Expansion microscopy of the chick embryo neural tube to overcome molecular crowding at the centrosomes-cilia.

We describe an optimized protocol for application of expansion microscopy (ExM) on chick neural tube (NT) which enables different oriented nanoscale resolution imaging of the centrosomes/cilia. We explain embryo NT transversal sections and open-book preparations, immunohistochemistry for labeling, and sample preparation for 5-fold tissue expansion. Further, we detail sample orientation and Fast Airyscan confocal acquisition and show that NT-ExM retains fluorescence signals and overcomes biomolecules crowding in structural features that to date were only imaged with electron microscopy on tissues.

Disruption of mitochondrial dynamics triggers muscle inflammation through interorganellar contacts and mitochondrial DNA mislocation.

Some forms of mitochondrial dysfunction induce sterile inflammation through mitochondrial DNA recognition by intracellular DNA sensors. However, the involvement of mitochondrial dynamics in mitigating such processes and their impact on muscle fitness remain unaddressed. Here we report that opposite mitochondrial morphologies induce distinct inflammatory signatures, caused by differential activation of DNA sensors TLR9 or cGAS. In the context of mitochondrial fragmentation, we demonstrate that mitochondria-endosome contacts mediated by the endosomal protein Rab5C are required in TLR9 activation in cells. Skeletal muscle mitochondrial fragmentation promotes TLR9-dependent inflammation, muscle atrophy, reduced physical performance and enhanced IL6 response to exercise, which improved upon chronic anti-inflammatory treatment. Taken together, our data demonstrate that mitochondrial dynamics is key in preventing sterile inflammatory responses, which precede the development of muscle atrophy and impaired physical performance. Thus, we propose the targeting of mitochondrial dynamics as an approach to treating disorders characterized by chronic inflammation and mitochondrial dysfunction.

Proteostasis failure and mitochondrial dysfunction leads to aneuploidy-induced senescence.

Aneuploidy, an unbalanced number of chromosomes, is highly deleterious at the cellular level and leads to senescence, a stress-induced response characterized by permanent cell-cycle arrest and a well-defined associated secretory phenotype. Here, we use a Drosophila epithelial model to delineate the pathway that leads to the induction of senescence as a consequence of the acquisition of an aneuploid karyotype. Whereas aneuploidy induces, as a result of gene dosage imbalance, proteotoxic stress and activation of the major protein quality control mechanisms, near-saturation functioning of autophagy leads to compromised mitophagy, accumulation of dysfunctional mitochondria, and the production of radical oxygen species (ROS). We uncovered a role of c-Jun N-terminal kinase (JNK) in driving senescence as a consequence of dysfunctional mitochondria and ROS. We show that activation of the major protein quality control mechanisms and mitophagy dampens the deleterious effects of aneuploidy, and we identify a role of senescence in proteostasis and compensatory proliferation for tissue repair.

CK1δ restrains lipin-1 induction, lipid droplet formation and cell proliferation under hypoxia by reducing HIF-1α/ARNT complex formation.

Proliferation of cells under hypoxia is facilitated by metabolic adaptation, mediated by the transcriptional activator Hypoxia Inducible Factor-1 (HIF-1). HIF-1α, the inducible subunit of HIF-1 is regulated by oxygen as well as by oxygen-independent mechanisms involving phosphorylation. We have previously shown that CK1δ phosphorylates HIF-1α in its N-terminus and reduces its affinity for its heterodimerization partner ARNT. To investigate the importance of this mechanism for cell proliferation under hypoxia, we visually monitored HIF-1α interactions within the cell nucleus using the in situ proximity ligation assay (PLA) and fluorescence recovery after photobleaching (FRAP). Both methods show that CK1δ-dependent modification of HIF-1α impairs the formation of a chromatin binding HIF-1 complex. This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation. Inhibition of CK1δ increases lipid droplet formation and proliferation of both cancer and normal cells specifically under hypoxia and in an HIF-1α- and lipin-1-dependent manner. These data reveal a novel role for CK1δ in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.