Targeting UGCG Overcomes Resistance to Lysosomal Autophagy Inhibition.

Lysosomal autophagy inhibition (LAI) with hydroxychloroquine or DC661 can enhance cancer therapy, but tumor regrowth is common. To elucidate LAI resistance, proteomics and immunoblotting demonstrated that LAI induced lipid metabolism enzymes in multiple cancer cell lines. Lipidomics showed that LAI increased cholesterol, sphingolipids, and glycosphingolipids. These changes were associated with striking levels of GM1+ membrane microdomains (GMM) in plasma membranes and lysosomes. Inhibition of cholesterol/sphingolipid metabolism proteins enhanced LAI cytotoxicity. Targeting UDP-glucose ceramide glucosyltransferase (UGCG) synergistically augmented LAI cytotoxicity. Although UGCG inhibition decreased LAI-induced GMM and augmented cell death, UGCG overexpression led to LAI resistance. Melanoma patients with high UGCG expression had significantly shorter disease-specific survival. The FDA-approved UGCG inhibitor eliglustat combined with LAI significantly inhibited tumor growth and improved survival in syngeneic tumors and a therapy-resistant patient-derived xenograft. These findings nominate UGCG as a new cancer target, and clinical trials testing UGCG inhibition in combination with LAI are warranted. SIGNIFICANCE: We discovered UGCG-dependent lipid remodeling drives resistance to LAI. Targeting UGCG with a drug approved for a lysosomal storage disorder enhanced LAI antitumor activity without toxicity. LAI and UGCG inhibition could be tested clinically in multiple cancers. This article is highlighted in the In This Issue feature, p. 247.

cDC1 coordinate innate and adaptive responses in the omentum required for T cell priming and memory.

In the peritoneal cavity, the omentum contains fat-associated lymphoid clusters (FALCs) whose role in response to infection is poorly understood. After intraperitoneal immunization with Toxoplasma gondii, conventional type 1 dendritic cells (cDC1s) were critical to induce innate sources of IFN-γ and cellular changes in the FALCs. Unexpectedly, infected peritoneal macrophages that migrated into the FALCs primed CD8+ T cells. Although T cell priming was cDC1 independent, these DCs were required for maximal CD8+ T cell expansion. An agent-based computational model and experimental data highlighted that cDC1s affected the magnitude of the proliferative burst and promoted CD8+ T cell expression of nutrient uptake receptors and cell survival. Thus, although FALCs lack the organization of secondary lymphoid organs, cDC1s resident in this tissue coordinate innate responses to microbial challenge and provide secondary signals required for T cell expansion and memory formation.

A Platelet-Rich Plasma-Derived Biologic Clears Staphylococcus aureus Biofilms While Mitigating Cartilage Degeneration and Joint Inflammation in a Clinically Relevant Large Animal Infectious Arthritis Model.

The leading cause of treatment failure in Staphylococcus aureus infections is the development of biofilms. Biofilms are highly tolerant to conventional antibiotics which were developed against planktonic cells. Consequently, there is a lack of antibiofilm agents in the antibiotic development pipeline. To address this problem, we developed a platelet-rich plasma (PRP)-derived biologic, termed BIO-PLY (for the BIOactive fraction of Platelet-rich plasma LYsate) which has potent in vitro bactericidal activity against S. aureus synovial fluid free-floating biofilm aggregates. Additional in vitro studies using equine synoviocytes and chondrocytes showed that BIO-PLY protected these cells of the joint from inflammation. The goal of this study was to test BIO-PLY for in vivo efficacy using an equine model of infectious arthritis. We found that horses experimentally infected with S. aureus and subsequently treated with BIO-PLY combined with the antibiotic amikacin (AMK) had decreased bacterial concentrations within both synovial fluid and synovial tissue and exhibited lower systemic and local inflammatory scores compared to horses treated with AMK alone. Most importantly, AMK+BIO-PLY treatment reduced the loss of infection-associated cartilage proteoglycan content in articular cartilage and decreased synovial tissue fibrosis and inflammation. Our results demonstrate the in vivo efficacy of AMK+BIO-PLY and represents a new approach to restore and potentiate antimicrobial activity against synovial fluid biofilms.

