THE ROUTE TO THE LYSOSOME _ THE EGF RECEPTOR
TRANCYTOSIS _ MDCK CELLS AND THE POLYMERIC IgA RECEPTOR
covered in the CSPS course.
It is made by Harvard scientists and is scientifically accurate;
http://www.studiodaily.com/main/technique/tprojects/6850.html
Key
References
J. Cell Biol., 56.
206-229, 1973
EMBO J. 11, 1717-1722, 1992.
Transport
Vesicles
Golgi
maturation visualized in living Yeast Nature 441, 939-940, 2006
Rab conversion as a mechanism of progression from
early to late endosomes.
Cell. 2005 ;122:735-49.
Modregger J, Uttenweiler-Joseph S, Wilm M, Nystuen
A, Frankel WN, Solimena M, De
Camilli P, Zerial M.
An enzymatic cascade of Rab5 effectors regulates
phosphoinositide turnover in
the endocytic pathway.
J Cell Biol. 2005;170:607-18.
H, Zerial M.
Modulation of receptor recycling and degradation by
the endosomal kinesin
KIF16B.
Cell. 2005 ;121:437-50.
Mol Biol Cell 16:1744-55, 2005
Murray
RZ, Kay JG, Sangermani DG, Stow JL. A role for the
phagosome in cytokine secretion. Science. 310:1492-1495, 2005
Caveolae
Caveolin-stabilized membrane domains as
multifunctional transport and sorting
devices in endocytic membrane traffic.
Cell. 2004 ;118:767-80.
Kinase-regulated quantal assemblies and
kiss-and-run recycling of caveolae.
Nature. 2005; 436:128-33.
Technique
Imaging intracellular fluorescent proteins at
nanometer resolution.
Science. 15:1642- 1645.2006
Seet B. T., Dikic I., Zhou, M, -M., and Pawson T.
Reading protein modifications with interaction domains.
Nature Rev. Mol. Cell Biol. 7,473-483, 2006
Pelkmans L, Fava E, Grabner H, Hannus M, Habermann B, Krausz E, Zerial M.
Genome-wide analysis of human kinases in clathrin-
and caveolae/raft-mediated
endocytosis.
Nature. 2005 ;436:78-86.
Viral glycoproteins destined for apical or
basolateral plasma membrane domains traverse the same Golgi apparatus during
their intracellular transport in doubly infected Madin-Darby canine kidney
cells.
J Cell Biol. 98:1304-19; 1984
Coated Pits and Membrane invagination
Nature 441;
528 – 531; 2006
GTPase activity of dynamin and resulting
conformation change are essential for endocytosis.
Nature 410; 231-235, 2001
Di Paolo G. and De Camilli P Phosphoinositides in Cell Regulation and Membrane Dynamics - A Review
Nature 443, 605 - 722 2006 (12th October issue)
For background revision look up the following diagrams in
Alberts et al, Molecular Biology of the Cell (4th Edition);
Figs. 9-10, 9-43, 12-7, 13-10, 13-11, 13-12, 13-14, http://13-17, 13-20,
13-21, 13-26 (pts 1&2), 13-36, 13-29, 13-30.
1955 to 1965: Modern era of molecular Cell Biology founded by George Palade, Keith Porter, Philip Siekevitz, Christian de Duve,
Gunter Blobel and David Sabatini working at the Rockefeller Institute in New York
They developed subcellular fractionation of membrane compartments, electron microscopy and cell free transport assays
Differential vs equilibrium centrifugation for separating cell fractions
"Rough Microsome" fraction viewed by electron microscopy
Cellular Pathways: biosynthetic route to the plasma membrane begins with the rough endoplasmic reticulum
(Palade electron micrograph)
Biosynthetic route: Regulated (secretory granule) route Biosynthetic route "constitutive" route
Endocytotic route: plasma membrane towards lysosome
Biosynthetic route to endocytotic compartments as followed by Mannose-6-phosphate receptor
Back to Contents
Plasma cell in lymphoid tissue, packed with rough endoplasmic reticulum (rer)
and with Golgi stack (G) just above the nucleus
Golgi and rer in pancreatic acinar cell
(by George Palade)
Vesicle fusion promoted by SNAREs
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COP II Vesicles
Randy Schekman mutagenized yeast (Saccharomyces cerevisiae) and selected mutants that failed to secrete invertase.
