scholarly journals Fusion of Lysosomes with Late Endosomes Produces a Hybrid Organelle of Intermediate Density and Is NSF Dependent

1998 ◽  
Vol 140 (3) ◽  
pp. 591-601 ◽  
Author(s):  
Barbara M. Mullock ◽  
Nicholas A. Bright ◽  
Clare W. Fearon ◽  
Sally R. Gray ◽  
J. Luzio
Keyword(s):  
Rat Liver ◽  
Gradient Centrifugation ◽  
Rab Protein ◽  
Late Endosomes ◽  
A Cell ◽  
Enzyme Formation ◽  
And Function ◽  
Gdp Dissociation Inhibitor ◽  
Brain Cytosol ◽  
Direct Fusion

Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37°C, late endosome–lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide–sensitive factor– dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.

scholarly journals The Role of Intraorganellar Ca2+In Late Endosome–Lysosome Heterotypic Fusion and in the Reformation of Lysosomes from Hybrid Organelles

2000 ◽  
Vol 149 (5) ◽  
pp. 1053-1062 ◽  
Author(s):  
Paul R. Pryor ◽  
Barbara M. Mullock ◽  
Nicholas A. Bright ◽  
Sally R. Gray ◽  
J. Paul Luzio
Keyword(s):  
Dynamic Equilibrium ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Free System ◽  
Rab Protein ◽  
Late Endosomes ◽  
The Reformation ◽  
A Cell ◽  
Gdp Dissociation Inhibitor

We have investigated the requirement for Ca2+ in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca2+. Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca2+ reduced the amount of fusion. The requirement for Ca2+ for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome–lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca2+ plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.

Rab22a affects the morphology and function of the endocytic pathway

2001 ◽  
Vol 114 (22) ◽  
pp. 4041-4049 ◽  
Author(s):  
Rosana Mesa ◽  
Cristina Salomón ◽  
Marcelo Roggero ◽  
Philip D. Stahl ◽  
Luis S. Mayorga
Keyword(s):  
Fluorescent Protein ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Rab Proteins ◽  
Rab Protein ◽  
Late Endosomes ◽  
And Function ◽  
Negative Mutant

Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.

Relationship between endosomes and lysosomes

2001 ◽  
Vol 29 (4) ◽  
pp. 476-480 ◽  
Author(s):  
J. P. Luzio ◽  
B. M. Mullock ◽  
P. R. Pryor ◽  
M. R. Lindsay ◽  
D. E. James ◽  
...  
Keyword(s):  
Acid Hydrolases ◽  
Attachment Protein ◽  
Intact Cells ◽  
Vesicular Traffic ◽  
Vacuole Fusion ◽  
Late Endosomes ◽  
Sensitive Factor ◽  
A Cell ◽  
Hydrolysis Of ◽  
Direct Fusion

Delivery of endocytosed macromolecules to lysosomes occurs by means of direct fusion of late endosomes with lysosomes. This has been formally demonstrated in a cell-free content mixing assay using late endosomes and lysosomes from rat liver. There is evidence from electron microscopy studies that the same process occurs in intact cells. The fusion process results in the formation of hybrid organelles from which lysosomes are reformed. The discovery of the hybrid organelle has opened up three areas of investigation: (i) the mechanism of direct fusion of late endosomes and lysosomes, (ii) the mechanism of re-formation of lysosomes from the hybrid organelle, and (iii) the function of the hybrid organelle. Fusion has analogies with homotypic vacuole fusion in yeast. It requires syntaxin 7 as part of the functional trans-SNARE [SNAP receptor, where SNAP is soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein] complex and the release of lumenal calcium to achieve membrane fusion. Reformation of lysosomes from the hybrid organelle occurs by a maturation process involving condensation of lumenal content and probably removal of some membrane proteins by vesicular traffic. Lysosomes may thus be regarded as a type of secretory granule, storing acid hydrolases in between fusion events with late endosomes. The hybrid organelle is predicted to function as a ‘cell stomach’, acting as a major site of hydrolysis of endocytosed macromolecules.

scholarly journals Reconstitution of an endosome-lysosome interaction in a cell-free system.

1989 ◽  
Vol 108 (6) ◽  
pp. 2093-2099 ◽  
Author(s):  
B M Mullock ◽  
W J Branch ◽  
M van Schaik ◽  
L K Gilbert ◽  
J P Luzio
Keyword(s):  
Intravenous Injection ◽  
Rat Liver ◽  
Specific Interaction ◽  
Free System ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Free Model ◽  
A Cell ◽  
Isopycnic Centrifugation

A cell-free model for the transfer of endocytosed material to lysosomes is described. Rat liver late endosomes, loaded in vivo with radiolabeled ligand by intravenous injection shortly before killing the animal, showed a specific interaction with lysosomes when incubated at 37 degrees C in the presence of cytosol and an ATP regenerating system. The location of the ligand, generally asialofetuin, was analyzed by isopycnic centrifugation on Nycodenz gradients. Appearance of radiolabel in the lysosomal position on such gradients was maximal after approximately 30 min at 37 degrees C and required the provision of undamaged cytosol, lysosomes, and an ATP regenerating system. It could not be accounted for by nonspecific bulk aggregation of membranes. Transfer occurred only from late endosomes; radiolabel in early endosomes was unaffected. Digestion of the asialofetuin, as shown by the appearance of TCA-soluble radioactivity, occurred on incubation at 37 degrees C and was increased by the provision of an ATP regenerating system.

