MAPL loss dysregulates bile and liver metabolism in mice.

Mitochondrial and peroxisomal anchored protein ligase (MAPL) is a dual ubiquitin and small ubiquitin-like modifier (SUMO) ligase with roles in mitochondrial quality control, cell death and inflammation in cultured cells. Here, we show that MAPL function in the organismal context converges on metabolic control, as knockout mice are viable, insulin-sensitive, and protected from diet-induced obesity. MAPL loss leads to liver-specific activation of the integrated stress response, inducing secretion of stress hormone FGF21. MAPL knockout mice develop fully penetrant spontaneous hepatocellular carcinoma. Mechanistically, the peroxisomal bile acid transporter ABCD3 is a primary MAPL interacting partner and SUMOylated in a MAPL-dependent manner. MAPL knockout leads to increased bile acid production coupled with defective regulatory feedback in liver in vivo and in isolated primary hepatocytes, suggesting cell-autonomous function. Together, our findings establish MAPL function as a regulator of bile acid synthesis whose loss leads to the disruption of bile acid feedback mechanisms. The consequences of MAPL loss in liver, along with evidence of tumor suppression through regulation of cell survival pathways, ultimately lead to hepatocellular carcinogenesis.

From a Chemotherapeutic Drug to a High-Performance Nanocatalyst: A Fast Colorimetric Test for Cisplatin Detection at ppb Level.

A rapid point-of-care method for the colorimetric detection of cisplatin was developed, exploiting the efficient conversion of the chemotherapeutic drug into a high-performance nanocatalyst with peroxidase enzyme mimics. This assay provides high specificity and ppb-detection sensitivity with the naked eye or a smartphone-based readout, outperforming many standard laboratory-based techniques. The nanocatalyst-enabled colorimetric assay can be integrated with machine-learning methods, providing accurate quantitative measurements. Such a combined approach opens interesting perspectives for the on-site monitoring of both chemotherapeutic patients to achieve optimal treatments and healthcare workers to prevent their unsafe exposure.

A new functional role of mitochondria-anchored protein ligase in peroxisome morphology in mammalian cells.

Mitochondria and peroxisomes are metabolically interconnected and functionally active subcellular organelles. These two dynamic organelles, share a number of common biochemical functions such as ß-oxidation of fatty acids and detoxification of peroxides. The biogenesis and morphology of both these organelles in the mammalian cells is controlled by common transcription factors like PGC1α, and by a common fission machinery comprising of fission proteins like DRP1, Mff, and hFis1, respectively. In addition, the outer membrane mitochondria-anchored protein ligase (MAPL), the first mitochondrial SUMO E3 ligase with a RING-finger domain, also regulates mitochondrial morphology inducing mitochondrial fragmentation upon its overexpression. This fragmentation is dependent on both the RING domain of MAPL and the presence of the mitochondrial fission GTPase dynamin-related protein-1 (DRP1). Earlier studies have demonstrated that mitochondrial-derived vesicles are formed independently of the known mitochondrial fission GTPase, DRP1 are enriched for MAPL and are targeted to peroxisomes. The current study shows that MAPL regulates morphology of peroxisomes in a cell-type specific manner. Fascinatingly, the peroxisome elongation caused either due to silencing of DRP1 or by addition of polyunsaturated fatty acid, docosahexaenoic acid was blocked by overexpressing MAPL in mammalian cell lines. Furthermore, the transfection and colocalisation studies of MAPL with peroxisome membrane marker, PMP70, in different cell lines clearly revealed a cell-type specificity of transport of MAPL to peroxisomes. Previous work has placed the Vps35 (retromer component) as vital for delivery of MAPL to peroxisomes, placing the retromer as critical for the formation of MAPL-positive mitochondrial-derived vesicles. The results of polyethylene glycol-based cell-cell fusion assay signified that the enrichment of MAPL in peroxisomes is through vesicles and a retromer dependent phenomenon. Thus, a novel function for MAPL in peroxisomes is established to regulate peroxisome elongation and morphology under growth conditions and thus possibly modulate peroxisome fission.

