Kinetic coupling of the respiratory chain with ATP synthase, but not proton gradients, drives ATP production in cristae membranes.

Mitochondria have a characteristic ultrastructure with invaginations of the inner membrane called cristae that contain the protein complexes of the oxidative phosphorylation system. How this particular morphology of the respiratory membrane impacts energy conversion is currently unknown. One proposed role of cristae formation is to facilitate the establishment of local proton gradients to fuel ATP synthesis. Here, we determined the local pH values at defined sublocations within mitochondria of respiring yeast cells by fusing a pH-sensitive GFP to proteins residing in different mitochondrial subcompartments. Only a small proton gradient was detected over the inner membrane in wild type or cristae-lacking cells. Conversely, the obtained pH values did barely permit ATP synthesis in a reconstituted system containing purified yeast F1F0 ATP synthase, although, thermodynamically, a sufficiently high driving force was applied. At higher driving forces, where robust ATP synthesis was observed, a P-side pH value of 6 increased the ATP synthesis rate 3-fold compared to pH 7. In contrast, when ATP synthase was coreconstituted with an active proton-translocating cytochrome oxidase, ATP synthesis readily occurred at the measured, physiological pH values. Our study thus reveals that the morphology of the inner membrane does not influence the subcompartmental pH values and is not necessary for robust oxidative phosphorylation in mitochondria. Instead, it is likely that the dense packing of the oxidative phosphorylation complexes in the cristae membranes assists kinetic coupling between proton pumping and ATP synthesis.

Spatial orchestration of mitochondrial translation and OXPHOS complex assembly.

Oxidative phosphorylation (OXPHOS) is vital for the regeneration of the vast majority of ATP in eukaryotic cells 1 . OXPHOS is carried out by large multi-subunit protein complexes in the cristae membranes, which are invaginations of the mitochondrial inner membrane. The OXPHOS complexes are a mix of subunits encoded in the nuclear and mitochondrial genomes. Thus, the assembly of these dual-origin complexes is an enormous logistical challenge for the cell. Using super-resolution microscopy (nanoscopy) and quantitative cryo-immunogold electron microscopy, we determined where specific transcripts are translated and where distinct assembly steps of the dual-origin complexes in the yeast Saccharomyces cerevisiae occur. Our data indicate that the mitochondrially encoded proteins of complex III and complex IV are preferentially inserted in different sites of the inner membrane than those of complex V. We further demonstrate that the early, but not the late, assembly steps of complex III and complex IV occur preferentially in the inner boundary membrane. By contrast, all steps of complex V assembly occur mainly in the cristae membranes. Thus, OXPHOS complex assembly is spatially well orchestrated, probably representing an unappreciated regulatory layer in mitochondrial biogenesis.

Characterisation of tumour-derived microvesicles in cancer patients' blood and correlation with clinical outcome.

To evaluate whether tumour-derived microvesicles (T-MV), originating from the plasma membrane, represent suitable cancer biomarkers, we isolated MV from peripheral blood samples of cancer patients with locally advanced and/or metastatic solid tumours (n = 330, including 79 head & neck cancers, 74 lung cancers, 41 breast cancers, 28 colorectal cancers and 108 with other cancer forms) and controls (n = 103). Whole MV preparations were characterised using flow cytometry. While MV carrying the tumour-associated proteins MUC1, EGFR and EpCAM were found to be enhanced in a tumour-subtype-specific way in patients' blood, expression of the matrix metalloproteinase inducer EMMPRIN was increased independent of tumour type. Higher levels of EMMPRIN+-MV correlated significantly with poor overall survival, whereas the other markers were prognostic only in specific tumour subgroups. By combining all four tumour-associated antigens, cancer patients were separated from healthy controls with an AUC of up to 0.85. Ex vivo, whole MV preparations from cancer patients, in contrast to those of controls, induced a tumour-supporting phenotype in macrophages and increased tumour cell invasion, which was dependent on the highly glycosylated isoform of EMMPRIN. In conclusion, the detection of T-MV in whole blood, even in minor amounts, is feasible with standard techniques, proves functionally relevant and correlates with clinical outcome.

AP-1/σ1A and AP-1/σ1B adaptor-proteins differentially regulate neuronal early endosome maturation via the Rab5/Vps34-pathway.

