Quantitative subcellular reconstruction reveals a lipid mediated inter-organelle biogenesis network.

The structures and functions of organelles in cells depend on each other but have not been systematically explored. We established stable knockout cell lines of peroxisomal, Golgi and endoplasmic reticulum genes identified in a whole-genome CRISPR knockout screen for inducers of mitochondrial biogenesis stress, showing that defects in peroxisome, Golgi and endoplasmic reticulum metabolism disrupt mitochondrial structure and function. Our quantitative total-organelle profiling approach for focussed ion beam scanning electron microscopy revealed in unprecedented detail that specific organelle dysfunctions precipitate multi-organelle biogenesis defects, impair mitochondrial morphology and reduce respiration. Multi-omics profiling showed a unified proteome response and global shifts in lipid and glycoprotein homeostasis that are elicited when organelle biogenesis is compromised, and that the resulting mitochondrial dysfunction can be rescued with precursors for ether-glycerophospholipid metabolic pathways. This work defines metabolic and morphological interactions between organelles and how their perturbation can cause disease.

Immunofluorescence-Based Measurement of Autophagosome Formation During Mitophagy.

Damaged, dysfunctional, or excess mitochondria are removed from cells via a selective form of macroautophagy termed mitophagy. The clearance of mitochondria during mitophagy is mediated by double-membrane vesicles called autophagosomes, which encapsulate mitochondria that have been tagged for mitophagic removal before delivering them to lysosomes for degradation. A variety of different mitophagy pathways exist that differ in their mechanisms of initiation but share a common pathway of autophagosome formation. Autophagosome biogenesis is regulated by a number of autophagy factors which translocate from the cytosol to spatially defined focal points (foci) on the mitochondrial surface after mitophagy has been initiated. The functional analysis of autophagosome biogenesis requires the use of microscopy-based techniques which assess the recruitment of autophagy factors to mitophagic foci representing autophagosome formation sites. Here, we describe a routine method for the quantitative 3D analysis of mitophagic foci in PINK1/Parkin mitophagy immunofluorescence samples through the application of object-based image analysis (OBIA) to 3D confocal imaging datasets. The approach enables unbiased high-throughput characterisation of autophagosome biogenesis during mitophagy.

STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain.

Following the detection of cytosolic double-stranded DNA from viral or bacterial infection in mammalian cells, cyclic dinucleotide activation of STING induces interferon ß expression to initiate innate immune defenses. STING activation also induces LC3B lipidation, a classical but equivocal marker of autophagy, that promotes a cell-autonomous antiviral response that arose before evolution of the interferon pathway. We report that STING activation induces LC3B lipidation onto single-membrane perinuclear vesicles mediated by ATG16L1 via its WD40 domain, bypassing the requirement of canonical upstream autophagy machinery. This process is blocked by bafilomycin A1 that binds and inhibits the vacuolar ATPase (V-ATPase) and by SopF, a bacterial effector that catalytically modifies the V-ATPase to inhibit LC3B lipidation via ATG16L1. These results indicate that activation of the cGAS-STING pathway induces V-ATPase-dependent LC3B lipidation that may mediate cell-autonomous host defense, an unanticipated mechanism that is distinct from LC3B lipidation onto double-membrane autophagosomes.

Ablation of tau causes an olfactory deficit in a murine model of Parkinson's disease.

Parkinson's disease is diagnosed upon the presentation of motor symptoms, resulting from substantial degeneration of dopaminergic neurons in the midbrain. Prior to diagnosis, there is a lengthy prodromal stage in which non-motor symptoms, including olfactory deficits (hyposmia), develop. There is limited information about non-motor impairments and there is a need for directed research into these early pathogenic cellular pathways that precede extensive dopaminergic death in the midbrain. The protein tau has been identified as a genetic risk factor in the development of sporadic PD. Tau knockout mice have been reported as an age-dependent model of PD, and this study has demonstrated that they develop motor deficits at 15-months-old. We have shown that at 7-month-old tau knockout mice present with an overt hyposmic phenotype. This olfactory deficit correlates with an accumulation of α-synuclein, as well as autophagic impairment, in the olfactory bulb. This pathological feature becomes apparent in the striatum and substantia nigra of 15-month-old tau knockout mice, suggesting the potential for a spread of disease. Initial primary cell culture experiments have demonstrated that ablation of tau results in the release of α-synuclein enriched exosomes, providing a potential mechanism for disease spread. These alterations in α-synuclein level as well as a marked autophagy impairment in the tau knockout primary cells recapitulate results seen in the animal model. These data implicate a pathological role for tau in early Parkinson's disease.

BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.

Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that induce mitochondrial outer membrane permeabilization, leading to cytochrome c release and activation of apoptotic caspases. In the absence of active caspases, mitochondrial DNA (mtDNA) triggers the innate immune cGAS/STING pathway, causing dying cells to secrete type I interferon. How cGAS gains access to mtDNA remains unclear. We used live-cell lattice light-sheet microscopy to examine the mitochondrial network in mouse embryonic fibroblasts. We found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner mitochondrial membrane to herniate into the cytosol, carrying with it mitochondrial matrix components, including the mitochondrial genome. Apoptotic caspases did not prevent herniation but dismantled the dying cell to suppress mtDNA-induced innate immune signaling.

