POLYCYSTIC RENAL DISEASE!
1 in 500 autopsies ! 1 in 3000 hospital admissions! Accounts for ≈10% of end-stage renal failure ! Autosomal dominant inheritance
!
! !
CYSTIC FIBROSIS!
1/2000 births in white Americans! Median age for survival is about 20-25 years ! Autosomal recessive inheritance
!
! !
EPITHELIAL CELL!
JUNCTIONAL COMPLEXES! micr ov illi
tight junct ion zonula occludens belt desmosome zonula adher ens junctional complex spot desmosome mac ula adherens
keratin filaments
gap junc tion
hemidesmosome
basal lamina
LIMITING JUNCTION! interacting plasma membranes
intracellular space
0.6 µM
Occludin Claudin(s)
cytoplasmic half of lipid bilayer
LIMITING JUNCTION!
Mariano C., H. Sasaki, D. Brites, and M.A. Brito. Eur J Cell Biol. 90:787-96, 2011!
TJ PLASMA MEMBRANE PROTEINS!
Tetra span proteins: ! !Occludin (65 kDa; ≈500 aa) !
! !Claudins (23-30 kDa; 211-260 aa, 27 identified)!
! !Tricellulin (64 kD; ≈550 aa, 4 splice isoforms identified)!
TJ PLASMA MEMBRANE PROTEINS!
Single span proteins: ! !
!!
!Junctional Adhesion Molecule (JAM) 3 proteins ! ! ! !identified (JAM1-3) 43 kDa; Ig Superfamily ! ! ! !cyto PDZ domain binds ZO-1, PAR-3, cingulin !! ! !role in endothelial leukocyte exit! ! ! ! ! !Coxsackievirus & Adenovirus Receptor (CAR) ! ! !Ig-like domain, cyto PDZ domain binds ZO-1!
A. Escudero-Esparza, W.G. Wen, Jiang, T.A. Martin. Frontiers in Bioscience 16: 1069-1083, 2011
Mariano C., H. Sasaki, D. Brites, and M.A. Brito. Eur J Cell Biol. 90:787-96, 2011!
CLAUDINS!
CLAUDINS!
TJ CYTOPLASMIC PROTEINS!
Membrane-Associated Guanylate Kinase proteins:!
!
!ZO-1 (210-250 kDa), ZO-2 (160 kDa), ZO-3 (130 kDa)! !
!3 PDZ; Src homology, SH-3; !
!
!guanylate kinase-like, GUK domains!
!!
!Cingulin- actin, myosin binding! !! !ZONAB- ZO-1 Associated Nucleic Acid Binding; ! ! ! !Y-box transcription factor!
SOME PDZ PROTEINS FOUND AT THE TJ!
PROTEIN INTERACTIONS AT THE ZO!
BELT DESMOSOMES! actin filaments inside microvillus
LUMEN microvilli extending from apical surface
tight junction
adhesion belt bundle of actin filaments
lateral plasma membranes of adjacent epithelial cells
basal surface
PLASMA MEMBRANE ADHEREN JUNCTION PROTEINS ! E-Cadherin- Cadependent binding! ! Nectin- Ig Subfamily; CaIndependent binding! ! Vezatin- Myosin binding!
CADHERIN SUPERFAMILY! Subfamily
!
Examples
!
Distinguishing Features!
! 1. Classical cadherins ! ! ! ! ! 2. Desmosmal cadherins ! ! 3. Protocadherins ! 4. Cadherin-like
E-, N-, C-, R-, P- ! VE-cadherins !
desmogleins, desmocollins !
! ! !
! !-, "-, #-subclasses ! ! protocadherins-1, -11!
!5 EC domains; one TM domain; conserved cytosolic! !domains connected to actin cytoskeleton via catenins!
!5 EC domains; one TM domain; cytosolic domains! !conserved within two subclasses; connected to intermediate ! !filaments via plaque proteins ! !6-7 EC domains; one TM domain; conserved cytosolic domains! !within subclasses, different cytosolic binding partners!
