Conference on the Endocytosis

Conference on the Endocytosis plasma membrane IDl receptors Nucleus CYTOSOL ENDOCYTOStS UNCOATING early eodosome RETURN OF LDL RECEPTORS TO PLASMA MEMBRANE BULK FLO BUODING OFF Of TRANSPORT VESICLES FUSION WITH ENDOSOME \ I / free cholesterol •• % ••.* •• •. „•••••• •••• •• .%•* ••••• •• • •••••• •• • • • • •• •• • • lysosome hydrolytic enrymes TRANSPORT OF MACROMOLECULES: ENDOCYTOSIS 1. FLUID (OR BULK) FLOWS 2. RECEPTOR-MEDIATED BULK FLO Plasmalemmal vesicles ENDOCYTOSIS – – – SPECIFIC RECEPTOR COATES PITS (CLATHRIN) LYSOSOMAL FUSION WITH ENDOSOMES -- Recycling of receptors plasma membrane LDL receptors CYTOSOL ENDOCYTOSIS coated UNCOATING vesicle early endosome RETURN OF LDL RECEPTORS TO PLASMA MEMBRANE BUDDING OFF OF TRANSPORT VESICLES FUSION WITH ENDOSOME \ J free cholesterol . ••• ••••*••• .. •• • • •• • ••• • •••• • • •• . / . # X hydrolytic ' enzymes lysosome Conference on Endocytosis: Receptor-mediated endocytosis Objectives: • To reinforce the interplay among receptors, cargo, and actions of the apical portion of the cell by considering defects in the interplay • To use techniques we have discussed to analyze the process of endocytosis • To reinforce the idea that each step of a process is an opportunity for a disease endosome cytosol lysosome Golgi apparatus peroxisome mitochondrion endoplasmic reticulum with membrane-bound polyribosomes free nucleus polyribosomes •• plasma membrane % r* • •• t • 44 4 # 4 m • • ;• •• •• •• # 4 •• . 4 4 * # 4 4 4 . •• ••• ••• •, • • .• • •• • 4 •* • •4 • 4 4 4 4 4 4 4 4 4 4 •• • 4 4 4 . • •* • rnm 4 4 4 4 • 4# • •• 4 • 4* • 4 4 • 4 4 • 4 ••4 4* 4 4 4 4 4 4 4 4 4 • •• 4 4 « 4 4 •• 9 4 4 4 4 • 4 4 4 4 4 4 4 4 « •• • • • 4 • # 4 4 «* 4 * 4 4 4 4 ' 0 4 4 4 4 4 . •• •• 4 4 • •: 4 * •# •» 4 4 4 •• * 4 •• 4 •• • 4 •• • 4 4 4 4 4 A 4 4 . •.• ,* 4 * 4 * •• •• • • * *• 4 4 4 4 1 Random insertion \ i 4 1 Clustering r y Internalization 4 Invagination t - Receptor binding site A - Internalization site Coated vesicle k •• • N 4 4 4 • •• • • •• 4 4 4 . .. • • •:• *•• v : ?',' •• •• • • : ••• •••• •• f / f • 4 4 4 4 4 4 4 4 # :. 4 # 4 4 4 •4• • • • 4 •• • 4 4 • V • •' • 1 4 4 4 4 4 4 •• f Random insertion t - Receptor binding site A - Internalization site Coated vesicle completed transport coat subunits membrane coated region of membrane vesicle u COAT DISASSEMBLY organelle lumen ’ COAT ASSEMBLY BUD FORMATION VESICLE FORMATION CYTOSOL veucle containing cargo dathrin coated transport nI UNCOATING t\ * DOCKING V BUDDING A * £ t r X i > f-\ W i l i membrane of Golgi network FUSION ffsnt delivered cargo r *- clathitn acceptor FUSION docking marker cargo receptor /» l T c ' 1 BUDDING fa a UNCOAflNG lipid bilaycr A cargo molecule - docking marker acceptor j empty clathrin coated transport vesicle clathrin molecule Insulin receptors do not recycle endolysosome endosome s < *— \ f ) > endocytosis I ( tans Golgi i * network network r\ i v % membrane recycling c stack * ) * r\ r J J constitutive secretion f / ( plasma '' w u v o zf\ r COAT DISASSEMBLY organelle lumen VESICLE 'A • * trmne < ER membrane * clathrin coated vesicle (dots label clathrin coat) uncoated vesicle (no dots) *5L | * •uff* 1 . > o- < Insulin receptors do not recycle Fluorescent compound stackt Peroxidase particles particles clathrin coated vesicle (dots label clathrin coat) uncoated vesicle (no dots) * MANNOSE-6-PHOSPHTAE = SIGNAL FOR SORTING ENZYME TO LYSOSOME VIA RECEPTORS lysosomal hydrolase precursor RECEPTOR DEPENDENT ATP TRANSPORT from ER BINDING TO M6P RECEPTOR H* ADDITION OF PHOSPHATE mannose 6- to jhosphate M6P) —* clathnn coat fc transport DISSOCIATION AT ACIDIC pH vesicle V REMOVAL OF PHOSPHATE m f mature lysosomal hydrolase RECEPTOR RECYCLING cis Golgi network trans Golgi network j M6P receptor in budding vesicle late endosome 1. recycling apical plasma membrane early endosome \ O transport tight / junction vesicles 3. transcytosis r TRANSCYTOSIS method to get antibodies into secretions or body fluids like milk, nasal fluid, or tears 2. degradation lysosome basolateral plasma membrane i. nucleus EPITHELIAL CELL mm «9 LUMEN IgA dimer secretoq comporn TRANSCYTOSIS membrane- bound Pf <7 •rtf //r Wk> ( EM 4c -* * jy ' 7W ££// )* ; S3 r > « <#&* * " •r * Intestinal absorptive cell . &- ^ • wi ^ T - f * A . ' * ,r A . kT Ms A V A / i v / -* ' * - I ^0K A’W ‘^ OAft: o - : ' A -CrfrCJ 0 . r x j 4 ' Si *? /y *» / V* . r gy ** . >£ a r: esttJsQ > N .1 ^•./ ^o i - 0 •^ : ? 4‘ > Nmm M . * Cut AO, *T ?« coated vesicle 0 “ > . m* // / > i£ , » 4/ / ' - -A i V' ' V, * ' -* M nucleus - rr. v> ^7 r*> o . £ - ;< • ? 0 « coated vesicle 0 v: •vs A « ^** r rc >i * '. / mitochondnon / * j’ . 1 V V - - w ."V w >/ . . .