Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo–electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic β-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells

Nucleoporin mRNA localization and Annulate Lamellae biosynthesis during Drosophila melanogaster oogenesis

Nuclear pore complexes (NPCs) are large protein assemblies that connect the eukaryotic nucleus with the cytoplasm, thus facilitating all transport between them. Besides the nuclear envelope (NE), NPCs also occur in parallel stacks of cytoplasmic membranes called Annulate Lamellae (AL) that can serve as storage, facilitating rapid nuclear growth via NE insertion during fruit fly embryogenesis. How and when AL are assembled is largely unknown. In this work, I established that AL are already abundant in late stage oocytes, and progressively accumulate throughout oogenesis specifically in the oocyte. By screening the localization of 39 nucleoporin and related mRNAs, I detected the specific enrichment of two nucleoporin and three importin encoding transcripts to AL, the NE, and previously unidentified nucleoporin granules throughout the egg chamber. Perturbation experiments revealed a dependence on active translation, but independence of an intact microtubule network on mRNA localization. Generation of GFP::Nup358 transgenic flies revealed granules with distinct partial nucleoporin contents, that are subject to microtubule-dependent transport and interactions among them. Their spatiotemporal abundance distribution is indicative of NPC precursors, and they contain partial accumulations of pore complexes within internal membranes. These granules further displayed characteristics of biomolecular condensates, including fast intra-granule dynamics, exclusion of cytoplasmic constituents, and sensitivity to 1,6-hexanediol. Both condensation state and AL assembly were dependent on Ran, a protein also fundamental for NPC assembly at the NE. Its nucleotide status throughout this is likely controlled by differential localization of its modulators RanGAP and Rcc1 to granules and cytoplasm respectively. This work thus established a molecular framework and basic sequence of events that leads to the assembly of AL, which are crucial during early development, and might have broader implications for NPC assembly also at the NE

Conformational dynamics of coatomer : functional and structural studies

In my PhD thesis I have investigated molecular mechanisms in the biogenesis of membrane vesicles. Formation of transport vesicles involves polymerization of cytoplasmic coat proteins. In COPI vesicle biogenesis, the heptameric complex coatomer is recruited to donor membranes by the interaction of multiple coatomer subunits with the budding machinery. Specific binding to the trunk domain of coatomer's subunit gamma-COP of the Golgi membrane protein p23 induces a conformational change in the gamma-subunit, leading to polymerization of the complex in vitro. Using a combination of biochemical assays and an assay based on single-molecule, singlepair fluorescence resonance energy transfer, we find that this conformational change is only induced by dimers of the p24-family proteins p23 and p24, and neither by the other p24-family members nor by cargo proteins. This conformational change takes place in individual coatomer complexes, independent of each other, and the rearrangement induced in gamma-COP is transmitted within the complex to its alpha-subunit. Alpha-COP is one of coatomer's subunits capable of binding to dibasic cargo motifs, and also shows analogy to the Clathrin molecule. We propose a model in which capture of membrane protein machinery triggers cage formation in the COPI system. At the nanometer resolution I started investigating the structure of the lattice of conformationally changed coatomer on COPI vesicles generated in vitro from purified Golgi membranes and coating machinery, using cryo electron tomography. Initial data on coated vesicles and coated buds is presented

Lysosome Turnover in the Retinal Pigment Epithelium in Health, Ageing and Age- related Macular Degeneration

The Retinal Pigment Epithelium (RPE) phagocytoses and degrades spent Photoreceptor Outer Segments (POS) every day, placing an unparalleled burden on lysosomes in RPE cells, which require well-regulated turnover to maintain degradative capacity. With age and in Age-related Macular Degeneration (AMD), debris accumulates inside and outside RPE cells leading to the hypothesis that lysosome dysfunction could contribute to AMD pathology. This project aimed to identify the functional relevance of different subpopulations of lysosome within the heterogenous lysosomal compartment of RPE cells. Models of lysosome dysfunction were then generated in order to determine whether they recapitulate hallmarks of aging and AMD. Electron microscopy (EM) and electron tomography of primary porcine RPE (pRPE) revealed morphologically distinct subpopulations of lysosomes that form a complex network. Pulse-chase experiments showed that multilamellar Cathepsin D-rich lysosomes transition to electron dense lysosomes that stained poorly for Cathepsin D, suggesting that these subpopulations represent different stages of the lysosome cycle. Tracking of phagocytosed POS suggests that POS degradation occurs by both full fusion and kiss-and-run interactions between phagosomes and lysosomes. Correlative light and electron microscopy, and high content imaging, demonstrated that POS loading induces the gradual formation of lipofuscin-like autofluorescent granules. Loading cells with degradation- resistant UV-irradiated POS induced the formation of more and larger autofluorescent granules, thus recapitulating one of the hallmarks of aging and AMD. Treatment with the acidotropic compound chloroquine caused accumulation of undigested phagocytic, endocytic and autophagic cargo, consistent with lysosome dysfunction. This was accompanied by accumulation of lamellar lysosomes containing unprocessed lysosomal Cathepsin D, a feature 4 recapitulated by direct protease inhibition and prevented by inhibiting protein synthesis. Together, the findings in this PhD project shed new light on the identity, function and fate of lysosomes in the RPE and suggest that lysosome dysfunction could contribute to the pathology of AMD, as well as to chloroquine-induced retinopathy

Microscopy Conference 2017 (MC 2017) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2017", die vom 21. bis 25.08.2017, in Lausanne stattfand