T-RexNet-A Hardware-Aware Neural Network for Real-Time Detection of Small Moving Objects.

This paper presents the T-RexNet approach to detect small moving objects in videos by using a deep neural network. T-RexNet combines the advantages of Single-Shot-Detectors with a specific feature-extraction network, thus overcoming the known shortcomings of Single-Shot-Detectors in detecting small objects. The deep convolutional neural network includes two parallel paths: the first path processes both the original picture, in gray-scale format, and differences between consecutive frames; in the second path, differences between a set of three consecutive frames is only handled. As compared with generic object detectors, the method limits the depth of the convolutional network to make it less sensible to high-level features and easier to train on small objects. The simple, Hardware-efficient architecture attains its highest accuracy in the presence of videos with static framing. Deploying our architecture on the NVIDIA Jetson Nano edge-device shows its suitability to embedded systems. To prove the effectiveness and general applicability of the approach, real-world tests assessed the method performances in different scenarios, namely, aerial surveillance with the WPAFB 2009 dataset, civilian surveillance using the Chinese University of Hong Kong (CUHK) Square dataset, and fast tennis-ball tracking, involving a custom dataset. Experimental results prove that T-RexNet is a valid, general solution to detect small moving objects, which outperforms in this task generic existing object-detection approaches. The method also compares favourably with application-specific approaches in terms of the accuracy vs. speed trade-off.

Golgi-derived PI(4)P-containing vesicles drive late steps of mitochondrial division.

Mitochondrial plasticity is a key regulator of cell fate decisions. Mitochondrial division involves Dynamin-related protein-1 (Drp1) oligomerization, which constricts membranes at endoplasmic reticulum (ER) contact sites. The mechanisms driving the final steps of mitochondrial division are still unclear. Here, we found that microdomains of phosphatidylinositol 4-phosphate [PI(4)P] on trans-Golgi network (TGN) vesicles were recruited to mitochondria-ER contact sites and could drive mitochondrial division downstream of Drp1. The loss of the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) or its effector, phosphatidylinositol 4-kinase IIIß [PI(4)KIIIß], in different mammalian cell lines prevented PI(4)P generation and led to a hyperfused and branched mitochondrial network marked with extended mitochondrial constriction sites. Thus, recruitment of TGN-PI(4)P-containing vesicles at mitochondria-ER contact sites may trigger final events leading to mitochondrial scission.

SIM-ELM: Connecting the ELM model with similarity-function learning.

This paper moves from the affinities between two well-known learning schemes that apply randomization in the training process, namely, Extreme Learning Machines (ELMs) and the learning framework using similarity functions. These paradigms share a common approach involving data remapping and linear separators, but differ in the role of randomization within the respective learning algorithms. The paper presents an integrated approach connecting the two models, which ultimately yields a new variant of the basic ELM. The resulting learning scheme is characterized by an analytical relationship between the dimensionality of the remapped space and the learning abilities of the eventual predictor. Experimental results confirm that the new learning scheme can improve over conventional ELM in terms of the trade-off between classification accuracy and predictor complexity (i.e., the dimensionality of the remapped space).

MAPL SUMOylation of Drp1 Stabilizes an ER/Mitochondrial Platform Required for Cell Death.

There has been evidence that mitochondrial fragmentation is required for apoptosis, but the molecular links between the machinery regulating dynamics and cell death have been controversial. Indeed, activated BAX and BAK can form functional channels in liposomes, bringing into question the contribution of mitochondrial dynamics in apoptosis. We now demonstrate that the activation of apoptosis triggers MAPL/MUL1-dependent SUMOylation of the fission GTPase Drp1, a process requisite for cytochrome c release. SUMOylated Drp1 functionally stabilizes ER/mitochondrial contact sites that act as hotspots for mitochondrial constriction, calcium flux, cristae remodeling, and cytochrome c release. The loss of MAPL does not alter the activation and assembly of BAX/BAK oligomers, indicating that MAPL is activated downstream of BAX/BAK. This work demonstrates how interorganellar contacts are dynamically regulated through active SUMOylation during apoptosis, creating a stabilized platform that signals cytochrome c release.

