The Complex Ultrastructure of the Endolysosomal System

Live-cell imaging reveals the endolysosomal system as a complex and highly dynamic network of interacting compartments. Distinct types of endosomes are discerned by kinetic, molecular, and morphological criteria. Although none of these criteria, or combinations thereof, can capture the full complexity of the endolysosomal system, they are extremely useful for experimental purposes. Some membrane domain specializations and specific morphological characteristics can only be seen by ultrastructural analysis after preparation for electron microscopy (EM). Immuno-EM allows a further discrimination of seemingly identical compartments by their molecular makeup. In this review we provide an overview of the ultrastructural characteristics and membrane organization of endosomal compartments, along with their organizing machineries.The endolysosomal network is required for multiple functions and control of cell homeostasis. It is not only reached by endocytic cargo but also by biosynthetic cargoes. It is an intermediate to degradation, but also essential for recycling, signaling, cell polarity, cilia formation, cytokinesis, and migration (Gould and Lippincott-Schwartz 2009; Taguchi 2013). This multitude of functions can only be ensured by an extremely organized ultrastructure. With the increased understanding of how cellular machinery defines endolysosomal subdomains, the nomenclature of the endolysosomal system has also increased in complexity. We start this review, therefore, with a brief introduction of the terminology of the endolysosomal system.Coated pits and vesicles were described in 1964 (Roth and Porter 1964), and lysosomes were first described by De Duve and Novikoff in the mid-1950s (Novikoff et al. 1956), but the range of organelles in between these beginning and ending stages of endocytosis was only described later (Bhisey and Freed 1971). Electron microscopy (EM) studies by Allen and coworkers on the unicellular ciliate Paramecium caudatum revealed the existence of intracellular compartments that could be loaded with the endocytic marker horseradish peroxidase (HRP) (Allen and Fok 1980). These were named “endosomes.” Parallel studies in mammalian cells, by Pastan, Willingham, and colleagues, also using HRP, described intracellular vacuoles and tubules involved in the transport of transferrin receptor (TfR) (Gonatas et al. 1977; Goud et al. 1981; Willingham and Pastan 1983). These were called “receptosomes” (Willingham and Pastan 1980). Geuze, Slot, and collaborators introduced immunogold labeling, allowing the quantitative localization of multiple proteins within one EM sample (Geuze et al. 1981). When they localized the recycling asialoglycoprotein receptor together with its ligand destined for lysosomal degradation (Geuze et al. 1983), they identified compartments consisting of a vacuole and multiple associated tubules. These were called compartments involved in the uncoupling of receptors and ligands (CURLs) because the vacuoles accumulated the ligand (for degradation) and the tubules the receptor (for recycling). Today the CURL is known as the “early endosome” (EE), which in addition to receptors and ligands is now known to be reached by virtually all components internalized from the cell surface (see Mayor et al. 2014; Cossart and Helenius 2014).In the current literature, different nomenclatures are still used to describe the endolysosomal system, which can sometimes cause some confusion. In this review, based on combined ultrastructural and functional knowledge, we propose the following nomenclature: We refer to the vacuolar domains of EEs as sorting endosomes (SEs) and the tubules emerging from SEs as recycling endosomes (REs). Although in some cells (e.g., melanocytes) (see Delevoye et al. 2009), the RE tubules may stay attached while functioning in recycling, more typically they detach from the SE to form a tubular endosomal network (TEN). The term “endosomal recycling compartment” (ERC) is used to designate the peri-centriolar compartment that can be observed only in some cell types. Late endosomes (LEs), also referred to as multivesicular bodies (MVBs), are rounded compartments filled with intraluminal vesicles (ILVs). Lysosomes are the final compartments of the endocytic pathway, with different morphologies depending on the cell type (schematic representation in Fig. 1). Moreover, in the literature, these terms are differently used because most studies involve light microscopy, which does not provide sufficient resolution to detect all of the distinct domains.Open in a separate windowFigure 1.Schematic and simplified representation of the endolysosomal system showing the different organelles described in this article. Sorting endosomes (SE) are vacuolar compartments often bearing bilayered, flat clathrin coats (brown). Tubules emanate from SE that form the recycling endosomes (RE). The RE may localize to the peri-Golgi area forming the endocytic recycling compartment (ERC) or distribute to the cell periphery. The RE network is complex with multiple sorting sites, thereby the tubular sorting endosome (TSE) or tubular endosomal network (TEN) is also represented. AP1 (red) and AP3 (green) coated buds on RE (ERC/TSE/TEN) are shown. Late endosomes correspond to multivesicular bodies (MVBs) filled with intraluminal vesicles (ILVs). MVBs are fated for fusion with lysosomes. In some cells, a population of MVBs fuse with the plasma membrane, a process during which the ILVs are secreted as exosomes. Gray arrows indicate directions of transport/maturation of compartments. Blue arrows indicate invagination at the endosomal membrane of SE and MVBs required for ILV formation.     