Oncolytic Myxoma virus infects and damages the tegument of the human parasitic flatworm Schistosoma mansoni.

Schistosomiasis is a devastating disease caused by parasitic flatworms of the genus Schistosoma. Praziquantel (PZQ), the current treatment of choice, is ineffective against immature worms and cannot prevent reinfection. The continued reliance on a single drug for treatment increases the risk of the development of PZQ-resistant parasites. Reports of PZQ insusceptibility lends urgency to the need for new therapeutics. Here, we report that Myxoma virus (MYXV), an oncolytic pox virus which is non-pathogenic in all mammals except leporids, infects and replicates in S. mansoni schistosomula, juveniles, and adult male and female worms. MYXV infection results in the shredding of the tegument and reduced egg production in vitro, identifying MYXV as the first viral pathogen of schistosomes. MYXV is currently in preclinical studies to manage multiple human cancers, supporting its use in human therapeutics. Our findings raise the exciting possibility that MYXV virus represents a novel and safe class of potential anthelmintic therapeutics.

SCF ubiquitin E3 ligase regulates DNA double-strand breaks in early meiotic recombination.

Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for SKP1, a constitutive subunit of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase, in early meiotic processes. SKP1 restrains accumulation of HORMAD1 and the pre-DSB complex (IHO1-REC114-MEI4) on the chromosome axis in meiotic germ cells. Loss of SKP1 prior to meiosis leads to aberrant localization of DSB repair proteins and a failure in synapsis initiation in meiosis of both males and females. Furthermore, SKP1 is crucial for sister chromatid cohesion during the pre-meiotic S-phase. Mechanistically, FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and degradation in HEK293T cells. Our results support a model wherein the SCF ubiquitin E3 ligase prevents hyperactive DSB formation through proteasome-mediated degradation of HORMAD1 and subsequent modulation of the pre-DSB complex during meiosis.

WWOX-Mediated Degradation of AMOTp130 Negatively Affects Egress of Filovirus VP40 Virus-Like Particles.

Ebola virus (EBOV) and Marburg virus (MARV) continue to emerge and cause severe hemorrhagic disease in humans. A comprehensive understanding of the filovirus-host interplay will be crucial for identifying and developing antiviral strategies. The filoviral VP40 matrix protein drives virion assembly and egress, in part by recruiting specific WW domain-containing host interactors via its conserved PPxY late (L) domain motif to positively regulate virus egress and spread. In contrast to these positive regulators of virus budding, a growing list of WW domain-containing interactors that negatively regulate virus egress and spread have been identified, including BAG3, YAP/TAZ, and WWOX. In addition to host WW domain regulators of virus budding, host PPxY-containing proteins also contribute to regulating this late stage of filovirus replication. For example, angiomotin (AMOT) is a multi-PPxY-containing host protein that functionally interacts with many of the same WW domain-containing proteins that regulate virus egress and spread. In this report, we demonstrate that host WWOX, which negatively regulates egress of VP40 virus-like particles (VLPs) and recombinant vesicular stomatitis virus (VSV) M40 virus, interacts with and suppresses the expression of AMOT. We found that WWOX disrupts AMOT's scaffold-like tubular distribution and reduces AMOT localization at the plasma membrane via lysosomal degradation. In sum, our findings reveal an indirect and novel mechanism by which modular PPxY-WW domain interactions between AMOT and WWOX regulate PPxY-mediated egress of filovirus VP40 VLPs. A better understanding of this modular network and competitive nature of protein-protein interactions will help to identify new antiviral targets and therapeutic strategies. IMPORTANCE Filoviruses (Ebola virus [EBOV] and Marburg virus [MARV]) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we reveal a novel mechanism by which host proteins WWOX and AMOTp130 interact with each other and with the filovirus matrix protein VP40 to regulate VP40-mediated egress of virus-like particles (VLPs). Our results highlight the biological impact of competitive interplay of modular virus-host interactions on both the virus life cycle and the host cell.

Schistosome TRPML channels play a role in neuromuscular activity and tegumental integrity.