Computational intelligence techniques for tactile sensing systems.

Tactile sensing helps robots interact with humans and objects effectively in real environments. Piezoelectric polymer sensors provide the functional building blocks of the robotic electronic skin, mainly thanks to their flexibility and suitability for detecting dynamic contact events and for recognizing the touch modality. The paper focuses on the ability of tactile sensing systems to support the challenging recognition of certain qualities/modalities of touch. The research applies novel computational intelligence techniques and a tensor-based approach for the classification of touch modalities; its main results consist in providing a procedure to enhance system generalization ability and architecture for multi-class recognition applications. An experimental campaign involving 70 participants using three different modalities in touching the upper surface of the sensor array was conducted, and confirmed the validity of the approach.

A vesicular transport pathway shuttles cargo from mitochondria to lysosomes.

Mitochondrial respiration relies on electron transport, an essential yet dangerous process in that it leads to the generation of reactive oxygen species (ROS). ROS can be neutralized within the mitochondria through enzymatic activity, yet the mechanism for steady-state removal of oxidized mitochondrial protein complexes and lipids is not well understood. We have previously characterized vesicular profiles budding from the mitochondria that carry selected cargo. At least one population of these mitochondria-derived vesicles (MDVs) targets the peroxisomes; however, the fate of the majority of MDVs was unclear. Here, we demonstrate that MDVs carry selected cargo to the lysosomes. Using a combination of confocal and electron microscopy, we observe MDVs in steady state and demonstrate that they are stimulated as an early response to oxidative stress, the extent of which is determined by the respiratory status of the mitochondria. Delivery to the lysosomes does not require mitochondrial depolarization and is independent of ATG5 and LC3, suggesting that vesicle delivery complements mitophagy. Consistent with this, ultrastructural analysis of MDV formation revealed Tom20-positive structures within the vesicles of multivesicular bodies. These data characterize a novel vesicle transport route between the mitochondria and lysosomes, providing insights into the basic mechanisms of mitochondrial quality control.

Vps35 mediates vesicle transport between the mitochondria and peroxisomes.

Mitochondria-derived vesicles (MDVs) have been shown to transport cargo from the mitochondria to the peroxisomes. Mitochondria and peroxisomes share common functions in the oxidation of fatty acids and the reduction of damaging peroxides. Their biogenesis is also linked through both the activation of master transcription factors such as PGC-1alpha and the common use of fission machinery, including DRP1, Mff, and hFis1. We have previously shown that MDVs are formed independently of the known mitochondrial fission GTPase Drp1 and are enriched for a mitochondrial small ubiquitin-like modifier (SUMO) E3 ligase called MAPL (mitochondrial-anchored protein ligase). Here, we demonstrate that the retromer complex, a known component of vesicle transport from the endosome to the Golgi apparatus, regulates the transport of MAPL from mitochondria to peroxisomes. An unbiased screen shows that Vps35 and Vps26 are found in complex with MAPL, and confocal imaging reveals Vps35 recruitment to mitochondrial vesicles. Silencing of Vps35 or Vps26A leads to a significant reduction in the delivery of MAPL to peroxisomes, placing the retromer within a novel intracellular trafficking route and providing insight into the formation of MAPL-positive MDVs.

A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators.

