Francesc Baixauli

Sorting it out: Regulation of exosome loading

Extracellular vesicles (EVs), a term that includes both exosomes of endocytic origin and vesicles derived from plasma membranes, are continuously secreted by cells to the extracellular environment, and represent a novel vehicle for cell-cell communication. Exosomes contain specific repertoires of proteins and RNAs, indicating the existence of mechanisms that control the sorting of molecules into them. Although the molecular mechanisms that regulate the loading of proteins into exosomes have been studied for years, the sorting of RNA has been elusive until recently. Here we review the molecular mechanisms that control the sorting of molecules into exosomes, with special attention to the sorting of RNA. We also discuss how the cellular context affects the composition of exosomes, and thus the outcome of the communication between the exosome-producer and recipient cells, with particular focus on the communication between tumor cells and with cells of the tumor microenvironment.

Exosomes and autophagy: coordinated mechanisms for the maintenance of cellular fitness.

Conditions resulting from loss of cellular homeostasis, including oxidative stress, inflammation, protein aggregation, endoplasmic reticulum stress, metabolic stress, and perturbation of mitochondrial function, are common to many pathological disorders and contribute to aging. Cells face these stress situations by engaging quality control mechanisms aimed to restore cellular homeostasis and preserve cell viability. Among them, the autophagy-lysosomal pathway mediates the specific degradation of damaged proteins and organelles, and its proper function is related to cellular protection and increased life span in many model organisms. Besides autophagy, increasing evidence underscores a role for exosomes in the selective secretion of harmful/damaged proteins and RNAs and thus in the maintenance of cellular fitness. In this perspective article, we discuss the emerging function of exosomes as a means of alleviating intracellular stress conditions, and how secretion of harmful or unwanted material in exosomes, in coordination with the autophagy-lysosomal pathway, is essential to preserve intracellular protein and RNA homeostasis. Finally, we provide an overview about the consequences of the spreading of the exosome content in physiological and pathological situations, and suggest putative therapeutic strategies for these exosome-mediated alterations.

The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse

During antigen‐specific T‐cell activation, mitochondria mobilize towards the vicinity of the immune synapse. We show here that the mitochondrial fission factor dynamin‐related protein 1 (Drp1) docks at mitochondria, regulating their positioning and activity near the actin‐rich ring of the peripheral supramolecular activation cluster (pSMAC) of the immune synapse. Mitochondrial redistribution in response to T‐cell receptor engagement was abolished by Drp1 silencing, expression of the phosphomimetic mutant Drp1S637D and the Drp1‐specific inhibitor mdivi‐1. Moreover, Drp1 knockdown enhanced mitochondrial depolarization and T‐cell receptor signal strength, but decreased myosin phosphorylation, ATP production and T‐cell receptor assembly at the central supramolecular activation cluster (cSMAC). Our results indicate that Drp1‐dependent mitochondrial positioning and activity controls T‐cell activation by fuelling central supramolecular activation cluster assembly at the immune synapse.

Mitochondrial Respiration Controls Lysosomal Function during Inflammatory T Cell Responses

The endolysosomal system is critical for the maintenance of cellular homeostasis. However, how endolysosomal compartment is regulated by mitochondrial function is largely unknown. We have generated a mouse model with defective mitochondrial function in CD4(+) T lymphocytes by genetic deletion of the mitochondrial transcription factor A (Tfam). Mitochondrial respiration deficiency impairs lysosome function, promotes p62 and sphingomyelin accumulation, and disrupts endolysosomal trafficking pathways and autophagy, thus linking a primary mitochondrial dysfunction to a lysosomal storage disorder. The impaired lysosome function in Tfam-deficient cells subverts T cell differentiation toward proinflammatory subsets and exacerbates the in vivo inflammatory response. Restoration of NAD(+) levels improves lysosome function and corrects the inflammatory defects in Tfam-deficient T cells. Our results uncover a mechanism by which mitochondria regulate lysosome function to preserve T cell differentiation and effector functions, and identify strategies for intervention in mitochondrial-related diseases.

Immune synapse: conductor of orchestrated organelle movement

To ensure proper cell function, intracellular organelles are not randomly distributed within the cell, but polarized and highly constrained by the cytoskeleton and associated adaptor proteins. This relationship between distribution and function was originally found in neurons and epithelial cells; however, recent evidence suggests that it is a general phenomenon occurring in many highly specialized cells including T lymphocytes. Recent studies reveal that the orchestrated redistribution of organelles is dependent on antigen-specific activation of and immune synapse (IS) formation by T cells. This review highlights the functional implications of organelle polarization in early T cell activation and examines recent findings on how the IS sets the rhythm of organelle motion and the spread of the activation signal to the nucleus.