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma. Mono-therapeutic treatment of this disease with the drug praziquantel, presents challenges such as inactivity against immature worms and inability to prevent reinfection. Importantly, ion channels are important targets for many current anthelmintics. Transient receptor potential (TRP) channels are important mediators of sensory signals with marked effects on cellular functions and signaling pathways. TRPML channels are a class of Ca2+-permeable TRP channels expressed on endolysosomal membranes. They regulate lysosomal function and trafficking, among other functions. Schistosoma mansoni is predicted to have a single TRPML gene (SmTRPML) with two splice variants differing by 12 amino acids. This study focuses on exploring the physiological properties of SmTRPML channels to better understand their role in schistosomes. In mammalian cells expressing SmTRPML, TRPML activators elicit a rise in intracellular Ca2+. In these cells, SmTRPML localizes both to lysosomes and the plasma membrane. These same TRPML activators elicit an increase in adult worm motility that is dependent on SmTRPML expression, indicating a role for these channels in parasite neuromuscular activity. Suppression of SmTRPML in adult worms, or exposure of adult worms to TRPML inhibitors, results in tegumental vacuolations, balloon-like surface exudates, and membrane blebbing, similar to that found following TRPML loss in other organisms. Together, these findings indicate that SmTRPML may regulate the function of the schistosome endolysosomal system. Further, the role of SmTRPML in neuromuscular activity and in parasite tegumental integrity establishes this channel as a candidate anti-schistosome drug target.

C2CD6 regulates targeting and organization of the CatSper calcium channel complex in sperm flagella.

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here, we report identification of an uncharacterized protein, C2CD6, as a subunit of the mouse CatSper complex. C2CD6 contains a calcium-dependent, membrane-targeting C2 domain. C2CD6 associates with the CatSper calcium-selective, core-forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. CatSper currents are present but at a significantly lower level in C2CD6-deficient sperm. Transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 facilitates incorporation of the CatSper complex into the flagellar plasma membrane and may function as a calcium sensor. The identification of C2CD6 may enable the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

YTHDC2 is essential for pachytene progression and prevents aberrant microtubule-driven telomere clustering in male meiosis.

Mechanisms driving the prolonged meiotic prophase I in mammals are poorly understood. RNA helicase YTHDC2 is critical for mitosis to meiosis transition. However, YTHDC2 is highly expressed in pachytene cells. Here we identify an essential role for YTHDC2 in meiotic progression. Specifically, YTHDC2 deficiency causes microtubule-dependent telomere clustering and apoptosis at the pachytene stage of prophase I. Depletion of YTHDC2 results in a massively dysregulated transcriptome in pachytene cells, with a tendency toward upregulation of genes normally expressed in mitotic germ cells and downregulation of meiotic transcripts. Dysregulation does not correlate with m6A status, and YTHDC2-bound mRNAs are enriched in genes upregulated in mutant germ cells, revealing that YTHDC2 primarily targets mRNAs for degradation. Furthermore, altered transcripts in mutant pachytene cells encode microtubule network proteins. Our results demonstrate that YTHDC2 regulates the pachytene stage by perpetuating a meiotic transcriptome and preventing microtubule network changes that could lead to telomere clustering.

Characterization of the Canine Retinal Vasculature With Optical Coherence Tomography Angiography: Comparisons With Histology and Fluorescein Angiography.

Purpose: To present a methodology for quantification of the canine retinal vasculature imaged by optical coherence tomography angiography (OCTA) and validate this approach by comparison with fluorescein angiography (FA) and confocal imaging of retinal wholemounts labelled by immunohistochemistry (IHC). Methods: Six normal adult dogs underwent retinal OCTA imaging in both eyes. The images extracted from the different microvascular plexuses at eight retinal locations spanning the central and mid-peripheral fundus were analyzed using the AngioTool software. FA was performed in one eye and was compared to the OCTA images. Six eyes from three dogs were processed by IHC to examine the retinal vasculature. Results: A total of four retinal plexuses were identified by OCTA in the canine retina, and their density and topographical pattern varied with eccentricity. OCTA offered improved resolution over FA with the advantage of allowing imaging of the individual plexuses. Detection by OCTA of small vessels within the deep capillary plexus was possible and approached the level of resolution achieved with ex vivo imaging of the retinal vasculature by confocal microscopy/IHC. The plexuses herein described are analogous to human retinal vasculature. Conclusion: OCTA can be used to image and quantify non-invasively the vascular retinal networks of the canine retina. We provide normative data in eight different retinal locations that can be imaged non-invasively with this technology. This could support analysis of retinal vascular changes associated with disease and following therapeutic intervention.

