scholarly journals Delivery of ligands from sorting endosomes to late endosomes occurs by maturation of sorting endosomes

1992 ◽  
Vol 117 (2) ◽  
pp. 301-310 ◽  
Author(s):  
KW Dunn ◽  
FR Maxfield
Keyword(s):  
Cho Cells ◽  
Digital Image Analysis ◽  
Compartment Model ◽  
Half Time ◽  
Chase Period ◽  
Cell Biol ◽  
Late Endosomes ◽  
Pass Through ◽  
Endocytic Vesicles ◽  
To Receive

After endocytosis, lysosomally targeted ligands pass through a series of endosomal compartments. The endocytic apparatus that accomplishes this passage may be considered to take one of two forms: (a) a system in which lysosomally targeted ligands pass through preexisting, long-lived early sorting endosomes and are then selectively transported to long-lived late endosomes in carrier vesicles, or (b) a system in which lysosomally targeted ligands are delivered to early sorting endosomes which themselves mature into late endosomes. We have previously shown that sorting endosomes in CHO cells fuse with newly formed endocytic vesicles (Dunn, K. W., T. E. McGraw, and F. R. Maxfield. 1989. J. Cell Biol. 109:3303-3314) and that previously endocytosed ligands lose their accessibility to fusion with a half-time of approximately 8 min (Salzman, N. H., and F. R. Maxfield. 1989. J. Cell Biol. 109:2097-2104). Here we have studied the properties of individual endosomes by digital image analysis to distinguish between the two mechanisms for entry of ligands into late endosomes. We incubated TRVb-1 cells (derived from CHO cells) with diO-LDL followed, after a variable chase, by diI-LDL, and measured the diO content of diI-containing endosomes. As the chase period was lengthened, an increasing percentage of the endosomes containing diO-LDL from the initial incubation had no detectable diI-LDL from the second incubation, but those endosomes that contained both probes showed no decrease in the amount of diO-LDL per endosomes. These results indicate that (a) a pulse of fluorescent LDL is retained by individual sorting endosomes, and (b) with time sorting endosomes lose the ability to fuse with primary endocytic vesicles. These data are inconsistent with a preexisting compartment model which predicts that the concentration of ligand in sorting endosomes will decline during a chase interval, but that the ability of the stable sorting endosome to receive newly endocytosed ligands will remain high. These data are consistent with a maturation mechanism in which the sorting endosome retains and accumulates lysosomally directed ligands until it loses its ability to fuse with newly formed endocytic vesicles and matures into a late endosome. We also find that, as expected according to the maturation model, new sorting endosomes are increasingly labeled during the chase period indicating that new sorting endosomes are continuously formed to replace those that have matured into late endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Hidden Technologies of the Great Siberian Migration: Newcomers Breaking Into the Old Residents Community (Kharlova Village Conflict of 1893)

2018 ◽  
Vol 19 (4) ◽  
pp. 479-513 ◽  
Author(s):  
Alexey Kirillov ◽  
Anastasiya Karavayeva
Keyword(s):  
Case Study Method ◽  
Narrow Channels ◽  
Chain Migration ◽  
Mass Migration ◽  
Tide Height ◽  
Voronezh Region ◽  
To Come ◽  
Pass Through ◽  
To Receive

Peasant migration to Siberia in the second half of the 19th - the first half of the 20th century was a chronological parallel to the mass migration of Europeans across the Atlantics. One of the issues of the Great Siberian migration is the reasons for which it did not reach the proportions sufficient to defuse the land crisis in European Russia. The authors of the article are trying to solve this problem by studying the conflicts between the old Siberian residents and the migrants. By applying the case study method, the authors draw attention to one particular case, a clash in Kharlova village (Altai District of Cabinet of His Majesty Emperor) in 1893. It is one of the few conflicts described in detail. The mechanism of the conflict origination is discovered by confronting mutually exclusive statements of both parties and reconstructing hidden facts. It is proved that the resettlement of the Voronezh region peasants to the Altai village was a bright example of chain migration. New migrants would come on the advice of their predecessors. Thus, a group of the new old residents sympathetic to the newcomers was formed among the peasants belonging to the Kharlova community. The immediate reason for the conflict was an attempt of a big group of migrants to get a right to live in Kharlova village by cheating. A delegate of this group obtained the community council permission to come with a couple more of adult peasants and returned next year with six dozen of his compatriots. Though untypical, this method of penetration into an old residents community highlights a common issue: the ground for the conflicts was created by the two peasant groups contradiction of interests. It was important for the newcomers to start new life with the help of those who had already put down roots in Siberia; but the old residents were ready to receive only a small number of new neighbors. The rising tide of peasant migration could not spread evenly over the Siberian expanse; it had to pass through narrow channels of the already inhabited places - which considerably restricted the tide height.

scholarly journals Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome.

