RESUMEN
Microtubule-associated protein 1 light chain 3 gamma (MAP1LC3C or LC3C) is a member of the microtubule-associated family of proteins that are essential in the formation of autophagosomes and lysosomal degradation of cargo. LC3C has tumor-suppressing activity, and its expression is dependent on kidney cancer tumor suppressors, such as von Hippel-Lindau protein and folliculin. Recently, we demonstrated that LC3C autophagy is regulated by noncanonical upstream regulatory complexes and targets for degradation postdivision midbody rings associated with cancer cell stemness. Here, we show that loss of LC3C leads to peripheral positioning of the lysosomes and lysosomal exocytosis (LE). This process is independent of the autophagic activity of LC3C. Analysis of isogenic cells with low and high LE shows substantial transcriptomic reprogramming with altered expression of zinc (Zn)-related genes and activity of polycomb repressor complex 2, accompanied by a robust decrease in intracellular Zn. In addition, metabolomic analysis revealed alterations in amino acid steady-state levels. Cells with augmented LE show increased tumor initiation properties and form aggressive tumors in xenograft models. Immunocytochemistry identified high levels of lysosomal-associated membrane protein 1 on the plasma membrane of cancer cells in human clear cell renal cell carcinoma and reduced levels of Zn, suggesting that LE occurs in clear cell renal cell carcinoma, potentially contributing to the loss of Zn. These data indicate that the reprogramming of lysosomal localization and Zn metabolism with implication for epigenetic remodeling in a subpopulation of tumor-propagating cancer cells is an important aspect of tumor-suppressing activity of LC3C.
Asunto(s)
Carcinoma de Células Renales , Exocitosis , Neoplasias Renales , Lisosomas , Proteínas Asociadas a Microtúbulos , Zinc , Animales , Humanos , Autofagia , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Zinc/metabolismo , Complejo Represivo Polycomb 2 , Epigénesis GenéticaRESUMEN
LC3s are canonical proteins necessary for the formation of autophagosomes. We have previously established that two paralogs, LC3B and LC3C, have opposite activities in renal cancer, with LC3B playing an oncogenic role and LC3C a tumor-suppressing role. LC3C is an evolutionary late gene present only in higher primates and humans. Its most distinct feature is a C-terminal 20-amino acid peptide cleaved in the process of glycine 126 lipidation. Here, we investigated mechanisms of LC3C-selective autophagy. LC3C autophagy requires noncanonical upstream regulatory complexes that include ULK3, UVRAG, RUBCN, PIK3C2A, and a member of ESCRT, TSG101. We established that postdivision midbody rings (PDMBs) implicated in cancer stem-cell regulation are direct targets of LC3C autophagy. LC3C C-terminal peptide is necessary and sufficient to mediate LC3C-dependent selective degradation of PDMBs. This work establishes a new noncanonical human-specific selective autophagic program relevant to cancer stem cells.
Asunto(s)
Autofagosomas/genética , Autofagia/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Relacionadas con la Autofagia/genética , Proteínas de Unión al ADN , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Células HeLa , Humanos , Péptidos/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , Factores de Transcripción , Proteínas Supresoras de Tumor/genéticaRESUMEN
Autophagy promotes tumor growth by generating nutrients from the degradation of intracellular structures. Here we establish, using shRNAs, a dominant-negative mutant, and a pharmacologic inhibitor, mefenamic acid (MFA), that the Transient Receptor Potential Melastatin 3 (TRPM3) channel promotes the growth of clear cell renal cell carcinoma (ccRCC) and stimulates MAP1LC3A (LC3A) and MAP1LC3B (LC3B) autophagy. Increased expression of TRPM3 in RCC leads to Ca(2+) influx, activation of CAMKK2, AMPK, and ULK1, and phagophore formation. In addition, TRPM3 Ca(2+) and Zn(2+) fluxes inhibit miR-214, which directly targets LC3A and LC3B. The von Hippel-Lindau tumor suppressor (VHL) represses TRPM3 directly through miR-204 and indirectly through another miR-204 target, Caveolin 1 (CAV1).
Asunto(s)
Autofagia , Carcinoma de Células Renales/patología , Neoplasias Renales/patología , MicroARNs/fisiología , Canales Catiónicos TRPM/genética , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Caveolina 1/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Neoplasias Renales/genética , Ratones Desnudos , Trasplante de Neoplasias , Oncogenes , Interferencia de ARN , Canales Catiónicos TRPM/metabolismo , Carga Tumoral , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
The von Hippel-Lindau tumor-suppressor gene (VHL) is lost in most clear cell renal cell carcinomas (ccRCC). Here, using human ccRCC specimens, VHL-deficient cells, and xenograft models, we show that miR-204 is a VHL-regulated tumor suppressor acting by inhibiting macroautophagy, with MAP1LC3B (LC3B) as a direct and functional target. Of note, higher tumor grade of human ccRCC was correlated with a concomitant decrease in miR-204 and increase in LC3B levels, indicating that LC3B-mediated macroautophagy is necessary for RCC progression. VHL, in addition to inducing endogenous miR-204, triggered the expression of LC3C, an HIF-regulated LC3B paralog, that suppressed tumor growth. These data reveal a function of VHL as a tumor-suppressing regulator of autophagic programs.
