RESUMEN
The main cardiovascular alteration in Marfan syndrome (MFS) is the formation of aortic aneurysms in which augmented TGF-ß signaling is reported. However, the primary role of TGF-ß signaling as a molecular link between the genetic mutation of fibrillin-1 and disease onset is controversial. The compartmentalization of TGF-ß endocytic trafficking has been shown to determine a signaling response in which clathrin-dependent internalization leads to TGF-ß signal propagation, and caveolin-1 (CAV-1) associated internalization leads to signal abrogation. We here studied the contribution of endocytic trafficking compartmentalization to increased TGF-ß signaling in vascular smooth muscle cells (VSMC) from MFS patients. We examined molecular components involved in clathrin- (SARA, SMAD2) and caveolin-1- (SMAD7, SMURF2) dependent endocytosis. Marfan VSMC showed higher recruitment of SARA and SMAD2 to membranes and their increased interaction with TGF-ß receptor II, as well as higher colocalization of SARA with the early endosome marker EEA1. We assessed TGF-ß internalization using a biotinylated ligand (b-TGF-ß), which colocalized equally with either EEA1 or CAV-1 in VSMC from Marfan patients and controls. However, in Marfan cells, colocalization of b-TGF-ß with SARA and EEA1 was increased and accompanied by decreased colocalization with CAV-1 at EEA1-positive endosomes. Moreover, Marfan VSMC showed higher transcriptional levels and membrane enrichment of RAB5. Our results indicate that increased RAB5-associated SARA localization to early endosomes facilitates its TGF-ß receptor binding and phosphorylation of signaling mediator SMAD2 in Marfan VSMC. This is accompanied by a reduction of TGF-ß sorting into multifunctional vesicles containing cargo from both internalization pathways.
Asunto(s)
Endocitosis , Síndrome de Marfan/metabolismo , Miocitos del Músculo Liso/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo , Adulto , Caveolina 1/metabolismo , Clatrina/metabolismo , Citosol/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/citología , Fosforilación , Transporte de Proteínas , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína Smad2/metabolismo , Adulto Joven , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
The inhibition of phosphatidic acid phosphatase (PAP) activity by propanolol indicates that diacylglycerol (DAG) is required for the formation of transport carriers at the Golgi and for retrograde trafficking to the ER. Here we report that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER-Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER-Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Fosfatidato Fosfatasa/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Diglicéridos/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Transporte de Proteínas , Vías Secretoras , Toxina Shiga/metabolismo , Células 3T3 Swiss , Proteínas de Unión al GTP rab/metabolismoRESUMEN
The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in trafficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers.
Asunto(s)
Actinas/metabolismo , Aparato de Golgi/metabolismo , Animales , Aparato de Golgi/ultraestructura , Humanos , Modelos BiológicosRESUMEN
The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DCs) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single-particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Células Dendríticas/metabolismo , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , VIH-1/fisiología , Actinas/metabolismo , Liposomas/metabolismo , Ligandos , Gangliósidos/metabolismoRESUMEN
Cross-presentation, in which exogenous antigens are presented via MHC I complexes, is involved both in the generation of anti-infectious and anti-tumoral cytotoxic CD8(+) T cells and in the maintenance of immune tolerance. While cross-presentation was described almost four decades ago and while it is now established that some dendritic cell (DC) subsets are better than others in processing and cross-presenting internalized antigens, the involved molecular mechanisms remain only partially understood. Some of the least explored molecular mechanisms in cross-presentation concern the origin of cross-presenting MHC I molecules and the cellular compartments where antigenic peptide loading occurs. This review focuses on MHC I molecules and their intracellular trafficking. We discuss the source of cross-presenting MHC I in DCs as well as the role of the endocytic pathway in their recycling from the cell surface. Next, we describe the importance of the TAP peptide transporter for delivering peptides to MHC I during cross-presentation. Finally, we highlight the impact of innate immunity mechanisms on specific antigen cross-presentation mechanisms in which TLR activation modulates MHC I trafficking and TAP localization.
RESUMEN
Dendritic cells (DCs) have the unique ability to pick up dead cells carrying antigens in tissue and migrate to the lymph nodes where they can cross-present cell-associated antigens by MHC class I to CD8(+) T cells. There is strong in vivo evidence that the mouse XCR1(+) DCs subset acts as a key player in this process. The intracellular processes underlying cross-presentation remain controversial and several pathways have been proposed. Indeed, a wide number of studies have addressed the cellular process of cross-presentation in vitro using a variety of sources of antigen and antigen-presenting cells. Here, we review the in vivo and in vitro evidence supporting the current mechanistic models and disscuss their physiological relevance to the cross-presentation of cell-associated antigens by DCs subsets.
RESUMEN
Phago-lysosome formation is important for cell-autonomous immunity to intracellular pathogens, antigen presentation and metabolism. A hallmark feature of phago-lysosomal compartments is that they undergo progressive luminal acidification controlled by the activation of vacuolar V-ATPase. Acidification is required for many enzymatic processes taking place in phago-lysosomes, like proteolysis, and supports the microbicidal activity of macrophages. Here we present a new quantitative methodology to assess phagosome acidification by flow cytometry based on the use of bi-fluorescent particles. This method relies on the use of UV polystyrene beads labelled with the acid sensor pHrodo-succinimidyl ester (pHrodo(TM) SE red) and enables us to dissociate particle association with phagocytes from their engulfment in acidified compartments. This methodology is well suited to monitor the acidification of phagosomes formed in vivo after fluorescent bead administration.