Unit+3+endocytosis+slides

Lecture'Unit'3:''Endocytosis'

Exocy3c,'Endocy3c','Recycling'Flow'Routes'

Modes'of'endocytosis'

Macropino>cytosis'

Clathrin)Mediated)endocytosis)

Pinocytosis'

Caveolar'medidated'

Phagocytosis'

Phagocytosis'of'erythrocytes'by'lung'macrophage.'

SEM'image.'

Arrows'indicate'edge'of'engulging'phagocy3c'cup.''

Ac3n'filament'filled'extensions'

TEM'image'

Neutrophil'phagocytosing'a'bacteria;'note'the'bacteria'is'coated'with'an3>bug'an3bodies'which'are'recognized'by'IgG'FC'receptors'on'neutrophil'cell'surface.''

Lysosomes'

Note'meshwork'of'ac3n'filaments'underlying'phagocy3c'cup;'Beads'can'be'mechanically'isolated'with'aQached'membrane.'

coated'latex'bead'

Steps&in&phagocytosis:&1.  Phagocyte'

recogni3on'of'foreign'par3cle'(e.g.'bacterium).'

2.  Recruitment'of'ac3n'filament'machinery'to'extend'membrane'around'par3cle.''

3.  Forma3on'of'a'phagosome'vesicle;'removal'/disassembly'of'ac3n'

4.''Primary'and'secondary'Lysosome'fusion'with'phagosome'and'degrada3on'of'contents.''Note,'unlike'other'endocy3c'vesicles,'phagosomes'do'not'first'fuse'with'the'endosome.'''

thin'sec3on'and'deep>etch'rotary>shadowed'images'of'caveolae'

Caveolar'mediated'transcytosis'of'fluid'across'capillary'endothelial'cell.''''

Organelles'of'the'Endocy3c'Pathway'(post'endocytosis/internaliza3on):'

Endocy3c'vesicle'Early''late'endosome'Lyososome'Trans>Golgi'network'

1.  Fluid'phase'2.  Endocy3c'membrane'3.  Receptor'4.  Ligand'

Components'of'an'Endocy3c'Vesicle'

1'

2'3'

4'

'Endosome'

Early'late'endosome'matura3on'Note'decrease'in'pH'during'endosome'matura3on.''Note'that'recycling'of'membrane'can'occur'directly'from'endosome'or'through'TGN/recycling'endosome'compartments'

Thin'Sec3on'EM'of'fibroblast'showing'endosomes,'

pinocy3c'vacuoles'and'lysosomes'

Pseudo>colored'TEM'of'early'(EE)'and''late'(LE)'endosomes,'lysosomes'(LY)'and'clathrin'coated'pits'(CP).'

Experimental'demonstra3on'that'internalized'endocy3c'membrane'components'are'recycled:''

1.  Prepare'beads'with'covalently'linked'lactoperoxidase.'

2.  “Feed”'beads'to'two'aliquots'of'macrophages'and'wait'10'minutes'to'allow'endocy3c'internaliza3on;''

3.  chill'cells'to'4oC;'add'125>I'and'H2O2;''(This'covalently'links'125>I'(iodina3on)'to'any'proteins'in'contact'with'the'bead'surface)'

4.  Fix'one'aliquot'and''process'for'EM'autoradiography.'

5.  Warm'the'other'batch'of'cells'to'37o'and'incubate'for'10'minutes;'fix'and'process'for'EM'AR.'

'0o'cell' Cell'warmed'to'37o'for'10'min.'''

Result:''Iodinated,'internalized'membrane'proteins'have'recycled'back'to'plasma'membrane'ader'warming.''

Acid'ac3vated'hydrolases'of'the'lysosome'

Imagine)the)consequences)of)secre7ng)these)enzymes)into)the)extracellular)compartments)of)your)body.'

Lysosomes'and'autophagic'degrada3on'of'“damaged”'organelles'

Clathrin/receptor'>mediated'endocytosis'

An'example'of'clathrin'mediated'receptor'endocytosis:'uptake'of'LDL''

The'“machinery”'involved'in'clathrin'mediated'endocytosis:'

Cytoplasmic'adaptor'targe3ng'domains'of'receptors'Adaptors'that'bind'to'receptor'target'domains'Clathrin'triskelions'to'form'clathrin'“basket”/'coated'pits'Dynamin'to'effect'scission'of'coated'vesicle''Uncoa3ng'chaparones'to'remove'clathrin'coat'

The'3'types'of'carrier'vesicle'coats'

Coated'vesicle'uptake'of'yolk'protein'(from'blood'meal)'by'mosquito'oocytes:'The'first'visualiza3on'of'clathrin>coated'pits/vesicles'(early'60’s)'

Quick>freeze>deep'etch,'rotary'shadowed'TEM'of'coated'pits;'J.'Heuser.'

Stereo'pair'images'of'coated'pits'at'various'stages'of'forma3on/closure.'by'J.'Heuser'

Progressive'stages'of'clathrin'coated'pit'closure'

Coated'vesicles'can'be'readily'purified'by'cell'frac3ona3on/'differen3al'sedimenta3on'allowing'purifica3on'of'clathrin.'

rotary>enhancement'of'coated'vesicle'structure;'nega3vely'stained'prepara3on.'