Bacteriophage Transcytosis Provides a Mechanism To Cross Epithelial Cell Layers.

Bacterial viruses are among the most numerous biological entities within the human body. These viruses are found within regions of the body that have conventionally been considered sterile, including the blood, lymph, and organs. However, the primary mechanism that bacterial viruses use to bypass epithelial cell layers and access the body remains unknown. Here, we used in vitro studies to demonstrate the rapid and directional transcytosis of diverse bacteriophages across confluent cell layers originating from the gut, lung, liver, kidney, and brain. Bacteriophage transcytosis across cell layers had a significant preferential directionality for apical-to-basolateral transport, with approximately 0.1% of total bacteriophages applied being transcytosed over a 2-h period. Bacteriophages were capable of crossing the epithelial cell layer within 10 min with transport not significantly affected by the presence of bacterial endotoxins. Microscopy and cellular assays revealed that bacteriophages accessed both the vesicular and cytosolic compartments of the eukaryotic cell, with phage transcytosis suggested to traffic through the Golgi apparatus via the endomembrane system. Extrapolating from these results, we estimated that 31 billion bacteriophage particles are transcytosed across the epithelial cell layers of the gut into the average human body each day. The transcytosis of bacteriophages is a natural and ubiquitous process that provides a mechanistic explanation for the occurrence of phages within the body.IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria. They cannot infect eukaryotic cells but can penetrate epithelial cell layers and spread throughout sterile regions of our bodies, including the blood, lymph, organs, and even the brain. Yet how phages cross these eukaryotic cell layers and gain access to the body remains unknown. In this work, epithelial cells were observed to take up and transport phages across the cell, releasing active phages on the opposite cell surface. Based on these results, we posit that the human body is continually absorbing phages from the gut and transporting them throughout the cell structure and subsequently the body. These results reveal that phages interact directly with the cells and organs of our bodies, likely contributing to human health and immunity.

Deciphering the Molecular Signals of PINK1/Parkin Mitophagy.

Functional mitochondria are critically important for the maintenance of cellular integrity and survival. Mitochondrial dysfunction is a major contributor to neurodegenerative diseases including Parkinson's disease (PD). Two gene products mutated in familial Parkinsonism, PINK1 and Parkin, function together to degrade damaged mitochondria through a selective form of autophagy termed mitophagy. PINK1 accumulates on the surface of dysfunctional mitochondria where it simultaneously recruits and activates Parkin's E3 ubiquitin ligase activity. This forms the basis of multiple signaling events that culminate in engulfment of damaged mitochondria within autophagosomes and degradation by lysosomes. This review discusses the molecular signals of PINK1/Parkin mitophagy and the ubiquitin code that drives not only Parkin recruitment and activation by PINK1 but also the downstream signaling events of mitophagy.

Live-cell CLEM of subcellular targets: an optimized procedure for polymer-based imaging substrates.

Live-cell correlative light and electron microscopy permits the visualization of ultrastructure details associated with dynamic biological processes. On the optical level, fluorescence microscopy can be further combined with functional studies of intracellular processes and manipulation of biological samples using laser light. However, the major challenge is to relocate intracellular compartments in three dimensions after the sample has undergone an extensive EM sample preparation process. Here, we describe a detailed protocol for live-cell CLEM that provides easy guidance for 3D relocalization. Based on the use of the novel polymer film TOPAS as direct imaging substrate, we provide a setup that uses highly visible toner particles for tracking the region of interest in 2D and fiducial markers for the 3D relocation of intracellular structures. An example is given where a single mitochondria is targeted by laser microirradiation in live-cell fluorescence microscopy. After relocating the same structure in 3D in serial EM sections, the changes to the mitochondrial ultrastructure are observed by TEM. The method is suitable for correlation of live-cell microscopy of cells and can be performed using any inverted optical microscope.

An improved procedure for subcellular spatial alignment during live-cell CLEM.

Live-cell correlative light and electron microscopy (CLEM) offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope.

The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells.

Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.

Multimodal analysis of PEI-mediated endocytosis of nanoparticles in neural cells.

Polymer nanoparticles are widely used as a highly generalizable tool to entrap a range of different drugs for controlled or site-specific release. However, despite numerous studies examining the kinetics of controlled release, the biological behavior of such nanoparticles remains poorly understood, particularly with respect to endocytosis and intracellular trafficking. We synthesized polyethylenimine-decorated polymer nanospheres (ca. 100-250 nm) of the type commonly used for drug release and used correlated electron microscopy, fluorescence spectroscopy and microscopy, and relaxometry to track endocytosis in neural cells. These capabilities provide insight into how polyethylenimine mediates the entry of nanoparticles into neural cells and show that polymer nanosphere uptake involves three distinct steps, namely, plasma membrane attachment, fluid-phase as well as clathrin- and caveolin-independent endocytosis, and progressive accumulation in membrane-bound intracellular vesicles. These findings provide detailed insight into how the intracellular delivery of nanoparticles is mediated by polyethylenimine, which is presently the most commonly used nonviral gene transfer agent. This fundamental knowledge may also assist in the preparation of next-generation nonviral vectors.