Mitochondria and AMP-activated Protein Kinase-dependent Mechanism of Efferocytosis

Defective clearance of apoptotic cells is frequently associated with perpetuation of inflammatory conditions. Our results show a rapid activation of AMP-activated kinase (AMPK) in macrophages upon exposure to apoptotic cells or lysophosphatidylcholine, a specific phospholipid that is produced and released from dying cells. AMPK activation resulted from inhibition of mitochondrial oxygen consumption and ATP production and further depended on Ca²⁺ mobilization and mitochondrial reactive oxygen species generation. Once activated, AMPK increased microtubule synthesis and chemokinesis and provided adaptation to energy demand during tracking and engulfment. Uptake of apoptotic cells was increased in lungs of mice that received lysophosphatidylcholine. Furthermore, inhibition of AMPK diminished clearance of apoptotic thymocytes in vitro and in dexamethasone-treated mice. Taken together, we conclude that the mitochondrial AMPK axis is a sensor and enhancer of tracking and removal of apoptotic cell, processes crucial to resolution of inflammatory conditions and a return to tissue homeostasis.     

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10⁴–10⁶ copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10³ copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.     

T-Cell Infiltration Correlates with CXCL10 Expression in Ganglia of Cynomolgus Macaques with Reactivated Simian Varicella Virus

Ganglia of monkeys with reactivated simian varicella virus (SVV) contained more CD8 than CD4 T cells around neurons. The abundance of CD8 T cells was greater less than 2 months after reactivation than that at later times and correlated with that of CXCL10 RNA but not with those of SVV protein or open reading frame 61 (ORF61) antisense RNA. CXCL10 RNA colocalized with T-cell clusters. After SVV reactivation, transient T-cell infiltration, possibly mediated by CXCL10, parallels varicella zoster virus (VZV) reactivation in humans.     

Characterization of the diffusion of epidermal growth factor receptor clusters by single particle tracking

A number of studies have shown that receptors of the epidermal growth factor receptor family (ErbBs) exist as higher-order oligomers (clusters) in cell membranes in addition to their monomeric and dimeric forms. Characterizing the lateral diffusion of such clusters may provide insights into their dynamics and help elucidate their functional relevance. To that end, we used single particle tracking to study the diffusion of clusters of the epidermal growth factor (EGF) receptor (EGFR; ErbB1) containing bound fluorescently-labeled ligand, EGF. EGFR clusters had a median diffusivity of 6.8 × 10^–11 cm²/s and were found to exhibit different modes of transport (immobile, simple, confined, and directed) similar to that previously reported for single EGFR molecules. Disruption of actin filaments increased the median diffusivity of EGFR clusters to 10.3 × 10^–11 cm²/s, while preserving the different modes of diffusion. Interestingly, disruption of microtubules rendered EGFR clusters nearly immobile. Our data suggests that microtubules may play an important role in the diffusion of EGFR clusters either directly or perhaps indirectly via other mechanisms. To our knowledge, this is the first report probing the effect of the cytoskeleton on the diffusion of EGFR clusters in the membranes of live cells.     

Starvation Resistance in a Stenothermal Species from the Indian Subcontinent: Mechanistic Basis of Clinal Variation

Parallel clines for starvation resistance and lipid content are well documented among drosophilids on the Indian subcontinent. However, the mechanistic basis of these clines has not been investigated so far. Here, we investigate the utilization of lipids during starvation as a function of duration of stress in D. ananassae. We found higher lipid content responsible for high starvation resistance at lower latitudes. Lipids were utilized during starvation only; not during any other climatic stresses like desiccation or thermal stresses. We also found a cline for consumption of total body lipids; as more content (out of total amount of lipids) was utilized by flies at lower latitudes and lesser at higher latitudes. But, there was no latitudinal cline for threshold lipid amount in the case of females while for males there was a positive cline. Lastly, parallel clines have evolved under contrasting climatic conditions i.e. drier and colder northern localities have flies with lower lipid and reduced starvation resistance while hot and humid localities favor flies with higher lipid levels and greater starvation tolerance. Thus, the evolution of clines associated with starvation and lipid content might have resulted due to specific ecological conditions i.e. humidity gradient on the Indian subcontinent.     

The FBXO4 Tumor Suppressor Functions as a Barrier to BrafV600E-Dependent Metastatic Melanoma

Cyclin D1–cyclin-dependent kinase 4/6 (CDK4/6) dysregulation is a major contributor to melanomagenesis. Clinical evidence has revealed that p16^INK4A, an allosteric inhibitor of CDK4/6, is inactivated in over half of human melanomas, and numerous animal models have demonstrated that p16^INK4A deletion promotes melanoma. FBXO4, a specificity factor for the E3 ligase that directs timely cyclin D1 proteolysis, has not been studied in melanoma. We demonstrate that Fbxo4 deficiency induces Braf-driven melanoma and that this phenotype depends on cyclin D1 accumulation in mice, underscoring the importance of this ubiquitin ligase in tumor suppression. Furthermore, we have identified a substrate-binding mutation, FBXO4 I377M, that selectively disrupts cyclin D1 degradation while preserving proteolysis of the other known FBXO4 substrate, TRF1. The I377M mutation and Fbxo4 deficiency result in nuclear accumulation of cyclin D1, a key transforming neoplastic event. Collectively, these data provide evidence that FBXO4 dysfunction, as a mechanism for cyclin D1 overexpression, is a contributor to human malignancy.