Biochemical analysis of distinct Rab5- and Rab11-positive endosomes along the transferrin pathway

1999 ◽  
Vol 112 (24) ◽  
pp. 4773-4783 ◽  
Author(s):  
M. Trischler ◽  
W. Stoorvogel ◽  
O. Ullrich
Keyword(s):  
Intracellular Transport ◽  
Protein Composition ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Vitro Assay ◽  
Recycling Endosomes ◽  
Morphological Differences ◽  
And Function ◽  
Cellular Compartments

Rab GTPases are associated with distinct cellular compartments and function as specific regulators of intracellular transport. In the endocytic pathway, it is well documented that Rab5 regulates transport from plasma membrane to early (sorting) endosomes. In contrast, little is known about the precise localization and function of Rab4 and Rab11, which are believed to control endocytic recycling. In the present study we have analysed the protein composition of Rab5- and Rab11-carrying endosomes to gain further insight into the compartmental organization of the endocytic and recycling pathway. Endosome populations of this transport route were purified by immunoadsorption from endosome-enriched subcellular fractions using antibodies directed against the cytoplasmic tail of the transferrin receptor, Rab5 or Rab11. Endocytosed transferrin moved sequentially through compartments that could be immunoadsorbed with anti-Rab5 and anti-Rab11, consistent with the theory that Rab5 and Rab11 localise to sorting and recycling endosomes, respectively. These compartments exhibited morphological differences, as determined by electron microscopy. Although their overall protein compositions were very similar, some proteins were found to be selectively enriched. While Rab4 was present on all endosome populations, Rab5 and Rab11 were strikingly segregated. Furthermore, the Rab11-positive endosomes were rich in annexin II, actin and the t-SNARE syntaxin 13, compared to Rab5-containing endosomes. In an in vitro assay, the Rab5 effector protein EEA1 was preferentially recruited by Rab5-positive endosomes. Taken together, our data suggest an organization of the transferrin pathway into distinct Rab5- and Rab11-positive compartments.

scholarly journals SNARE Function Is Not Involved in Early Endosome Docking

2008 ◽  
Vol 19 (12) ◽  
pp. 5327-5337 ◽  
Author(s):  
Ulf Geumann ◽  
Sina Victoria Barysch ◽  
Peer Hoopmann ◽  
Reinhard Jahn ◽  
Silvio O. Rizzoli
Keyword(s):  
Phosphatidyl Inositol ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Vitro Assay ◽  
Receptor Proteins ◽  
Transport Vesicles ◽  
Early Endosomes ◽  
Sensitive Factor ◽  
Rab Effector

Docking and fusion of transport vesicles constitute elementary steps in intracellular membrane traffic. While docking is thought to be initiated by Rab-effector complexes, fusion is mediated by SNARE (N-ethylmaleimide-sensitive factor [NSF] attachment receptor) proteins. However, it has been recently debated whether SNAREs also play a role in the establishment or maintenance of a stably docked state. To address this question, we have investigated the SNARE dependence of docking and fusion of early endosomes, one of the central sorting compartments in the endocytic pathway. A new, fluorescence-based in vitro assay was developed, which allowed us to investigate fusion and docking in parallel. Similar to homotypic fusion, docking of early endosomes is dependent on the presence of ATP and requires physiological temperatures. Unlike fusion, docking is insensitive to the perturbation of SNARE function by means of soluble SNARE motifs, SNARE-specific Fab fragments, or by a block of NSF activity. In contrast, as expected, docking is strongly reduced by interfering with the synthesis of phosphatidyl inositol (PI)-3 phosphate, with the function of Rab-GTPases, as well as with early endosomal autoantigen 1 (EEA1), an essential tethering factor. We conclude that docking of early endosomes is independent of SNARE function.

scholarly journals Isolation and preliminary characterization of the major membrane boundaries of the endocytic pathway in lymphocytes.

