Imaging the Dynamics of Endocytosis in Live Mammalian Tissues

In mammalian cells, endocytosis plays a pivotal role in regulating several basic cellular functions. Up to now, the dynamics and the organization of the endocytic pathways have been primarily investigated in reductionist model systems such as cell and organ cultures. Although these experimental models have been fully successful in unraveling the endocytic machinery at a molecular level, our understanding of the regulation and the role of endocytosis in vivo has been limited. Recently, advancements in intravital microscopy have made it possible to extend imaging in live animals to subcellular structures, thus revealing new aspects of the molecular machineries regulating membrane trafficking that were not previously appreciated in vitro. Here, we focus on the use of intravital microscopy to study endocytosis in vivo, and discuss how this approach will allow addressing two fundamental questions: (1) how endocytic processes are organized in mammalian tissues, and (2) how they contribute to organ physiopathology.Endocytosis is a fundamental process used by the cell to internalize molecules from the plasma membrane (Mellman 1996; Doherty and McMahon 2009), and its dysregulation is the cause of several pathological conditions, such as cancer and neurodegenerative, metabolic, and storage diseases (Lanzetti and Di Fiore 2008; Mosesson et al. 2008; Ballabio and Gieselmann 2009).In mammals, endocytosis has been primarily studied in cell culture, which has been instrumental in identifying various endocytic pathways and elucidating the trafficking of internalized molecules throughout the endolysosomal system (Conner and Schmid 2003; Maxfield and McGraw 2004; Donaldson et al. 2009; Hurley and Stenmark 2011). The degree of complexity in the organization and the regulation of the endocytic processes have been shown to substantially increase in polarized cells (Mostov et al. 2003; Folsch et al. 2009) and in organ cultures (Dunn et al. 1980; Kandimalla et al. 2009; Khandelwal et al. 2010), which recapitulate some of the architectural features of the intact tissue. The scenario is further complicated in live animals, where tissues are continuously exposed to a specific combination of cues coming from the vasculature, the central nervous system, and the extracellular environment, which are difficult to reconstitute accurately in vitro. Therefore, although our knowledge of the molecular machineries controlling mammalian endocytosis has substantially increased in the last decades, there are still fundamental issues that have not been explored yet, such as how endocytic pathways are organized and regulated in mammalian tissues. Specifically, it is fundamental to establish whether in vivo cells show the same regulation of endocytic pathways that has been reported in vitro, or how molecules are internalized and trafficked in the presence of physiological levels of ligands and regulatory molecules. Another question is what is the contribution of the endocytic pathways to the physiopathology of a specific tissue or organ. For example, it is of paramount importance to determine whether and how endocytic pathways are altered in epithelial and stromal cells during tumor development and progression, and which specific cell function is affected by their dysregulation.Investigations of endocytosis in live mammals (i.e., rodents) were extensively performed during the 1980s and 1990s by using conventional techniques (e.g., biochemical assays, EM, and indirect immunofluorescence). However, the advent of the green fluorescent protein (GFP) technology, which has enabled imaging subcellular organelles in real time, has significantly shifted the focus toward cell cultures.The recent advancements in intravital microscopy (IVM), which encompasses a series of light microscopy–based techniques, have now made possible imaging biological processes in live animals at a subcellular resolution (Weigert et al. 2013). In this perspective, we focus on reviewing most of the recent data on IVM and endocytosis and try to convey to the reader a sense of the potential, challenges, and limitations of this approach. However, before discussing the “heart of the matter,” we start by briefly pointing out the advantages of using animal models versus the more popular and well-established in vitro model systems.     

The crystal structure of archaeal serine hydroxymethyltransferase reveals idiosyncratic features likely required to withstand high temperatures

Serine hydroxymethyltransferases (SHMTs) play an essential role in one‐carbon unit metabolism and are used in biomimetic reactions. We determined the crystal structure of free (apo) and pyridoxal‐5′‐phosphate‐bound (holo) SHMT from Methanocaldococcus jannaschii, the first from a hyperthermophile, from the archaea domain of life and that uses H₄MPT as a cofactor, at 2.83 and 3.0 Å resolution, respectively. Idiosyncratic features were observed that are likely to contribute to structure stabilization. At the dimer interface, the C‐terminal region folds in a unique fashion with respect to SHMTs from eubacteria and eukarya. At the active site, the conserved tyrosine does not make a cation‐π interaction with an arginine like that observed in all other SHMT structures, but establishes an amide‐aromatic interaction with Asn257, at a different sequence position. This asparagine residue is conserved and occurs almost exclusively in (hyper)thermophile SHMTs. This led us to formulate the hypothesis that removal of frustrated interactions (such as the Arg‐Tyr cation‐π interaction occurring in mesophile SHMTs) is an additional strategy of adaptation to high temperature. Both peculiar features may be tested by designing enzyme variants potentially endowed with improved stability for applications in biomimetic processes. Proteins 2014; 82:3437–3449. © 2014 Wiley Periodicals, Inc.     