1989 ◽  
Vol 109 (6) ◽  
pp. 3303-3314 ◽  
Author(s):  
K W Dunn ◽  
T E McGraw ◽  
F R Maxfield
Keyword(s):  
Digital Image Analysis ◽  
High Efficiency ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Recycling Endosomes ◽  
Double Label ◽  
Fluorescent Ligands ◽  
Endocytic Vesicles ◽  
Endocytic Compartments ◽  
Very High

To study the fusion and separation of endocytic compartments, we have used digital image analysis to quantify the accumulation of fluorescent ligands in endosomes during continuous endocytosis for periods of 1-20 min. Fluorescently labeled transferrin (Tf) and low density lipoproteins (LDL) were used as markers of recycling receptors and lysosomally directed ligands respectively. By measuring the intensity of individual endosomes, we found that the amount of LDL per endosome increases 30-40-fold between 1 and 10 min and then plateaus. In contrast, the amount of Tf per endosome reaches a steady state within 2 min at a level that is only three to four times that at 1 min. We used pulse-chase double label methods to demonstrate that Tf cycles through the compartment in which the LDL accumulates. When both Tf and LDL are added to cells simultaneously for 2 min, nearly all endosomes contain both labels. With 2-4 min further incubation in the absence of external ligands, LDL-containing compartments become depleted of Tf as Tf is directed to para-Golgi recycling endosomes. However, if Tf is added to the medium 2-4 min after a pulse with LDL, most of the LDL-containing endosomes become labeled with Tf. The data indicate that at least 30-40 endocytic vesicles containing both Tf and LDL fuse with an endosomal compartment over a period of 5-10 min. LDL accumulates within this compartment and Tf is simultaneously removed. Simple mathematical models suggest that this type of iterative fractionation can lead to very high efficiency sorting.

Technologies for Optical Networking

2021 ◽  
pp. 23-132
Author(s):  
Debasish Datta
Keyword(s):  
Semiconductor Lasers ◽  
Optical Networks ◽  
Optical Fibers ◽  
Optical Signal ◽  
Wdm Optical Networks ◽  
Active Devices ◽  
Wide Range ◽  
Optical Domain ◽  
Pass Through ◽  
To Receive

The technologies used in optical networks have evolved seamlessly over the past six decades. Optical fibers with extremely low loss and enormous bandwidth are used as the transmission medium, while semiconductor lasers and LEDs serve as optical sources, and the photodetectors – pin and avalanche photodiodes – are used to receive the optical signal at the destination nodes. The transmitted optical signal has to pass through a variety of network elements, which in turn need a wide range of passive and active devices, carrying out the necessary networking functionalities. For WDM optical networks, many of these tasks need to be accomplished in the optical domain itself in a wavelength-selective manner, calling for various types of WDM-based networking elements. In this chapter, we present a comprehensive description of the optical and optoelectronic devices that are used in today’s optical networks. (137 words)

scholarly journals Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

1987 ◽  
Vol 105 (6) ◽  
pp. 2713-2721 ◽  
Author(s):  
D J Yamashiro ◽  
F R Maxfield
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Ph Value ◽  
Ph Regulation ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Wild Type ◽  
Cell Biol ◽  
Endocytic Compartments

