neuronal assembly: filopodial dynamics, axon branching, synapse formation and postsynaptic connections scale up or down accordingly. I observed that indeed all the cellular processes slow down at lower developmental temperature and vice versa, which changes the DCN wiring pattern accordingly. Interestingly, behavior of flies adapts to their developmental temperature, performing best at the temperature they have been raised at. This shows that optimal brain function is an adaptation of robust brain wiring patterns which are specified by noisy developmental processes. In conclusion, my doctoral work helps us better understand the developmental regulation of axon branching and synapse formation for establishing precise brain wiring pattern. We need all the cell intrinsic developmental processes to be highly regulated in space and time. It is infact a combinatorial effect of such stochastic processes and external factors that contribute to the final outcome, a functional and robust adult brain

    Dissecting Extracellular Matrix Internalisation Mechanisms using Functional Genomics

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    Breast and ovarian malignancies account for one third of female cancers. The role of the stroma in supporting invasive growth in breast cancer has become clear. Breast cancer cells interact and respond to the cues from the surrounding extracellular matrix (ECM). Integrins are main cell adhesion receptors and key players in invasive migration by linking the ECM to the actin cytoskeleton. In addition, integrins mediate distinctive biochemical and biomechanical signals to support cancer invasion. The role of matrix proteases in promoting ECM degradation and cancer dissemination has been extensively studied; however, cancer cells possess additional means to support those processes, such as integrin-mediated ECM endocytosis and consequent degradation in the lysosomes. Internalisation of the extracellular matrix is upregulated in invasive breast cancer. Nonetheless, the mechanisms by which cancer cells regulate this process are poorly understood. We developed a high throughput pH sensitive system to detect ECM uptake. Here, we show that MDA-MB-231 breast cancer cells converge in macropinocytosis to internalise diverse ECM components and we confirm that this process is modulated by PAK1. To unravel which ECM components breast cancer cells internalise in a complex environment (namely, cell derived matrices), we performed mass spectrometry. Proteomic analysis identified Annexin A6, Collagen VI, Tenascin C and fibronectin, among other matrisome proteins, to be internalised by invasive breast cancer cells. Following ECM endocytosis, ECM is targeted for lysosomal degradation. To unravel the molecular mechanisms behind this process, we performed a trafficking screen and identified the AP3 complex, VAMP7, Arf1 and ARFGEF2. Our results suggest that the AP3 complex may regulate ECM-integrin delivery to lysosomes. To gain more insight on the signalling pathways governing macropinocytosis in breast cancer cells, we performed a kinase and phosphatase screen that unravelled MAP3K1 and PPP2R1A, a subunit of protein phosphatase 2A (PP2A) as relevant regulators of ECM endocytosis. Furthermore, our data suggests that p38 mitogen-activated protein kinase (MAPK) activation upon binding to the ECM is required for ECM macropinocytosis. Outstandingly, inhibiting p38 MAPK led to profound changes in the ability of breast cancer cells to migrate in cell derived matrices. Previous work from the Rainero lab focused on characterising the receptors involved in ECM internalisation; α2β1 integrin was identified as the main regulator of ECM uptake in MDA-MB-231 cells. In particular, α2β1 integrin has been shown to activate p38 MAPK pathway. Taken together, we hypothesise that binding of ECM to α2β1 integrin results in the activation of PAK1 and MAP3K1, which in turn leads to ECM endocytosis. p38 MAPK activity may induce changes in actin polymerisation via PPP2R1A and/or focal adhesion turnover, which consequently promotes ECM macropinocytosis and invasive migration

    Adaptations of Cryptococcus to the host extracellular niche

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    Cryptococcus is an opportunistic human fungal pathogen with the potential to cause life- threatening infections of the central nervous system. Ubiquitous in the environment, Cryptococcus switches from a saprotrophic lifestyle in the environment to a pathogenic lifestyle in humans by altering key cellular functions required for adaptation and invasion of the host. Once inhaled, the host extracellular niche (particularly the lung mucosa) serves as a modulator for cryptococcal adaptive phenotypes which are critical for survival and proliferation. However, the cellular changes exhibited by Cryptococcus as it responds to the pulmonary environment is poorly understood. The surface of the lung mucosa is heavily loaded with secretions from host lung cells, such as Type I and II epithelial cells as well as alveolar macrophages. Upon inhalation, Cryptococcus comes into close contact with these secretions. The first part of this thesis probed and described the potency of mammalian cells secreted factors (including lung-associated cells) to stimulate phenotypic responses that are associated with Cryptococcus adaptive mechanisms. While C. neoformans responded to these secreted factors with rapid replication, its sister species C. gattii instead demonstrated a high capacity to form enormously enlarged titan-like cells. Within the lungs, Cryptococcus undergoes morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. In the second part of this thesis, a new in vitro titan-induction approach is introduced. Using this in vitro approach, I revealed a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterised strain-specific differences within the clade. In addition, this approach demonstrates for the first time that the cell-cycle-regulated phenotypes: cell size changes, DNA replication and budding, are not always synchronous during titanisation. i Titan cell formation is triggered by host-specific environmental conditions [such as physiological temperature (37°C) and CO2 level (5%) coupled with hypoxia, and nutrient limitation] and modulated by genetic regulators including those associated with cell cycle progression. The last part of this thesis established a strong correlation between progression of the cell cycle phenotypes (cell size, DNA replication and budding) and expression of cell cycle genes and identifies the role of cryptococcal quorum sensing peptide Qsp1 peptide and exogenous p-Aminobenzoic acid as a key inducer of titanisation in C. gattii

