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Autophagy is a key catabolic process responsible for the removal of waste and damaged cell components by lysosomal degradation. It plays an important role in fundamental cell processes, including ER stress mitigation, control of cell metabolism, and cell differentiation and proliferation, all of which are essential for the development and survival of bone and cartilage cells, and for skeletogenesis. Correspondingly, autophagy dysregulation has been implicated in several skeletal disorders, including the joint disease, osteoarthritis (OA). OA is the most common cause of arthritis globally and one the biggest causes of disability, affecting 15% of the UK population. It is characterised by the progressive degeneration of articular cartilage at the joint interface, leading to exposure of the underlying bone and joint misalignment, pain, and loss of function. In this thesis, the role of autophagy-related genes: atg13 and lmx1b, in skeletal development and joint function was explored using zebrafish lines mutant for these genes. Loss of autophagy activity in the atg13 mutants caused an acceleration to chondrocyte (cartilage cell) maturation, leading to changes in extra-cellular matrix (ECM) formation and restricted jaw joint mobility. These results are consistent with autophagy contributing to the regulation of chondrocyte maturation which supports joint formation and function. Meanwhile, characterisation of the lmx1b mutant lines demonstrated that the lmx1b paralogues, lmx1ba and lmx1bb have divergent roles in zebrafish development, affecting skeletal and neuronal development, and renal development, respectively. Whilst loss of both paralogues (lmx1b dKO) resulted in muscular abnormalities and reduced body growth. As adults, loss of lmx1b caused skeletal changes reminiscent of premature OA and behavioural changes, suggesting a role for lmx1b in skeletal and neuronal development and maintenance. Overall, these results expand our understanding of the roles played

Autophagy is a key catabolic process responsible for the removal of waste and damaged cell components by lysosomal degradation. It plays an important role in fundamental cell processes, including ER stress mitigation, control of cell metabolism, and cell differentiation and proliferation, all of which are essential for the development and survival of bone and cartilage cells, and for skeletogenesis. Correspondingly, autophagy dysregulation has been implicated in several skeletal disorders, including the joint disease, osteoarthritis (OA). OA is the most common cause of arthritis globally and one the biggest causes of disability, affecting 15% of the UK population. It is characterised by the progressive degeneration of articular cartilage at the joint interface, leading to exposure of the underlying bone and joint misalignment, pain, and loss of function. In this thesis, the role of autophagy-related genes: atg13 and lmx1b, in skeletal development and joint function was explored using zebrafish lines mutant for these genes. Loss of autophagy activity in the atg13 mutants caused an acceleration to chondrocyte (cartilage cell) maturation, leading to changes in extra-cellular matrix (ECM) formation and restricted jaw joint mobility. These results are consistent with autophagy contributing to the regulation of chondrocyte maturation which supports joint formation and function. Meanwhile, characterisation of the lmx1b mutant lines demonstrated that the lmx1b paralogues, lmx1ba and lmx1bb have divergent roles in zebrafish development, affecting skeletal and neuronal development, and renal development, respectively. Whilst loss of both paralogues (lmx1b dKO) resulted in muscular abnormalities and reduced body growth. As adults, loss of lmx1b caused skeletal changes reminiscent of premature OA and behavioural changes, suggesting a role for lmx1b in skeletal and neuronal development and maintenance. Overall, these results expand our understanding of the roles played

Understanding the role of autophagy in cancer has been limited by the inability to measure this dynamic process in formalin-fixed tissue. We considered that 3-dimensional models including ex vivo tumor, such as we have developed for studying mesothelioma, would provide valuable insights. Using these models, in which we could use lysosomal inhibitors to measure the autophagic flux, we sought a marker of autophagy that would be valid in formalin-fixed tumor and be used to assess the role of autophagy in patient outcome. Autophagy was studied in mesothelioma cell lines, as 2-dimensional (2D) monolayers and 3-dimensional (3D) multicellular spheroids (MCS), and in tumor from 25 chemonaive patients, both as ex vivo 3D tumor fragment spheroids (TFS) and as formalin-fixed tissue. Autophagy was evaluated as autophagic flux by detection of the accumulation of LC3 after lysosomal inhibition and as autophagy initiation by detection of ATG13 puncta. We found that autophagic flux in 3D, but not in 2D, correlated with ATG13 positivity. In each TFS, ATG13 positivity was similar to that of the original tumor. When tested in tissue microarrays of 109 chemonaive patients, higher ATG13 positivity correlated with better prognosis and provided information independent of known prognostic factors. Our results show that ATG13 is a static marker of the autophagic flux in 3D models of mesothelioma and may also reflect autophagy levels in formalin-fixed tumor. If confirmed, this marker would represent a novel prognostic factor for mesothelioma, supporting the notion that autophagy plays an important role in this cancer.