This approach identified 21 (sec) genes required for secretion. EM showed vesicles often accumulated at specific locations along biosynthetic path.
See "Vesicle budding in the endoplasmic reticulum" by Shimoni Y. and Schekman R in Methods Enzymol., 351. 258-278, 2002
Advantages of yeast system:
New genes can be identified
Complexity of the system can be assessed (e. g. in yeast there is only one set of coatomer genes)
Disadvantages:
Morphology of Yeast Golgi not as complex as Mammalian
Transport signals often different (yeast uses ubiquitin more often and clathrin is not essential
As with COPI vesiculation begins with activation of a GTP-binding protein. The activation of this protein (Sar1p) is by the GEF (Sec 12p) which is distributed all over the RER; specificity thought, in turn to be controlled by Sec 16p and Sed 4p.
GTP binding of Sar1p causes a conformational change exposing a fatty acid chain that anchors it to the RER bilayer.
Activated Sar1p induces binding of Sec 23/24 complex from the cytosol. This complex has a banana-shape with the concave face bearing +ve charged amino acids - similar to the BAR domains discussed in relation to amphiphysin.
Into the forming coat cargo and SNARE proteins (Sec 22p, Bet1p and Bos1p) are also recruited.
Sec 23/24p complex also recruits Sec 13/31p to the COPII coat and, since this complex has GTPase activity it probably brings about coat dis-assembly.
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The transport pathway between the RER and the Golgi Complex can be followed at high resolution using an electron-opaque tracer
Horseradish peroxidase-chimaera (HRP-KDEL) identifying its site of synthesis
in the RER (and nuclear envelope)
HRP-chimaera chased out of RER is packaged into transport vesicles
(note some vesicles don't contain HRP - they are probably returning to the RER)
KDEL-peroxidase in cis cisternae during a prolonged chase incubation
(this tracer does not reach the Golgi stack because it is recycled to the RER)
THE GOLGI COMPLEX
First described by Camillio Golgi (1905)
Definitively described by electron microscopy (George Palade 1957)
Characteristic stack of flat, saucer-shaped cisternae.
Stability of stack depends upon tethering proteins (Golgin 45, Giantin, GM 130, GRASP 46, 55, 65).
Membrane thickness increases across the stack in a cis-trans direction. Bretscher and Munro (Bretscher, M and Munro S. Science 261, 1280-1281, 1993) showed that VSVG with transmembrane domain of only 14 residues (usually 23) was transported only as far as the cis Golgi.
The fungal metabolite Brefeldin (BFA) inhibits a family of ARF GEFs (see below). These GEFs include BIG1 and BIG2 (Golgi-associated Brefeldin A- inhibited GEFs).
BIG1 mediates recruitment of COP I whereas BIG2 mediates recruitment of GGAs (see below).
BFA treatment causes Golgi stack to disassemble and membrane proteins such as glycosidases return to the RER.
Large polymeric proteins like Procollagen and some algal scales are shown to transfer sequentially up the Golgi stack supporting the view that the stack is a dynamic collection through which the cisternay move in a cis-trans direction.
ENDOCYTOSIS
The plasma membrane internalizes an area equal to the entire cell surface every 90 minutes.
Functions of endocytic ligand uptake:
Uptake of nutrients and waste products:
Transferrin receptor: takes up iron-transporter Transferrin
Asialoglycoprotein receptor takes up serum glycoproteins with terminal sugars (e.g. fucose ) after terminal sialic acid removed.