Performance evaluation of a mesophilic anaerobic fluidized-bed reactor treating wastewater derived from the production of proteins from extracted sunflower flour

A study of the anaerobic digestion of wastewater derived from the production of protein isolates from extracted sunflower flour was carried out in a laboratory-scale, mesophilic (35 degrees C) fluidized-bed reactor with saponite as bacterial support. Chemical oxygen demand (COD) removal efficiencies in the range of 98.3-80.0% were achieved in the reactor at organic loading rates (OLR) of between 0.6 and 9.3 g COD/I d, hydraulic retention times (HRT) of between 20.0 and 1.1 d and average feed COD concentration of 10.6 g/l. Eighty percent of feed COD could be removed up to OLR of 9.3 g COD/l d. The yield coefficient of methane production was 0.33 l of methane (at STP) per gram of COD removed and was virtually independent of the OLR applied. Because the buffering capacity of the experimental system was maintained at favorable levels with excess total alkalinity present at all loadings, the rate of methanogenesis was not affected by loading. The experimental data indicated that a total alkalinity in the range of 2,000-2,460 mg/l as CaCO3 was sufficient to prevent the pH from dropping to below 7.0 for OLR of up to 9.3 g COD/l d. The volatile fatty acid (VFA) levels and the VFA/alkalinity ratio were lower than the suggested limits for digester failure (0.3-0.4) for OLR and HRT up to 9.3 g COD/l d and 1.1 d, respectively. For a HRT of 0.87 d (OLR of 12.1 g COD/l d) the start of acidification was observed in the reactor.     

Synchronization of secretory protein traffic in populations of cells

To dissect secretory traffic, we developed the retention using selective hooks (RUSH) system. RUSH is a two-state assay based on the reversible interaction of a hook protein fused to core streptavidin and stably anchored in the donor compartment with a reporter protein of interest fused to streptavidin-binding peptide (SBP). Biotin addition causes a synchronous release of the reporter from the hook. Using the RUSH system, we analyzed different transport characteristics of various Golgi and plasma membrane reporters at physiological temperature in living cells. Using dual-color simultaneous live-cell imaging of two cargos, we observed intra- and post-Golgi segregation of cargo traffic, consistent with observation in other systems. We show preliminarily that the RUSH system is usable for automated screening. The system should help increase the understanding of the mechanisms of trafficking and enable screens for molecules that perturb pathological protein transport.     

Pollen and seed flow patterns of Carapa guianensis Aublet. (Meliaceae) in two types of Amazonian forest

Various factors affect spatial genetic structure in plant populations, including adult density and primary and secondary seed dispersal mechanisms. We evaluated pollen and seed dispersal distances and spatial genetic structure of Carapa guianensis Aublet. (Meliaceae) in occasionally inundated and terra firme forest environments that differed in tree densities and secondary seed dispersal agents. We used parentage analysis to obtain contemporary gene flow estimates and assessed the spatial genetic structure of adults and juveniles. Despite the higher density of adults (diameter at breast height ≥ 25 cm) and spatial aggregation in occasionally inundated forest, the average pollen dispersal distance was similar in both types of forest (195 ± 106 m in terra firme and 175 ± 87 m in occasionally inundated plots). Higher seed flow rates (36.7% of juveniles were from outside the plot) and distances (155 ± 84 m) were found in terra firme compared to the occasionally inundated plot (25.4% and 114 ± 69 m). There was a weak spatial genetic structure in juveniles and in terra firme adults. These results indicate that inundation may not have had a significant role in seed dispersal in the occasionally inundated plot, probably because of the higher levels of seedling mortality.     

Abundance and Genetic Diversity of nifH Gene Sequences in Anthropogenically Affected Brazilian Mangrove Sediments

Although mangroves represent ecosystems of global importance, the genetic diversity and abundance of functional genes that are key to their functioning scarcely have been explored. Here, we present a survey based on the nifH gene across transects of sediments of two mangrove systems located along the coast line of São Paulo state (Brazil) which differed by degree of disturbance, i.e., an oil-spill-affected and an unaffected mangrove. The diazotrophic communities were assessed by denaturing gradient gel electrophoresis (DGGE), quantitative PCR (qPCR), and clone libraries. The nifH gene abundance was similar across the two mangrove sediment systems, as evidenced by qPCR. However, the nifH-based PCR-DGGE profiles revealed clear differences between the mangroves. Moreover, shifts in the nifH gene diversities were noted along the land-sea transect within the previously oiled mangrove. The nifH gene diversity depicted the presence of nitrogen-fixing bacteria affiliated with a wide range of taxa, encompassing members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and also a group of anaerobic sulfate-reducing bacteria. We also detected a unique mangrove-specific cluster of sequences denoted Mgv-nifH. Our results indicate that nitrogen-fixing bacterial guilds can be partially endemic to mangroves, and these communities are modulated by oil contamination, which has important implications for conservation strategies.     

