QATS: an ImageJ plugin for the quantification of toroidal nuclei in biological images.

MOTIVATION: The toroidal nucleus is a novel chromosomal instability (CIN) biomarker which complements the micronucleus. Understanding the specific biological stresses leading to the formation of each CIN-associated phenotype requires the evaluation of large panels of biological images collected from different genetic backgrounds and environmental conditions. However, the quantification of toroidal nuclei is currently a manual process which is unviable on a large scale. RESULTS: Here, we present QATS (QuAntification of Toroidal nuclei in biological imageS), a tool that automates the identification of toroidal nuclei, minimizing false positives while highly agreeing with the manual quantifications. Additionally, QATS identifies micronuclei for a convenient comparison of both CIN biomarkers. QATS is an open-source ImageJ plugin with a user-friendly interface that enables a wide scientific community to easily assess the frequency of CIN biomarkers for the determination of CIN levels in cellular models. AVAILABILITY AND IMPLEMENTATION: QATS is an ImageJ plugin freely available at http://www.toroidalnucleus.org/qats. The user manual and the images used for the evaluation of QATS are included in the website. Supplementary data are available at Bioinformatics online.

α4-α5 Helices on Surface of KRAS Can Accommodate Small Compounds That Increase KRAS Signaling While Inducing CRC Cell Death.

KRAS is the most frequently mutated oncogene associated with the genesis and progress of pancreatic, lung and colorectal (CRC) tumors. KRAS has always been considered as a therapeutic target in cancer but until now only two compounds that inhibit one specific KRAS mutation have been approved for clinical use. In this work, by molecular dynamics and a docking process, we describe a new compound (P14B) that stably binds to a druggable pocket near the α4-α5 helices of the allosteric domain of KRAS. This region had previously been identified as the binding site for calmodulin (CaM). Using surface plasmon resonance and pulldown analyses, we prove that P14B binds directly to oncogenic KRAS thus competing with CaM. Interestingly, P14B favors oncogenic KRAS interaction with BRAF and phosphorylated C-RAF, and increases downstream Ras signaling in CRC cells expressing oncogenic KRAS. The viability of these cells, but not that of the normal cells, is impaired by P14B treatment. These data support the significance of the α4-α5 helices region of KRAS in the regulation of oncogenic KRAS signaling, and demonstrate that drugs interacting with this site may destine CRC cells to death by increasing oncogenic KRAS downstream signaling.

Emerging roles of mitotic autophagy.

Lysosomes exert pleiotropic functions to maintain cellular homeostasis and degrade autophagy cargo. Despite the great advances that have boosted our understanding of autophagy and lysosomes in both physiology and pathology, their function in mitosis is still controversial. During mitosis, most organelles are reshaped or repurposed to allow the correct distribution of chromosomes. Mitotic entry is accompanied by a reduction in sites of autophagy initiation, supporting the idea of an inhibition of autophagy to protect the genetic material against harmful degradation. However, there is accumulating evidence revealing the requirement of selective autophagy and functional lysosomes for a faithful chromosome segregation. Degradation is the most-studied lysosomal activity, but recently described alternative functions that operate in mitosis highlight the lysosomes as guardians of mitotic progression. Because the involvement of autophagy in mitosis remains controversial, it is important to consider the specific contribution of signalling cascades, the functions of autophagic proteins and the multiple roles of lysosomes, as three entangled, but independent, factors controlling genomic stability. In this Review, we discuss the latest advances in this area and highlight the therapeutic potential of targeting autophagy for drug development.

Detection of Nuclear Biomarkers for Chromosomal Instability.

Chromosomal instability (CIN) is a hallmark of cancer, which is characterized by the gain or loss of chromosomes as well as the rearrangement of the genetic material during cell division. Detection of mitotic errors such as misaligned chromosomes or chromosomal bridges (also known as lagging chromosomes) is challenging as it requires the analysis and manual discrimination of chromosomal aberrations in mitotic cells by molecular techniques. In interphase cells, more frequent in the cell population than mitotic cells, two distinct nuclear phenotypes are associated with CIN: the micronucleus and the toroidal nucleus. Several methods are available for the detection of micronuclei, but none for toroidal nuclei. Here, we provide a method to quantify the presence of both nuclear biomarkers for the evaluation of CIN status in non-mitotic cells particularly suited for genotoxicity screens.