! DN-, DN-cadherin; Ret; !Variable number of EC domains and other non-EC domains; ! ! Tat; Flamingo cadherins ! none to several TM domains; different cytosolic domains and! ! ! ! ! binding partners! ! ! ! !! E, epithelial; N, neuronal; C, compactson; P, placental; R, retinal; VE, vascular endothelial; ! DN, Drosophila neuronal; DE, Drosophila epithelial; EC, extracellular cadherin; TM, transmembrane.!
CADHERIN ZIPPER!
5
5
5
4
4
4
3
3
3
2
2
2
EC1
EC1
EC1
EC1
EC1
EC1
2
2
2
3
3
3
4
4
4
5
5
5
CATENIN COMPLEXES!
Cadherin
ß-catenin !-catenin actin
Wnt/ß-CATENIN PATHWAY!
ARCHITECTURAL COMPONENTS OF ! ADHERENS JUNCTIONS!
ARCHITECTURAL COMPONENTS OF ! ADHERENS JUNCTIONS!
Representative Signaling Pathways! During the Formation of AJs and TJs!
SPOT AND HEMI- DESMOSOMES! d e sm o glei n s
ker a ti n fi l am e n ts
spot des mos om e
cy top l a sm ic p l a que m a de of d e sm o plak ins
ker a ti n fi l am e n ts a n cho red to cy top l a sm ic p l a que
i n te rc e llul a r spac e
i n te rac ti ng p l a sm a me mbr a nes b a sal l a min a 0 .3 µ m
h e mid e sm o so me
DESMOSOMAL PLASMA MEMBRANE PROTEINS! E-cadherin
Desmocollin-1a
Desmoglein-1 E1 E2 E3 E4 EA
IA ICS
E1 E2 E3 E4 EA
Desmocollin-1b
E1
E1
E2
E2
E3
E3
E4
E4
EA
EA
IA
IA
ICS
ICS
IPL RUD DTD
DSI
IA ICS
3 Isoforms of DSG and DSC; Two splice variants!
DSI
DESMOSOMAL PLAQUE PROTEINS!
ß-catenin related proteins: Plakoglobin and ! ! ! ! ! Plakophilin! ! Plakin Family: Desmoplakin- most abundant! ! ! ! !
! ! !
!Dumbbell shaped-links plaque !to intermediate filaments! !phosphorylated by PKA!
DESMOSOMAL PLAQUE PROTEINS! PLAKOGLOBIN
PLAKOPHILIN
DESMOPLAKIN PLECTIN
MOLECULAR ORGANIZATION OF DESMOSOMES! !
MAJOR PROTEINS OF DESMOSOMES ! AND HEMIDESMOSOMES !
D sg
3 00 kD IF AP
Pg DP
D sc a BP2 30
IF
BP1 80 3 00 kD IF AP ! 6ß 4 L am 5
ADHERING JUNCTIONS! Structure
!Intracellular !Plaque ! !Proteins!
!Intracellular Cytoskeletal Attachment
! Transmembrane ! Link Protein ! ! !!
catenins ! ! ! ! !
actin filaments ! ! ! !
!
desmoplakin !plakoglobin !plakophilin !!
!Intermediate !filaments! ! !
!
desmoplakin ! BP230, !
!Intermediate filaments!
!
Extracellular! Ligand!
! Belt ! Desmosome ! ! Spot ! Desmosome
! Hemi- ! desmosome ! !
!
E-cadherin nectin-2 !
! !
E-cadherin ! nectin-2 in! adjacent cell!
cadherins: desmoglein desmocollin!
! !
cadherins in! adjacent cell!
! !
laminin & other! matrix proteins!
integrin BP180
HUMAN DISEASES INVOLVING DESMOSOMAL! MUTATIONS! ! Epidermolysis bullosa (EB): blistering disorders; 1 in 50,000 live births!
JUNCTIONAL COMPLEXES! micr ov illi
tight junct ion zonula occludens belt desmosome zonula adher ens junctional complex spot desmosome mac ula adherens
keratin filaments
gap junc tion
hemidesmosome
basal lamina
GAP JUNCTIONS!
I on s
Io ns
Prot ein
cAM P
CONNEXIN OLIGOMERIZATION!
GATING OF CONNEXONS!
Cx43-Binding Proteins
Biochem. J. (2006) 394, 527-543 !
