Cellubrevin is present in the basolateral endocytic compartment of hepatocytes and follows the transcytotic pathway after IgA internalization

The endocytic compartment of polarized cells is organized in basolateral and apical endosomes plus those endocytic structures specialized in recycling and transcytosis, which are still poorly characterized. The complexity of the various populations of endosomes has been demonstrated by the exquisite repertoire of endogenous proteins. In this study we examined the distribution of cellubrevin in the endocytic compartment of hepatocytes, since its intracellular location and function in polarized cells are largely unknown. Highly purified rat liver endosomes were isolated from estradiol-treated rats, and the early/sorting endosomal fraction was further subfractionated in a multistep sucrose density gradient, and studied. Analysis of dissected endosomal fractions showed that cellubrevin was located in early/sorting endosomes, with Rab4, annexins II and VI, and transferrin receptor, but in a specific subpopulation of these early endosomes with the same density range as pIgA and Raf-1. Interestingly, only in those isolated endosomal fractions, endosomes enriched in transcytotic structures (of livers loaded with IgA), the polymeric immunoglobulin receptor specifically co-immunoprecipitated with cellubrevin. In addition, confocal and immuno-electron microscopy identification of cellubrevin in tubular structures underneath the sinusoidal plasma membrane together with the re-organization of cellubrevin, in the endocytic compartment, after the IgA loading, strongly suggest the involvement of cellubrevin in the transcytosis of pIgA

Polyethylene Glycol 35 as a perfusate additive for mitochondrial and glycocalyx protection in HOPE liver preservation

Organ transplantation is a multifactorial process in which proper graft preservation is a mandatory step for the success of the transplantation. Hypothermic preservation of abdominal organs is mostly based on the use of several commercial solutions, including UW, Celsior, HTK and IGL-1. The presence of the oncotic agents HES (in UW) and PEG35 (in IGL-1) characterize both solution compositions, while HTK and Celsior do not contain any type of oncotic agent. Polyethylene glycols (PEGs) are non-immunogenic, non-toxic and water-soluble polymers, which present a combination of properties of particular interest in the clinical context of ischemia-reperfusion injury (IRI): they limit edema and nitric oxide induction and modulate immunogenicity. Besides static cold storage (SCS), there are other strategies to preserve the organ, such as the use of machine perfusion (MP) in dynamic preservation strategies, which increase graft function and survival as compared to the conventional static hypothermic preservation. Here we report some considerations about using PEG35 as a component of perfusates for MP strategies (such as hypothermic oxygenated perfusion, HOPE) and its benefits for liver graft preservation. Improved liver preservation is closely related to mitochondria integrity, making this organelle a good target to increase graft viability, especially in marginal organs (e.g., steatotic livers). The final goal is to increase the pool of suitable organs, and thereby shorten patient waiting lists, a crucial problem in liver transplantation

Polyethylene glycols: an effective strategy for limiting liver ischemia reperfusion injury

Liver ischemia-reperfusion injury (IRI) is an inherent feature of liver surgery and liver transplantation in which damage to a hypoxic organ (ischemia) is exacerbated following the return of oxygen delivery (reperfusion). IRI is a major cause of primary non-function after transplantation and may lead to graft rejection, regardless of immunological considerations. The immediate response involves the disruption of cellular mitochondrial oxidative phosphorylation and the accumulation of metabolic intermediates during the ischemic period, and oxidative stress during blood flow restoration. Moreover, a complex cascade of inflammatory mediators is generated during reperfusion, contributing to the extension of the damage and finally to organ failure. A variety of pharmacological interventions (antioxidants, anti-cytokines, etc.) have been proposed to alleviate graft injury but their usefulness is limited by the local and specific action of the drugs and by their potential undesirable toxic effects. Polyethylene glycols (PEGs), which are non-toxic water-soluble compounds approved by the FDA, have been widely used as a vehicle or a base in food, cosmetics and pharmaceuticals, and also as adjuvants for ameliorating drug pharmacokinetics. Some PEGs are also currently used as additives in organ preservation solutions prior to transplantation in order to limit the damage associated with cold ischemia reperfusion. More recently, the administration of PEGs of different molecular weights by intravenous injection has emerged as a new therapeutic tool to protect liver grafts from IRI. In this review, we summarize the current knowledge concerning the use of PEGs as a useful target for limiting liver IRI