Original'model'proposed'for'vesicle'coat'structure.'

Clathrin'triskelion'structure;'heavy'chain'triskelions'are'stabilized'by'the'light'chains.'

The'polygonal'clathrin'triskelion'cage'surrounds'the'endocy3c'pit/vesicle'

1.'Adap3ns/adaptors'recognize'receptors,2.Adap3ns'recruit'clathrin'to'assemble'coated'pit'3.'Dynamin>GTPase''effects'vesicle'closure.'4.''HSC'70'(chaparone'ATPase)'plus'coat'binding'protein'auxilin'mediate'vesicle'uncoa3ng.'

Stages'of'coated'pit'forma3on,'closure,'coated'vesicle'release'and'vesicle'uncoa3ng.'

Clathrin'is'recruited'to'different'compartments'by'compartment'specific'adaptors.'

Note,'for'this'unit,'we'will'only'consider'clathrin’s'role'in'receptor'mediated'endocytosis'

Adaptors'recognize'specific'signals'on'cytoplasmic'domain'of'receptors.'

/adaptor'

Sor3ng'signals'using'contain'at'least'one'tyrosine'residue'

Example'of'an'internaliza3on'“signal”'recognized'by'adap3n'complex:''The'tyrosine'residue'is'crucial.'''

Scission'requires'GTP'hydrolysis'by'dynamin'

Mutant'dynamin'causes'the'forma3on'of'extremely'elongated'coated'pits'

Forma3on'of'dynamin>GTP>gamma>S'(nonhydrolyzable')'induced'collars'in'isolated'synap3c'membranes'in'vitro.'

This'demonstrates'that'dynamin>GTP'binds'to'the'pit'“neck”'to'form'the'constric3on'collar'but'closure'of'the'collar'to''pinch'of'the'vesicle'requires'GTP'hydrolysis.'

not'shown:'dephosphoryla3on'of'innositol'lipids'

Chaparones'

Classes'of'receptors'internalized'by'clathrin'mediated'endocytosis.'

>>Non'recycled'receptors'(e.g.'growth'factor'receptors);'Internaliza3on'of'these'receptors'requires'ligand'binding.'

>>Recycled'receptors'(e.g.'“nutrient”'receptors);'these'receptors'are'internalized'with'or'without'bound'ligand.''

Other'components'internalized'by'clathrin'mediated'endocytosis:''exocytosed'membrane,'certain'viruses.'

Why'might'some'receptors'recycle'back'to'the'cell'surface'(e.g.'LDL'receptor)''while'others'(e.g.'growth'factor'receptors)'are'not?'

pH&dependence&of&receptor4ligand&binding:'pH'resistant'binding'results'in'targe3ng'of'receptor'and'ligand'to'late'endosome/lysosome;'pH'sensi3ve'(acid'dissocia3on)'binding'results'in'ligand'dissocia3on'and'recycling'of'receptor'to'plasma'membrane.''

pH'sensi3ve'receptors:''LDL'receptor','Fe2+'bound'to'transferrin'on'transferrin'receptor.'

pH'insensi3ve'receptors:''EGF'receptor'

Text'from'Lecture'outline'Unit'3'

11'

12'

2,3'

4'

5'

61,2'63''ligand>receptor'trancytosis'not'shown'

8'

7'

7'

Transcytosis:''Endocy3c'movement'of'cargo'from'one'plasma'membrane'(e.g.'apical'domain)'to'another'plasma'membrane'domain'(e.g.'basolateral'domain'of'an'epithelial'cell'

pH'6'

pH'7.4:'IgG'dissociates'from'FC'receptor''

IgG'binds'to'Fc'receptor'at'~'acid'pH'of'gut'lumen.'

Uptake'of'maternal'an3bodies'in'neonate'intes3ne'via'clathrin'mediated'transcytosis'of'the'FC'receptor.''

coated'vesicle>FC'receptor'mediated'uptake'of'maternal'IgG'in'intes3ne'of'neonate.''Note'the'numerous'coated'pits'(far'fewer'in'adult)'

Experimental'dissec3on'of'the'LDL'uptake'pathway:'From'clinical'studies'of'pa3ents'with'severe'heart'disease'to'dissec3on'of'the'cell'and'molecular'mechanics'of'clathrin/receptor'mediated'endocytosis.'Brown,'Goldstein'(Anderson):'Nobel'Prize'in'Medicine.'1985.'

Low'density'lipoprotein'par3cle'(LDL):'

“bad”'serum'cholesterol.'LDL'receptor'binds'to'the'protein'component,'apoprotein'B'

LDL'serum'concentra3ons'and'onset'of'heart'aQacks'in'pa3ents'homozygous'and'heterozygous'for''a'LDL'receptor'null'muta3on.'