The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5(GTP)-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation.

Nuclear Trapping through Inhibition of Exosomal Export by Indomethacin Increases Cytostatic Efficacy of Doxorubicin and Pixantrone.

PURPOSE: Although R-CHOP-based immunochemotherapy cures significant proportions of patients with aggressive B-cell lymphoma, tumor cell susceptibility to chemotherapy varies, with mostly fatal outcome in cases of resistant disease. We and others have shown before that export of cytostatic drugs contributes to drug resistance. Now we provide a novel approach to overcome exosome-mediated drug resistance in aggressive B-cell lymphomas. EXPERIMENTAL DESIGN: We used well-established centrifugation protocols to purify exosomes from DLBCL cell lines and detected anthracyclines using FACS and HPLC. We used shRNA knockdown of ABCA3 to determine ABCA3 dependence of chemotherapy susceptibility and monitored ABCA3 expression after indomethacin treatment using qPCR. Finally, we established an in vivo assay using a chorioallantoic membrane (CAM) assay to determine the synergy of anthracycline and indomethacin treatment. RESULTS: We show increased efficacy of the anthracycline doxorubicin and the anthracenedione pixantrone by suppression of exosomal drug resistance with indomethacin. B-cell lymphoma cells in vitro efficiently extruded doxorubicin and pixantrone, in part compacted in exosomes. Exosomal biogenesis was critically dependent on the expression of the ATP-transporter A3 (ABCA3). Genetic or chemical depletion of ABCA3 augmented intracellular retention of both drugs and shifted the subcellular drug accumulation to prolonged nuclear retention. Indomethacin increased the cytostatic efficacy of both drugs against DLBCL cell lines in vitro and in vivo in a CAM assay. CONCLUSIONS: We propose pretreatment with indomethacin toward enhanced antitumor efficacy of anthracyclines and anthracenediones.

The GTPase Rab26 links synaptic vesicles to the autophagy pathway.

Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles.

Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN.

Tumor cells secrete not only a variety of soluble factors, but also extracellular vesicles that are known to support the establishment of a favorable tumor niche by influencing the surrounding stroma cells. Here we show that tumor-derived microvesicles (T-MV) also directly influence the tumor cells by enhancing their invasion in a both autologous and heterologous manner. Neither the respective vesicle-free supernatant nor MV from benign mammary cells mediate invasion. Uptake of T-MV is essential for the proinvasive effect. We further identify the highly glycosylated form of the extracellular matrix metalloproteinase inducer (EMMPRIN) as a marker for proinvasive MV. EMMPRIN is also present at high levels on MV from metastatic breast cancer patients in vivo. Anti-EMMPRIN strategies, such as MV deglycosylation, gene knockdown, and specific blocking peptides, inhibit MV-induced invasion. Interestingly, the effect of EMMPRIN-bearing MV is not mediated by matrix metalloproteinases but by activation of the p38/MAPK signaling pathway in the tumor cells. In conclusion, T-MV stimulate cancer cell invasion via a direct feedback mechanism dependent on highly glycosylated EMMPRIN.

Populational equilibrium through exosome-mediated Wnt signaling in tumor progression of diffuse large B-cell lymphoma.

Tumors are composed of phenotypically heterogeneous cell populations. The nongenomic mechanisms underlying transitions and interactions between cell populations are largely unknown. Here, we show that diffuse large B-cell lymphomas possess a self-organized infrastructure comprising side population (SP) and non-SP cells, where transitions between clonogenic states are modulated by exosome-mediated Wnt signaling. DNA methylation modulated SP-non-SP transitions and was correlated with the reciprocal expressions of Wnt signaling pathway agonist Wnt3a in SP cells and the antagonist secreted frizzled-related protein 4 in non-SP cells. Lymphoma SP cells exhibited autonomous clonogenicity and exported Wnt3a via exosomes to neighboring cells, thus modulating population equilibrium in the tumor.

Induction and transport of Wnt 5a during macrophage-induced malignant invasion is mediated by two types of extracellular vesicles.