Analysis of Autophagic Vesicles in Mitotic Cells.

The detection of autophagic vesicles in interphase cells is well characterized with markers such as LC3, SQSTM1 (also known as p62) and LAMP2, which are commonly used in immunofluorescence and biochemistry assays to evaluate the status of autophagy in adherent cells. During mitosis, cells undergo important morphological changes which alter the position of the central plane, therefore the imaging of dividing cells has to be specifically designed. Here, we describe a method to label and image autophagic vesicles in mitotic cells to systematically analyze their number, morphology and distribution.

Lysosomal degradation ensures accurate chromosomal segregation to prevent chromosomal instability.

Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability.Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H+-translocating ATPase; WAPL: WAPL cohesion release factor.

Nucleotide depletion reveals the impaired ribosome biogenesis checkpoint as a barrier against DNA damage.

Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21-mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC-mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability.

Phosphofructokinases Axis Controls Glucose-Dependent mTORC1 Activation Driven by E2F1.

Cancer cells rely on mTORC1 activity to coordinate mitogenic signaling with nutrients availability for growth. Based on the metabolic function of E2F1, we hypothesize that glucose catabolism driven by E2F1 could participate on mTORC1 activation. Here, we demonstrate that glucose potentiates E2F1-induced mTORC1 activation by promoting mTORC1 translocation to lysosomes, a process that occurs independently of AMPK activation. We showed that E2F1 regulates glucose metabolism by increasing aerobic glycolysis and identified the PFKFB3 regulatory enzyme as an E2F1-regulated gene important for mTORC1 activation. Furthermore, PFKFB3 and PFK1 were found associated to lysosomes and we demonstrated that modulation of PFKFB3 activity, either by substrate accessibility or expression, regulates the translocation of mTORC1 to lysosomes by direct interaction with Rag B and subsequent mTORC1 activity. Our results support a model whereby a glycolytic metabolon containing phosphofructokinases transiently interacts with the lysosome acting as a sensor platform for glucose catabolism toward mTORC1 activity.

Exploring Autophagy in Drosophila.

Autophagy is a catabolic process in eukaryotic cells promoting bulk or selective degradation of cellular components within lysosomes. In recent decades, several model systems were utilized to dissect the molecular machinery of autophagy and to identify the impact of this cellular "self-eating" process on various physiological and pathological processes. Here we briefly discuss the advantages and limitations of using the fruit fly Drosophila melanogaster, a popular model in cell and developmental biology, to apprehend the main pathway of autophagy in a complete animal.

Autophagosomes take the Klp98-A train.

The intracellular movement of membrane-bound vesicles is closely tied to their formation, maturation and ultimate function within the cell. Motor proteins and their associated cytoskeletal networks are critical for vesicle transport, but whether these factors play a more direct role in vesicle biogenesis is unclear. In recent work, we found that the Drosophila kinesin proteins Khc and Klp98A are both required for the normal anterograde movement of autophagosomes and autolysosomes during starvation-induced autophagy. In addition, Klp98A has a transport-independent function of promoting autophagosome-lysosome fusion, a key step in the maturation of autophagic vesicles. This function correlates with the association of Klp98A with the autophagosomal protein Atg8 and with the endolysosomal protein Rab14, suggesting that Klp98A may promote vesicle fusion by physically linking these vesicle surface proteins. These findings demonstrate how the delivery of vesicles to their proper destination can be coordinated with additional steps in their life cycle through molecular motor-based interactions.

Coordination of autophagosome-lysosome fusion and transport by a Klp98A-Rab14 complex in Drosophila.

Degradation of cellular material by autophagy is essential for cell survival and homeostasis, and requires intracellular transport of autophagosomes to encounter acidic lysosomes through unknown mechanisms. Here, we identify the PX-domain-containing kinesin Klp98A as a new regulator of autophagosome formation, transport and maturation in Drosophila. Depletion of Klp98A caused abnormal clustering of autophagosomes and lysosomes at the cell center and reduced the formation of starvation-induced autophagic vesicles. Reciprocally, overexpression of Klp98A redistributed autophagic vesicles towards the cell periphery. These effects were accompanied by reduced autophagosome-lysosome fusion and autophagic degradation. In contrast, depletion of the conventional kinesin heavy chain caused a similar mislocalization of autophagosomes without perturbing their fusion with lysosomes, indicating that vesicle fusion and localization are separable and independent events. Klp98A-mediated fusion required the endolysosomal GTPase Rab14, which interacted and colocalized with Klp98A, and required Klp98A for normal localization. Thus, Klp98A coordinates the movement and fusion of autophagic vesicles by regulating their positioning and interaction with the endolysosomal compartment.

