scholarly journals Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia

2019 ◽  
Author(s):  
Lenka Cernikova ◽  
Carmen Faso ◽  
Adrian B. Hehl
Keyword(s):  
Giardia Lamblia ◽  
Fluid Phase ◽  
Membrane Lipid ◽  
Model Systems ◽  
Coated Vesicle ◽  
New Members ◽  
Partial Conservation ◽  
Parasitic Protist ◽  
Organelle Morphology ◽  
Endocytic Compartments

AbstractPhosphorylated derivatives of phosphatidylinositol (PIPs), are key membrane lipid residues involved in clathrin-mediated endocytosis (CME). CME relies on PI(4,5)P2 to mark endocytic sites at the plasma membrane (PM) associated to clathrin-coated vesicle (CCV) formation. The highly diverged parasitic protist Giardia lamblia presents disordered and static clathrin assemblies at PM invaginations, contacting specialized endocytic organelles called peripheral vacuoles (PVs). The role for clathrin assemblies in fluid phase uptake and their link to internal membranes via PIP-binding adaptors is unknown.Here we provide evidence for a robust link between clathrin assemblies and fluid-phase uptake in G. lamblia mediated by proteins carrying predicted PX, FYVE and NECAP1 PIP-binding modules. We show that chemical and genetic perturbation of PIP-residue binding and turnover elicits novel uptake and organelle-morphology phenotypes. A combination of co-immunoprecipitation and in silico annotation techniques expands the initial PIP-binding network with addition of new members. Our data indicate that, despite the partial conservation of lipid markers and protein cohorts known to play important roles in dynamic endocytic events in well-characterized model systems, the Giardia lineage presents a strikingly divergent clathrin-centered network. This includes several PIP-binding modules, often associated to domains of currently unknown function that shape and modulate fluid-phase uptake at PVs.

scholarly journals Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia

2020 ◽  
Vol 16 (2) ◽  
pp. e1008317
Author(s):  
Lenka Cernikova ◽  
Carmen Faso ◽  
Adrian B. Hehl
Keyword(s):  
Giardia Lamblia ◽  
Binding Proteins ◽  
Parasitic Protist ◽  
Endocytic Compartments

scholarly journals Efficient Dilution-to-Extinction isolation of novel virus-host model systems for fastidious heterotrophic bacteria

2020 ◽  
Author(s):  
Holger H. Buchholz ◽  
Michelle Michelsen ◽  
Luis M. Bolaños ◽  
Emily Browne ◽  
Michael J. Allen ◽  
...  
Keyword(s):  
Heterotrophic Bacteria ◽  
Nutrient Recycling ◽  
Model Systems ◽  
Metagenomic Data ◽  
Culture Collections ◽  
Genomic Databases ◽  
New Members ◽  
Host Interactions ◽  
Infection Dynamics ◽  
Ecological Patterns

AbstractMicrobes and their associated viruses are key drivers of biogeochemical processes in marine and soil biomes. While viruses of phototrophic cyanobacteria are well-represented in model systems, challenges of isolating marine microbial heterotrophs and their viruses have hampered experimental approaches to quantify the importance of viruses in nutrient recycling. A resurgence in cultivation efforts has improved the availability of fastidious bacteria for hypothesis testing, but this has not been matched by similar efforts to cultivate their associated bacteriophages. Here, we describe a high-throughput method for isolating important virus-host systems for fastidious heterotrophic bacteria that couples advances in culturing of hosts with sequential enrichment and isolation of associated phages. Applied to six monthly samples from the Western English Channel, we first isolated one new member of the globally dominant bacterial SAR11 clade and three new members of the methylotrophic bacterial clade OM43. We used these as bait to isolate 117 new phages including the first known siphophage infecting SAR11, and the first isolated phage for OM43. Genomic analyses of 13 novel viruses revealed representatives of three new viral genera, and infection assays showed that the viruses infecting SAR11 have ecotype-specific host-ranges. Similar to the abundant human-associated phage ΦCrAss001, infection dynamics within the majority of isolates suggested either prevalent lysogeny or chronic infection, despite a lack of associated genes; or host phenotypic bistability with lysis putatively maintained within a susceptible subpopulation. Broader representation of important virus-host systems in culture collections and genomic databases will improve both our understanding of virus-host interactions, and accuracy of computational approaches to evaluate ecological patterns from metagenomic data.

scholarly journals Trafficking of Shigella Lipopolysaccharide in Polarized Intestinal Epithelial Cells

1999 ◽  
Vol 145 (4) ◽  
pp. 689-698 ◽  
Author(s):  
Wandy L. Beatty ◽  
Stéphane Méresse ◽  
Pierre Gounon ◽  
Jean Davoust ◽  
Joëlle Mounier ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junctions ◽  
Apical Membrane ◽  
Intestinal Epithelial Cells ◽  
Fluid Phase ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Electron Microscopic ◽  
Basolateral Side ◽  
Endocytic Compartments

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395–4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

Is fluid-phase endocytosis conserved in hepatocytes of species acclimated and adapted to different temperatures?