BACKGROUND: Mitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulate mitochondrial morphology and the mechanisms that govern mitochondrial dynamics are poorly understood. However, recent evidence indicates that the core machinery that governs mitochondrial dynamics is linked within complex intracellular signalling cascades, including apoptotic pathways, cell cycle transitions and nuclear factor kappa B activation. Given the emerging importance of mitochondrial plasticity in cell signalling pathways and metabolism, it is essential that we develop tools to quantitatively analyse the processes of fission and fusion. In terms of mitochondrial fusion, the field currently relies upon on semi-quantitative assays which, even under optimal conditions, are labour-intensive, low-throughput and require complex imaging techniques. RESULTS: In order to overcome these technical limitations, we have developed a new, highly quantitative cell-free assay for mitochondrial fusion in mammalian cells. This assay system has allowed us to establish the energetic requirements for mitochondrial fusion. In addition, our data reveal a dependence on active protein phosphorylation for mitochondrial fusion, confirming emerging evidence that mitochondrial fusion is tightly integrated within the global cellular response to signaling events. Indeed, we have shown that cytosol derived from cells stimulated with different triggers either enhance or inhibit the cell-free fusion reaction. CONCLUSIONS: The adaptation of this system to high-throughput analysis will provide an unprecedented opportunity to identify and characterize novel regulatory factors. In addition, it provides a framework for a detailed mechanistic analysis of the process of mitochondrial fusion and the various axis of regulation that impinge upon this process in a wide range of cellular conditions.See Commentary: http://www.biomedcentral.com/1741-7007/8/99.

Using unsupervised analysis to constrain generalization bounds for support vector classifiers.

A crucial issue in designing learning machines is to select the correct model parameters. When the number of available samples is small, theoretical sample-based generalization bounds can prove effective, provided that they are tight and track the validation error correctly. The maximal discrepancy (MD) approach is a very promising technique for model selection for support vector machines (SVM), and estimates a classifier's generalization performance by multiple training cycles on random labeled data. This paper presents a general method to compute the generalization bounds for SVMs, which is based on referring the SVM parameters to an unsupervised solution, and shows that such an approach yields tight bounds and attains effective model selection. When one estimates the generalization error, one uses an unsupervised reference to constrain the complexity of the learning machine, thereby possibly decreasing sharply the number of admissible hypothesis. Although the methodology has a general value, the method described in the paper adopts vector quantization (VQ) as a representation paradigm, and introduces a biased regularization approach in bound computation and learning. Experimental results validate the proposed method on complex real-world data sets.

Translocation of SenP5 from the nucleoli to the mitochondria modulates DRP1-dependent fission during mitosis.

The mechanisms that ensure an equal inheritance of cellular organelles during mitosis are an important area of study in cell biology. For the mitochondria fragment during mitosis, however, the cellular links that signal these changes are largely unknown. We recently identified a SUMO protease, SenP5, that deSUMOylates a number of mitochondrial targets, including the dynamin-related fission GTPase, DRP1. In interphase, SenP5 resides primarily within the nucleoli, in addition to a cytosolic pool. Here we report the relocalization of SenP5 from the nucleoli to the mitochondrial surface at G2/M transition prior to nuclear envelope breakdown. The recruitment of SenP5 results in a significant loss in mitochondrial SUMOylation, and a concomitant increase in the labile pool of DRP1 that drives mitochondrial fragmentation. Importantly, silencing of SenP5 leads to an arrest in the cell cycle precisely at the time when the protease is translocated to the mitochondria. These data indicate that transition of SenP5 to the mitochondria plays an important role in mitochondrial fragmentation during mitosis. The altered intracellular localization of SenP5 represents the first example of the mitochondrial recruitment of a SUMO protease and provides new insights into the mechanisms of interorganellar communication during the cell cycle.

MAPL is a new mitochondrial SUMO E3 ligase that regulates mitochondrial fission.

The modification of proteins by the small ubiquitin-like modifier (SUMO) is known to regulate an increasing array of cellular processes. SUMOylation of the mitochondrial fission GTPase dynamin-related protein 1 (DRP1) stimulates mitochondrial fission, suggesting that SUMOylation has an important function in mitochondrial dynamics. The conjugation of SUMO to its substrates requires a regulatory SUMO E3 ligase; however, so far, none has been functionally associated with the mitochondria. By using biochemical assays, overexpression and RNA interference experiments, we characterized the mitochondrial-anchored protein ligase (MAPL) as the first mitochondrial-anchored SUMO E3 ligase. Furthermore, we show that DRP1 is a substrate for MAPL, providing a direct link between MAPL and the fission machinery. Importantly, the large number of unidentified mitochondrial SUMO targets suggests a global role for SUMOylation in mitochondrial function, placing MAPL as a crucial component in the regulation of multiple conjugation events.