    Identification and characterization of N-degron pathways

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    Papel del transportoma en quimiorresistencia y quimiosensibilización en hepatoblastoma

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    [ES] Se profundiza en el estudio del MOC1 para identificar los transportadores de membrana plasmática responsables de la refractariedad a fármacos antitumorales del HB, lo que podría ser de gran utilidad para emplearlos como biomarcadores diagnósticos y pronósticos. Esto es especialmente importante en pacientes pediátricos por su mayor fragilidad y la inmadurez de sus órganos que pueden determinar en gran medida la eficacia de los fármacos antitumorales y sus efectos secundarios. Esta información es también crucial para el desarrollo de nuevas estrategias para el tratamiento del tumor, pues se podrían utilizar como dianas para quimiosensibilizar al HB frente a los fármacos antitumorales

    Investigating the regulation and putative interphase function of the condensin II subunit NCAPH2

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    Condensins are protein complexes imperative for the individualisation and rigidity of mitotic chromatids. The functions of these highly conserved proteins are essential for the successful inheritance of genetic material during cell division. Mutations in condensin subunits cause DNA damage and abnormal ploidy in cells, and tissue-specific phenotypes such as T-cell lymphoma and microcephaly when inherited through the mammalian germline. The kleisin subunit NCAPH2 is dose limiting for condensin II assembly, and has been suggested to function during interphase as well as mitosis. This thesis studies how the level of NCAPH2 protein, and thus presumably condensin II, is regulated, and explores possible interphase roles. Within the 5’UTR of NCAPH2 is an upstream open reading frame (uORF), a potential cis-regulator of NCAPH2 protein synthesis. Many uORFs have been characterised throughout the genome as negative regulators of translation efficiency. These 5’ UTR elements have been proposed to act through several mechanisms, which ultimately result in a reduction in the number of ribosomes commencing translation at their main ORF initiation codon. The human NCAPH2 uORF has been studied previously using transient over expression assays, which are limited in their ability to capture the true influence of the uORF in its endogenous context. When looking at ribosome occupancy at this putative translational regulatory element, analysis of public data show that the uORF is robustly occupied by ribosomes in a variety of mammalian cell types. Consequently, I hypothesised that this region could be important in regulating NCAPH2 protein levels. Using transient reporter assays designed to test the regulatory ability of both the mouse and human 5’ UTR of NCAPH2, I established that this conserved uORF was capable of regulating downstream translation in cis. Upon this finding, I then performed base-editing of the endogenous uORF locus in a NCAPH2AID:mCherry/AID:mCherry reporter cell line. The resulting ΔuORF cell line revealed that this element does regulate NCAPH2 protein levels at the endogenous locus. Further work to characterise the consequences of its loss on condensin II function found that ΔuORF cells were able to divide at similar rates to the parental cell line, and that the rate of de novo NCAPH2 synthesis was not detectably altered. Studies of condensin function during interphase have historically used RNAi or constitutive mutations to perturb protein function. Previous work in our lab generated novel CRISPR-engineered mice, in which auxin-inducible degron-tagged NCAPH2 can be rapidly depleted upon addition of the small molecule auxin. Using this system, experiments were performed ex vivo and in vivo to monitor the effects of rapid NCAPH2 loss on peripheral T-cells and thymic precursors. This enabled me to characterise the impact of rapid NCAPH2 loss at different stages of cellular differentiation, and at specific cell cycle phases. Contrary to previous reports, acute loss of NCAPH2 did not alter STAT5 target gene expression, ability to exit quiescence, or subnuclear heterochromatin distribution. Rather, it caused abnormalities consistent with known mitotic functions of condensin II. The Ncaph2AID tagged allele was subsequently combined with Ncaph2I15N, a germline mutation which causes a developmental block during thymopoiesis. By generating compound heterozygous animals combining AID-tagged alleles and germline I15N mutations, I conditionally depleted tagged NCAPH2 to study the consequences of the missense mutation in a thymus that had developed normally, without downstream consequences of prior defective cell divisions. Finally, previous work from our lab using Ncaph- and Ncaph2-AID tagged mice revealed that depletion of either subunit in precursor thymic T and bone marrow B cells resulted in a significant decrease in cell division efficiency, but not in mature peripheral lymphocytes. By combining Ncaph- and Ncaph2-AID tagged alleles, I found that simultaneous loss of both kleisin subunits severely impacts cell division efficiency in the periphery. These results support previous findings that different cell types have different vulnerabilities to condensin deficiencies, despite sharing the same genetic load
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