2000 ◽  
Vol 278 (2) ◽  
pp. R529-R536 ◽  
Author(s):  
David Padrón ◽  
Michael E. Bizeau ◽  
Jeffrey R. Hazel
Keyword(s):  
Lucifer Yellow ◽  
Fluid Phase ◽  
Membrane Lipid ◽  
Primary Objective ◽  
Chain Polyunsaturated Fatty Acid ◽  
Chow Diet ◽  
Sprague Dawley ◽  
Fluid Phase Endocytosis ◽  
Different Temperatures ◽  
Long Chain

Our primary objective was to determine if rates of fluid-phase endocytosis (FPE) were conserved in hepatocytes from organisms acclimated and adapted to different temperatures. To this aim, the fluorescent dye Lucifer yellow was employed to measure FPE at different assay temperatures (AT) in hepatocytes from 5°C- and 20°C-acclimated trout, Oncorhynchus mykiss (at 5 and 20°C AT), 22°C- and 35°C-acclimated tilapia, Oreochromis nilotica (at 22 and 35°C AT), and the Sprague-Dawley rat (at 10, 20, and 37°C AT). FPE was also studied in rats fed a long-chain polyunsaturated fatty acid (PUFA)-enriched diet (at 10°C AT). Despite being temperature dependent, endocytic rates (values in pl ⋅ cell− 1 ⋅ h− 1) in both species of fish were compensated after a period of acclimation. For example, in 20°C-acclimated trout, the rate of endocytosis declined from 1.84 to 1.07 when the AT was reduced from 20 to 5°C; however, after a period of acclimation at 5°C, the rate (at 5°C AT) was largely restored (1.80) and almost perfectly compensated (95%). In tilapia, endocytic rates were also temperature compensated, although only partially (36%). Relatively similar rates obtained at 5°C in 5°C-acclimated trout (1.8), at 20°C in 20°C-acclimated trout (1.84), and at 22°C in 22°C-acclimated tilapia (2.2) suggest that endocytic rates are somewhat conserved in these two species of fish. In contrast, the rate in rat measured at 37°C (16.83) was severalfold greater than in fish at their respective body temperatures. A role for lipids in determining rates of endocytosis was supported by data obtained at 10°C in hepatocytes isolated from rats fed a long-chain PUFA-enriched diet: endocytic rates were higher (5.35 pl ⋅ cell− 1 ⋅ h− 1) than those of rats fed a standard chow diet (2.33 pl ⋅ cell− 1 ⋅ h− 1). The conservation of endocytic rates in fish may be related to their ability to conserve other membrane characteristics (i.e., order or phase behavior) by restructuring their membrane lipid composition or by modulating the activities of proteins that regulate endocytosis and membrane traffic, whereas the lack of conservation between fish and rat may be due to differences in metabolic rate.

scholarly journals Effect of Starvation on the Endocytic Pathway in Dictyostelium Cells

2010 ◽  
Vol 9 (3) ◽  
pp. 387-392 ◽  
Author(s):  
Ewan W. Smith ◽  
Wanessa C. Lima ◽  
Steve J. Charette ◽  
Pierre Cosson
Keyword(s):  
Cell Surface ◽  
Functional Organization ◽  
General Structure ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Rich Medium ◽  
Content Type ◽  
Structure And Dynamics ◽  
Endocytic Compartments ◽  
Mutant Cells

ABSTRACT Dictyostelium discoideum amoebae have been used extensively to study the structure and dynamics of the endocytic pathway. Here, we show that while the general structure of the endocytic pathway is maintained in starved cells, its dynamics rapidly slow down. In addition, analysis of apm3 and lvsB mutants reveals that the functional organization of the endocytic pathway is profoundly modified upon starvation. Indeed, in these mutant cells, some of the defects observed in rich medium persist in starved cells, notably an abnormally slow transfer of endocytosed material between endocytic compartments. Other parameters, such as endocytosis of the fluid phase or the rate of fusion of postlysosomes to the cell surface, vary dramatically upon starvation. Studying the endocytic pathway in starved cells can provide a different perspective, allowing the primary (invariant) defects resulting from specific mutations to be distinguished from their secondary (conditional) consequences.

scholarly journals Endocytosis in filter-grown Madin-Darby canine kidney cells.