Understanding the role of autophagy in cancer has been limited by the inability to measure this dynamic process in formalin-fixed tissue. We considered that 3-dimensional models including ex vivo tumor, such as we have developed for studying mesothelioma, would provide valuable insights. Using these models, in which we could use lysosomal inhibitors to measure the autophagic flux, we sought a marker of autophagy that would be valid in formalin-fixed tumor and be used to assess the role of autophagy in patient outcome. Autophagy was studied in mesothelioma cell lines, as 2-dimensional (2D) monolayers and 3-dimensional (3D) multicellular spheroids (MCS), and in tumor from 25 chemonaive patients, both as ex vivo 3D tumor fragment spheroids (TFS) and as formalin-fixed tissue. Autophagy was evaluated as autophagic flux by detection of the accumulation of LC3 after lysosomal inhibition and as autophagy initiation by detection of ATG13 puncta. We found that autophagic flux in 3D, but not in 2D, correlated with ATG13 positivity. In each TFS, ATG13 positivity was similar to that of the original tumor. When tested in tissue microarrays of 109 chemonaive patients, higher ATG13 positivity correlated with better prognosis and provided information independent of known prognostic factors. Our results show that ATG13 is a static marker of the autophagic flux in 3D models of mesothelioma and may also reflect autophagy levels in formalin-fixed tumor. If confirmed, this marker would represent a novel prognostic factor for mesothelioma, supporting the notion that autophagy plays an important role in this cancer.

SummaryThe biogenesis of autophagosomes commences at the phagophore assembly site (PAS), a protein-vesicle ultrastructure that is organized by the Atg1 complex. The Atg1 complex consists of the Atg1 protein kinase, the intrinsically disordered region-rich Atg13, and the dimeric double crescent-shaped Atg17-Atg31-Atg29 subcomplex. We show that the PAS contains a relatively uniform ~28 copies of Atg17, and upon autophagy induction, similar numbers of Atg1 and Atg13 molecules. We then apply ensemble refinement of small angle x-ray scattering (SAXS) to determine the solution structures of the Atg1-Atg13 and Atg17-Atg31-Atg29 subcomplexes and the Atg1 complex, using a trimmed “mini-pentamer” tractable to biophysical studies. We observe tetramers of Atg1 pentamers that assemble via Atg17-Atg31-Atg29. This leads to a model for the higher organization of the Atg1 complex in PAS scaffolding.

SummaryThe biogenesis of autophagosomes commences at the phagophore assembly site (PAS), a protein-vesicle ultrastructure that is organized by the Atg1 complex. The Atg1 complex consists of the Atg1 protein kinase, the intrinsically disordered region-rich Atg13, and the dimeric double crescent-shaped Atg17-Atg31-Atg29 subcomplex. We show that the PAS contains a relatively uniform ~28 copies of Atg17, and upon autophagy induction, similar numbers of Atg1 and Atg13 molecules. We then apply ensemble refinement of small angle x-ray scattering (SAXS) to determine the solution structures of the Atg1-Atg13 and Atg17-Atg31-Atg29 subcomplexes and the Atg1 complex, using a trimmed “mini-pentamer” tractable to biophysical studies. We observe tetramers of Atg1 pentamers that assemble via Atg17-Atg31-Atg29. This leads to a model for the higher organization of the Atg1 complex in PAS scaffolding.

Autophagy is a conserved mechanism that is essential for cell survival in starvation. Moreover, autophagy maintains cellular health by clearing unneeded or harmful materials from cells. Autophagy proceeds by the engulfment of bulk cytosol and organelles by a cup-shaped double-membrane sheet known as the phagophore. The phagophore closes on itself to form the autophagosome, which delivers its contents to the vacuole or lysosome for degradation. A multiprotein complex comprising the protein kinase autophagy-related protein 1 (Atg1) together with Atg13, Atg17, Atg29, and Atg31 (ULK1, ATG13, FIP200, and ATG101 in humans) has a pivotal role in the earliest steps of this process. This review summarizes recent structural and ultrastructural analysis of the earliest step in autophagosome biogenesis and discusses a model in which the Atg1 complex clusters high-curvature vesicles containing the integral membrane protein Atg9, thereby initiating the phagophore.

Autophagy is a conserved mechanism that is essential for cell survival in starvation. Moreover, autophagy maintains cellular health by clearing unneeded or harmful materials from cells. Autophagy proceeds by the engulfment of bulk cytosol and organelles by a cup-shaped double-membrane sheet known as the phagophore. The phagophore closes on itself to form the autophagosome, which delivers its contents to the vacuole or lysosome for degradation. A multiprotein complex comprising the protein kinase autophagy-related protein 1 (Atg1) together with Atg13, Atg17, Atg29, and Atg31 (ULK1, ATG13, FIP200, and ATG101 in humans) has a pivotal role in the earliest steps of this process. This review summarizes recent structural and ultrastructural analysis of the earliest step in autophagosome biogenesis and discusses a model in which the Atg1 complex clusters high-curvature vesicles containing the integral membrane protein Atg9, thereby initiating the phagophore.