Analysis of a genome-wide association study-linked locus (CCR6) in Asian rheumatoid arthritis

A genome-wide association study in Japan identified the C-C chemokine receptor type 6 gene (CCR6) as associated with rheumatoid arthritis (RA). This finding has not been validated in other Asian populations. A case-control study involving 996 subjects, comprising 440 controls and 556 RA patients, was done to determine their anticyclic citrullinated peptide (anti-CCP) antibody status and CCR6 polymorphism (rs3093024) genotype. Three hundred eighty-seven patients were anti-CCP positive and 153 anti-CCP negative. Logistic regression showed that allele A was likely to increase the risk of developing RA among females via a recessive model (odds ratio [OR]=1.55, 95% confidence interval [CI]=1.01, 2.39), whereas the risk effect appeared to be reduced among males via an additive model (OR=0.60, 95% CI=0.42, 0.85). Considering only subjects who are anti-CCP positive, allele A increased RA risk among females via a recessive model (OR=1.68, 95% CI=1.07, 2.64) but decreased the risk among males via an additive model (OR=0.59, 95% CI=0.39, 0.89). We showed that CCR6 polymorphism was a risk factor among females but a protective factor among males. Functional studies are warranted to unravel the pathophysiological relevance of the gene variant and other linked variants with RA.     

The influence of turning angles on the success of non-oriented animal searches

Animal searches cover a full range of possibilities from highly deterministic to apparently completely random behaviors. However, even those stochastic components of animal movement can be adaptive, since not all random distributions lead to similar success in finding targets. Here we address the general problem of optimizing encounter rates in non-deterministic, non-oriented searches, both in homogeneous and patchy target landscapes. Specifically, we investigate how two different features related to turning angle distributions influence encounter success: (i) the shape (relative kurtosis) of the angular distribution and (ii) the correlations between successive relative orientations (directional memory). Such influence is analyzed in correlated random walk models using a proper choice of representative turning angle distributions of the recently proposed Jones and Pewsey class. We consider the cases of distributions with nearly the same shape but considerably distinct correlation lengths, and distributions with same correlation but with contrasting relative kurtosis. In homogeneous landscapes, we find that the correlation length has a large influence in the search efficiency. Moreover, similar search efficiencies can be reached by means of distinctly shaped turning angle distributions, provided that the resulting correlation length is the same. In contrast, in patchy landscapes the particular shape of the distribution also becomes relevant for the search efficiency, specially at high target densities. Excessively sharp distributions generate very inefficient searches in landscapes where local target density fluctuations are large. These results are of evolutionary interest. On the one hand, it is shown that equally successful directional memory can arise from contrasting turning behaviors, therefore increasing the likelihood of robust adaptive stochastic behavior. On the other hand, when target landscape is patchy, adequate tumbling may help to explore better local scale heterogeneities, being some details of the shape of the distribution also potentially adaptive.     

Mobility of metals in salt marsh sediments colonised by <Emphasis Type="Italic">Spartina maritima</Emphasis> (Tagus estuary,Portugal)

Chemical associations of Zn, Pb, Cu, Co and Cd were determined using a sequential extraction procedure in sediments colonised by S. maritima in three salt marshes within the Tagus estuary: Rosário, Corroios and Pancas. Concentrations of these metals were also analysed in above- and belowground parts of Spartina maritima, as well as in sediments colonised by the plant. The highest metal concentrations in sediments were found in the marshes near the industrial and urban areas, whereas metal concentrations in plants were not significantly different among sites. This was thought to be a consequence of differences observed in metal bioavailability: Metals in Pancas, the least polluted location, were largely associated to easily accessible fractions for plant uptake, probably as a result of low organic matter content and high sandy fraction in sediments. S. maritima was able to induce the concentration of metals between its roots in the three salt marshes. The results obtained in this study indicate that S. maritima could be useful to induce phytostabilisation of metals in sediments, although the effectiveness to modify chemical associations is highly dependent on existing sediment parameters, and thus different results could be obtained depending on site characteristics. Guest editors: J. Davenport, G. Burnell, T. Cross, M. Emmerson, R. McAllen, R. Ramsay & E. Rogan Challenges to Marine Ecosystems