Acidification of endocytic compartments is necessary for the proper sorting and processing of many ligands and their receptors. Robbins and co-workers have obtained Chinese hamster ovary (CHO) cell mutants that are pleiotropically defective in endocytosis and deficient in ATP-dependent acidification of endosomes isolated by density centrifugation (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308). In this and the following paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2723-2733) we describe detailed studies of endosome acidification in the mutant and wild-type CHO cells. Here we describe a new microspectrofluorometry method based on changes in fluorescein fluorescence when all cellular compartments are equilibrated to the same pH value. Using this method we measured the pH of endocytic compartments during the first minutes of endocytosis. We found in wild-type CHO cells that after 3 min, fluorescein-labeled dextran (F-Dex) was in endosomes having an average pH of 6.3. By 10 min, both F-Dex and fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) had reached acidic endosomes having an average pH of 6.0 or below. In contrast, endosome acidification in the CHO mutants DTG 1-5-4 and DTF 1-5-1 was markedly slowed. The average endosomal pH after 5 min was 6.7 in both mutant cell lines. At least 15 min was required for F-Dex and F-alpha 2M to reach an average pH of 6.0 in DTG 1-5-4. Acidification of early endocytic compartments is defective in the CHO mutants DTG 1-5-4 and DTF 1-5-1, but pH regulation of later compartments on both the recycling pathway and lysosomal pathway is nearly normal. The properties of the mutant cells suggest that proper functioning of pH regulatory mechanisms in early endocytic compartments is critical for many pH-mediated processes of endocytosis.

Phamacokinetics (PK) Substudy of Rituximab in a Prospective Clinical Trial for Pediatric Chronic Immune Thrombocytopenic Purpura (cITP).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1243-1243 ◽  
Author(s):  
Carolyn M. Bennett ◽  
James B. Bussel ◽  
Brigitta U. Mueller ◽  
Thomas C. Abshire ◽  
Hadi Sawaf ◽  
...  
Keyword(s):  
Analysis Of Variance ◽  
Pediatric Patients ◽  
Compartment Model ◽  
Volume Of Distribution ◽  
Half Time ◽  
Age Cohorts ◽  
Elimination Half ◽  
Significant Difference ◽  
Older Subjects ◽  
The Difference

Abstract Rationale: Rituximab dosing in pediatric patients is derived from body surface area-based “standard” adult lymphoma regimens. PK data for rituximab in children are lacking. Methods: We evaluated serum rituximab levels in a subset of 36 children and adolescents with severe cITP treated on a prospective phase I/II study. Patients received four weekly infusions, 375mg/m2/dose. Trough and 30-minute, 24- and 48-hour (hr) post-infusion levels were obtained with doses 1 and 4, and 7 days after dose 1. PK parameters were obtained separately for each patient at the 1-wk and 4-wk infusions by fitting a two-compartment model. Two-factor analysis of variance was employed to compare parameters between younger (2–9 years) and older (10–18 years) subjects and between responders and non-responders at the two infusion times. Results: Fourteen patients had PK measurements at week 1 and eleven at week 4 (Table). The median initial volume of distribution (V1), for all patients, calculated by back-extrapolation to t0, approximated plasma volume, 53 mL/kg. Kinetics fit a two-component model, with relatively rapid initial half-time (exit) reflecting distribution out of plasma space, and equilibration representing slow return into the vascular pool. The drug elimination half-time was too slow to observe in these 7-day periods. Apparent half-time for initial redistribution from the plasma space was significantly longer for younger subjects at week 4 than at week 1 and than for older subjects at week 4 (Table). By comparison, in studies of 14 adults with lymphoma treated with rituximab at the same dosage regimen, the manufacturer reports mean serum half-time of 76 hrs (range, 31–153) at week 1 and 205 hrs (range, 84 – 407) at week 4. In this PK sub-study, we found no significant difference in kinetic parameters between the 5 responders (defined by sustained platelet count >50,000/mm3 at week 12) and 11 non-responders. Conclusion: In this small number of pediatric patients with cITP treated with 4 weekly infusions of rituximab, the half-time was longer for younger subjects at week 4 than week 1 and than for older subjects at week 4. Trough levels at week 4 do not represent steady state because the elimination half-time is long. The reason for the difference between the two age cohorts is not readily apparent, but we propose that the shorter exit half-time in younger patients at week 1 compared to week 4 corresponds to a higher number of initially accessible CD20 binding sites (B cells) in young children. PK parameters of rituximab distribution at weeks 1 and 4 Age (yr) Week 1 n Week4 n Median (min, max) Median (min, max) *Age x week interaction significant in two-way analysis of variance, p<0.015 V1 (mL/kg) All 53 (35–76) 14 54 (35–74) 11 2–9 54 (43–76) 7 62 (54–74) 4 10–18 52 (35–73) 7 49 (35–62) 7 Exit Half-time (hr) All 49 (16–83) 14 83 (25–193) 11 2–9 45 (31–55) 7 100 (83–193)* 4 10–18 57 (16–83) 7 57 (25–97) 7

scholarly journals The Secretory Route of the Leaderless Protein Interleukin 1β Involves Exocytosis of Endolysosome-related Vesicles

