Continuing the resolution revolution: The living cell contains dynamic, spatially complex subassemblies that are sensitive to external perturbations. To minimize such perturbations, cells should be imaged in their native multicellular environments, under as gentle illumination as possible. However, achieving the spatiotemporal resolution needed to follow three-dimensional subcellular processes in detail under these conditions is challenging: Sample-induced aberrations degrade resolution and sensitivity, and high resolution usually requires intense excitation. Liu et al. combined noninvasive lattice light-sheet microscopy with aberration-correcting adaptive optics to study a variety of delicate subcellular events in vivo, including organelle remodeling during mitosis and growth cone dynamics during spinal cord development. Science , this issue p. eaaq1392

It has long been postulated, although never directly demonstrated, that mitochondria are strategically positioned in the cytoplasm to meet local requirements for energy production. Here we show that positioning of mitochondria in mouse embryonic fibroblasts (MEFs) determines the shape of intracellular energy gradients in living cells. Specifically, the ratio of ATP to ADP was highest at perinuclear areas of dense mitochondria and gradually decreased as more-peripheral sites were approached. Furthermore, the majority of mitochondria were positioned at the ventral surface of the cell, correlating with high ATP:ADP ratios close to the ventral membrane, which rapidly decreased toward the dorsal surface. We used cells deficient for the mitochondrial Rho-GTPase 1 (Miro1), an essential mediator of microtubule-based mitochondrial motility, to study how changes in mitochondrial positioning affect cytoplasmic energy distribution and cell migration, an energy-expensive process. The mitochondrial network in Miro1^−/− MEFs was restricted to the perinuclear area, with few mitochondria present at the cell periphery. This change in mitochondrial distribution dramatically reduced the ratio of ATP to ADP at the cell cortex and disrupted events essential for cell movement, including actin dynamics, lamellipodia protrusion, and membrane ruffling. Cell adhesion status was also affected by changes in mitochondrial positioning; focal adhesion assembly and stability was decreased in Miro1^−/−MEFs compared with Miro1^+/+ MEFs. Consequently Miro1^−/− MEFs migrated slower than control cells during both collective and single-cell migration. These data establish that Miro1-mediated mitochondrial positioning at the leading edge provides localized energy production that promotes cell migration by supporting membrane protrusion and focal adhesion stability.

Mitochondria are strategically trafficked throughout the cell by the action of microtubule motors, the actin cytoskeleton and adapter proteins. The intracellular positioning of mitochondria supports subcellular levels of ATP, Ca2+ and reactive oxygen species (ROS, i.e. hydrogen peroxide, H2O2). Previous work from our group showed that deletion of the mitochondrial adapter protein Miro1 leads to perinuclear clustering of mitochondria, leaving the cell periphery devoid of mitochondria which compromises peripheral energy status. Herein, we report that deletion of Miro1 significantly restricts subcellular H2O2 levels to the perinuclear space which directly affects intracellular responses to elevated mitochondrial ROS. Using the genetically encoded H2O2-responsive fluorescent biosensor HyPer7, we show that the highest levels of subcellular H2O2 map to sites of increased mitochondrial density. Deletion of Miro1 or disruption of microtubule dynamics with Taxol significantly reduces peripheral H2O2 levels. Following inhibition of mitochondrial complex 1 with rotenone we observe elevated spikes of H2O2 in the cell periphery and complementary oxidation of mitochondrial peroxiredoxin 3 (PRX3) and cytosolic peroxiredoxin 2 (PRX2). Conversely, in cells lacking Miro1, rotenone did not increase peripheral H2O2 or PRX2 oxidation but rather lead to increased nuclear H2O2 and an elevated DNA-damage response. Lastly, local levels of HyPer7 oxidation correlate with the size and abundance of focal adhesions (FAs) in MEFs and cells lacking Miro1 have significantly smaller focal adhesions and reduced phosphorylation levels of vinculin and p130Cas compared to Miro1+/+ MEFs. Together, we present evidence that the intracellular distribution of mitochondria influences subcellular H2O2 levels and local cellular responses dependent on mitochondrial ROS.

