Arp2/3 complex and the pentose phosphate pathway regulate late phases of neutrophil swarming.

Neutrophil swarming is an essential process of the neutrophil response to many pathological conditions. Resultant neutrophil accumulations are hallmarks of acute tissue inflammation and infection, but little is known about their dynamic regulation. Technical limitations to spatiotemporally resolve individual cells in dense neutrophil clusters and manipulate these clusters in situ have hampered recent progress. We here adapted an in vitro swarming-on-a-chip platform for the use with confocal laser-scanning microscopy to unravel the complexity of single-cell responses during neutrophil crowding. Confocal sectioning allowed the live visualization of subcellular components, including mitochondria, cell membranes, cortical actin, and phagocytic cups, inside neutrophil clusters. Based on this experimental setup, we identify that chemical inhibition of the Arp2/3 complex causes cell death in crowding neutrophils. By visualizing spatiotemporal patterns of reactive oxygen species (ROS) production in developing neutrophil swarms, we further demonstrate a regulatory role of the metabolic pentose phosphate pathway for ROS production and neutrophil cluster growth.

Metabolic rewiring tunes dermal macrophages in staphylococcal skin infection.

The skin needs to balance tolerance of colonizing microflora with rapid detection of potential pathogens. Flexible response mechanisms would seem most suitable to accommodate the dynamic challenges of effective antimicrobial defense and restoration of tissue homeostasis. Here, we dissected macrophage-intrinsic mechanisms and microenvironmental cues that tune macrophage signaling in localized skin infection with the colonizing and opportunistic pathogen Staphylococcus aureus. Early in skin infection, the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by γδ T cells and hypoxic conditions within the dermal microenvironment diverted macrophages away from a homeostatic M-CSF- and hypoxia-inducible factor 1α (HIF-1α)-dependent program. This allowed macrophages to be metabolically rewired for maximal inflammatory activity, which requires expression of Irg1 and generation of itaconate, but not HIF-1α. This multifactorial macrophage rewiring program was required for both the timely clearance of bacteria and for the provision of local immune memory. These findings indicate that immunometabolic conditioning allows dermal macrophages to cycle between antimicrobial activity and protection against secondary infections.

LC-MS-Based Targeted Metabolomics for FACS-Purified Rare Cells.

Metabolism plays a fundamental role in regulating cellular functions and fate decisions. Liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomic approaches provide high-resolution insights into the metabolic state of a cell. However, the typical sample size is in the order of 105-107 cells and thus not compatible with rare cell populations, especially in the case of a prior flow cytometry-based purification step. Here, we present a comprehensively optimized protocol for targeted metabolomics on rare cell types, such as hematopoietic stem cells and mast cells. Only 5000 cells per sample are required to detect up to 80 metabolites above background. The use of regular-flow liquid chromatography allows for robust data acquisition, and the omission of drying or chemical derivatization avoids potential sources of error. Cell-type-specific differences are preserved while the addition of internal standards, generation of relevant background control samples, and targeted metabolite with quantifiers and qualifiers ensure high data quality. This protocol could help numerous studies to gain thorough insights into cellular metabolic profiles and simultaneously reduce the number of laboratory animals and the time-consuming and costly experiments associated with rare cell-type purification.

TFEB induces mitochondrial itaconate synthesis to suppress bacterial growth in macrophages.

Successful elimination of bacteria in phagocytes occurs in the phago-lysosomal system, but also depends on mitochondrial pathways. Yet, how these two organelle systems communicate is largely unknown. Here we identify the lysosomal biogenesis factor transcription factor EB (TFEB) as regulator for phago-lysosome-mitochondria crosstalk in macrophages. By combining cellular imaging and metabolic profiling, we find that TFEB activation, in response to bacterial stimuli, promotes the transcription of aconitate decarboxylase (Acod1, Irg1) and synthesis of its product itaconate, a mitochondrial metabolite with antimicrobial activity. Activation of the TFEB-Irg1-itaconate signalling axis reduces the survival of the intravacuolar pathogen Salmonella enterica serovar Typhimurium. TFEB-driven itaconate is subsequently transferred via the Irg1-Rab32-BLOC3 system into the Salmonella-containing vacuole, thereby exposing the pathogen to elevated itaconate levels. By activating itaconate production, TFEB selectively restricts proliferating Salmonella, a bacterial subpopulation that normally escapes macrophage control, which contrasts TFEB's role in autophagy-mediated pathogen degradation. Together, our data define a TFEB-driven metabolic pathway between phago-lysosomes and mitochondria that restrains Salmonella Typhimurium burden in macrophages in vitro and in vivo.