1999 ◽  
Vol 10 (5) ◽  
pp. 1463-1475 ◽  
Author(s):  
Cristina Andrei ◽  
Cecilia Dazzi ◽  
Lavinia Lotti ◽  
Maria Rosaria Torrisi ◽  
Giovanna Chimini ◽  
...  
Keyword(s):  
Cathepsin D ◽  
Secretory Protein ◽  
Interleukin 1Β ◽  
Human Monocytes ◽  
Hypotonic Medium ◽  
High Concentration ◽  
Lysosomal Hydrolases ◽  
Late Endosomes ◽  
Medium Increase ◽  
Endocytic Vesicles

Interleukin 1β (IL-1β), a secretory protein lacking a signal peptide, does not follow the classical endoplasmic reticulum-to-Golgi pathway of secretion. Here we provide the evidence for a “leaderless” secretory route that uses regulated exocytosis of preterminal endocytic vesicles to transport cytosolic IL-1β out of the cell. Indeed, although most of the IL-1β precursor (proIL-1β) localizes in the cytosol of activated human monocytes, a fraction is contained within vesicles that cofractionate with late endosomes and early lysosomes on Percoll density gradients and display ultrastructural features and markers typical of these organelles. The observation of organelles positive for both IL-1β and the endolysosomal hydrolase cathepsin D or for both IL-1β and the lysosomal marker Lamp-1 further suggests that they belong to the preterminal endocytic compartment. In addition, similarly to lysosomal hydrolases, secretion of IL-1β is induced by acidotropic drugs. Treatment of monocytes with the sulfonylurea glibenclamide inhibits both IL-1β secretion and vesicular accumulation, suggesting that this drug prevents the translocation of proIL-1β from the cytosol into the vesicles. A high concentration of extracellular ATP and hypotonic medium increase secretion of IL-1β but deplete the vesicular proIL-1β content, indicating that exocytosis of proIL-1β–containing vesicles is regulated by ATP and osmotic conditions.

Evidence for retrograde traffic between terminal lysosomes and the prelysosomal/late endosome compartment

1994 ◽  
Vol 107 (1) ◽  
pp. 145-157 ◽  
Author(s):  
A. Jahraus ◽  
B. Storrie ◽  
G. Griffiths ◽  
M. Desjardins
Keyword(s):  
Light Microscope ◽  
Retrograde Transport ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Fusion Event ◽  
Microtubule Network ◽  
Cell Biol ◽  
Late Endosomes ◽  
Latex Beads ◽  
Retrograde Traffic

We have investigated the interactions occurring between the prelysosomal compartment, PLC/late endosome, and terminal lysosomes using an approach that allowed us to internalize and deliver specific tracers to these compartments, and look for evidence of their meeting. After internalization of sucrose, the lysosomes (sucrosomes), but not the PLC/late endosomes, became significantly swollen and visible in the light microscope. If invertase is then added to the medium it reaches the lysosomes where it cleaves sucrose into fructose and glucose. These sugars, unlike sucrose, can be transported into the cytosol, resulting in the disappearance of the sucrosomes. We previously showed that phagocytosed latex beads are delivered specifically to, and reside in, the PLC/late endosome, a stage earlier than the lysosomes in the endocytic pathway (Rabinowitz et al. (1992) J. Cell Biol. 116, 95–112). In the present study, we demonstrate that invertase conjugated to the latex beads, and thus immobilized in late endosomes, has access to the sucrose present in the more distal lysosomes. Experiments using nocodazole indicate that this retrograde fusion event requires the presence of an intact microtubule network. The simplest interpretation of our results is that the two compartments fuse, allowing for a retrograde transport of sucrose from the lysosomes to the PLC/late endosomes.

Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins

2021 ◽  
Vol 221 (1) ◽  
Author(s):  
Jan van der Beek ◽  
Cecilia de Heus ◽  
Nalan Liv ◽  
Judith Klumperman
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Functional Organization ◽  
Light And Electron Microscopy ◽  
Correlative Microscopy ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endocytic Vesicles ◽  
Endosomal System

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural–functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.

scholarly journals Inhibition of Endoplasmic Reticulum-Associated Degradation in CHO Cells Resistant to Cholera Toxin, Pseudomonas aeruginosa Exotoxin A, and Ricin

2002 ◽  
Vol 70 (11) ◽  
pp. 6172-6179 ◽  
Author(s):  
Ken Teter ◽  
Randall K. Holmes
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Endoplasmic Reticulum ◽  
Cholera Toxin ◽  
Cell Lines ◽  
Cho Cells ◽  
Mutant Cell ◽  
Resistant Cell ◽  
Exotoxin A ◽  
Surface Receptors ◽  
Pass Through

ABSTRACT Many plant and bacterial toxins act upon cytosolic targets and must therefore penetrate a membrane barrier to function. One such class of toxins enters the cytosol after delivery to the endoplasmic reticulum (ER). These proteins, which include cholera toxin (CT), Pseudomonas aeruginosa exotoxin A (ETA), and ricin, move from the plasma membrane to the endosomes, pass through the Golgi apparatus, and travel to the ER. Translocation from the ER to the cytosol is hypothesized to involve the ER-associated degradation (ERAD) pathway. We developed a genetic strategy to assess the role of mammalian ERAD in toxin translocation. Populations of CHO cells were mutagenized and grown in the presence of two lethal toxins, ETA and ricin. Since these toxins bind to different surface receptors and attack distinct cytoplasmic targets, simultaneous acquisition of resistance to both would likely result from the disruption of a shared trafficking or translocation mechanism. Ten ETA- and ricin-resistant cell lines that displayed unselected resistance to CT and continued sensitivity to diphtheria toxin, which enters the cytosol directly from acidified endosomes, were screened for abnormalities in the processing of a known ERAD substrate, the Z form of α1-antitrypsin (α1AT-Z). Compared to the parental CHO cells, the rate of α1AT-Z degradation was decreased in two independent mutant cell lines. Both of these cell lines also exhibited, in comparison to the parental cells, decreased translocation and degradation of a recombinant CTA1 polypeptide. These findings demonstrated that decreased ERAD function was associated with increased cellular resistance to ER-translocating protein toxins in two independently derived mutant CHO cell lines.

scholarly journals Distribution and Elimination of Ingested Mercuric Oxide in Mice

1983 ◽  
Vol 2 (4) ◽  
pp. 307-317
Author(s):  
G. G. Berg ◽  
B. S. Smith
Keyword(s):  
Body Burden ◽  
Compartment Model ◽  
Whole Body ◽  
Published Data ◽  
Mercuric Oxide ◽  
Half Time ◽  
The Brain ◽  
Intermediate Rate ◽  
Body Concentration ◽  
Concentration Ratios

Neutron-activated mercuric oxide was administered by gavage to female BALB/c mice. Counts of 197Hg and 203Hg in the whole body, urine, and feces were followed for up to 36 days. Elimination of mercury fitted a 3-compartment model. Nonpregnant mice eliminated approximately 87.5% of the dose at a fast rate (t1-2 = 9 hours), 12% at an intermediate rate (t>1/2 = 2 days), and 0.5% at a slow rate (t1/2 = 15 days). Each half-time was approximately 7 times shorter than the corresponding half-time fitted to published data on rats. Mice were also faster than humans in eliminating the ingested mercury. Pregnancy slowed down the intermediate rate of elimination. The total administered dose was recovered from feces and urine in a 9:1 ratio. Organ weights and mercury burdens were measured after serial sacrifice. Peak concentrations were reached within two days, with highest levels in kidneys followed by placentae and livers. In brains, peak concentrations were delayed and low. Subsequent losses of mercury differed widely in rate constants, with fastest overall rates in the brain, intestine, and integument, followed in order by whole body, liver, and kidneys. Ten days after dosing, mercury concentration ratios of placenta to 17-day old fetus were 20:1; 11 days after dosing, and with less than 2% of body burden remaining, while body concentration ratios of mother to neonate were 4:1. The data indicated that mice eliminated mercuric salts faster than had been reported for rats or humans, and that rapid elimination coupled with a placental barrier shielded fetuses from equlibrating with the peak concentrations of mercury found in dams after a single dose.

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