Low density lipoprotein receptor binds LDL particles which have a phospholipid shell containing a receptor-binding 500kDa protein enclosing ~1500 mols cholesterol.
The Endosome Compartment includes the Early Endosome, the late Endosome and Recycling Endosome sub-compartments.
Coated vesicles derived from clathrin coated pits deliver their endocytosed cargo to the Early Endosome.
Fluid Phase cargo is then transferred, via the Late Endosome to the lysosome.
Integral membrane proteins (such as the transferrin receptor ) can be selectively recycled,
via the recycling endosome, back to the cell surface.
Internalized EGF receptors are transferred to the inner vesicles in the late endosome.
When the late Endosome vacuole fuses with the lysosome the EGF receptors are degraded.
Constitutive Uptake: Transferrin receptor, asialoglycoprotein receptor, low density lipoprotein receptor.
Ligand-induced uptake: Hormones (e.g. insulin) and Growth factor (e.g. Epidermal Growth Factor) receptors. Ligand binding delivers receptor to site of signal transduction and to the site at which the signal is attenuated (down regulation).
Mechanisms of micropinocytosis at the plasma membrane
Coated Pit containing gold-labelled Transferrin Receptors
Surface view of plasma membrane, gold-labelled transferrin receptors shown as white dots
Transferrin Receptor trafficking via the fast recycling pathway
Cartoon showing receptors being loaded into a clathrin-coated pit, surface view shows distribution of receptors relative to clathrin triskelion
Internalization signals
Transferrin receptor (TR) has a single motif characterized by a tyrosine, charged residue (arginine) and large hydrophobic residue (phenylalanine).
CAVEOLAE
High Resolution Structure of Clathrin Triskelion from Kirkhausen
Go to the following hyperlink and click on "the birth of a clathrin coat" and "Simulation of how a clathrin coat forms;
http://www.cbr.med.harvard.edu/labs/kirchhausen/research.html
To see how real-time video studies of clathrin coated pit formation can be followed usin TIRF microscopy, see:
Merrifield, C Qualmann, B Kessels, M and Almers K
Invagination and dynamin recruitment of single coated pits. Nature Cell Biology 4. 691-698, 2002
and Merrifield et al Eur. J. Cell Biol. 83, 13-18 2004. N-WASP and ARP2/3 are recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts.
ADAPTOR PROTEINS
The AP2 adaptor is found only on the plasma membrane.
Other adaptors (AP1, AP3, AP3 and GGA) are found on internal membranes. Their role in sorting specific subsets of trafficking proteins can be shown by studies with mutants by knock-down studies (SRNAi) and by analyzing their ability to recognize specific sorting signals but their precise locations on trafficking pathways continue to be debated.
The sub-unit structure of AP2 and AP1 is similar
IMPORTANT TO NOTE:
Unlike AP2/clathrin coats, adaptors in coats on intracellular membranes are usually ARF-regulated.
GGA coats
So-called because they are Golgi-associated, Gamma sub unit-homologous, ARF-regulated coats.
Domains (From Pawson http://pawsonlab.mshri.on.ca )
ENTH domain EH domain
To
demonstrate the interactions at the
aqueous buffer surface and introduced
AMPHIPHYSIN
Contains a BAR domain which on dimerization can cause tubulation in liposome preparations. Bar domain also found in other clathrin accessory proteins (e.g. Endophillin)
There are no obvious phenotypes in knock-out mice devoid of amphiphysin other than susceptibility to siezures - suggesting that this accessory protein most important for clathrin-mediated retrieval of synaptic vesicle membrane.
High resolution structure of BAR domain of amphiphysin
For further discussion and relationship to COPII vesicle formation see;
"BAR domains go on a bender" by M. C. Lee and Randy Schekman in Science 303, 479-480, 2004
DYNAMIN _ Evidence that it is a mechanochemical enzyme
GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature. 2006; 441528-31
Roux A, Uyhazi K, Frost A, De Camilli P.
