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.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

What Can Epitope Specific Antibodies Tell us About the Organization of Caveolin in Cells?

2001 ◽  
Vol 7 (S2) ◽  
pp. 1030-1031
Author(s):  
J.M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Biochemical Characterization ◽  
Cell Types ◽  
Membrane Microdomains ◽  
Truncated Form ◽  
Coated Pits ◽  
N Terminus ◽  
Plasma Membrane Microdomains

There are three members of the caveolin (CAV) gene family that give rise to four polypeptides. These polypeptides are CAV-1α, CAV-1β, CAV-2, and CAV-3. The CAV-1β isoform is a truncated form of CAV-1α that lacks 31 amino acids at the N-terminus of the molecule. The CAV- 1β molecule arises through an alternative splicing mechanism.Caveolae are specialized plasma membrane microdomains that are expressed at high levels in some cell types (e.g., endothelium, adipocytes, fibroblasts). These specialized regions of the plasma membrane have a characteristic omega-shaped appearance with diameters ranging from 40-90 run. They are distinct from clathrin-coated pits since they lack the characteristic coated appearance in electron microscopy. Caveolae were among the first structures to be discovered by biological electron microscopy. However, biochemical characterization of these structures did not begin in earnest until a marker protein was identified. The initial marker was the 22-kDa protein known as caveolin.

scholarly journals The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway.

1996 ◽  
Vol 7 (1) ◽  
pp. 155-172 ◽  
Author(s):  
G P Leser ◽  
K J Ector ◽  
R A Lamb
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Simian Virus ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Immunogold Staining ◽  
Simian Virus 5 ◽  
Fusion Glycoprotein ◽  
Infected Cells ◽  
Endosomal System

The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic pathway.

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.

To bead or not to bead? Lens-specific intermediate filaments revisited

1997 ◽  
Vol 110 (21) ◽  
pp. 2629-2634 ◽  
Author(s):  
S.D. Georgatos ◽  
F. Gounari ◽  
G. Goulielmos ◽  
U. Aebi
Keyword(s):  
Plasma Membrane ◽  
Intermediate Filaments ◽  
Cultured Cells ◽  
Fiber Cells ◽  
New Information ◽  
Specific Proteins ◽  
Beaded Filaments ◽  
Nodular Appearance

For nearly three decades cytoplasmic intermediate filaments (IFs) have been described as 10 nm thick, unbranched ropes radiating from the cell nucleus and extending to the plasma membrane. This stereotype is now being challenged by the discovery and molecular characterization of the beaded filaments (BFs), a novel class of IFs composed of the lens-specific proteins filensin and phakinin. In contrast to ‘mainstream’ IFs, BFs have a distinctly nodular appearance and form a meshwork underneath the plasma membrane of the lens fiber cells. In vitro assembly studies, expression of filensin and phakinin in cultured cells, and analysis of the corresponding genes reveal that these proteins have evolved from two different subfamilies of IF proteins, thus yielding a unique structure. The new information provides a basis for understanding how the various forms of tissue-specific IF proteins might have developed adopting to the constraints of a specialized environment.

scholarly journals Sorting signals in the MHC class II invariant chain cytoplasmic tail and transmembrane region determine trafficking to an endocytic processing compartment.

1994 ◽  
Vol 126 (2) ◽  
pp. 317-330 ◽  
Author(s):  
C G Odorizzi ◽  
I S Trowbridge ◽  
L Xue ◽  
C R Hopkins ◽  
C D Davis ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mhc Class Ii ◽  
Cytoplasmic Tail ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Invariant Chain ◽  
Transmembrane Region ◽  
Coated Pits ◽  
Endocytic Compartment ◽  
Sorting Signals

Targeting of MHC class II molecules to the endocytic compartment where they encounter processed antigen is determined by the invariant chain (Ii). By analysis of Ii-transferrin receptor (TR) chimera trafficking, we have identified sorting signals in the Ii cytoplasmic tail and transmembrane region that mediate this process. Two non-tyrosine-based sorting signals in the Ii cytoplasmic tail were identified that mediate localization to plasma membrane clathrin-coated pits and promote rapid endocytosis. Leu7 and Ile8 were required for the activity of the signal most distal to the cell membrane whereas Pro15 Met16 Leu17 were important for the membrane-proximal signal. The same or overlapping non-tyrosine-based sorting signals are essential for delivery of Ii-TR chimeras, either by an intracellular route or via the plasma membrane, to an endocytic compartment where they are rapidly degraded. The Ii transmembrane region is also required for efficient delivery to this endocytic processing compartment and contains a signal distinct from the Ii cytoplasmic tail. More than 80% of the Ii-TR chimera containing the Ii cytoplasmic tail and transmembrane region is delivered directly to the endocytic pathway by an intracellular route, implying that the Ii sorting signals are efficiently recognized by sorting machinery located in the trans-Golgi.