Mitochondrial and Lipid Droplet Dynamics Regulate Intra- and Intercellular Fatty Acid Trafficking.

Imaging of fatty acid (FA) trafficking revealed that FAs stored in lipid droplets were delivered to mitochondria when the cells were starved. This delivery required cytoplasmic lipases and mitochondrial fusion activity, whereas lipid droplets were replenished with FAs supplied by autophagy. These findings have important implications for cancer.

Mitochondrial Dynamics at the Interface of Immune Cell Metabolism and Function.

Immune cell differentiation and function are crucially dependent on specific metabolic programs dictated by mitochondria, including the generation of ATP from the oxidation of nutrients and supplying precursors for the synthesis of macromolecules and post-translational modifications. The many processes that occur in mitochondria are intimately linked to their morphology that is shaped by opposing fusion and fission events. Exciting evidence is now emerging that demonstrates reciprocal crosstalk between mitochondrial dynamics and metabolism. Metabolic cues can control the mitochondrial fission and fusion machinery to acquire specific morphologies that shape their activity. We review the dynamic properties of mitochondria and discuss how these organelles interlace with immune cell metabolism and function.

Mitochondrial Priming by CD28.

T cell receptor (TCR) signaling without CD28 can elicit primary effector T cells, but memory T cells generated during this process are anergic, failing to respond to secondary antigen exposure. We show that, upon T cell activation, CD28 transiently promotes expression of carnitine palmitoyltransferase 1a (Cpt1a), an enzyme that facilitates mitochondrial fatty acid oxidation (FAO), before the first cell division, coinciding with mitochondrial elongation and enhanced spare respiratory capacity (SRC). microRNA-33 (miR33), a target of thioredoxin-interacting protein (TXNIP), attenuates Cpt1a expression in the absence of CD28, resulting in cells that thereafter are metabolically compromised during reactivation or periods of increased bioenergetic demand. Early CD28-dependent mitochondrial engagement is needed for T cells to remodel cristae, develop SRC, and rapidly produce cytokines upon restimulation-cardinal features of protective memory T cells. Our data show that initial CD28 signals during T cell activation prime mitochondria with latent metabolic capacity that is essential for future T cell responses.

Mitochondrial Dynamics Controls T Cell Fate through Metabolic Programming.

Activated effector T (TE) cells augment anabolic pathways of metabolism, such as aerobic glycolysis, while memory T (TM) cells engage catabolic pathways, like fatty acid oxidation (FAO). However, signals that drive these differences remain unclear. Mitochondria are metabolic organelles that actively transform their ultrastructure. Therefore, we questioned whether mitochondrial dynamics controls T cell metabolism. We show that TE cells have punctate mitochondria, while TM cells maintain fused networks. The fusion protein Opa1 is required for TM, but not TE cells after infection, and enforcing fusion in TE cells imposes TM cell characteristics and enhances antitumor function. Our data suggest that, by altering cristae morphology, fusion in TM cells configures electron transport chain (ETC) complex associations favoring oxidative phosphorylation (OXPHOS) and FAO, while fission in TE cells leads to cristae expansion, reducing ETC efficiency and promoting aerobic glycolysis. Thus, mitochondrial remodeling is a signaling mechanism that instructs T cell metabolic programming.

Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics.

Fatty acids (FAs) provide cellular energy under starvation, yet how they mobilize and move into mitochondria in starved cells, driving oxidative respiration, is unclear. Here, we clarify this process by visualizing FA trafficking with a fluorescent FA probe. The labeled FA accumulated in lipid droplets (LDs) in well-fed cells but moved from LDs into mitochondria when cells were starved. Autophagy in starved cells replenished LDs with FAs, increasing LD number over time. Cytoplasmic lipases removed FAs from LDs, enabling their transfer into mitochondria. This required mitochondria to be highly fused and localized near LDs. When mitochondrial fusion was prevented in starved cells, FAs neither homogeneously distributed within mitochondria nor became efficiently metabolized. Instead, FAs reassociated with LDs and fluxed into neighboring cells. Thus, FAs engage in complex trafficking itineraries regulated by cytoplasmic lipases, autophagy, and mitochondrial fusion dynamics, ensuring maximum oxidative metabolism and avoidance of FA toxicity in starved cells.