Familial'Hypercholesterolemia'

From'the'studies'of'Brown,'Goldstein'and'Anderson'

Chronic'uptake'of'LDL'results'in''down'regula3on'of'receptor'and'enzymes'for'cholesterol'synthesis;'up'regula3on'of'enzymes'for'cholesterol'storage.'

target'for'cholesterol'reduc3on'drugs'like'Lipitor.'

From'the'studies'of'Brown,'Goldstein'and'Anderson'

N>linked'glycosyl'groups'

N>linked'glycosyl'groups'

O>linked'glycosyl'groups'

Structure'of'the'LDL'Receptor'

Receptor'mediated'uptake'of'LDL'

Analysis'of'LDL'cell'surface'binding'at'4o'and'kine3cs'of'uptake'ader'warming'to'37o'

1.  Iodinate'LDL'(on'tyrosine'residues'of'apoprotein'B)'with'125'I'(radioac3ve).'

2.  Add'125>I>LDL'to'cells'at'4o.''Wash'out'unbound'LDL.'3.  Release'surface'bound'LDL'with'heparin'(a'compe33ve'

inhibitor)'and'quan3fy'releasable'125>I'LDL'par3cles'(=surface'bound)'

4. Warm'cells'to'37o'and'quan3fy'at'various'3mes'ader'warming''surface'bound'(heparin'releasible)'and'nonreleasible'(internalized),'TCA'precipitable'and'TCA'soluble'125'I.'''TCA'ppt='intact'protein''TCA'soluble=digested'small'125'I'tyrosine'contac3ng'pep3des'and'free'125>I'tyrosine.''

Low'density'lipoprotein'par3cle'(LDL):'

“bad”'serum'cholesterol.'LDL'receptor'binds'to'the'protein'component,'apoprotein'B'

125>I>'

LDL'cell'surface'binding'and'kine3cs'of'LDL'uptake'by'cells'with'wild'type'LDL'receptor.'''Why'is'there'a'gradual'increase'in'TCA'soluble'125>I'and'a'gradual'decrease'in'TCA'insoluble'(heparin'resistant)'125>I.''

Heparin'releases'LDL'from'receptors'on'cell'surface'but'not'internalized'LDL'(heparin'resistant)'

Uptake'of'ferri3n>labeled'LDL'by'coated'pits'(7,'8)'and'subsequent'targe3ng'to'lysosomes'(9).''This'was'performed'as'a'pulse>chase'experiment:'Cells'in'7,8'were'fixed'for'EM'1,2'minutes'ader''LDL'addi3on'at'4o'

Cell'in'9'was'fixed'ader'warming'cells'to'37o'for'~'10'minutes'to'allow'internaliza3on'and'targe3ng'to'lyosome.'

Experimental'demonstra3on'that'LDL'is'internalized'by'clathrin'coated'pits/vesicles'

Stereo'view'of'LDL'par3cles'on'outside'surface'of'coated'pit.'Quick'freeze'deep'etch'rotary'shadowed'replica'

Classes&of&LDL&receptor&(homozygous)&muta?ons&leading&to&familial&cholesterolemia:&

1.  null'muta3on:''no'LDL'surface'binding;'no'internaliza3on'

2.  Mutated'ligand'binding'domain:''No'LDL'binding'or'internaliza3on;'receptor'localizes'to'coated'pits;'cycles'through'endosome>>cell'surface'

3.  Truncated'cytoplasmic'domain:''Cell'surface'binding'of'LDL;'no'internaliza3on'

4.  Stop'codon'muta3on'before'transmembrane'domain:''no'binding'no'internaliza3on;'receptor'is'secreted/degraded.'

5.   TyrosineX&muta?on&in&cytoplasmic&domain;&normal&surface&binding;&no&recruitment&to&coated&pits;&no&internaliza?on.&&

WT'''#1'''''#2'''''#3'''''''''''''''#4'''''''#5'

Classes'of'LDL'receptor'muta3ons'iden3fied'by'Brown,'Goldstein'and'Anderson'

Class'5'LDL'receptor'muta3on''Tyrosine>>'X'

Replacement'of'Tyr'residue'

Muta3ons'in'LDL'receptor'result'in'high'levels'of'serum'LDL'and'early'onset'of'heart'disease.'

WT'

Class'5'muta3on'

Classes'of'LDL'receptor'muta3ons:'

1.  null'muta3on:''no'LDL'surface'binding;'no'internaliza3on'

2.  Mutated'ligand'binding'domain:''No'LDL'binding'or'internaliza3on;'receptor'localizes'to'coated'pits;'cycles'

3.  Truncated'cytoplasmic'domain:''Cell'surface'binding'of'LDL;'no'internaliza3on'

4.  Stop'codon'muta3on'before'transmembrane'domain:''no'binding,'no'internaliza3on'

5.  YX'muta3on'in'cytoplasmic'domain;'normal'surface'binding;'no'recruitment'to'coated'pits;'no'internaliza3on.''

Diagram'the'kine3cs'of'LDL'surface'binding'(at'4oC),''and'uptake,'degrada3on'ader'warming'cells'with'Class'1>5'LDL'receptor'muta3ons.'''