Role of nuclear localization in the regulation and function of T-bet and Eomes in exhausted CD8 T cells.

The transcription factors T-bet and Eomesodermin (Eomes) regulate CD8 T cell exhaustion through undefined mechanisms. Here, we show that the subcellular localization of T-bet and Eomes dictate their regulatory activity in exhausted T cells (TEXs). TEXs had a higher ratio of nuclear Eomes:T-bet than memory T cells (TMEMs) during chronic lymphocytic choriomeningitis virus (LCMV) infection in preclinical cancer models and in human tumors. Biochemically, T-bet and Eomes compete for the same DNA sequences, including the Pdcd1 T-box. High nuclear T-bet strongly represses Pdcd1 transcription in TMEM, whereas low nuclear T-bet in TEX leads to a dominant effect of Eomes that acts as a weaker repressor of Pdcd1. Blocking PD-1 signaling in TEXs increases nuclear T-bet, restoring stronger repression of Pdcd1, and driving T-bet-associated gene expression programs of chemotaxis, homing, and activation. These data identify a mechanism whereby the T-bet-Eomes axis regulates exhaustion through their nuclear localization, providing insights into how these transcription factors regulate TEX biology.

Angiomotin Counteracts the Negative Regulatory Effect of Host WWOX on Viral PPxY-Mediated Egress.

Filoviridae family members Ebola (EBOV) and Marburg (MARV) viruses and Arenaviridae family member Lassa virus (LASV) are emerging pathogens that can cause hemorrhagic fever and high rates of mortality in humans. A better understanding of the interplay between these viruses and the host will inform about the biology of these pathogens, and may lead to the identification of new targets for therapeutic development. Notably, expression of the filovirus VP40 and LASV Z matrix proteins alone drives assembly and egress of virus-like particles (VLPs). The conserved PPxY Late (L) domain motifs in the filovirus VP40 and LASV Z proteins play a key role in the budding process by mediating interactions with select host WW-domain containing proteins that then regulate virus egress and spread. To identify the full complement of host WW-domain interactors, we utilized WT and PPxY mutant peptides from EBOV and MARV VP40 and LASV Z proteins to screen an array of GST-WW-domain fusion proteins. We identified WW domain-containing oxidoreductase (WWOX) as a novel PPxY-dependent interactor, and we went on to show that full-length WWOX physically interacts with eVP40, mVP40 and LASV Z to negatively regulate egress of VLPs and of a live VSV/Ebola recombinant virus (M40). Interestingly, WWOX is a versatile host protein that regulates multiple signaling pathways and cellular processes via modular interactions between its WW-domains and PPxY motifs of select interacting partners, including host angiomotin (AMOT). Notably, we demonstrated recently that expression of endogenous AMOT not only positively regulates egress of VLPs, but also promotes egress and spread of live EBOV and MARV. Toward the mechanism of action, we show that the competitive and modular interplay among WWOX-AMOT-VP40/Z regulates VLP and M40 virus egress. Thus, WWOX is the newest member of an emerging group of host WW-domain interactors (e.g. BAG3; YAP/TAZ) that negatively regulate viral egress. These findings further highlight the complex interplay of virus-host PPxY/WW-domain interactions and their potential impact on the biology of both the virus and the host during infection.Author Summary Filoviruses (Ebola [EBOV] and Marburg [MARV]) and arenavirus (Lassa virus; LASV) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we identified host WW-domain containing protein WWOX as a novel interactor with VP40 and Z, and showed that WWOX inhibited budding of VP40/Z virus-like particles (VLPs) and live virus in a PPxY/WW-domain dependent manner. Our findings are important to the field as they expand the repertoire of host interactors found to regulate PPxY-mediated budding of RNA viruses, and further highlight the competitive interplay and modular virus-host interactions that impact both the virus lifecycle and the host cell.

Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes.

During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.