The lung is comprised of more than 40 distinct cell types that support a complex 3-dimensional (3D) architecture that is required for efficient lung function. Loss of this proper architecture can accommodate and promote lung disease, highlighting researchers’ growing need to analyze lung structures in detail. Additionally, in vivo cellular and molecular response to chemical and physical signals, along with the recapitulation of gene-expression patterns, can be lost during the transition from complex 3D tissues to 2D cell culture systems. Therefore, technologies that allow for the investigation of lung function under normal and disease states utilizing the entirety of the lung architecture are required to generate a complete understanding of these processes. Airway cell-derived organoids that can recapitulate lung structure and function ex vivo while being amenable to experimental manipulation, have provided a new and exciting model system to investigate lung biology. In this perspective, we discuss emerging technologies for culturing lung-derived organoids, techniques to visualize organoids using high-resolution microscopy and the resulting information extracted from organoids supporting research focused on lung function and diseases.

Mitochondria regulate a myriad of cellular functions. Dysregulation of mitochondrial control within airway epithelial cells has been implicated in the pro-inflammatory response to allergens in asthma patients. Because of their multifaceted nature, mitochondrial structure must be tightly regulated through fission and fusion. Dynamin Related Protein 1 (DRP1) is a key driver of mitochondrial fission. During allergic asthma, airway epithelial mitochondria appear smaller and structurally altered. The role of DRP1-mediated mitochondrial fission, however, has not been fully elucidated in epithelial response to allergens. We used a Human Bronchial Epithelial Cell line (HBECs), primary Mouse Tracheal Epithelial Cells (MTECs), and conditional DRP1 ablation in lung epithelial cells to investigate the impact of mitochondrial fission on the pro-inflammatory response to house dust mite (HDM) in vitro and in vivo. Our data suggest that, following HDM challenge, mitochondrial fission is rapidly upregulated in airway epithelial cells and precedes production of pro-inflammatory cytokines and chemokines. Further, deletion of Drp1 in lung epithelial cells leads to decreased fission and enhanced pro-inflammatory signaling in response to HDM in vitro, as well as enhanced airway hyper-responsiveness (AHR), inflammation, differential mucin transcription, and epithelial cell death in vivo. Mitochondrial fission, therefore, regulates the lung epithelial pro-inflammatory response to HDM.

A central hallmark of tumorigenesis is metabolic alterations that increase mitochondrial reactive oxygen species (mROS). In response, cancer cells upregulate their antioxidant capacity and redox-responsive signaling pathways. A promising chemotherapeutic approach is to increase ROS to levels incompatible with tumor cell survival. Mitochondrial peroxiredoxin 3 (PRX3) plays a significant role in detoxifying hydrogen peroxide (H₂O₂). PRX3 is a molecular target of thiostrepton (TS), a natural product and FDA-approved antibiotic. TS inactivates PRX3 by covalently adducting its two catalytic cysteine residues and crosslinking the homodimer. Using cellular models of malignant mesothelioma, we show here that PRX3 expression and mROS levels in cells correlate with sensitivity to TS and that TS reacts selectively with PRX3 relative to other PRX isoforms. Using recombinant PRXs 1–5, we demonstrate that TS preferentially reacts with a reduced thiolate in the PRX3 dimer at mitochondrial pH. We also show that partially oxidized PRX3 fully dissociates to dimers, while partially oxidized PRX1 and PRX2 remain largely decameric. The ability of TS to react with engineered dimers of PRX1 and PRX2 at mitochondrial pH, but inefficiently with wild-type decameric protein at cytoplasmic pH, supports a novel mechanism of action and explains the specificity of TS for PRX3. Thus, the unique structure and propensity of PRX3 to form dimers contribute to its increased sensitivity to TS-mediated inactivation, making PRX3 a promising target for prooxidant cancer therapy.