JUNCTIONAL COMPLEXES! micr ov illi
tight junct ion zonula occludens belt desmosome zonula adher ens junctional complex spot desmosome mac ula adherens
keratin filaments
gap junc tion
hemidesmosome
basal lamina
TYPES OF EPITHELIA! Na+-Transporting Epithelia! Examples of Na+-transporting epithelia include the distal segments (distal tubule and cortical collecting tubule) of the renal tubule, colon, amphibian skin, and amphibian and mammalian urinary bladder. !
! Cl¯ Transporting Epithelia
! Examples include: Regions involved in Cl¯ absorption such as the thick segments of the loop of Henle in the mammalian kidney and the diluting segment of amphibian renal tubule and tissues involve in Cl¯ secretion such as the trachea, corneal epithelium and the rectal gland of some fishes. !
! H+-Transporting Epithelia! Predominant function of this epithelia is to secrete H+. Transport of other ions is observed, and depending on the mechanism of H+ transport can be directly coupled (H,K-ATPase) or independent (HATPase) from H+ secretion. ! Examples include: gastric epithelium, medullary renal collecting tubule, and reptilian urinary bladder. ! !
K+-Transporting Epithelia! Transport of K+ is the predominant function. Large gradients are often established and maintained indicating a low ionic permeability. The side from which K+ is transported is negative. Examples include: stria vascularis epithelium of the inner ear that transports K+ into the endolymph and the insect midgut that secretes K+ into the midgut lumen.! !
PROTEIN MEDIATED MEMBRANE TRANSPORT! • PRIMARY ACTIVE! • SECONDARY ACTIVE TRANSPORT! • FACILITATED DIFFUSION! • ENDOCYTOSIS/TRANSCYTOSIS!
TRANSPORT OF MOLECULES THROUGH MEMBRANES! transported molecule
lipid bilayer
electrochemical gradient
PASSIVE TRANSPORT (FACILITATED DIFFUSION)
GY
carriermediated diffusion
ER
channelmediated diffusion
EN
simple diffusion
ACTIVE TRANSPORT
TRANSEPITHELIAL TRANSPORT!
A
B Na+
Na+ K+
glucose
glucose
COMMON MEMBRANE PROPERTIES OF EPITHELIA! 1. !Generally the Na,K-ATPase (Na,K pump) is located exclusively on the basolateral membrane.! 2. !K+ is accumulated intracellularly by the Na,K-ATPase and the basolateral membrane is predominately K+ permeable; therefore the membrane potential is typically close to the K+ diffusion potential. ! 3. !Na+ activity is much lower in the cell than in the extracellular fluid. In addition to the approximate 10 fold concentration ratio, the cell negative membrane potential provides an additional driving force for Na+ entry. Therefore, Na+, using its electrochemical gradient can drive the accumulation of an uncharged solute, producing up to a 100 fold concentration ratio.!
USSING CHAMBER!
NA TRANSPORT IN FROG SKIN! outer barrier
Na
Na entry is by diffusion Vo = f
Naout Nacell
inner barrier
Na
Na
K
K
Na extrusion is by a Na,K exchange pump Vi = f
Kcell Kout
VOLTAGE SCANNING GALL BLADDER EPITHELIUM!
A
∆V
X
XX XX X X
X X X
X
X
J
J J CC J C
J
J
C
A
epithelium
∆V (mV)
+ C C
0 -1
0
50
100
150
Position of electrode (µm)
200
STANDING OSMOTIC GRADIENT! salt sweeping away diffusion
osmolarity
H2O
iso
Length
AQUAPORIN STRUCTURE!
ADH Stimulated Water Permeability!
AQP2 Movement to the Apical Membrane of the Collecting Duct Principal Cells in Response to ADH!
-ADH!
+ADH!
Epithelial Cell Transport Mechanisms
!
Absorptive Cell
!
!
!
Secretory Cell!
CELLULAR MECHANISMS FOR INFLUENCING TRANSPORT ACTIVITY! • Expression of Specific Isoforms! • Assembly of Different Isoform Subunits! • Exocytosis/Endocytosis! • Specific Regulation by Protein Kinases! • Modification Through Inhibitors ! • Assembly with Accessory/Regulatory Proteins! • Changes in Rate of Synthesis! • Changes in Rate of Degradation!