1990 ◽  
Vol 111 (5) ◽  
pp. 1811-1823 ◽  
Author(s):  
B D Beaumelle ◽  
A Gibson ◽  
C R Hopkins
Keyword(s):  
Plasma Membrane ◽  
Protein Composition ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Selective Labeling ◽  
Specific Proteins ◽  
T Lymphoma ◽  
Cellular Compartments ◽  
Density Shift

Plasma membrane, coated pits, endosomes, and lysosomes were isolated from a mouse T lymphoma cell line using a density shift protocol in which these compartments were selectively loaded with gold conjugates. The plasma membrane was prepared after selective labeling for 1 h at 2 degrees C with gold-ricin and gave a yield of 40% according to enzymatic and antigenic markers. Endosomes were obtained by loading the cells for 2 h at 22 degrees C with gold complexed to an antimouse transferrin receptor mAb. Coated pits were isolated using a similar procedure, but after an incubation at 10 degrees C, which allowed deep invagination of the pits but prevented internalization. The yield (calculated using the recovery of [125I]transferrin) was 32% for endosomes and 10% for coated pits. Finally lysosomes were prepared by loading the cells for 18 h at 37 degrees C with gold low density lipoproteins (LDLs) followed by a 3-h chase at 37 degrees C with LDL alone. The final lysosome yield (based on the recovery of lysosomal enzymes) was 16%. Studies of the protein composition of these cellular compartments on two-dimensional gels showed that while some major proteins are present throughout the pathway, specific proteins can be identified in each of the isolated fractions. The greatest change in the pattern of protein constituents seen along the pathway was between endosomal and lysosomal preparations.

scholarly journals Acute-phase protein synthesis: a key feature of innate immune functions of the liver

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Christian Ehlting ◽  
Stephanie D. Wolf ◽  
Johannes G. Bode
Keyword(s):  
Acute Phase ◽  
Acute Phase Proteins ◽  
Protein Composition ◽  
Systemic Circulation ◽  
Central Component ◽  
Immune Functions ◽  
Main Site ◽  
And Function

Abstract The expression of acute-phase proteins (APP’s) maintains homeostasis and tissue repair, but also represents a central component of the organism’s defense strategy, especially in the context of innate immunity. Accordingly, an inflammatory response is accompanied by significant changes in the serum protein composition, an aspect that is also used diagnostically. As the main site of APP synthesis the liver is constantly exposed to antigens or pathogens via blood flow, but also to systemic inflammatory signals originating either from the splanchnic area or from the circulation. Under both homeostatic and acute-phase response (APR) conditions the composition of APP’s is determined by the pattern of regulatory mediators derived from the systemic circulation or from local cell populations, especially liver macrophages. The key regulators mentioned here most frequently are IL-1β, IL-6 and TNF-α. In addition to a variety of molecular mediators described mainly on the basis of in vitro studies, recent data emphasize the in vivo relevance of cellular key effectors as well as molecular key mediators and protein modifications for the regulation and function of APP’s. These are aspects, on which the present review is primarily focused.

scholarly journals Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase

2013 ◽  
Vol 3 (1) ◽  
Author(s):  
Divyanshu Mahajan ◽  
Boon Kim Boh ◽  
Yan Zhou ◽  
Li Chen ◽  
Tobias Carl Cornvik ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Active Form ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
And Function ◽  
Golgi Network

Abstract Arl1 is a member of Arf family small GTPases that is essential for the organization and function of Golgi complex. Mon2/Ysl2, which shares significant homology with Sec7 family Arf guanine nucleotide exchange factors, was poorly characterized in mammalian cells. Here, we report the first in depth characterization of mammalian Mon2. We found that Mon2 localized to trans-Golgi network which was dependent on both its N and C termini. The depletion of Mon2 did not affect the Golgi localized or cellular active form of Arl1. Furthermore, our in vitro assay demonstrated that recombinant Mon2 did not promote guanine nucleotide exchange of Arl1. Therefore, our results suggest that Mon2 could be neither necessary nor sufficient for the guanine nucleotide exchange of Arl1. We demonstrated that Mon2 was involved in endosome-to-Golgi trafficking as its depletion accelerated the delivery of furin and CI-M6PR to Golgi after endocytosis.