Recently, we have shown that macrophage (MΦ)-induced invasion of breast cancer cells requires upregulation of Wnt 5a in MΦ leading to activation of ß-Catenin-independent Wnt signaling in the tumor cells. However, it remained unclear, how malignant cells induce Wnt 5a in MΦ and how it is transferred back to the cancer cells. Here we identify two types of extracellular particles as essential for this intercellular interaction in both directions. Plasma membrane-derived microvesicles (MV) as well as exosomes from breast cancer cells, although biologically distinct populations, both induce Wnt 5a in MΦ. In contrast, the particle-free supernatant and vesicles from benign cells, such as platelets, have no such effect. Induction is antagonized by the Wnt inhibitor Dickkopf-1. Subsequently, Wnt 5a is shuttled via responding MΦ-MV and exosomes to the tumor cells enhancing their invasion. Wnt 5a export on both vesicle fractions depends at least partially on the cargo protein Evenness interrupted (Evi). Its knockdown leads to Wnt 5a depletion of both particle populations and reduced vesicle-mediated invasion. In conclusion, MV and exosomes are critical for MΦ-induced invasion of cancer cells since they are responsible for upregulation of MΦ-Wnt 5a as well as for its delivery to the recipient cells via a reciprocal loop. Although of different biogenesis, both populations share common features regarding function and Evi-dependent secretion of non-canonical Wnts.

Immunostimulatory activity of murine keratinocyte-derived exosomes.

It has long been known that keratinocytes influence cutaneous immunity through secretion of soluble factors. Exosomes, small membrane vesicles of endocytotic origin, have been implicated in intercellular communication processes such as the transfer of tumor cell antigens and the activation of recipient dendritic cells (DC). However, little is known about immunomodulatory functions of keratinocyte-derived exosomes. To address this question, we analysed exosome secretion of the murine keratinocyte cell line MPEK under steady state as well as inflammatory conditions (+/- IFNγ). These exosomes were readily taken up by bone marrow-derived DC (BMDC) in vitro resulting in a matured phenotype, as evidenced by increased CD40 expression as well as by the production of large amounts of IL-6, IL-10 and IL-12. When the transfer of antigen-specific information through exosomes was investigated, it was found that keratinocytes took up antigen (ovalbumin) and transferred it to their exosomes. However, these antigen-harbouring exosomes failed to induce antigen-specific T cell responses via BMDC. Together, this novel biological function suggests that keratinocytes are able to direct unspecific immune processes but do not elicit specific immune responses.

STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria.

The mitochondrial inner membrane organizing system (MINOS) is a conserved large hetero-oligomeric protein complex in the mitochondrial inner membrane, crucial for the maintenance of cristae morphology. MINOS has been suggested to represent the core of an extended protein network that controls mitochondrial function and structure, and has been linked to several human diseases. The spatial arrangement of MINOS within mitochondria is ill-defined, however. Using super-resolution stimulated emission depletion (STED) microscopy and immunogold electron microscopy, we determined the distribution of three known human MINOS subunits (mitofilin, MINOS1, and CHCHD3) in mammalian cells. Super-resolution microscopy revealed that all three subunits form similar clusters within mitochondria, and that MINOS is more abundant in mitochondria around the nucleus than in peripheral mitochondria. At the submitochondrial level, mitofilin, a core MINOS subunit, is preferentially localized at cristae junctions. In primary human fibroblasts, mitofilin labeling uncovered a regularly spaced pattern of clusters arranged in parallel to the cell growth surfaces. We suggest that this array of MINOS complexes might explain the observed phenomenon of largely horizontally arranged cristae junctions that connect the inner boundary membrane to lamellar cristae. The super-resolution images demonstrate an unexpectedly high level of regularity in the nanoscale distribution of the MINOS complex in human mitochondria, supporting an integrating role of MINOS in the structural organization of the organelle.

Mitochondrial SIRT4-type proteins in Caenorhabditis elegans and mammals interact with pyruvate carboxylase and other acetylated biotin-dependent carboxylases.