Advanced light microscopy techniques

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32166This article summarizes the basic principles of light microscopy, with examples of applications in biomedicine that illustrate the capabilities of the technique

Protein KinaseCdelta-Calmodulin Crosstalk Regulates Epidermal Growth Factor Receptor Exit from Early Endosomes

We have recently shown that calmodulin antagonist W13 interferes with the trafficking of the epidermal growth factor receptor (EGFR) and regulates the mitogen-activated protein kinase (MAPK) signaling pathway. In the present study, we demonstrate that in cells in which calmodulin is inhibited, protein kinase C (PKC) inhibitors rapidly restore EGFR and transferrin trafficking through the recycling compartment, although onward transport to the degradative pathway remains arrested. Analysis of PKC isoforms reveals that inhibition of PKCδ with rottlerin or its down-modulation by using small interfering RNA is specifically responsible for the release of the W13 blockage of EGFR trafficking from early endosomes. The use of the inhibitor Go 6976, specific for conventional PKCs (α, β, and γ), or expression of dominant-negative forms of PKCλ, ζ, or e did not restore the effects of W13. Furthermore, in cells treated with W13 and rottlerin, we observed a recovery of brefeldin A tubulation, as well as transport of dextran-fluorescein isothiocyanate toward the late endocytic compartment. These results demonstrate a specific interplay between calmodulin and PKCδ in the regulation of the morphology of and trafficking from the early endocytic compartment

Defective TNF-alpha-mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice

This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-alpha-mediated hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH) became sensitive to TNF-alpha, undergoing a time-dependent apoptotic cell death preceded by mitochondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A treatment rescued mGSH-depleted hepatocytes from TNF-alpha-induced cell death. In contrast, mGSH-depleted hepatocytes deficient in ASMase were resistant to TNF-alpha-mediated cell death but sensitive to exogenous ASMase. Furthermore, although in vivo administration of TNF-alpha or LPS to galactosamine-pretreated ASMase(+/+) mice caused liver damage, ASMase(-/-) mice exhibited minimal hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylceramide synthetase inhibition on TNF-alpha-mediated apoptosis. This approach, which blunted glycosphingolipid generation by TNF-alpha, protected mGSH-depleted ASMase(+/+) hepatocytes from TNF-alpha despite enhancement of TNF-alpha-stimulated ceramide formation. To further test the involvement of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase(+/+) but not in ASMase(-/-) hepatocytes. However, treatment of ASMase(-/-) hepatocytes with exogenous ASMase induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-alpha-induced hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids

MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration

Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes

A clathrin-dependent pathway leads to KRas signaling on late endosomes en route to lysosomes

Ras proteins are small guanosine triphosphatases involved in the regulation of important cellular functions such as proliferation, differentiation, and apoptosis. Understanding the intracellular trafficking of Ras proteins is crucial to identify novel Ras signaling platforms. In this study, we report that epidermal growth factor triggers Kirsten Ras (KRas) translocation onto endosomal membranes (independently of calmodulin and protein kinase C phosphorylation) through a clathrin-dependent pathway. From early endosomes, KRas but not Harvey Ras or neuroblastoma Ras is sorted and transported to late endosomes (LEs) and lysosomes. Using yellow fluorescent protein¿Raf1 and the Raichu-KRas probe, we identified for the first time in vivo¿active KRas on Rab7 LEs, eliciting a signal output through Raf1. On these LEs, we also identified the p14¿MP1 scaffolding complex and activated extracellular signal-regulated kinase 1/2. Abrogation of lysosomal function leads to a sustained late endosomal mitogen-activated protein kinase signal output. Altogether, this study reveals novel aspects about KRas intracellular trafficking and signaling, shedding new light on the mechanisms controlling Ras regulation in the cell