Bafilomycin A1 disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion.

Autophagosome-lysosome fusion and autolysosome acidification constitute late steps in the autophagic process necessary to maintain functional autophagic flux and cellular homeostasis. Both of these steps are disrupted by the V-ATPase inhibitor bafilomycin A1, but the mechanisms potentially linking them are unclear. We recently revisited the role of lysosomal acidification in autophagosome-lysosome fusion, using an in vivo approach in Drosophila. By genetically depleting individual subunits of the V-ATPase, we confirmed its role in lysosomal acidification and autophagic cargo degradation. Surprisingly, vesicle fusion remained active in V-ATPase-depleted cells, indicating that autophagosome-lysosome fusion and autolysosome acidification are 2 separable processes. In contrast, bafilomycin A1 inhibited both acidification and fusion, consistent with its effects in mammalian cells. Together, these results imply that this drug inhibits fusion independently of its effect on V-ATPase-mediated acidification. We identified the ER-calcium ATPase Ca-P60A/dSERCA as a novel target of bafilomycin A1. Autophagosome-lysosome fusion was defective in Ca-P60A/dSERCA-depleted cells, and bafilomycin A1 induced a significant increase in cytosolic calcium concentration and disrupted Ca-P60A/SERCA-mediated fusion. Thus, bafilomycin A1 disrupts autophagic flux by independently inhibiting V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion.

Autophagosome-lysosome fusion is independent of V-ATPase-mediated acidification.

The ATP-dependent proton pump V-ATPase ensures low intralysosomal pH, which is essential for lysosomal hydrolase activity. Based on studies with the V-ATPase inhibitor BafilomycinA1, lysosomal acidification is also thought to be required for fusion with incoming vesicles from the autophagic and endocytic pathways. Here we show that loss of V-ATPase subunits in the Drosophila fat body causes an accumulation of non-functional lysosomes, leading to a block in autophagic flux. However, V-ATPase-deficient lysosomes remain competent to fuse with autophagosomes and endosomes, resulting in a time-dependent formation of giant autolysosomes. In contrast, BafilomycinA1 prevents autophagosome-lysosome fusion in these cells, and this defect is phenocopied by depletion of the Ca(2+) pump SERCA, a secondary target of this drug. Moreover, activation of SERCA promotes fusion in a BafilomycinA1-sensitive manner. Collectively, our results indicate that lysosomal acidification is not a prerequisite for fusion, and that BafilomycinA1 inhibits fusion independent of its effect on lysosomal pH.

Assays to monitor autophagy in Drosophila.

The term autophagy refers to the engulfment and degradation of cytoplasmic components within the lysosome. This process can benefit cells and organisms by removing damaged, superfluous, or harmful cellular components, and by generating a supply of recycled macromolecules that can support biosynthesis or energy production. Recent interest in autophagy has been driven by its potential role in several disease-related phenomena including neurodegeneration, cancer, immunity and aging. Drosophila provides a valuable animal model context for these studies, and work in this system has also begun to identify novel developmental and physiological roles of autophagy. Here, we provide an overview of methods for monitoring autophagy in Drosophila, with a special emphasis on the larval fat body. These methods can be used to investigate whether observed vesicles are of autophagic origin, to determine a relative rate of autophagic degradation, and to identify specific step(s) in the autophagic process in which a given gene functions.

Mfn2 modulates the UPR and mitochondrial function via repression of PERK.