Cargo-selected transport from the mitochondria to peroxisomes is mediated by vesicular carriers.

Mitochondria and peroxisomes share a number of common biochemical processes, including the beta oxidation of fatty acids and the scavenging of peroxides. Here, we identify a new outer-membrane mitochondria-anchored protein ligase (MAPL) containing a really interesting new gene (RING)-finger domain. Overexpression of MAPL leads to mitochondrial fragmentation, indicating a regulatory function controlling mitochondrial morphology. In addition, confocal- and electron-microscopy studies of MAPL-YFP led to the observation that MAPL is also incorporated within unique, DRP1-independent, 70-100 nm diameter mitochondria-derived vesicles (MDVs). Importantly, vesicles containing MAPL exclude another outer-membrane marker, TOM20, and vesicles containing TOM20 exclude MAPL, indicating that MDVs selectively incorporate their cargo. We further demonstrate that MAPL-containing vesicles fuse with a subset of peroxisomes, marking the first evidence for a direct relationship between these two functionally related organelles. In contrast, a distinct vesicle population labeled with TOM20 does not fuse with peroxisomes, indicating that the incorporation of specific cargo is a primary determinant of MDV fate. These data are the first to identify MAPL, describe and characterize MDVs, and define a new intracellular transport route between mitochondria and peroxisomes.

Bax/Bak promote sumoylation of DRP1 and its stable association with mitochondria during apoptotic cell death.

Dynamin-related protein 1 (DRP1) plays an important role in mitochondrial fission at steady state and during apoptosis. Using fluorescence recovery after photobleaching, we demonstrate that in healthy cells, yellow fluorescent protein (YFP)-DRP1 recycles between the cytoplasm and mitochondria with a half-time of 50 s. Strikingly, during apoptotic cell death, YFP-DRP1 undergoes a transition from rapid recycling to stable membrane association. The rapid cycling phase that characterizes the early stages of apoptosis is independent of Bax/Bak. However, after Bax recruitment to the mitochondrial membranes but before the loss of mitochondrial membrane potential, YFP-DRP1 becomes locked on the membrane, resulting in undetectable fluorescence recovery. This second phase in DRP1 cycling is dependent on the presence of Bax/Bak but independent of hFis1 and mitochondrial fragmentation. Coincident with Bax activation, we detect a Bax/Bak-dependent stimulation of small ubiquitin-like modifier-1 conjugation to DRP1, a modification that correlates with the stable association of DRP1 with mitochondrial membranes. Altogether, these data demonstrate that the apoptotic machinery regulates the biochemical properties of DRP1 during cell death.

The SUMO protease SENP5 is required to maintain mitochondrial morphology and function.

Mitochondria are dynamic organelles that undergo regulated fission and fusion events that are essential to maintain metabolic stability. We previously demonstrated that the mitochondrial fission GTPase DRP1 is a substrate for SUMOylation. To further understand how SUMOylation impacts mitochondrial function, we searched for a SUMO protease that may affect mitochondrial dynamics. We demonstrate that the cytosolic pool of SENP5 catalyzes the cleavage of SUMO1 from a number of mitochondrial substrates. Overexpression of SENP5 rescues SUMO1-induced mitochondrial fragmentation that is partly due to the downregulation of DRP1. By contrast, silencing of SENP5 results in a fragmented and altered morphology. DRP1 was stably mono-SUMOylated in these cells, suggesting that SUMOylation leads to increased DRP1 mediated fission. In addition, the reduction of SENP5 levels resulted in a significant increase in the production of free radicals. Reformation of the mitochondrial tubules by expressing the dominant interfering DRP1 or by RNA silencing of endogenous DRP1 protein rescued both the morphological aberrations and the increased production of ROS induced by downregulation of SENP5. These data demonstrate the importance of SENP5 as a new regulator of SUMO1 proteolysis from mitochondrial targets, impacting mitochondrial morphology and metabolism.

Phosphorylation and cleavage of presenilin-associated rhomboid-like protein (PARL) promotes changes in mitochondrial morphology.