Asunto(s)
Autofagia/genética , Carcinoma de Células Renales/genética , Neoplasias Renales/genética , MicroARNs/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Animales , Carcinoma de Células Renales/patología , Humanos , Neoplasias Renales/patología , Ratones , MicroARNs/genética , Trasplante Heterólogo/patología , Células Tumorales CultivadasRESUMEN
The present study sought to examine the function of membrane lipid rafts in adherence-dependent oxidant production in human neutrophils. Rafts are membrane domains that are rich in glycosphingolipids and cholesterol and are thought to be the foci for formation of signaling complexes in a variety of cells. Disruption of lipid rafts by depletion of membrane cholesterol with the chelating agent methyl-beta-cyclodextrin (MbetaCD) has been widely used to examine the function of lipid rafts. Here, we report that treatment of human neutrophils with MbetaCD unexpectedly caused priming of these cells, manifested as enhanced adherence-dependent oxidant production. Treatment of neutrophils with MbetaCD dose-dependently increased oxidant production after adhesion to fibronectin-coated plates. This priming effect was associated with recruitment of CD11b- and CD66b-rich raft domains from the specific granules, as determined by immunoblot and flow cytometry. Confocal microscopy showed that MbetaCD caused otherwise untreated neutrophils to rapidly adhere and spread on fibronectin-coated plates. Furthermore, three-dimensional reconstruction microscopy studies showed that MbetaCD caused expansion and coalescence of raft domains that covered most of the cell surface. These large raft domains expressed CD11b primarily in the core of these regions. Our studies demonstrate that cholesterol depletion with MbetaCD results in neutrophil priming manifested as enhanced adherence-dependent oxidant production. These studies caution against assumption that any observed MbetaCD effects are a function of reduced raft formation.
Asunto(s)
Colesterol/metabolismo , Gránulos Citoplasmáticos/metabolismo , Microdominios de Membrana/metabolismo , Neutrófilos/metabolismo , Antígenos CD/metabolismo , Antígeno CD11b/metabolismo , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Proteínas Ligadas a GPI , Humanos , Peróxido de Hidrógeno/metabolismo , Reacción de Inmunoadherencia , Microdominios de Membrana/efectos de los fármacos , beta-Ciclodextrinas/farmacologíaRESUMEN
The mushroom bodies are brain centers involved in complex behaviors such as learning and orientation. Here we examine the organization of mushroom bodies in ants, focusing on visual input. We describe the structure of visual neurons and compare the volume of brain structures involved in visual processing, especially the optic lobes and parts of the mushroom bodies receiving visual input in males, winged females, and workers of carpenter ants (Camponotus). A relatively small number of neurons connect the medulla with the mushroom bodies, and these neurons have relatively large dendritic fields in the medulla, suggesting low spatial resolution in ants. These neurons terminate in different yet overlapping strata in the mushroom bodies' collar region. While males have larger optic lobes than workers, their collar region is smaller than in females. Male ants have an additional type of medulla-mushroom body neuron with dendrites probing the distal medulla. These neurons are absent in female and worker ants. Most mushroom body Kenyon cells that are postsynaptic to visual input neurons appear to integrate visual as well as antennal input. This is in contrast to honey bees, where visual input to the mushroom bodies is more prominent and where Kenyon cells are not known to combine visual and antennal input.
Asunto(s)
Hormigas/citología , Interneuronas/citología , Cuerpos Pedunculados/citología , Lóbulo Óptico de Animales no Mamíferos/citología , Vías Visuales/citología , Percepción Visual/fisiología , Animales , Hormigas/fisiología , Abejas/citología , Abejas/fisiología , Conducta Animal/fisiología , Dendritas/fisiología , Dendritas/ultraestructura , Femenino , Interneuronas/fisiología , Masculino , Cuerpos Pedunculados/fisiología , Neurópilo/citología , Neurópilo/fisiología , Vías Olfatorias/fisiología , Lóbulo Óptico de Animales no Mamíferos/fisiología , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Caracteres Sexuales , Olfato/fisiología , Transmisión Sináptica/fisiología , Vías Visuales/fisiologíaRESUMEN
Insect mushroom bodies are brain regions that receive multisensory input and are thought to play an important role in learning and memory. In most neopteran insects, the mushroom bodies receive direct olfactory input. In addition, the calyces of Hymenoptera receive substantial direct input from the optic lobes. We describe visual inputs to the calyces of the mushroom bodies of the honeybee Apis mellifera, the neurons' dendritic fields in the optic lobes, the medulla and lobula, and the organization of their terminals in the calyces. Medulla neurons terminate in the collar region of the calyx, where they segregate into five layers that receive alternating input from the dorsal or ventral medulla, respectively. A sixth, innermost layer of the collar receives input from lobula neurons. In the basal ring region of the calyx, medulla neuron terminals are restricted to a small, distal part. Lobula neurons are more prominent in the basal ring, where they terminate in its outer half. Although the collar and basal ring layers generally receive segregated input from both optic neuropils, some overlap occurs at the borders of the layers. At least three different types of mushroom body input neurons originate from the medulla: (a) neurons with narrow dendritic fields mainly restricted to the vicinity of the medulla's serpentine layer and found throughout the medulla; (b) neurons restricted to the ventral half of the medulla and featuring long columnar dendritic branches in the outer medulla; and (c) a group of neurons whose dendrites are restricted to the most ventral part of the medulla and whose axons form the anterior inferior optic tract. Most medulla neurons (groups a and b) send their axons via the anterior superior optic tract to the mushroom bodies. Neurons connecting the lobula with the mushroom bodies have their dendrites in a defined dorsal part of the lobula. Their axons form a third tract to the mushroom bodies, here referred to as the lobula tract. Our findings match the anatomy of intrinsic mushroom body neurons (Strausfeld, 2002) and together indicate that the mushroom bodies may be composed of many more functional subsystems than previously suggested.