See brief discussion on the importance of this data in "Twisting Endocytosis" Journal of Cell Biology173, 456, 2006
To start video move cursor over rectangle below
Coats and adaptors on intracellular membranes
A highly plastic intracellular compartment of vacuoles and tubules continuously changing shape forming long, often branching tubules and fusing and budding vacuoles.
Loading cells with tracers in the fluid phase (often called "bulk phase") such as fluorescent dextran or free horse radish peroxidase shows uptake into early endosomes (2 to 5 mins) then transfer, via late endosomes, to the lysosome. Relatively little fluid phase tracer enters tubules (and recycles).
Integral membrane proteins like the transferrin receptor enter the early endosome and distributes throughout its tubules. Two recycling routes back to the cell surface are available; a fast (2 to 3 min) and a slower (up to 20 min) route. The latter travels via a group of tubules in the pericentriolar area - the pericentriolar recycling endosome.
Amongs the receptors recycling out of the early endosome there are at least two alternative destinations (1) the plasma membrane and (2) the biosynthetic pathway. The path to the cell surface as followed by the transferrin receptor is thought not to require further signal information (a default pathway) . The route to the biosynthetic pathway, as followed by the mannose-6-phosphate receptor begins in tubules coated with the cytosolic nexin protein SNX1.
Early Endosome Fusion Assay
Confluent BHK-21 cells grown on 10-cm diameter Petri dishes were washed with 37°C phosphate-buffered saline, pH 7.4. Subsequently, either 4 mg/ml avidin or 1.8 mg/ml biotinylated horseradish peroxidase (HRP) in Dulbecco’s phosphate-buffered saline supplemented with 1 mM CaCl2 and 1 mM MgCl2 were incubated with the cells for 9 min at 37°C. This incubation was followed by extensive washing at 4°C before removal of cells from the dish with a cell scraper and homogenization in homogenization buffer (3 mM imidazole/HCl pH 7.4, 250 mM sucrose, 1 μg/ml pepstatin, 2 μg/ml aprotinin, 2 μg/ml leupeptin) by several passages through a 23-gauge needle. Postnuclear supernatants were obtained by centrifugation at 1,500 × g for 15 min.
Postnuclear fractions containing avidin or biotin-HRP–loaded endosomes were then combined in a mixture (total volume 217 μl) containing 10 mM HEPES-KOH, pH 7.0, 1.2 mM MgCl2, 50 mM KOAc, 0.8 mM DTT, biotin-insulin (to quench any free avidin) and ATP-regenerating system, to which had been added the various factors being assessed. This mixture was then incubated at 37°C for up to 10 minutes. Alternatively, postnuclear supernatants containing avidin or biotin-HRP–labeled early endosomes were aliquoted and separately preincubated for 10 min at 37°C with these factors. Preincubations were performed in the absence of ATP-regenerating system and salts. In all procedures the appropriate buffer was used to substitute volume additions of added reagents. The samples were then placed on ice before mixing in fusion assays as above. Fusion assays were stopped by lysis on ice for 15 min with 0.25% wt/vol Triton X-100. Fusion results in the formation of an avidin–biotin–HRP complex, which was immunoprecipitated with anti-avidin antibodies bound to protein A sepharose. The relative amounts of immunoprecipitated HRP were quantified by determination of the HRP activity with o-dianisidine as substrate or on gels.
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HOMOTYPIC FUSION OF EARLY ENDOSOMES MEDIATED BY EEA1 AND ACTIVATED Rab 5
Rab 5 in the GTP-bound form activates PI3kinase in the endosome membrane and recruits EEA1
EEA! binds PI3P via FYVE domains
FYVE finger domains include Zn and bind specifically to PtdIns(3)P through a basic pocket and hydrophobic surface.