Alcohol-induced CYP2E1, mitochondrial dynamics and retrograde signaling in human hepatic 3D organoids.

Alcohol toxicity is a significant health problem with ~3 million estimated deaths per year globally. Alcohol is metabolized to the toxic metabolite, acetaldehyde by alcohol dehydrogenase or CYP2E1 in the hepatic tissue, and also induces reactive oxygen species (ROS), which together play a pivotal role in cell and tissue damage. Our previous studies with COS-7 cells transduced with unique human CYP2E1 variants that mostly localize to either microsomes or mitochondria revealed that mitochondrially-localized CYP2E1 drives alcohol toxicity through the generation of higher levels of ROS, which has a consequent effect on cytochrome c oxidase (CcO) and mitochondrial oxidative function. Alcohol treatment of human hepatocyte cell line, HepaRG, in monolayer cultures increased ROS, affected CcO activity/stability, and induced mitophagy. Alcohol treatment of 3D organoids of HepaRG cells induced higher levels of CYP2E1 mRNA and activated mitochondrial stress-induced retrograde signaling, and also induced markers of hepatic steatosis. Knock down of CYP2E1 mRNA using specific shRNA, FK506, a Calcineurin inhibitor, and Mdivi-1, a DRP1 inhibitor, ameliorated alcohol-induced mitochondrial retrograde signaling, and hepatic steatosis. These results for the first time present a mechanistic link between CYP2E1 function and alcohol mediated mitochondrial dysfunction, retrograde signaling, and activation of hepatic steatosis in a 3D organoid system that closely recapitulates the in vivo liver response.

Angiomotin regulates budding and spread of Ebola virus.

The Ebola virus (EBOV) VP40 matrix protein (eVP40) orchestrates assembly and budding of virions in part by hijacking select WW-domain-bearing host proteins via its PPxY late (L)-domain motif. Angiomotin (Amot) is a multifunctional PPxY-containing adaptor protein that regulates angiogenesis, actin dynamics, and cell migration/motility. Amot also regulates the Hippo signaling pathway via interactions with the WW-domain-containing Hippo effector protein Yes-associated protein (YAP). In this report, we demonstrate that endogenous Amot is crucial for positively regulating egress of eVP40 virus-like particles (VLPs) and for egress and spread of authentic EBOV. Mechanistically, we show that ectopic YAP expression inhibits eVP40 VLP egress and that Amot co-expression rescues budding of eVP40 VLPs in a dose-dependent and PPxY-dependent manner. Moreover, results obtained with confocal and total internal reflection fluorescence microscopy suggested that Amot's role in actin organization and dynamics also contributes to promoting eVP40-mediated egress. In summary, these findings reveal a functional and competitive interplay between virus and host proteins involving the multifunctional PPxY-containing adaptor Amot, which regulates both the Hippo pathway and actin dynamics. We propose that our results have wide-ranging implications for understanding the biology and pathology of EBOV infections.

SKP1 drives the prophase I to metaphase I transition during male meiosis.

The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic events is poorly understood. Here, we identify an essential role for SKP1, a core subunit of the SKP1-Cullin-F-box (SCF) ubiquitin E3 ligase, in the PI/MI transition. SKP1 localizes to synapsed chromosome axes and evicts HORMAD proteins from these regions in meiotic spermatocytes. SKP1-deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, γH2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. SKP1-deficient oocytes exhibit desynapsis, chromosome misalignment, and progressive postnatal loss. Therefore, SKP1 maintains synapsis in meiosis of both sexes. Furthermore, our results support a model where SKP1 functions as the long-sought intrinsic metaphase competence factor to orchestrate MI entry during male meiosis.

Modular mimicry and engagement of the Hippo pathway by Marburg virus VP40: Implications for filovirus biology and budding.