scholarly journals Stimulation of the neurotrophin receptor TrkB on astrocytes drives nitric oxide production and neurodegeneration

2012 ◽  
Vol 209 (3) ◽  
pp. 521-535 ◽  
Author(s):  
Emanuela Colombo ◽  
Chiara Cordiglieri ◽  
Giorgia Melli ◽  
Jia Newcombe ◽  
Markus Krumbholz ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
White Matter Lesions ◽  
Neurotrophin Receptor ◽  
Autoimmune Encephalomyelitis ◽  
Vitro Assay ◽  
Neurodegenerative Process ◽  
The Central Nervous System ◽  
And Function ◽  
Experimental Autoimmune

Neurotrophin growth factors support neuronal survival and function. In this study, we show that the expression of the neurotrophin receptor TrkB is induced on astrocytes in white matter lesions in multiple sclerosis (MS) patients and mice with experimental autoimmune encephalomyelitis (EAE). Surprisingly, mice lacking TrkB specifically in astrocytes were protected from EAE-induced neurodegeneration. In an in vitro assay, astrocytes stimulated with the TrkB agonist brain-derived neurotrophic factor (BDNF) released nitric oxide (NO), and conditioned medium from activated astrocytes had detrimental effects on the morphology and survival of neurons. This neurodegenerative process was amplified by NO produced by neurons. NO synthesis in the central nervous system during EAE depended on astrocyte TrkB. Together, these findings suggest that TrkB expression on astrocytes may represent a new target for neuroprotective therapies in MS.

Cold storage of peripheral nerves: An in vitro assay of cell viability and function

Glia ◽  
1994 ◽  
Vol 10 (2) ◽  
pp. 121-131 ◽  
Author(s):  
Allan D. O. Levi ◽  
Peter J. Evans ◽  
Susan E. Mackinnon ◽  
Richard P. Bunge
Keyword(s):  
Cell Viability ◽  
Cold Storage ◽  
Peripheral Nerves ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
And Function

scholarly journals A novel in vitro assay reveals SNARE topology and the role of Ykt6 in autophagosome fusion with vacuoles

2018 ◽  
Vol 217 (10) ◽  
pp. 3670-3682 ◽  
Author(s):  
Jieqiong Gao ◽  
Fulvio Reggiori ◽  
Christian Ungermann
Keyword(s):  
Test Tube ◽  
In Vitro Assay ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
Vacuole Fusion ◽  
Nucleotide Exchange Factor ◽  
Plant Vacuoles ◽  
And Function

Autophagy is a catabolic pathway that delivers intracellular material to the mammalian lysosomes or the yeast and plant vacuoles. The final step in this process is the fusion of autophagosomes with vacuoles, which requires SNARE proteins, the homotypic vacuole fusion and protein sorting tethering complex, the RAB7-like Ypt7 GTPase, and its guanine nucleotide exchange factor, Mon1-Ccz1. Where these different components are located and function during fusion, however, remains to be fully understood. Here, we present a novel in vitro assay to monitor fusion of intact and functional autophagosomes with vacuoles. This process requires ATP, physiological temperature, and the entire fusion machinery to tether and fuse autophagosomes with vacuoles. Importantly, we uncover Ykt6 as the autophagosomal SNARE. Our assay and findings thus provide the tools to dissect autophagosome completion and fusion in a test tube.

scholarly journals Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

1996 ◽  
Vol 7 (3) ◽  
pp. 355-367 ◽  
Author(s):  
D J Spiro ◽  
W Boll ◽  
T Kirchhausen ◽  
M Wessling-Resnick
Keyword(s):  
Transferrin Receptor ◽  
Kinase Inhibitor ◽  
Morphological Changes ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
Phosphatidylinositol 3 Kinase ◽  
Vitro Assay ◽  
Phosphatidylinositol 3