Mechanisms of mitochondria and autophagy crosstalk.

Autophagy is a cellular survival pathway that recycles intracellular components to compensate for nutrient depletion and ensures the appropriate degradation of organelles. Mitochondrial number and health are regulated by mitophagy, a process by which excessive or damaged mitochondria are subjected to autophagic degradation. Autophagy is thus a key determinant for mitochondrial health and proper cell function. Mitophagic malfunction has been recently proposed to contribute to progressive neuronal loss in Parkinson's disease. In addition to autophagy's significance in mitochondrial integrity, several lines of evidence suggest that mitochondria can also substantially influence the autophagic process. The mitochondria's ability to influence and be influenced by autophagy places both elements (mitochondria and autophagy) in a unique position where defects in one or the other system could increase the risk to various metabolic and autophagic related diseases.

Together we are stronger: fusion protects mitochondria from autophagosomal degradation.

Starvation induces a protective process of self-cannibalization called autophagy that is thought to mediate nonselective degradation of cytoplasmic material. We recently reported that mitochondria escape autophagosomal degradation through extensive fusion into mitochondrial networks upon certain starvation conditions. The extent of mitochondrial elongation is dependent on the type of nutrient deprivation, with amino acid depletion having a particularly strong effect. Downregulation of the mitochondrial fission protein Drp1 was determined to be important in bringing about starvation-induced mitochondrial fusion. The formation of mitochondrial networks during nutrient depletion selectively blocked their autophagic degradation, presumably allowing cells to sustain efficient ATP production and thereby survive starvation.

Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation.

Mitochondria are highly dynamic organelles that mediate essential cell functions such as apoptosis and cell-cycle control in addition to their role as efficient ATP generators. Mitochondrial morphology changes are tightly regulated, and their shape can shift between small, fragmented units and larger networks of elongated mitochondria. We demonstrate that mitochondrial elements become significantly elongated and interconnected shortly after nutrient depletion. This mitochondrial morphological shift depends on the type of starvation, with an additive effect observed when multiple nutrients are depleted simultaneously. We further show that starvation-induced mitochondrial elongation is mediated by down-regulation of dynamin-related protein 1 (Drp1) through modulation of two Drp1 phosphorylation sites, leading to unopposed mitochondrial fusion. Finally, we establish that mitochondrial tubulation upon nutrient deprivation protects mitochondria from autophagosomal degradation, which could permit mitochondria to maximize energy production and supply autophagosomal membranes during starvation.

Fuse or die: Shaping mitochondrial fate during starvation.

Mitochondria continuously change their shape and thereby influence different cellular processes like cell death or development. Recently, we showed that during starvation mitochondria fuse into a highly connected network. The change in mitochondrial shape was dependent on inactivation of the fission protein Drp1, through targeting of two different phosphorylation sites. This rapid inhibition of mitochondrial fission led to unopposed fusion, protecting mitochondria from starvation-induced degradation and enabling the cell to survive nutrient scarce conditions.

Mitochondria supply membranes for autophagosome biogenesis during starvation.

Starvation-induced autophagosomes engulf cytosol and/or organelles and deliver them to lysosomes for degradation, thereby resupplying depleted nutrients. Despite advances in understanding the molecular basis of this process, the membrane origin of autophagosomes remains unclear. Here, we demonstrate that, in starved cells, the outer membrane of mitochondria participates in autophagosome biogenesis. The early autophagosomal marker, Atg5, transiently localizes to punctae on mitochondria, followed by the late autophagosomal marker, LC3. The tail-anchor of an outer mitochondrial membrane protein also labels autophagosomes and is sufficient to deliver another outer mitochondrial membrane protein, Fis1, to autophagosomes. The fluorescent lipid NBD-PS (converted to NBD-phosphotidylethanolamine in mitochondria) transfers from mitochondria to autophagosomes. Photobleaching reveals membranes of mitochondria and autophagosomes are transiently shared. Disruption of mitochondria/ER connections by mitofusin2 depletion dramatically impairs starvation-induced autophagy. Mitochondria thus play a central role in starvation-induced autophagy, contributing membrane to autophagosomes.

Targeting of the prion protein to the cytosol: mechanisms and consequences.