Ebola (EBOV) and Marburg (MARV) are members of the Filoviridae family, which continue to emerge and cause sporadic outbreaks of hemorrhagic fever with high mortality rates. Filoviruses utilize their VP40 matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW-domain-bearing host proteins via its conserved PPxY Late (L) domain motif. Here, we screened an array of 115 mammalian, bacterially expressed and purified WW-domains using a PPxY-containing peptide from MARV VP40 (mVP40) to identify novel host interactors. Using this unbiased approach, we identified Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) as novel mVP40 PPxY interactors. YAP and TAZ function as downstream transcriptional effectors of the Hippo signaling pathway that regulates cell proliferation, migration and apoptosis. We demonstrate that ectopic expression of YAP or TAZ along with mVP40 leads to significant inhibition of budding of mVP40 VLPs in a WW-domain/PPxY dependent manner. Moreover, YAP colocalized with mVP40 in the cytoplasm, and inhibition of mVP40 VLP budding was more pronounced when YAP was localized predominantly in the cytoplasm rather than in the nucleus. A key regulator of YAP nuclear/cytoplasmic localization and function is angiomotin (Amot); a multi-PPxY containing protein that strongly interacts with YAP WW-domains. Interestingly, we found that expression of PPxY-containing Amot rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Importantly, using a stable Amot-knockdown cell line, we found that expression of Amot was critical for efficient egress of mVP40 VLPs as well as egress and spread of authentic MARV in infected cell cultures. In sum, we identified novel negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress, that likely function in part, via competition between host and viral PPxY motifs binding to modular host WW-domains. These findings not only impact our mechanistic understanding of virus budding and spread, but also may impact the development of new antiviral strategies.

Esophageal 3D organoids of MPV17-/- mouse model of mitochondrial DNA depletion show epithelial cell plasticity and telomere attrition.

Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with late-stage detection and poor prognosis. This emphasizes the need to identify new markers for early diagnosis and treatment. Altered mitochondrial genome (mtDNA) content in primary tumors correlates with poor patient prognosis. Here we used three-dimensional (3D) organoids of esophageal epithelial cells (EECs) from the MPV17-/- mouse model of mtDNA depletion to investigate the contribution of reduced mtDNA content in ESCC oncogenicity. To test if mtDNA defects are a contributing factor in ESCC, we used oncogenic stimuli such as ESCC carcinogen 4-nitroquinoline oxide (4-NQO) treatment, or expressing p53R175H oncogenic driver mutation. We observed that EECs and 3D-organoids with mtDNA depletion had cellular, morphological and genetic alterations typical of an oncogenic transition. Furthermore, mitochondrial dysfunction induced cellular transformation is accompanied by elevated mitochondrial fission protein, DRP1 and pharmacologic inhibition of mitochondrial fission by mDivi-1 in the MPV17-/- organoids reversed the phenotype to that of normal EEC organoids. Our studies show that mtDNA copy number depletion, activates a mitochondrial retrograde response, potentiates telomere defects, and increases the oncogenic susceptibility towards ESCC. Furthermore, mtDNA depletion driven cellular plasticity is mediated via altered mitochondrial fission-fusion dynamics.

Dramatic changes in gene expression in different forms of Crithidia fasciculata reveal potential mechanisms for insect-specific adhesion in kinetoplastid parasites.

Kinetoplastids are a group of parasites that includes several medically-important species. These human-infective species are transmitted by insect vectors in which the parasites undergo specific developmental transformations. For each species, this includes a stage in which parasites adhere to insect tissue via a hemidesmosome-like structure. Although this structure has been described morphologically, it has never been molecularly characterized. We are using Crithidia fasciculata, an insect parasite that produces large numbers of adherent parasites inside its mosquito host, as a model kinetoplastid to investigate both the mechanism of adherence and the signals required for differentiation to an adherent form. An advantage of C. fasciculata is that adherent parasites can be generated both in vitro, allowing a direct comparison to cultured swimming forms, as well as in vivo within the mosquito. Using RNAseq, we identify genes associated with adherence in C. fasciculata. As almost all of these genes have orthologs in other kinetoplastid species, our findings may reveal shared mechanisms of adherence, allowing investigation of a crucial step in parasite development and disease transmission. In addition, dual-RNAseq allowed us to explore the interaction between the parasites and the mosquito. Although the infection is well-tolerated, anti-microbial peptides and other components of the mosquito innate immune system are upregulated. Our findings indicate that C. fasciculata is a powerful model system for probing kinetoplastid-insect interactions.

Correction to: HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression.

[This corrects the article DOI: 10.1038/celldisc.2016.45.].