The biological and enzymatic function of SIRT4 is largely uncharacterized. We show that the Caenorhabditis elegans SIR-2.2 and SIR-2.3 orthologs of SIRT4 are ubiquitously expressed, also localize to mitochondria and function during oxidative stress. Further, we identified conserved interaction with mitochondrial biotin-dependent carboxylases (PC, PCC, MCCC), key enzymes in anaplerosis and ketone body formation. The carboxylases were found acetylated on multiple lysine residues and detailed analysis of mPC suggested that one of these residues, K748ac, might regulate enzymatic activity. Nevertheless, no changes in mPC acetylation levels and enzymatic activity could be detected upon overexpression or loss of functional SIRT4.

The inner-mitochondrial distribution of Oxa1 depends on the growth conditions and on the availability of substrates.

The Oxa1 protein is a well-conserved integral protein of the inner membrane of mitochondria. It mediates the insertion of both mitochondrial- and nuclear-encoded proteins from the matrix into the inner membrane. We investigated the distribution of budding yeast Oxa1 between the two subdomains of the contiguous inner membrane--the cristae membrane (CM) and the inner boundary membrane (IBM)--under different physiological conditions. We found that under fermentable growth conditions, Oxa1 is enriched in the IBM, whereas under nonfermentable (respiratory) growth conditions, it is predominantly localized in the CM. The enrichment of Oxa1 in the CM requires mitochondrial translation; similarly, deletion of the ribosome-binding domain of Oxa1 prevents an enrichment of Oxa1 in the CM. The predominant localization in the IBM under fermentable growth conditions is prevented by inhibiting mitochondrial protein import. Furthermore, overexpression of the nuclear-encoded Oxa1 substrate Mdl1 shifts the distribution of Oxa1 toward the IBM. Apparently, the availability of nuclear- and mitochondrial-encoded substrates influences the inner-membrane distribution of Oxa1. Our findings show that the distribution of Oxa1 within the inner membrane is dynamic and adapts to different physiological needs.

Novel roles of Caenorhabditis elegans heterochromatin protein HP1 and linker histone in the regulation of innate immune gene expression.

Linker histone (H1) and heterochromatin protein 1 (HP1) are essential components of heterochromatin which contribute to the transcriptional repression of genes. It has been shown that the methylation mark of vertebrate histone H1 is specifically recognized by the chromodomain of HP1. However, the exact biological role of linker histone binding to HP1 has not been determined. Here, we investigate the function of the Caenorhabditis elegans H1 variant HIS-24 and the HP1-like proteins HPL-1 and HPL-2 in the cooperative transcriptional regulation of immune-relevant genes. We provide the first evidence that HPL-1 interacts with HIS-24 monomethylated at lysine 14 (HIS-24K14me1) and associates in vivo with promoters of genes involved in antimicrobial response. We also report an increase in overall cellular levels and alterations in the distribution of HIS-24K14me1 after infection with pathogenic bacteria. HIS-24K14me1 localization changes from being mostly nuclear to both nuclear and cytoplasmic in the intestinal cells of infected animals. Our results highlight an antimicrobial role of HIS-24K14me1 and suggest a functional link between epigenetic regulation by an HP1/H1 complex and the innate immune system in C. elegans.

Exosomal evasion of humoral immunotherapy in aggressive B-cell lymphoma modulated by ATP-binding cassette transporter A3.

Targeting the surface of malignant cells has evolved into a cornerstone in cancer therapy, paradigmatically introduced by the success of humoral immunotherapy against CD20 in malignant lymphoma. However, tumor cell susceptibility to immunochemotherapy varies, with mostly a fatal outcome in cases of resistant disease. Here, we show that lymphoma exosomes shield target cells from antibody attack and that exosome biogenesis is modulated by the lysosome-related organelle-associated ATP-binding cassette (ABC) transporter A3 (ABCA3). B-cell lymphoma cells released exosomes that carried CD20, bound therapeutic anti-CD20 antibodies, consumed complement, and protected target cells from antibody attack. ABCA3, previously shown to mediate resistance to chemotherapy, was critical for the amounts of exosomes released, and both pharmacological blockade and the silencing of ABCA3 enhanced susceptibility of target cells to antibody-mediated lysis. Mechanisms of cancer cell resistance to drugs and antibodies are linked in an ABCA3-dependent pathway of exosome secretion.

Epigenetics in C. elegans: facts and challenges.

