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.

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.

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.

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.

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.

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.

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.

Neuron to glia signaling triggers myelin membrane exocytosis from endosomal storage sites.

During vertebrate brain development, axons are enwrapped by myelin, an insulating membrane produced by oligodendrocytes. Neuron-derived signaling molecules are temporally and spatially required to coordinate oligodendrocyte differentiation. In this study, we show that neurons regulate myelin membrane trafficking in oligodendrocytes. In the absence of neurons, the major myelin membrane protein, the proteolipid protein (PLP), is internalized and stored in late endosomes/lysosomes (LEs/Ls) by a cholesterol-dependent and clathrin-independent endocytosis pathway that requires actin and the RhoA guanosine triphosphatase. Upon maturation, the rate of endocytosis is reduced, and a cAMP-dependent neuronal signal triggers the transport of PLP from LEs/Ls to the plasma membrane. These findings reveal a fundamental and novel role of LEs/Ls in oligodendrocytes: to store and release PLP in a regulated fashion. The release of myelin membrane from LEs/Ls by neuronal signals may represent a mechanism to control myelin membrane growth.

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.

AP-1 and AP-3 mediate sorting of melanosomal and lysosomal membrane proteins into distinct post-Golgi trafficking pathways.

The adaptor complexes AP-1 and AP-3 are localized to endosomes and/or the trans Golgi network (TGN). Because of limitations in analysing intracellular adaptor function directly, their site of function is a matter of ongoing uncertainty. To overcome this problem and to analyse adaptor sorting at the TGN, we reconstituted vesicle formation from Golgi/TGN-enriched membranes in a novel in vitro budding assay. Melanocytes were metabolically labelled followed by a 19 degrees C temperature block to accumulate newly synthesized proteins in Golgi membranes, which were then enriched by subcellular fractionation and used as donor membranes for vesicle formation in vitro. The incorporation of the melanosomal proteins tyrosinase and tyrosinase-related protein 1 (TRP-1) as well as Lamp-1 and 46 kDa mannose-6-phosphate receptor (MPR46) into Golgi/TGN-derived vesicles was temperature, nucleotide, cytosol, ADP ribosylation factor 1 and adaptor dependent. We show that sorting of TRP-1 and MPR46 was AP-1 dependent, while budding of tyrosinase and Lamp-1 required AP-3. Depletion of clathrin inhibited sorting of all four cargo proteins, suggesting that AP-1 and AP-3 are involved in the formation of distinct types of clathrin-coated vesicles, each of which is characterized by the incorporation of specific cargo membrane proteins.

Limiting transport steps and novel interactions of Connexin-43 along the secretory pathway.

Connexins are four-transmembrane-domain proteins expressed in all vertebrates which form permeable gap junction channels that connect cells. Here, we analysed Connexin-43 (Cx43) transport to the plasma membrane and studied the effects of small GTPases acting along the secretory pathway. We show that both GTP- and GDP-restricted Sar1 prevents exit of Cx43 from the endoplasmic reticulum (ER), but only GTP-restricted Sar1 arrests Cx43 in COP II-coated ER exit sites and accumulates 14-3-3 proteins in the ER fraction. FRET-FLIM data confirm that already in ER exit sites Cx43 exists in oligomeric form, suggesting an in vivo role for 14-3-3 in Cx43 oligomerization. Exit of Cx43 from the ER can be blocked by other factors--such as expression of the beta subunit of the COP I coat or p50/dynamitin that acts on the microtubule-based dynein motor complex. GTP-restricted Arf1 blocks Cx43 in the Golgi. Lastly, we show that GTP-restricted Arf6 removes Cx43 gap junction plaques from the cell-cell interface and targets them to degradation. These data provide a molecular explanation of how small GTPases act to regulate Cx43 transport through the secretory pathway, facilitating or abolishing cell-cell communication through gap junctions.

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.

A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis.

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within mu2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant mu2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant mu2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to mu2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.

Members of a mammalian SNARE complex interact in the endoplasmic reticulum in vivo and are found in COPI vesicles.

Retrograde traffic between the Golgi apparatus and the endoplasmic reticulum (ER) is largely mediated by COPI-coated transport vesicles. In mammalian cells, retrograde traffic can pass through an intermediate compartment. Here, we report that the mammalian soluble N-ethylmaleimide-sensitive factor (NSF) attachment receptor (SNARE) proteins mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 form a quaternary SNARE complex. Fluorescence resonance energy transfer (FRET) experiments prove that these interactions occur in the ER of living cells. In addition, mUse1/D12 and mSec20/BNIP1 form homo-oligomers in vivo. Furthermore, we show that mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 are recruited into COPI-coated vesicles formed in vitro. Immunogold electron microscopy confirmed that these SNARE proteins colocalize with the KDEL receptor ERD2 in COPI-coated vesicles. Moreover, both FRET and immunoprecipitation experiments reveal interactions of these SNAREs with both ERD2 and COPI subunits. We conclude that the SNAREs described here are sorted via interaction with components of the COPI-dependent budding complex into Golgi-to-ER retrograde COPI vesicles and function in retrograde transport from the Golgi to the ER Golgi intermediate compartment (ERGIC) or the ER.