Mitofusin 2 (Mfn2) is a key protein in mitochondrial fusion and it participates in the bridging of mitochondria to the endoplasmic reticulum (ER). Recent data indicate that Mfn2 ablation leads to ER stress. Here we report on the mechanisms by which Mfn2 modulates cellular responses to ER stress. Induction of ER stress in Mfn2-deficient cells caused massive ER expansion and excessive activation of all three Unfolded Protein Response (UPR) branches (PERK, XBP-1, and ATF6). In spite of an enhanced UPR, these cells showed reduced activation of apoptosis and autophagy during ER stress. Silencing of PERK increased the apoptosis of Mfn2-ablated cells in response to ER stress. XBP-1 loss-of-function ameliorated autophagic activity of these cells upon ER stress. Mfn2 physically interacts with PERK, and Mfn2-ablated cells showed sustained activation of this protein kinase under basal conditions. Unexpectedly, PERK silencing in these cells reduced ROS production, normalized mitochondrial calcium, and improved mitochondrial morphology. In summary, our data indicate that Mfn2 is an upstream modulator of PERK. Furthermore, Mfn2 loss-of-function reveals that PERK is a key regulator of mitochondrial morphology and function.

DOR undergoes nucleo-cytoplasmic shuttling, which involves passage through the nucleolus.

DOR is a bi-functional protein that regulates transcription and enhances starvation-induced autophagy. While autophagy has been mostly described as a stress-response mechanism, cells also need autophagy to maintain homeostasis in basal conditions. However, the mechanisms regulating basal autophagy still remain unknown. Our results show that DOR acts in basal autophagy. Indeed, DOR already undergoes nucleo-cytoplasmic shuttling in basal conditions and, surprisingly, DOR exits continuously the nucleus and traverses the nucleolus. However, the nucleolus integrity is not essential for both DOR nucleo-cytoplasmic shuttling and DOR function on basal autophagy. Taken together, we propose that DOR exit from the nucleus is essential for basal autophagy stimulation even under nucleolus disruption.

DOR/Tp53inp2 and Tp53inp1 constitute a metazoan gene family encoding dual regulators of autophagy and transcription.

Human DOR/TP53INP2 displays a unique bifunctional role as a modulator of autophagy and gene transcription. However, the domains or regions of DOR that participate in those functions have not been identified. Here we have performed structure/function analyses of DOR guided by identification of conserved regions in the DOR gene family by phylogenetic reconstructions. We show that DOR is present in metazoan species. Invertebrates harbor only one gene, DOR/Tp53inp2, and in the common ancestor of vertebrates Tp53inp1 may have arisen by gene duplication. In keeping with these data, we show that human TP53INP1 regulates autophagy and that different DOR/TP53INP2 and TP53INP1 proteins display transcriptional activity. The use of molecular evolutionary information has been instrumental to determine the regions that participate in DOR functions. DOR and TP53INP1 proteins share two highly conserved regions (region 1, aa residues 28-42; region 2, 66-112 in human DOR). Mutation of conserved hydrophobic residues in region 1 of DOR (that are part of a nuclear export signal, NES) reduces transcriptional activity, and blocks nuclear exit and autophagic activity under autophagy-activated conditions. We also identify a functional and conserved LC3-interacting motif (LIR) in region 1 of DOR and TP53INP1 proteins. Mutation of conserved acidic residues in region 2 of DOR reduces transcriptional activity, impairs nuclear exit in response to autophagy activation, and disrupts autophagy. Taken together, our data reveal DOR and TP53INP1 as dual regulators of transcription and autophagy, and identify two conserved regions in the DOR family that concentrate multiple functions crucial for autophagy and transcription.

The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells.

The regulation of autophagy in metazoans is only partly understood, and there is a need to identify the proteins that control this process. The diabetes- and obesity-regulated gene (DOR), a recently reported nuclear cofactor of thyroid hormone receptors, is expressed abundantly in metabolically active tissues such as muscle. Here, we show that DOR shuttles between the nucleus and the cytoplasm, depending on cellular stress conditions, and re-localizes to autophagosomes on autophagy activation. We demonstrate that DOR interacts physically with autophagic proteins Golgi-associated ATPase enhancer of 16 kDa (GATE16) and microtubule-associated protein 1A/1B-light chain 3. Gain-of-function and loss-of-function studies indicate that DOR stimulates autophagosome formation and accelerates the degradation of stable proteins. CG11347, the DOR Drosophila homologue, has been predicted to interact with the Drosophila Atg8 homologues, which suggests functional conservation in autophagy. Flies lacking CG11347 show reduced autophagy in the fat body during pupal development. All together, our data indicate that DOR regulates autophagosome formation and protein degradation in mammalian and Drosophila cells.

Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells.

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for beta-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.