Unconventional method based on circular dichroism to detect peanut DNA in food by means of a PNA probe and a cyanine dye

In this paper we report an innovative and unconventional method based on circular dichroism for the identification of peanut DNA in food, which can be detected after PCR amplification at the nanomolar level by using an achiral PNA probe complementary to a tract of the peanut Ara h 2 gene and an achiral 3,3'-diethylthiadicarbocyanine dye [DiSC(2)(5)]. Peanuts are one of the most common causes of severe allergic reactions to foods and are particularly dangerous when they are "hidden" (undeclared) in food. For better protection of consumers, detection methods are required to specifically detect the presence of hidden allergens in a wide variety of food items. Alternative to the detection of the proteins is the determination of species-specific DNA, which is more resistant to technological treatments. PNAs are very specific probes able to recognize DNA sequences with high affinity and evidence for the binding can be obtained by using the DiSC(2)(5) dye, which aggregates onto the PNA-DNA duplex giving rise to a characteristic visibile band at 540 nm. Because the PNA-DNA duplex is in a right-handed helical conformation, the aggregation of the dye to the duplex gives also rise to a strong CD signal in the 500-600 nm region with a strong exciton coupling due to the formation of multimeric species, since the handedness of the helix is transferred to the dye aggregate. The dye does not interact with the free single-stranded DNA and although aggregating on the achiral PNA, this interaction is obviously not detectable by circular dichroism. Thus, only the formation of the PNA-DNA duplex, which takes place only upon specific Watson-Crick hydrogen binding between the PNA and the DNA bases, is detected, ensuring a very high specificity and sensitivity. The method has been optimized in a model system by using a synthetic oligonucleotide complementary to the PNA probe, showing that the intensity of the signal is linearly related to the amount of the DNA. The optimized method has been applied to the identification and quantitation of DNA extracted and amplified by PCR from peanuts and from peanut-containing foods, allowing for a very sensitive detection at a very low level (few pmol).     

Interfacial properties of most monofluorinated bile acids deviate markedly from the natural congeners: studies with the Langmuir-Pockels surface balance

We characterized the air-water interfacial properties of four monofluorinated bile acids alone and in binary mixtures with a common lecithin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), using an automated Langmuir-Pockels surface balance. We compared 7alpha-fluoromurocholic acid (FMCA), 7alpha-fluorohyodeoxycholic acid (FHDCA), 6alpha-fluoroursodeoxycholic acid (FUDCA), and 6alpha-fluorochenodeoxycholic acid (FCDCA) with their natural dihydroxy homologs, murocholic acid (MCA), hyodeoxycholic acid (HDCA), ursodeoxycholic acid (UDCA), and chenodeoxycholic acid (CDCA). For further comparison, two trihydroxy bile acids, 3alpha,6beta,7alpha-trihydroxycholanoic acid [alpha-muricholic acid (alpha-MCA)] and 3alpha,6alpha,7beta-trihydroxycholanoic acid [omega-muricholic acid (omega-MCA)], with isologous OH polar functions to FMCA and FUDCA were also studied. Pressure-area isotherms of MCA, HDCA, UDCA, CDCA, and FMCA displayed sharp collapse points. In contrast, FHDCA, FUDCA, and FCDCA formed monolayers that were less stable than the trihydroxy bile acids, displaying second-order phase transitions in their isotherms. All natural and fluorinated bile acids condensed mixed monolayers with POPC, with maximal effects at molar bile acid concentrations between 30 and 50 mol%. Examination of molecular models revealed that the 7alpha-F atom of the interfacially stable FMCA projects away from the 6beta-OH function, resulting in minimal steric interactions, whereas in FHDCA, FUDCA, and FCDCA, close vicinal interactions between OH and F polar functions result in progressive bulk solubility upon monolayer compression. These results provide a framework for designing F-modified bile acids to mimic or diverge from the natural compounds in vivo.     

Drug-Eluting versus Bare-Metal Stent for Treatment of Saphenous Vein Grafts: A Meta-Analysis

Background

Saphenous vein grafts develop an aggressive atherosclerotic process and the efficacy of drug eluting stents (DES) in treating saphenous vein graft (SVG) lesions has not been convincingly demonstrated. The aim of this study was to review and analyze the current literature for controlled studies comparing DES versus bare metal stents (BMS) for treatment of SVG stenoses.

Methodology/Principal Findings

We searched several scientific databases and conference proceedings up to March 15, 2010 for controlled studies comparing target vessel revascularization (TVR) between DES and BMS. Summary odds ratios (OR) for the primary endpoint TVR and secondary endpoints infarction, stent thrombosis and death were calculated using random-effect models. A total of 29 studies (3 randomized controlled trials RCT) involving 7549 (202 in RCT) patients were included. The need for target vessel revascularization in the DES group tended to be lower compared to BMS for the 3 RCT (OR 0.50 [0.24–1.00]; p = 0.051) and for observational studies (0.62 [0.49–0.79]; p<0.001). There was no significant difference in the risk for myocardial infarction in the RCT (OR 1.25 [0.22–6.99]; p = 0.250) but a lower risk for DES based on the observational studies 0.68 [0.49–0.95]; p = 0.023. The risk for stent thrombosis was found to be non-different in the RCT (OR 0.78 [0.03–21.73], p = 0.885) while it was in favor of DES in the observational studies (0.58 [0.38 – 0.84]; p<0.001). The mortality was not significantly different between DES and BMS in the RCT''s (OR 2.22 [0.17 – 29.50]; p = 0.546) while the observation studies showed a decreased mortality in the DES group (0.69 [0.55–0.85]; p<0.001).