They are so-called because they are found in the Fab-1, YoTB, Vac-1 and EEA1 proteins in the endocytic pathway.
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Morphology of the endosome compartment in a fibroblast showing the Peri-centriolar subcompartment
Experiment by Jennifer Stow's group demonstrating that newly synthesized TNF alpha
transits via the endosome en-route for the plasma membrane
A Role for the Phagosome in Cytocrine secretion by R. Z. Murray, J.S.Kay, D.G. Sangermani and J.L.Stow, Science 310, 1492-1495, 2005
Vacuoles
and vesicles containing transferrin demonstrating
they belong to the endosome compartment
Newly synthesized TNF alpha was shown to travel from the RER via the Golgi complex to the plasma membrane.
VAMP 3/GFP was shown to be distributed on the plasma membrane and the transferrin-containing endosome
The Golgi Q SNAREs Syntaxin 6 and 7 and VTi!b could be co-immunoprecipitated with the R SNARE VAMP 3
A dominant/negative fragment of VAMP 3 prevented TNF alpha travelling further than the Golgi
CONCLUSION:
Transport of TNF alpha from the RER to the plasma membrane is a THREE step process.
For the last two steps transport vesicles from the Golgi employ Syntaxins 6 and 7/VTi1b and Vamp 3 to dock and fuse with the endosome
and then vesicles from the endosome employ Syntaxin 4, SNAP 23 and VAMP 3 to fuse with the plasma membrane
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THE ROUTE TO THE LYSOSOME - THE EGF RECEPTOR
The Epidermal Growth Factor Receptor (EGFR) is delivered to the early endosome by transport vesicles derived from clathrin coated pits. They can recycle but the majority concentrate in the vacuolar regions of the early endosome where the EGFR kinase phosphorylates a new sub-set of cytosoloc proteins. These include HRS-2 (which has a FYVE domain) ESCRT complex proteins (e.g. TSG 101) and clathrin (heavy chain only - no adaptors) which together form discrete domains identifiable by electron microscopy.
Following their sequestration in the early endosome EGFR are transferred (mechanism unknown) to invaginating domains on the perimeter membrane which pinch off small (30 to 50 nm dia. ) vesicles into the lumen of the vacuole. This invagination process requires the participation of a Wortmannin- sensitive lipid kinase (PIP3 kinase) and the activity of the EGFR kinase which phosphorylates the cytosolic protein Annexin 1. Wortmannin inhibits the formation of inner vesicles in early endosomes and in knock-out mice lacking annexin 1 the numbers of these vesicles are much reduced.
The major substartes for the EGFR kinase are the receptor itself (1 to 3 mins), HRS-2 (3 to 5 mins) and Annexin 1 (5 to 15 mins).
Late endosomes containing EGFR-loaded internal vesicles fuse with lysosomes in a Rab 7 regulated manner. The EGFR ( whose signal transduction has been attenuated by invagination, and thus its removal, from its substrates in the cytosol) is completely degraded by lysosomal hydrolases.
TRANSCYTOSIS MDCK CELLS AND THE POLYMERIC IgA RECEPTOR
References for Trancytosis of the Polymeric IgA receptor in Polarised Epithelial Cells
von Bonsdorff, C., Fuller, S. D. and Simons, K (1985). Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters. EMBO J 4, 2781-92
Apodaca, G., Katz, L. A. and Mostov, K. E (1994). Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes. J. Cell Biol 125, 67-86
Gibson, A, Futter, C, Maxwell, S, Shipman, M, Kreahenbull, J-P, Trowbridge, I S. and Hopkins C.R.Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarised MDCK cells. J. Cell Biol 98, 81 - 94, 1998
Lock J. G. and
Mol
Biol Cell. 16:1744-55. 2005
Rachael Z.
Murray, Jason G. Kay, Daniele G. Sangermani, Jennifer L. StowA
Role for the Phagosome in Cytokine Secretion
Science 310. 1492 – 1495. 2005