scholarly journals Separation and characterization of caveolae subclasses in the plasma membrane of primary adipocytes; segregation of specific proteins and functions

FEBS Journal ◽  
2006 ◽  
Vol 273 (14) ◽  
pp. 3381-3392 ◽  
Author(s):  
Unn Ortegren ◽  
Lan Yin ◽  
Anita Ost ◽  
Helen Karlsson ◽  
Fredrik H. Nystrom ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Specific Proteins

scholarly journals Characterization of the EYE2 Gene Required for Eyespot Assembly in Chlamydomonas reinhardtii

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1037-1049
Author(s):  
Douglas G W Roberts ◽  
Mary Rose Lamb ◽  
Carol L Dieckmann
Keyword(s):  
Plasma Membrane ◽  
Chlamydomonas Reinhardtii ◽  
De Novo ◽  
Pigment Granule ◽  
Site Directed Mutagenesis ◽  
Insertion Mutagenesis ◽  
Isolation And Characterization ◽  
Specialized Structure ◽  
Cellular Compartments

Abstract The unicellular biflagellate green alga Chlamydomonas reinhardtii can perceive light and respond by altering its swimming behavior. The eyespot is a specialized structure for sensing light, which is assembled de novo at every cell division from components located in two different cellular compartments. Photoreceptors and associated signal transduction components are localized in a discrete patch of the plasma membrane. This patch is tightly packed against an underlying sandwich of chloroplast membranes and carotenoid-filled lipid granules, which aids the cell in distinguishing light direction. In a prior screen for mutant strains with eyespot defects, the EYE2 locus was defined by the single eye2-1 allele. The mutant strain has no eyespot by light microscopy and has no organized carotenoid granule layers as judged by electron microscopy. Here we demonstrate that the eye2-1 mutant is capable of responding to light, although the strain is far less sensitive than wild type to low light intensities and orients imprecisely. Therefore, pigment granule layer assembly in the chloroplast is not required for photoreceptor localization in the plasma membrane. A plasmid-insertion mutagenesis screen yielded the eye2-2 allele, which allowed the isolation and characterization of the EYE2 gene. The EYE2 protein is a member of the thioredoxin superfamily. Site-directed mutagenesis of the active site cysteines demonstrated that EYE2 function in eyespot assembly is redox independent, similar to the auxiliary functions of other thioredoxin family members in protein folding and complex assembly.

c-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route

2001 ◽  
Vol 114 (11) ◽  
pp. 2167-2178 ◽  
Author(s):  
Annemieke A. de Melker ◽  
Gerda van der Horst ◽  
Jero Calafat ◽  
Hans Jansen ◽  
Jannie Borst
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinase ◽  
Egf Receptor ◽  
Ring Finger ◽  
Endocytic Pathway ◽  
Immunogold Electron Microscopy ◽  
Coated Pits ◽  
Receptor System ◽  
Regulatory Capacity ◽  
Ubiquitin Conjugating Enzymes

Cbl family members have an evolutionarily conserved role in attenuating receptor tyrosine kinase function. Their negative regulatory capacity depends on a Ring finger domain that interacts with ubiquitin conjugating enzymes. Cbl molecules constitute a novel type of E3 or ubiquitin ligase family that is recruited to phosphotyrosine motifs. Ubiquitination of the receptor system is coupled to its downregulation, but it is unclear at which point in the endocytic pathway Cbl molecules come into play. Using low temperature and a dynamin mutant, we find that c-Cbl associates with and ubiquitinates the activated epidermal growth factor (EGF) receptor at the plasma membrane in the absence of endocytosis. With the aid of confocal microscopy and immunogold electron microscopy, we could demonstrate that c-Cbl associates with the EGF receptor at the plasma membrane prior to receptor recruitment into clathrin-coated pits and remains associated throughout the clathrin-mediated endocytic pathway. c-Cbl and the EGF receptor also colocalize in internal vesicles of multivesicular endosomes. Our data are consistent with a role for c-Cbl in clathrin-mediated endocytosis of tyrosine kinase receptors, as well as their intracellular sorting.

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

2015 ◽  
Vol 57 ◽  
pp. 189-201 ◽  
Author(s):  
Jay Shankar ◽  
Cecile Boscher ◽  
Ivan R. Nabi
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cancer Cell ◽  
Cellular Response ◽  
Spatial Organization ◽  
Essential Feature ◽  
Cell Signalling ◽  
Coated Pits ◽  
Signalling Molecules ◽  
Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

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