Remodeling of mitochondria is a dynamic process coordinated by fusion and fission of the inner and outer membranes of the organelle, mediated by a set of conserved proteins. In metazoans, the molecular mechanism behind mitochondrial morphology has been recruited to govern novel functions, such as development, calcium signaling, and apoptosis, which suggests that novel mechanisms should exist to regulate the conserved membrane fusion/fission machinery. Here we show that phosphorylation and cleavage of the vertebrate-specific Pbeta domain of the mammalian presenilin-associated rhomboid-like (PARL) protease can influence mitochondrial morphology. Phosphorylation of three residues embedded in this domain, Ser-65, Thr-69, and Ser-70, impair a cleavage at position Ser(77)-Ala(78) that is required to initiate PARL-induced mitochondrial fragmentation. Our findings reveal that PARL phosphorylation and cleavage impact mitochondrial dynamics, providing a blueprint to study the molecular evolution of mitochondrial morphology.

Activated mitofusin 2 signals mitochondrial fusion, interferes with Bax activation, and reduces susceptibility to radical induced depolarization.

Mitochondrial fusion in higher eukaryotes requires at least two essential GTPases, Mitofusin 1 and Mitofusin 2 (Mfn2). We have created an activated mutant of Mfn2, which shows increased rates of nucleotide exchange and decreased rates of hydrolysis relative to wild type Mfn2. Mitochondrial fusion is stimulated dramatically within heterokaryons expressing this mutant, demonstrating that hydrolysis is not requisite for the fusion event, and supporting a role for Mfn2 as a signaling GTPase. Although steady-state mitochondrial fusion required the conserved intermembrane space tryptophan residue, this requirement was overcome within the context of the hydrolysis-deficient mutant. Furthermore, the punctate localization of Mfn2 is lost in the dominant active mutants, indicating that these sites are functionally controlled by changes in the nucleotide state of Mfn2. Upon staurosporine-stimulated cell death, activated Bax is recruited to the Mfn2-containing puncta; however, Bax activation and cytochrome c release are inhibited in the presence of the dominant active mutants of Mfn2. The dominant active form of Mfn2 also protected the mitochondria against free radical-induced permeability transition. In contrast to staurosporine-induced outer membrane permeability transition, pore opening induced through the introduction of free radicals was dependent upon the conserved intermembrane space residue. This is the first evidence that Mfn2 is a signaling GTPase regulating mitochondrial fusion and that the nucleotide-dependent activation of Mfn2 concomitantly protects the organelle from permeability transition. The data provide new insights into the critical relationship between mitochondrial membrane dynamics and programmed cell death.

Sumo1 conjugates mitochondrial substrates and participates in mitochondrial fission.

Mitochondrial fission requires the evolutionarily conserved dynamin related protein (DRP1), which is recruited from the cytosol to the mitochondrial outer membrane to coordinate membrane scission. Currently, the mechanism of recruitment and assembly of DRP1 on the mitochondria is unclear. Here, we identify Ubc9 and Sumo1 as specific DRP1-interacting proteins and demonstrate that DRP1 is a Sumo1 substrate. In addition, a surprising number of Sumo1 conjugates were observed in the mitochondrial fractions, suggesting that sumoylation is a common mitochondrial modification. Video microscopy demonstrates that YFP:Sumo1 is often found at the site of mitochondrial fission and remains tightly associated to the tips of fragmented mitochondria. Consistent with this, fluorescence microscopy revealed that a portion of total cytosolic YFP:Sumo1 colocalizes with endogenous mitochondrial DRP1. Finally, transient transfection of Sumo1 dramatically increases the level of mitochondrial fragmentation. Analysis of endogenous DRP1 levels indicates that overexpression of Sumo1 specifically protects DRP1 from degradation, resulting in a more stable, active pool of DRP1, which at least partially accounts for the excess fragmentation. Together, these data are the first to identify a function for Sumo1 on the mitochondria and suggest a novel role for the participation of Sumo1 in mitochondrial fission.