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

scholarly journals Septins promote macropinosome maturation and traffic to the lysosome by facilitating membrane fusion

2016 ◽  
Vol 214 (5) ◽  
pp. 517-527 ◽  
Author(s):  
Lee Dolat ◽  
Elias T. Spiliotis
Keyword(s):  
Cell Metabolism ◽  
Extracellular Fluid ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Septin Gtpases ◽  
And Function ◽  
Large Clusters ◽  
Blocking Antibodies

Macropinocytosis, the internalization of extracellular fluid and material by plasma membrane ruffles, is critical for antigen presentation, cell metabolism, and signaling. Macropinosomes mature through homotypic and heterotypic fusion with endosomes and ultimately merge with lysosomes. The molecular underpinnings of this clathrin-independent endocytic pathway are largely unknown. Here, we show that the filamentous septin GTPases associate preferentially with maturing macropinosomes in a phosphatidylinositol 3,5-bisphosphate–dependent manner and localize to their contact/fusion sites with macropinosomes/endosomes. Septin knockdown results in large clusters of docked macropinosomes, which persist longer and exhibit fewer fusion events. Septin depletion and overexpression down-regulates and enhances, respectively, the delivery of fluid-phase cargo to lysosomes, without affecting Rab5 and Rab7 recruitment to macropinosomes/endosomes. In vitro reconstitution assays show that fusion of macropinosomes/endosomes is abrogated by septin immunodepletion and function-blocking antibodies and is induced by recombinant septins in the absence of cytosol and polymerized actin. Thus, septins regulate fluid-phase cargo traffic to lysosomes by promoting macropinosome maturation and fusion with endosomes/lysosomes.

scholarly journals Rab39a and Rab39b Display Different Intracellular Distribution and Function in Sphingolipids and Phospholipids Transport

2019 ◽  
Vol 20 (7) ◽  
pp. 1688 ◽  
Author(s):  
Julián Gambarte Tudela ◽  
Julio Buonfigli ◽  
Agustín Luján ◽  
Mariano Alonso Bivou ◽  
Ignacio Cebrián ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Biology ◽  
Small Gtpases ◽  
Intracellular Distribution ◽  
Endocytic Pathway ◽  
Lipid Transport ◽  
Amino Acid Sequence Similarity ◽  
Multivesicular Bodies ◽  
Rab Gtpases ◽  
And Function

Rab GTPases define the identity and destiny of vesicles. Some of these small GTPases present isoforms that are expressed differentially along developmental stages or in a tissue-specific manner, hence comparative analysis is difficult to achieve. Here, we describe the intracellular distribution and function in lipid transport of the poorly characterized Rab39 isoforms using typical cell biology experimental tools and new ones developed in our laboratory. We show that, despite their amino acid sequence similarity, Rab39a and Rab39b display non-overlapping intracellular distribution. Rab39a localizes in the late endocytic pathway, mainly at multivesicular bodies. In contrast, Rab39b distributes in the secretory network, at the endoplasmic reticulum/cis-Golgi interface. Therefore, Rab39a controls trafficking of lipids (sphingomyelin and phospholipids) segregated at multivesicular bodies, whereas Rab39b transports sphingolipids biosynthesized at the endoplasmic reticulum-Golgi factory. Interestingly, lyso bis-phosphatidic acid is exclusively transported by Rab39a, indicating that both isoforms do not exert identical functions in lipid transport. Conveniently, the requirement of eukaryotic lipids by the intracellular pathogen Chlamydia trachomatis rendered useful for dissecting and distinguishing Rab39a- and Rab39b-controlled trafficking pathways. Our findings provide comparative insights about the different subcellular distribution and function in lipid transport of the two Rab39 isoforms.

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