1989 ◽  
Vol 109 (6) ◽  
pp. 3243-3258 ◽  
Author(s):  
M Bomsel ◽  
K Prydz ◽  
R G Parton ◽  
J Gruenberg ◽  
K Simons
Keyword(s):  
Cell Layer ◽  
Mdck Cells ◽  
Fluid Phase ◽  
Perinuclear Region ◽  
Apical Side ◽  
Early Endosomes ◽  
Basolateral Side ◽  
Endocytic Compartments ◽  
Kinetics Of ◽  
Endocytic Pathways

In this paper, we have characterized the apical and basolateral endocytic pathways of epithelial MDCK cells grown on filters. The three-dimensional organization of the endocytic compartments was analyzed by confocal microscopy after internalization of a fluorescent fluid-phase marker from either side of the cell layer. After 5 min of internalization, distinct sets of apical and basolateral early endosomes were observed lining the plasma membrane domain from which internalization had occurred. At later time points, the apical and the basolateral endocytic pathways were shown to converge in the perinuclear region. Mixing of two different fluorescent markers could be detected after their simultaneous internalization from opposite sides of the cell layer. The extent of the meeting was quantitated by measuring the amount of complex formed intracellularly between avidin internalized from the apical side and biotinylated horseradish peroxidase (HRP) from the basolateral side. After 15 min, 14% of the avidin marker was complexed with the biotinylated HRP and this value increased to 50% during a subsequent chase of 60 min in avidin-free medium. We also determined the kinetics of fluid internalization, recycling, transcytosis, and intracellular retention using HRP as a marker. Fluid was internalized with the same rates from either surface domain (1.2 x 10(-4) microns 3/min per microns 2 of surface area). However, significant differences were observed for each pathway in the amounts and kinetics of marker recycled and transcytosed. The content of apical early endosomes was primarily recycled and transcytosed (45% along Bach route after 1 h internalization), whereas delivery to late endocytic compartments was favored from the basolateral early endosome (77% after 1 h). Our results demonstrate that early apical and basolateral endosomes are functionally and topologically distinct, but that the endocytic pathways converge at later stages in the perinuclear region of the cell.

scholarly journals Intracellular fusion of sequentially formed endocytic compartments.

1988 ◽  
Vol 106 (4) ◽  
pp. 1083-1091 ◽  
Author(s):  
N H Salzman ◽  
F R Maxfield
Keyword(s):  
Cho Cells ◽  
Culture Medium ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Vesicle Traffic ◽  
Intracellular Vesicle ◽  
Surface Time ◽  
Golgi Region ◽  
Fluid Phase Endocytosis ◽  
Endocytic Compartments

A polyclonal anti-fluorescein antibody (AFA) which quenches fluorescein fluorescence has been used to distinguish between two models of intracellular vesicle traffic. These models address the question of whether sequentially endocytosed probes will mix intracellularly or whether they are carried through the cell in a sequential, isolated manner. Using transferrin (Tf) as a recycling receptor marker, we incubated Chinese hamster ovary (CHO) cells with fluorescein-Tf (F-Tf) which is rapidly endocytosed. After the F-Tf was completely cleared from the surface, AFA was added to the incubation medium and entered endocytic compartments by fluid phase endocytosis. Fusion of a vesicle containing AFA with the compartment containing F-Tf results in binding of AFA to fluorescein and the quenching of fluorescein fluorescence. When AFA was added to the culture medium 2 min after clearance of F-Tf from the surface, time dependent fluorescence quenching occurred. After 20 min, 67% saturation of F-Tf with AFA was observed. When the interval between F-Tf clearance and AFA addition was increased to 5 min only 41% saturation of F-Tf was found. These data indicate that there are some compartments which are accessible for mixing with subsequently endocytosed molecules, but the efficiency of mixing falls off rapidly as the interval between pulses is increased. In CHO cells Tf swiftly segregates to a collection of vesicles or tubules in the para-Golgi region, and at steady state most of the F-Tf is in this compartment. Using digital image analysis to quantify quenching in this region, we have found that F-Tf/AFA mixing is occurring either within this compartment or before transferrin enters it.

