RESUMEN
After mammalian cell invasion, the parasite Toxoplasma multiplies in a self-made membrane-bound compartment, the parasitophorous vacuole (PV). We previously showed that Toxoplasma interacts with many host cell organelles, especially from recycling pathways, and sequestrates Rab11A and Rab11B vesicles into the PV. Here, we examine the specificity of host Rab11 vesicle interaction with the PV by focusing on the recruitment of subpopulations of Rab11 vesicles characterized by different effectors, for example, Rab11-family interacting roteins (FIPs) or Arf6. Our quantitative microscopic analysis illustrates the presence of intra-PV vesicles with FIPs from class I (FIP1C, FIP2, FIP5) and class II (FIP3, FIP4) but to various degrees. The intra-PV delivery of vesicles with class I, but not class II, FIPs is dependent on Rab11 binding. Cell depletion of Rab11A results in a significant decrease in intra-PV FIP5, but not FIP3 vesicles. Class II FIPs also bind to Arf6, and we observe vesicles associated with FIP3-Rab11A or FIP3-Arf6 complexes concomitantly within the PV. Abolishing FIP3 binding to both Rab11 and Arf6 reduces the number of intra-PV FIP3 vesicles. These data point to a selective process of mammalian Rab11 vesicle recognition and scavenging mediated by Toxoplasma, suggesting that specific parasite PV proteins may be involved in these processes.
Asunto(s)
Toxoplasma , Factor 6 de Ribosilación del ADP , Animales , Endosomas/metabolismo , Células HeLa , Humanos , Mamíferos/metabolismo , Unión Proteica , Toxoplasma/metabolismo , Vacuolas/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Fluorescence microscopy and image analysis are powerful techniques to examine the distribution and interactions of different cellular compartments, including mammalian organelles with intravacuolar pathogens. Toxoplasma gondii is an obligate intracellular protozoan parasite that forms a membrane-bound compartment, the parasitophorous vacuole (PV), upon invasion of mammalian cells. From within the PV, the parasite interacts with many host organelles (without fusion), redirects host vesicles decorated with Rab GTPases to the PV, and internalizes many of these nutrient-filled Rab vesicles into the PV. Here, we report a method to distinguish the host Rab vesicles that are exclusively trapped in the Toxoplasma PV from those localized along the edge of the vacuole. Such a discrimination between the two Rab vesicle populations (inside versus outside of the PV) allows the selective characterization of the intra-PV Rab vesicles, for example, number per PV, volume, and distance from the PV centroid, as well as comparisons between wild-type and mutant Toxoplasma.
Asunto(s)
Toxoplasma , Animales , Interacciones Huésped-Parásitos , Humanos , Microscopía Fluorescente , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Vacuolas/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Many intracellular pathogens subvert host membrane trafficking pathways to promote their replication. Toxoplasma multiplies in a membrane-bound parasitophorous vacuole (PV) that interacts with mammalian host organelles and intercepts Golgi Rab vesicles to acquire sphingolipids. The mechanisms of host vesicle internalization and processing within the PV remain undefined. We demonstrate that Toxoplasma sequesters a broad range of Rab vesicles into the PV. Correlative light and electron microscopy analysis of infected cells illustrates that intravacuolar Rab1A vesicles are surrounded by the PV membrane, suggesting a phagocytic-like process for vesicle engulfment. Rab11A vesicles concentrate to an intravacuolar network (IVN), but this is reduced in Δgra2 and Δgra2Δgra6 parasites, suggesting that tubules stabilized by the TgGRA2 and TgGRA6 proteins secreted by the parasite within the PV contribute to host vesicle sequestration. Overexpression of a phospholipase TgLCAT, which is localized to the IVN, results in a decrease in the number of intravacuolar GFP-Rab11A vesicles, suggesting that TgLCAT controls lipolytic degradation of Rab vesicles for cargo release.
Asunto(s)
Vesículas Citoplasmáticas/metabolismo , Interacciones Huésped-Parásitos , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Toxoplasma/metabolismo , Vacuolas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Células CHO , Chlorocebus aethiops , Cricetulus , Vesículas Citoplasmáticas/ultraestructura , Fibroblastos/metabolismo , Fibroblastos/parasitología , Fibroblastos/ultraestructura , Regulación de la Expresión Génica , Genes Reporteros , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Fagocitosis , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Esfingolípidos/metabolismo , Toxoplasma/ultraestructura , Vacuolas/ultraestructura , Células Vero , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
We report that a single growth factor, NM23-H1, enables serial passaging of both human ES and iPS cells in the absence of feeder cells, their conditioned media or bFGF in a fully defined xeno-free media on a novel defined, xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more "naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1, we override the stem cell's inherent programming that turns off pluripotency and trick the cells into continuously replicating as pluripotent stem cells. Dimeric NM23-H1 binds to and dimerizes the extra cellular domain of the MUC1* transmembrane receptor which stimulates growth and promotes pluripotency. Inhibition of the NM23-H1/MUC1* interaction accelerates differentiation and causes a spike in miR-145 expression which signals a cell's exit from pluripotency.