Rapid phagosome isolation enables unbiased multiomic analysis of human microglial phagosomes.

Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis-and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions.

Neutrophil trapping and nexocytosis, mast cell-mediated processes for inflammatory signal relay.

Neutrophils are sentinel immune cells with essential roles for antimicrobial defense. Most of our knowledge on neutrophil tissue navigation derived from wounding and infection models, whereas allergic conditions remained largely neglected. Here, we analyzed allergen-challenged mouse tissues and discovered that degranulating mast cells (MCs) trap living neutrophils inside them.

Functional multi-organelle units control inflammatory lipid metabolism of macrophages.

Eukaryotic cells contain several membrane-separated organelles to compartmentalize distinct metabolic reactions. However, it has remained unclear how these organelle systems are coordinated when cells adapt metabolic pathways to support their development, survival or effector functions. Here we present OrgaPlexing, a multi-spectral organelle imaging approach for the comprehensive mapping of six key metabolic organelles and their interactions.

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 have hampered recent progress.

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 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.

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 10-10 cells and thus not compatible with rare cell populations, especially in the case of a prior flow cytometry-based purification step.

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.

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.

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.

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.

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.

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.

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.

Cell biology. SevERing mitochondria.

The endoplasmic reticulum is an active participant in the division of another organelle, the mitochondrion.

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.

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.

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.

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.

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.

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.

Semisynthetic murine prion protein equipped with a GPI anchor mimic incorporates into cellular membranes.

Conversion of cellular prion protein (PrP(C)) into the pathological conformer (PrP(Sc)) has been studied extensively by using recombinantly expressed PrP (rPrP). However, due to inherent difficulties of expressing and purifying posttranslationally modified rPrP variants, only a limited amount of data is available for membrane-associated PrP and its behavior in vitro and in vivo. Here, we present an alternative route to access lipidated mouse rPrP (rPrP(Palm)) via two semisynthetic strategies.