scholarly journals Effect of nucleoside triphosphate and magnesium ion concentration on the stability and function of rat liver polysomes in vitro

1968 ◽  
Vol 110 (4) ◽  
pp. 783-788 ◽  
Author(s):  
A. W. Pronczuk ◽  
B. S. Baliga ◽  
H. N. Munro
Keyword(s):  
Rat Liver ◽  
Ion Concentration ◽  
Nucleoside Triphosphate ◽  
Free Protein ◽  
A Cell ◽  
The Stability ◽  
And Function ◽  
Polysome Stability ◽  
Hydrolysis Of

The effects of different concentrations of ATP, GTP, UTP and CTP on polysome stability and function in a cell-free protein-synthesizing system prepared from rat liver were studied. Increasing the concentration of ATP in the incubation medium to 15mm resulted in progressive disaggregation of the polysomes; at ATP concentrations above 2mm their capacity to incorporate amino acids into peptide chains diminished. The same disaggregation phenomenon could be produced by incubating polysomes in a buffered medium containing 5mm-Mg2+ and increasing concentrations of ATP. Although the disaggregating action of ATP could be prevented by increasing Mg2+ concentration, the amino acid incorporation in the cell-free protein-synthesizing system remained impaired. The effects of different concentrations of GTP, UTP and CTP on polysome stability were similar to those of ATP. Increasing the concentrations of each nucleoside triphosphate also inhibited the hydrolysis of GTP in the cell-free protein-synthesizing system.

CD38 as an immunomodulator in cancer

2020 ◽  
Vol 16 (34) ◽  
pp. 2853-2861
Author(s):  
Yanli Li ◽  
Rui Yang ◽  
Limo Chen ◽  
Sufang Wu
Keyword(s):  
Multiple Myeloma ◽  
Cell Activation ◽  
Human Tissues ◽  
Transmembrane Glycoprotein ◽  
Cell Activation Marker ◽  
Research Findings ◽  
A Cell ◽  
And Function ◽  
Human Molecule

CD38 is a transmembrane glycoprotein that is widely expressed in a variety of human tissues and cells, especially those in the immune system. CD38 protein was previously considered as a cell activation marker, and today monoclonal antibodies targeting CD38 have witnessed great achievements in multiple myeloma and promoted researchers to conduct research on other tumors. In this review, we provide a wide-ranging review of the biology and function of the human molecule outside the field of myeloma. We focus mainly on current research findings to summarize and update the findings gathered from diverse areas of study. Based on these findings, we attempt to extend the role of CD38 in the context of therapy of solid tumors and expand the role of the molecule from a simple marker to an immunomodulator.

scholarly journals Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease

2021 ◽  
Author(s):  
Vitalii Kryvenko ◽  
Olga Vagin ◽  
Laura A. Dada ◽  
Jacob I. Sznajder ◽  
István Vadász
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Signaling ◽  
Loss Of Function ◽  
Α Subunit ◽  
Rate Limiting Step ◽  
Atpase Subunits ◽  
A Cell ◽  
Health And Disease ◽  
And Function ◽  
Rate Limiting

Abstract The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:β-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions. Graphic Abstract

scholarly journals Combined Effects of Methylated Cytosine and Molecular Crowding on the Thermodynamic Stability of DNA Duplexes

2021 ◽  
Vol 22 (2) ◽  
pp. 947
Author(s):  
Mitsuki Tsuruta ◽  
Yui Sugitani ◽  
Naoki Sugimoto ◽  
Daisuke Miyoshi
Keyword(s):  
Molecular Crowding ◽  
Dna Duplexes ◽  
Crowding Effect ◽  
Backbone Flexibility ◽  
Cpg Dinucleotides ◽  
Key Factor ◽  
Dna Backbone ◽  
A Cell ◽  
Stabilization Effect ◽  
And Function

Methylated cytosine within CpG dinucleotides is a key factor for epigenetic gene regulation. It has been revealed that methylated cytosine decreases DNA backbone flexibility and increases the thermal stability of DNA. Although the molecular environment is an important factor for the structure, thermodynamics, and function of biomolecules, there are few reports on the effects of methylated cytosine under a cell-mimicking molecular environment. Here, we systematically investigated the effects of methylated cytosine on the thermodynamics of DNA duplexes under molecular crowding conditions, which is a critical difference between the molecular environment in cells and test tubes. Thermodynamic parameters quantitatively demonstrated that the methylation effect and molecular crowding effect on DNA duplexes are independent and additive, in which the degree of the stabilization is the sum of the methylation effect and molecular crowding effect. Furthermore, the effects of methylation and molecular crowding correlate with the hydration states of DNA duplexes. The stabilization effect of methylation was due to the favorable enthalpic contribution, suggesting that direct interactions of the methyl group with adjacent bases and adjacent methyl groups play a role in determining the flexibility and thermodynamics of DNA duplexes. These results are useful to predict the properties of DNA duplexes with methylation in cell-mimicking conditions.

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