G1 phase-dependent nucleolar accumulation of human histone H1x.

BACKGROUND INFORMATION: H1 histones are a protein family comprising several subtypes. Although specific functions of the individual subtypes could not be determined so far, differential roles are indicated by varied nuclear distributions as well as differential expression patterns of the H1 subtypes. Although the group of replication-dependent H1 subtypes is synthesized during S phase, the replacement H1 subtype, H1 degrees , is also expressed in a replication-independent manner in non-proliferating cells. Recently we showed, by protein biochemical analysis, that the ubiquitously expressed subtype H1x is enriched in the micrococcal nuclease-resistant part of chromatin and that, although it shares common features with H1 degrees , its expression is differentially regulated, since, in contrast to H1 degrees , growth arrest or induction of differentiation did not induce an accumulation of H1x. RESULTS: In the present study, we show that H1x exhibits a cell-cycle-dependent change of its nuclear distribution. This H1 subtype showed a nucleolar accumulation during the G(1) phase, and it was evenly distributed in the nucleus during S phase and G(2). Immunocytochemical analysis of the intranucleolar distribution of H1x indicated that it is located mainly in the condensed nucleolar chromatin. In addition, we demonstrate that the amount of H1x protein remained nearly unchanged during S phase progression, which is in contrast to the replication-dependent subtypes. CONCLUSION: These results suggest that the differential localization of H1x provides a mechanism for a control of H1x activity by means of shuttling between nuclear subcompartments instead of a controlled turnover of the protein.

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.

Drebrin is a novel connexin-43 binding partner that links gap junctions to the submembrane cytoskeleton.

BACKGROUND: Connexins form gap junctions that mediate the transfer of ions, metabolites, and second messengers between contacting cells. Many aspects of connexin function, for example cellular transport, plaque assembly and stability, and channel conductivity, are finely tuned and likely involve proteins that bind to connexins' cytoplasmic domains. However, little is known about such regulatory proteins. To identify novel proteins that interact with the COOH-terminal domain of Connexin-43 (Cx43), the most widely expressed connexin family member, we applied a proteomics approach to screen fractions of mouse tissue homogenates for binding partners. RESULTS: Drebrin was recovered as a binding partner of the Cx43 COOH-terminal domain from mouse brain homogenate. Drebrin had previously been described as an actin binding protein that diminishes in brains during Alzheimer's disease. The novel Drebrin-Cx43 interaction identified by proteomics was confirmed by colocalization of endogenous proteins in astrocytes and Vero cells, coimmunoprecipitation, electron microscopy, electrophysiology, coexpression of both proteins with fluorescent tags, and live-cell FRET analysis. Depletion of Drebrin in cells with siRNA results in impaired cell-cell coupling, internalization of gap junctions, and targeting of Cx43 to a degradative pathway. CONCLUSIONS: We conclude that Drebrin is required for maintaining Cx43-containing gap junctions in their functional state at the plasma membrane. It is thus possible that Drebrin may interact with gap junctions in zones of cell-cell contacts in a regulated fashion in response to extracellular signals. The rearrangement or disruption of interactions between connexins and the Drebrin-containing submembrane cytoskeleton directs connexins to degradative cellular pathways.

Deletion of the SNARE vti1b in mice results in the loss of a single SNARE partner, syntaxin 8.

SNARE proteins participate in recognition and fusion of membranes. A SNARE complex consisting of vti1b, syntaxin 8, syntaxin 7, and endobrevin/VAMP-8 which is required for fusion of late endosomes in vitro has been identified recently. Here, we generated mice deficient in vti1b to study the function of this protein in vivo. vti1b-deficient mice had reduced amounts of syntaxin 8 due to degradation of the syntaxin 8 protein, while the amounts of syntaxin 7 and endobrevin did not change. These data indicate that vti1b is specifically required for the stability of a single SNARE partner. vti1b-deficient mice were viable and fertile. Most vti1b-deficient mice were indistinguishable from wild-type mice and did not display defects in transport to the lysosome. However, 20% of the vti1b-deficient mice were smaller. Lysosomal degradation of an endocytosed protein was slightly delayed in hepatocytes derived from these mice. Multivesicular bodies and autophagic vacuoles accumulated in hepatocytes of some smaller vti1b-deficient mice. This suggests that other SNAREs can compensate for the reduction in syntaxin 8 and for the loss of vti1b in most mice even though vti1b shows only 30% amino acid identity with its closest relative.

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.