« SA \ S / V % ^^. 1 . f yj iii _ » I ¥ ••r j iti ; Hr » . OC --/ <4 ; > y ./>- V-1 ' • *\ budding RER ). i* r itestinal absorptive cell (super nuclear regio jW 7 ir- "'v x Vf i. .. m TT ^- XvH v. iO • f f v• A «% • N \ « ** . coated vesicle^ > •« A ' > ' •• fe • • r ‘ >' o ^. - 51 iw // rmi / on f§j \ iI P s / V4 ' / : W u : # . ? if ;ent V cells n v * f •• O* :V # ff > , 4L/S . budding RER 9 v* 2y coated vesicle or f ‘% 4 m n O C) f) i V ' ft# 1 22 nm cholesterol molecule phospholipid molecule 5 cholesteryl este' molecule surface protrusion on protein molecule Figure 6 —75 A low-density lipoprotein ( LDL ) particle seen in crosssection. Each spherical particle has a mass of 3 x io * daltons. It contains about 1500 molecules of cholesteivl ester in its con* ( surrounded by a lipid monolayer composed of about 800 phospholipid and 500 unesterified cholesterol molecules. A single molecule of a plasma membrane LDL receptors CYTOSOL ENDOCYTOSIS coated vesicle UNCOATING early endosome RETURN OF LDL RECEPTORS TO PLASMA MEMBRANE BUDDING OFF OF TRANSPORT VESICLES FUSION WITH ENDOSOME \ i / / free cholesterol •• •• • • •••• • •V •• . .. • • *• *• * • • •••• ••• hydrolytic enzymes lysosome clathrin and other . coat -associated proteins LDL-binding site i * ini' , , , * , *\ i CYTOSOL LDL receptor protein coated-pitbinding site \ clathrin-coated pit .. * •• . •. • • • • • •J * • •• •• % ( B) .. v x \ . X * LDL receptor protein with defective coated-pit-binding site plasma membrani # * A - d9 Rat B - Adult Rat D - Adult LD Hamster C F - Adult SD + 4 d FSH E - Adult SD Hamster FIG. 1 . FSH induces Sertoli cell prolif era tion in the adult Djungarian hamster. These micrographs illustrate the reactivity of a proliferation marker (PCNA; red ) and Sertoli cell nuclear marker (GATA4; green) in testis sections, as assessed by confocal immunoHuores ence, with coexpression appearing vejjowj I he immature id*T' rat ( A ) served as a ^ nriciti VP rnnfrnl ITPT .TI KP sprtnl i rpIi «; — d5 Hamster nrp Considerations • Which cellular components are involved in receptor-mediated endocytosis? • What materials are required to double label cellular components? • Why do receptors recycle and why do some types of receptors not recycle? • How does pH influence receptor-cargo binding? CONFERENCE CONFERENCE ON ENDOCYTOSIS Familial hypercholesterolemia is an autosomal dominant genetic disease that affects cell surface receptors for low density lipoprotein (LDL). Three different abnormal phenotypes have been characterized for this disease: a) Keceptor negative, where the receiptors are unable to bind LUL or are not present on the cell surface. b} Receptor defective where the receptors bind lower than normal amounts of LC _. c) Internalization defective where receptors withnormal binding capacity are unable to internalize the bound LC _. 1. If you doublestain a normalcell , a receptor defective cel I . or an internalization defective cell with both fluorescein labelled anti- LC _ receptor antibody and rhodamine labelled anti- clatlirin antibody, what would be the difference in the staining pattern amongthese types of cells ? How could you confirm these findings by electron microscopy ? 2. 3. , , In studying a receptor negative mutant you find that there are many LC _ receptors which can bind LC _ intheER and Golgi region of the cell . What is the functional defect in this mutant receptor ? , , The drug monensin inhibits the recycling of LDL receptors in normal cells by causing them to be trap p ed i nside th e celI. W hat res u Its wou Id y o u ex pect if y o u treated internalization d efective cells with monensin ? Why do LUL receptors recycle in normal cells ? 4. I ne emission intensity otfluorescein is proportional to pH when it is excited at a wavelength of 490Hm however emission intensity does not depend on pH if it is excited at a wavelength of 450 nm . At pH 5.0. 490/ 450 emission ratio is 1 : at pH 7.0 it is 4. If yuti allow normal cells to take up briefly fluorescein I a belled LDL. you record an emission ratio of 1 in endocy ticvesicle: however if you do the same experiment inthe presence of monensin. you record an emission ratio of 4. H o w d o y o u ex plain these findings regarding recycling ? Explain how these results might be related to the effects of monensin on receptor recycling ? , , Conference on Endocytosis: Receptor-mediated endocytosis Objectives: • To reinforce the interplay among receptors, cargo, and actions of the apical portion of the cell by considering defects in the interplay • To use techniques we have discussed to analyze the process of endocytosis • To reinforce the idea that each step of a process is an opportunity for a disease