Cellular mechanism of aminoglycoside tolerance in long-term gentamicin treatment

1997 ◽  
Vol 272 (4) ◽  
pp. C1309-C1318 ◽  
Author(s):  
D. P. Sundin ◽  
C. Meyer ◽  
R. Dahl ◽  
A. Geerdes ◽  
R. Sandoval ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Fluid Phase ◽  
Selective Inhibition ◽  
Membrane Lipid ◽  
Immunofluorescent Staining ◽  
Morphological Studies ◽  
Gentamicin Treatment ◽  
Apical Membranes

In the rat, nephrotoxicity results from uptake of gentamicin at the apical membrane of proximal tubule (PT) cells. However, during continuous gentamicin treatment, the PT epithelium has been shown to recover. The mechanism(s) of cellular recovery and development of tolerance remains unknown. Therefore, we undertook studies designed to characterize cellular adaptations that occur during long-term gentamicin (LTG) treatment. After 19 days of gentamicin treatment, electron microscopy morphological evaluation revealed cellular recovery with an apparent mild decrease in height and number of microvilli. Enzymatic analysis of LTG PT membranes showed that apical and basolateral membranes had essentially returned to normal. Analysis of apical membrane lipid content revealed persistent statistically significant (P < 0.01) elevations in phosphatidylinositol (PI). In vivo immunogold morphological studies and biochemical studies in LTG rats revealed that endocytosis of gentamicin was selectively reduced, whereas the markers of fluid-phase (horseradish peroxidase) and receptor-mediated (beta2-microglobulin) endocytoses were unaffected or increased. Biochemical analysis showed that, although gentamicin binding to apical membranes isolated from LTG rats increased greater than twofold (P < 0.05) over membranes from untreated rats, in vivo cellular uptake, quantified with [3H]gentamicin, was reduced. Western blot analysis of LTG apical membranes and immunofluorescent staining of perfusion-fixed LTG kidneys showed no change in megalin levels or its apical membrane localization. These data imply that recovery of PT cells from and tolerance to LTG treatment involve a selective inhibition of gentamicin uptake across the apical membrane. They indicate that the mediators of gentamicin endocytosis were affected differently: PI levels increased, whereas megalin levels did not change. We conclude that selective inhibition of gentamicin uptake during LTG treatment is not affected by a reduction in PI or megalin levels. We postulate that trafficking of gentamicin and/or gentamicin-containing endocytic structures is reduced in LTG rats, allowing cells to develop tolerance to gentamicin.

scholarly journals Shape-driven solid–solid transitions in colloids

2017 ◽  
Vol 114 (20) ◽  
pp. E3892-E3899 ◽  
Author(s):  
Chrisy Xiyu Du ◽  
Greg van Anders ◽  
Richmond S. Newman ◽  
Sharon C. Glotzer
Keyword(s):  
Phase Transitions ◽  
Self Assembly ◽  
Thermal Activation ◽  
Colloidal Particles ◽  
Solid Phase ◽  
Control Variable ◽  
Fluid Phase ◽  
Model Systems ◽  
Coordination Polyhedra ◽  
System A

Solid–solid phase transitions are the most ubiquitous in nature, and many technologies rely on them. However, studying them in detail is difficult because of the extreme conditions (high pressure/temperature) under which many such transitions occur and the high-resolution equipment needed to capture the intermediate states of the transformations. These difficulties mean that basic questions remain unanswered, such as whether so-called diffusionless solid–solid transitions, which have only local particle rearrangement, require thermal activation. Here, we introduce a family of minimal model systems that exhibits solid–solid phase transitions that are driven by changes in the shape of colloidal particles. By using particle shape as the control variable, we entropically reshape the coordination polyhedra of the particles in the system, a change that occurs indirectly in atomic solid–solid phase transitions via changes in temperature, pressure, or density. We carry out a detailed investigation of the thermodynamics of a series of isochoric, diffusionless solid–solid phase transitions within a single shape family and find both transitions that require thermal activation or are “discontinuous” and transitions that occur without thermal activation or are “continuous.” In the discontinuous case, we find that sufficiently large shape changes can drive reconfiguration on timescales comparable with those for self-assembly and without an intermediate fluid phase, and in the continuous case, solid–solid reconfiguration happens on shorter timescales than self-assembly, providing guidance for developing means of generating reconfigurable colloidal materials.

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