Miro-1 links mitochondria and microtubule dynein motors to control lymphocyte migration and polarity

ABSTRACT The recruitment of leukocytes to sites of inflammation is crucial for a functional immune response. In the present work, we explored the role of mitochondria in lymphocyte adhesion, polarity, and migration. We show that during adhesion to the activated endothelium under physiological flow conditions, lymphocyte mitochondria redistribute to the adhesion zone together with the microtubule-organizing center (MTOC) in an integrin-dependent manner. Mitochondrial redistribution and efficient lymphocyte adhesion to the endothelium require the function of Miro-1, an adaptor molecule that couples mitochondria to microtubules. Our data demonstrate that Miro-1 associates with the dynein complex. Moreover, mitochondria accumulate around the MTOC in response to the chemokine CXCL12/SDF-1α; this redistribution is regulated by Miro-1. CXCL12-dependent cell polarization and migration are reduced in Miro-1-silenced cells, due to impaired myosin II activation at the cell uropod and diminished actin polymerization. These data point to a key role of Miro-1 in the control of lymphocyte adhesion and migration through the regulation of mitochondrial redistribution.

ROS-Triggered Phosphorylation of Complex II by Fgr Kinase Regulates Cellular Adaptation to Fuel Use

Electron flux in the mitochondrial electron transport chain is determined by the superassembly of mitochondrial respiratory complexes. Different superassemblies are dedicated to receive electrons derived from NADH or FADH2, allowing cells to adapt to the particular NADH/FADH2 ratio generated from available fuel sources. When several fuels are available, cells adapt to the fuel best suited to their type or functional status (e.g., quiescent versus proliferative). We show that an appropriate proportion of superassemblies can be achieved by increasing CII activity through phosphorylation of the complex II catalytic subunit FpSDH. This phosphorylation is mediated by the tyrosine-kinase Fgr, which is activated by hydrogen peroxide. Ablation of Fgr or mutation of the FpSDH target tyrosine abolishes the capacity of mitochondria to adjust metabolism upon nutrient restriction, hypoxia/reoxygenation, and T cell activation, demonstrating the physiological relevance of this adaptive response.

ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins

Exosomes are vesicles secreted to the extracellular environment through fusion with the plasma membrane of specific endosomes called multivesicular bodies (MVB) and mediate cell-to-cell communication in many biological processes. Posttranslational modifications are involved in the sorting of specific proteins into exosomes. Here we identify ISGylation as a ubiquitin-like modification that controls exosome release. ISGylation induction decreases MVB numbers and impairs exosome secretion. Using ISG15-knockout mice and mice expressing the enzymatically inactive form of the de-ISGylase USP18, we demonstrate in vitro and in vivo that ISG15 conjugation regulates exosome secretion. ISG15 conjugation triggers MVB co-localization with lysosomes and promotes the aggregation and degradation of MVB proteins. Accordingly, inhibition of lysosomal function or autophagy restores exosome secretion. Specifically, ISGylation of the MVB protein TSG101 induces its aggregation and degradation, being sufficient to impair exosome secretion. These results identify ISGylation as a novel ubiquitin-like modifier in the control of exosome production.

End-binding protein 1 controls signal propagation from the T Cell Receptor

The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unresolved. Here, we show that T cell activation requires the growth of MTs mediated by the plus‐end specific protein end‐binding 1 (EB1). A direct interaction of the T cell receptor (TCR) complex with EB1 provides the molecular basis for EB1 activity promoting TCR encounter with signalling vesicles at the IS. EB1 knockdown alters TCR dynamics at the IS and prevents propagation of the TCR activation signal to LAT, thus inhibiting activation of PLCγ1 and its localization to the IS. These results identify a role for EB1 interaction with the TCR in controlling TCR sorting and its connection with the LAT/PLCγ1 signalosome.

Mitochondria Know No Boundaries: Mechanisms and Functions of Intercellular Mitochondrial Transfer.

Mitochondria regulate multiple cell processes, including calcium signaling, apoptosis and cell metabolism. Mitochondria contain their own circular genome encoding selected subunits of the oxidative phosphorylation complexes. Recent findings reveal that, in addition to being maternally inherited, mitochondria can traverse cell boundaries and thus be horizontally transferred between cells. Although, the physiological relevance of this phenomenon is still under debate, mitochondria uptake rescues mitochondrial respiration defects in recipient cells and regulates signaling, proliferation or chemotherapy resistance in vitro and in vivo. In this review, we outline the pathophysiological consequences of horizontal mitochondrial transfer and offer a perspective on the cellular and molecular mechanisms mediating their intercellular transmission, including tunneling nanotubes, extracellular vesicles, cellular fusion, and GAP junctions. The physiological relevance of mitochondrial transfer and the potential therapeutic application of this exchange for treating mitochondrial-related diseases are discussed.