Conclusion

DES may decrease TVR rate in treatment of SVG stenoses. No differences in reinfarction rate, stent thrombosis or mortality was found between the DES and BMS groups in the RCT''s while the observational data showed lower risk for myocardial infarction, stent thrombosis and death in the DES group. This may be a result of patient selection bias in the observational studies or represent a true finding that was not the detected in the RCT analysis due to limited statistical power.     

Molecular modeling of the human eukaryotic translation initiation factor 5A (eIF5A) based on spectroscopic and computational analyses

The eukaryotic translation initiation factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to eIF5A has been reported in the literature. The discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. In this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance.     

A screen for genes of heme uptake identifies the FLC family required for import of FAD into the endoplasmic reticulum

Although Candida albicans and Saccharomyces cerevisiae express very similar systems of iron uptake, these species differ in their capacity to use heme as a nutritional iron source. Whereas C. albicans efficiently takes up heme, S. cerevisiae grows poorly on media containing heme as the sole source of iron. We identified a gene from C. albicans that would enhance heme uptake when expressed in S. cerevisiae. Overexpression of CaFLC1 (for flavin carrier 1) stimulated the growth of S. cerevisiae on media containing heme iron. In C. albicans, deletion of both alleles of CaFLC1 resulted in a decrease in heme uptake activity, whereas overexpression of CaFLC1 resulted in an increase in heme uptake. The S. cerevisiae genome contains three genes with homology to CaFLC1, and two of these, termed FLC1 and FLC2, also stimulated growth on heme when overexpressed in S. cerevisiae. The S. cerevisiae Flc proteins were detected in the endoplasmic reticulum and the FLC genes encoded an essential function, as strains deleted for either FLC1 or FLC2 were viable, but deletion of both FLC1 and FLC2 was synthetically lethal. FLC gene deletion resulted in pleiotropic phenotypes related to defects in cell wall integrity. High copy suppressors of this synthetic lethality included three mannosyltransferases, VAN1, KTR4, and HOC1. FLC deletion strains exhibited loss of cell wall mannose phosphates, defects in cell wall assembly, and delayed maturation of carboxypeptidase Y. Permeabilized cells lacking FLC proteins exhibited dramatic loss of FAD import activity. We propose that the FLC genes are required for import of FAD into the lumen of the endoplasmic reticulum, where it is required for disulfide bond formation.     

PD-L1 Blockade Differentially Impacts Regulatory T Cells from HIV-Infected Individuals Depending on Plasma Viremia

Blocking the PD-1/PD-L1 pathway has emerged as a potential therapy to restore impaired immune responses in human immunodeficiency virus (HIV)-infected individuals. Most reports have studied the impact of the PD-L1 blockade on effector cells and neglected possible effects on regulatory T cells (Treg cells), which play an essential role in balancing immunopathology and antiviral effector responses. The aim of this study was to define the consequences of ex vivo PD-L1 blockade on Treg cells from HIV-infected individuals. We observed that HIV infection led to an increase in PD-1+ and PD-L1+ Treg cells. This upregulation correlated with disease progression and decreased under antiretroviral treatment. Treg cells from viremic individuals had a particularly high PD-1 expression and impaired proliferative capacity in comparison with Treg cells from individuals under antiretroviral treatment. PD-L1 blockade restored the proliferative capacity of Treg cells from viremic individuals but had no effect on its suppressive capacity. Moreover, it increased the viral production in cell cultures from viremic individuals. This increase in viral production correlated with an increase in Treg cell percentage and a reduction in the CD4/Treg and CD8/Treg cell ratios. In contrast to the effect of the PD-L1 blockade on Treg cells from viremic individuals, we did not observe a significant effect on the proliferative capacity of Treg cells from individuals in whom viremia was controlled (either spontaneously or by antiretroviral treatment). However, PD-L1 blockade resulted in an increased proliferative capacity of HIV-specific-CD8 T cells in all subjects. Taken together, our findings suggest that manipulating PD-L1 in vivo can be expected to influence the net gain of effector function depending on the subject’s plasma viremia.     

Translational research impacting on crop productivity in drought-prone environments

Conventional breeding for drought-prone environments (DPE) has been complemented by using exotic germplasm to extend crop gene pools and physiological approaches that consider water uptake (WU), water-use efficiency (WUE), and harvest index (HI) as drivers of yield. Drivers are associated with proxy genetic markers, such as carbon-isotope discrimination for WUE, canopy temperature for WU, and anthesis-silking interval for HI in maize. Molecular markers associated with relevant quantitative trait loci are being developed. WUE has also been increased through combining understanding of root-to-shoot signaling with deficit irrigation. Impacts in DPE will be accelerated by combining proven technologies with promising new strategies such as marker-assisted selection, and genetic transformation, as well as conservation agriculture that can increase WU while averting soil degradation.