HORMONE
H
Active Receptor
H
R
Active complex
H
R
H
R D N A
ANTAGONIST
A
A
R'
Inactive Receptor
A R'
mRNA
Inactive complex
Specific Protein
Cell polarity:Asymmetry is a defining feature of eukaryotic cells!
Other examples of! constitutively polarized! cells:! hepatocytes, neurons,! osteoclasts, photo-! receptor rods and cones!
BASOLATERAL AND APICAL MEMBRANES HAVE DIFFERENT PROTEIN AND LIPID COMPOSITIONS! nonpolarized!
polarized!
Lipids:!
BL: enriched in phosphatidylcholine; Apical: reduced PC, high sphingomyelin!
!
EMBO J. 1982; 1(7): 847-852
ENVELOPED RNA VIRUSES!
i n flue n za vi r u s b u ds o nl y fr om th e a p ic al m em br a n e
ve si cu l a r s to ma ti ti s vi r u s b u d s o n ly fr o m th e b a so l at er a l me mb r an e
Na,K-ATPase Localization in MDCK Cells!
Direct and Indirect Sorting Pathways !
Step 2:! Step 1:!
Selective labeling ! of post-TGN vesicles:! ! pulse with 35S-met! then chase with! 20°C block (TGN)! in cycloheximide!
Step 3:! (HA - apical; VSVG - basolateral)!
Protein profiles of immunoisolated apical and ! basolateral vesicles !
• Shaded forms are proteins preferentially associated with one type of vesicle!
• Filled forms are represented in both
! IEF!
SDS-! PAGE
Conclude that transport vesicles for HA and VSVG are different,! therefore direct sorting at TGN can explain polarity!
Hepatocytes- Apical proteins delivered to basolateral surface- transcytosed! ! MDCK Cells- Most apical and basolateral proteins delivered efficiently! ! CaCo-2 Cells- Some apical proteins delivered basolateral surface- transcytosed! !
APICAL SORTING SIGNALS!
!
Type!
!
!
Location !
!
Examples!
Glycosylphosphatidyl ! Membrane, ! Inositol (GPI) anchor ! luminal leaflet ! ! !!
! Placental alkaline phosphatase! ! Thy-1!
N-, O- glycosylation ! !
! !
Luminal !
! !
! gp-80! ! Erythropoietin!
! !
Membrane !
! !
! !
! Transmembrane domain! !
Influenza virus neurominidase! Influenza virus hemagglutinin!
GLYCOSYLPHOSPHATIDYL INOSITOL ANCHOR! N
Protein
O C-NH CH 2
ethanolamine
CH 2 P GLYCAN-GlcNH 2 O O C=O C=O
BASOLATERAL SORTING SIGNALS! Tyrosine-Dependent Basolateral Sorting Signals LDLR LAP HA Y543 ASGPR H1 Igp120 TGN38
9NSINFDNPVYQKTTEDEVHICHN 405RMQAQPPGYRHVADGEDHA 538NGSLQYRICI 1MTKEYQDLQML 1RKRSHAGYQTI 5VTRRPKASDYQRLNLKL
Di-Hydrophobic Basolateral Sorting Signals FcRII-B2 22NTITYSLLKH MHC II/Ii 1MSSQRDLISNNEQLPMLGRRPGAPESKCSR
BASOLATERAL SIGNALS# IN THE CYTOPLASMIC DOMAIN OF THE LDL RECEPTOR! PROXIMAL SIGNA L
DISTAL SIGNAL
KNWRLKNINSINFDNPVYQKTTEDEVHICHNQDGYSYPSRQMVSLEDDVA CLATHRIN-COATED PIT SIGNAL
CLATHRIN ADAPTOR PROTEIN COMPLEXES!
SORTING SIGNALS
!
CONCLUSIONS! 1. ! 2. ! 3.
! 4. ! 5.
!Proteins can be specifically delivered to either the basolateral ! !or apical membrane surface.! !Different epithelial cells emphasize different pathways in the !sorting mechanism. ! !Anchoring of polypeptides to the membrane through !glycosylphosphatidyl inositol may be an important sorting !signal to send some polypeptides to the apical surface. ! !Protein-based basolateral sorting signals within the !cytoplasmic domain of some proteins have been identified. ! !The sorting mechanisms for secreted and membrane proteins !may be different. !