Epigenetics is defined as the study of heritable changes in gene expression that are not accompanied by changes in the DNA sequence. Epigenetic mechanisms include histone post-translational modifications, histone variant incorporation, non-coding RNAs, and nucleosome remodeling and exchange. In addition, the functional compartmentalization of the nucleus also contributes to epigenetic regulation of gene expression. Studies on the molecular mechanisms underlying epigenetic phenomena and their biological function have relied on various model systems, including yeast, plants, flies, and cultured mammalian cells. Here we will expose the reader to the current understanding of epigenetic regulation in the roundworm C. elegans. We will review recent models of nuclear organization and its impact on gene expression, the biological role of enzymes modifying core histones, and the function of chromatin-associated factors, with special emphasis on Polycomb (PcG) and Trithorax (Trx-G) group proteins. We will discuss how the C. elegans model has provided novel insight into mechanisms of epigenetic regulation as well as suggest directions for future research.

Far-field autofluorescence nanoscopy.

We demonstrate far-field optical imaging at the nanoscale with unlabeled samples. Subdiffraction resolution images of autofluorescent samples are obtained by depleting the ground state of natural fluorophores by transferring them to a metastable dark state and simultaneously localizing those fluorophores that are transiently returning. Our approach is based on the insight that nanoscopy methods relying on stochastic single-molecule switching require only a single fluorescence on-off cycle to yield an image, a condition fulfilled by various biomolecules. The method is exemplified by recording label-free nanoscopy images of thylakoid membranes of spinach chloroplasts.

Synaptic scaffolding protein SYD-2 clusters and activates kinesin-3 UNC-104 in C. elegans.

Kinesin-3 motor UNC-104/KIF1A is essential for transporting synaptic precursors to synapses. Although the mechanism of cargo binding is well understood, little is known how motor activity is regulated. We mapped functional interaction domains between SYD-2 and UNC-104 by using yeast 2-hybrid and pull-down assays and by using FRET/fluorescence lifetime imaging microscopy to image the binding of SYD-2 to UNC-104 in living Caenorhabditis elegans. We found that UNC-104 forms SYD-2-dependent axonal clusters (appearing during the transition from L2 to L3 larval stages), which behave in FRAP experiments as dynamic aggregates. High-resolution microscopy reveals that these clusters contain UNC-104 and synaptic precursors (synaptobrevin-1). Analysis of motor motility indicates bi-directional movement of UNC-104, whereas in syd-2 mutants, loss of SYD-2 binding reduces net anterograde movement and velocity (similar after deleting UNC-104's liprin-binding domain), switching to retrograde transport characteristics when no role of SYD-2 on dynein and conventional kinesin UNC-116 motility was found. These data present a kinesin scaffolding protein that controls both motor clustering along axons and motor motility, resulting in reduced cargo transport efficiency upon loss of interaction.

ABC transporter A3 facilitates lysosomal sequestration of imatinib and modulates susceptibility of chronic myeloid leukemia cell lines to this drug.

BACKGROUND: Inhibition of BCR-ABL tyrosine kinase activity has evolved as a mainstay of therapy for patients with chronic myeloid leukemia. However, a fraction of leukemic cells persists under targeted therapy and can lead to disease progression on cessation of treatment. DESIGN AND METHODS: We analyzed bone marrow progenitor cells with the side population phenotype, and characterized the role of the intracellular ABC transporter A3 in imatinib detoxification. RESULTS: BCR-ABL-positive leukemic cells contribute to the side population cell compartment in untreated patients. Such leukemic side population cells, as well as CD34-positive progenitors from chronic myeloid leukemia samples, strongly express the intracellular ABCA3. Functionally, ABCA3 levels are critical for the susceptibility of chronic myeloid leukemia blast cell lines to specific BCR-ABL inhibition by imatinib. The transporter is localized in the limiting membrane of lysosomes and multivesicular bodies, and intracellular [(14)C]-labeled imatinib accumulates in such organelles. The lysosomal storage capacity increases with ABCA3 expression, thus regulating imatinib sequestration. CONCLUSIONS: The intracellular ABC transporter A3 is expressed in chronic myeloid leukemia progenitor cells and may contribute to intrinsic imatinib resistance by facilitating lysosomal sequestration in chronic myeloid leukemia cells.