Prion diseases are characterized by the conformational transition of the cellular prion protein (PrP(C)) into an aberrant protein conformer, designated scrapie-prion protein (PrP(Sc)). A causal link between protein misfolding and neurodegeneration has been established for a variety of neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease and polyglutamine diseases, but there is an ongoing debate about the nature of the neurotoxic species and how non-native conformers can damage neuronal populations. PrP is normally imported into the endoplasmic reticulum (ER) and targeted to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. However, several conditions, such as ER stress or some pathogenic mutations in the PrP gene, can induce the mislocalization of PrP in the cytosol, where it has a neurotoxic potential as demonstrated in cell culture and transgenic mouse models. In this review we focus on intrinsic factors and cellular pathways implicated in the import of PrP into the ER and its mistargeting to the cytosol. The findings summarized here not only reveal a complex regulation of the biogenesis of PrP, but also provide interesting new insight into toxic activities of pathogenic protein conformers and quality control pathways of ER-targeted proteins.

alpha-Helical domains promote translocation of intrinsically disordered polypeptides into the endoplasmic reticulum.

Co-translational import into the endoplasmic reticulum (ER) is primarily controlled by N-terminal signal sequences that mediate targeting of the ribosome-nascent chain complex to the Sec61/translocon and initiate the translocation process. Here we show that after targeting to the translocon the secondary structure of the nascent polypeptide chain can significantly modulate translocation efficiency. ER-targeted polypeptides dominated by unstructured domains failed to efficiently translocate into the ER lumen and were subjected to proteasomal degradation via a co-translocational/preemptive pathway. Productive ER import could be reinstated by increasing the amount of alpha-helical domains, whereas more effective ER signal sequences had only a minor effect on ER import efficiency of unstructured polypeptides. ER stress and overexpression of p58(IPK) promoted the co-translocational degradation pathway. Moreover polypeptides with unstructured domains at their N terminus were specifically targeted to proteasomal degradation under these conditions. Our study indicates that extended unstructured domains are signals to dispose ER-targeted proteins via a co-translocational, preemptive quality control pathway.

Green tea extracts interfere with the stress-protective activity of PrP and the formation of PrP.

A hallmark in prion diseases is the conformational transition of the cellular prion protein (PrP(C)) into a pathogenic conformation, designated scrapie prion protein (PrP(Sc)), which is the essential constituent of infectious prions. Here, we show that epigallocatechin gallate (EGCG) and gallocatechin gallate, the main polyphenols in green tea, induce the transition of mature PrP(C) into a detergent-insoluble conformation distinct from PrP(Sc). The PrP conformer induced by EGCG was rapidly internalized from the plasma membrane and degraded in lysosomal compartments. Isothermal titration calorimetry studies revealed that EGCG directly interacts with PrP leading to the destabilizing of the native conformation and the formation of random coil structures. This activity was dependent on the gallate side chain and the three hydroxyl groups of the trihydroxyphenyl side chain. In scrapie-infected cells EGCG treatment was beneficial; formation of PrP(Sc) ceased. However, in uninfected cells EGCG interfered with the stress-protective activity of PrP(C). As a consequence, EGCG-treated cells showed enhanced vulnerability to stress conditions. Our study emphasizes the important role of PrP(C) to protect cells from stress and indicate efficient intracellular pathways to degrade non-native conformations of PrP(C).

Stress-protective signalling of prion protein is corrupted by scrapie prions.

Studies in transgenic mice revealed that neurodegeneration induced by scrapie prion (PrP(Sc)) propagation is dependent on neuronal expression of the cellular prion protein PrP(C). On the other hand, there is evidence that PrP(C) itself has a stress-protective activity. Here, we show that the toxic activity of PrP(Sc) and the protective activity of PrP(C) are interconnected. With a novel co-cultivation assay, we demonstrate that PrP(Sc) can induce apoptotic signalling in PrP(C)-expressing cells. However, cells expressing PrP mutants with an impaired stress-protective activity were resistant to PrP(Sc)-induced toxicity. We also show that the internal hydrophobic domain promotes dimer formation of PrP and that dimerization of PrP is linked to its stress-protective activity. PrP mutants defective in dimer formation did not confer enhanced stress tolerance. Moreover, in chronically scrapie-infected neuroblastoma cells the amount of PrP(C) dimers inversely correlated with the amount of PrP(Sc) and the resistance of the cells to various stress conditions. Our results provide new insight into the mechanism of PrP(C)-mediated neuroprotection and indicate that pathological PrP conformers abuse PrP(C)-dependent pathways for apoptotic signalling.