EPITHELIAL CELLS

Bob Mercer [email protected] http://www.unicefusa.org/news/releases/unicef-and-who-launch-report.html EPITHELIAL CELL JUNCTIONAL COMPLEXES

microvilli

zonula occludens belt zonula adherens junctional complex spot desmosome macula adherens

keratin filaments

LIMITING JUNCTION/TIGHT JUNCTION TIGHT JUNCTION

Mariano C., H. Sasaki, D. Brites, and M.A. Brito. Eur J Cell Biol. 90:787-96, 2011 TJ PLASMA MEMBRANE Tetra span proteins: TJ-associated marvel (TAMP): (marvelD1; 65 kDa; 522 aa; 4 splice isoforms identified)

marvel: myelin and lymphocyte protein and ṟelated proteins for vesicle trafficking and membrane ḻink (23-30 kDa; 211-260 aa, 27 identified)

Tricellulin (marvelD2; 64 kD; ≈550 aa, 4 splice isoforms identified) MarvelD3 (2 splice isoforms, 410, 401 aa) TJ PLASMA MEMBRANE PROTEINS

Single span proteins:

Junctional Adhesion Molecule (JAM) 3 proteins identified (JAM1-3) 43 kDa; Ig Superfamily cyto PDZ (PSD-95, discs large, ZO-1) domain binds ZO-1, PAR-3/PAR-6/aPKC, cingulin

role in endothelial leukocyte exit

Coxsackievirus & Adenovirus Receptor (CAR) Ig-like domain, cyto PDZ domain binds ZO-1 TIGHT JUNCTIONAL PROTEINS

Molecular Cell Biology, Seventh Edition ©2013 W.H. Freeman and Company OCCLUDIN AND TRICELLULIN EXPRESSION

Molecular Cell Biology, Seventh Edition ©2013 W.H. Freeman and Company 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 OCCLUDIN PHOSPHORYLATION SITES

Dorfel and Hubner, J Biomed Biotechnol. 2012; 2012:807356 CLAUDINS

CLAUDINS

Gupta IR, Ryan AK. Clin. Genet. (2010) 77:314-325. CLAUDINS

EXPRESSION OF DIFFERENT CLAUDINS IN THE HUMAN INTESTINE AND HEPATOBILIARY TRACT

Barmeyer, Schulzke, and Fromm. (2015). Seminars in Cell & Developmental Biology, 42:30-38 LOCALIZATION OF CLAUDINS ALONG THE ADULT MAMMALIAN RENAL TUBULE

Yu, A. (2015) JASN 26: 11-19 Cancer genetic alteration profiles of OCCLUDIN and -1

Runkle and Mu.Cancer Letters 337, 2013, 41-48 TRICELLULIN- Nonsyndromic hearing loss

Absence of tricellulin may lead to paracellular permeability barrier defects. The intramembranous particles formed by bicellular tight junction proteins are shown between the plasma membrane lipid bilayers. These proteins form lateral associations with junctional proteins in the adjacent cell obliterating the intercellular space. (A) At tricellular junctions in Tric+/+ mice, tricellulin and other proteins from the 3 cells associate with each other to form the central sealing elements, where the bicellular junction strands unite with it. (B) In the absence of tricellulin, the tricellular junctions are no longer continuous and the disconnected particles are possibly formed by other as yet unknown proteins. These strands are no longer able to associate with the elements of the bicellular junctions, potentially resulting in “channels” or conduits for paracellular permeability and barrier defect in the TricR497X/R497X mice. The arrows depict paracellular leak of ions or small signaling molecules through the “channels” at the tricellular junctions of TricR497X/R497X mice. From: Nayak G, et al,. J Clin Invest. 2013 Sep 3;123(9): 4036-49 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

PAR-3/PAR-6/aPKC Complex Important in oriented cell division PROTEIN INTERACTIONS AT THE ZO ZO-1 Immunofluorescence in MDCK Cells BELT actin filaments LUMEN inside 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-- Ca- dependent binding

Nectin- Ig Subfamily; Ca- Independent binding

Vezatin- Myosin binding CADHERIN SUPERFAMILY

Subfamily Examples Distinguishing Features

1. Classical E-, N-, C-, R-, P- 5 EC domains; one TM domain; conserved cytosolic VE-cadherins domains connected to actin via catenins

2. Desmosmal cadherins , 5 EC domains; one TM domain; cytosolic domains desmocollins conserved within two subclasses; connected to intermediate filaments via plaque proteins

3. Protocadherins α-, β-, γ-subclasses 6-7 EC domains; one TM domain; conserved cytosolic domains protocadherins-1, -11 within subclasses, different cytosolic binding partners

4. Cadherin-like 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 E-CADHERIN MEDIATES Ca2+-DEPENDENT ADHESION OF L CELLS GFP-Cadherin in cultured epithelial cells CATENIN COMPLEXES METAZOAN EVOLUTION OF CATENINS

Hulpiau, P., I.S. Gul, F. Roy. (2013) Progress in Molecular Biology and Translational Science, 116: 71–94 CADHEREN/CATENIN INTERACTIONS

αE-catenin M1-M3 domains are autoinhibited, which prevents them from interacting with vinculin (VCL). An open form of αE-catenin in which the M2-M3 interface is disrupted unfurls the M1 domain to expose the VCL-binding site. The αE-catenin-VCL complex binds to more actin filaments than autoinhibited αE-catenin. Bhatt, et. al, (2013). Cell Communication & Adhesion 20:189-199. 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

desm oglei ns ker atin filaments spot des mos ome cy top lasm ic plaque m ade of desm oplak ins

ker atin filaments ancho red to interc ellul ar cy top lasm ic p laque spac e

interac ting plasm a membr anes basal l amin a hemid esm osome 0.3 µ m DESMOSOMAL PLASMA MEMBRANE PROTEINS

E-cadherin Desmocollin-1a -1 Desmocollin-1b E1 E1 E1 3 Isoforms of DSG and E1 DSC; Two splice E2 E2 E2 E2 variants E3 E3 E3 E3 E4 E4 E4 E4 EA EA EA EA

IA IA IA IA DSI DSI ICS ICS ICS ICS IPL

RUD

DTD DESMOSOMAL PLAQUE PROTEINS

Catenin related proteins: and Plakophilin

Plakin Family: - 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

300kD IF AP Dsg

Pg DP Dsc a BP230

IF BP180 300kD IF AP

α6ß 4 Lam 5 ADHERING JUNCTIONS

Structure Intracellular Intracellular Transmembrane Extracellular Plaque Cytoskeletal Link Protein Ligand Proteins Attachment

Belt catenins actin filaments E-cadherin E-cadherin Desmosome nectin-2 nectin-2 in adjacent cell

Spot desmoplakin Intermediate cadherins: cadherins in Desmosome plakoglobin filaments desmoglein adjacent cell plakophilin desmocollin

Hemi- desmoplakin Intermediate integrin laminin & other desmosome BP230, filaments BP180 matrix proteins HUMAN DISEASES INVOLVING DESMOSOMAL MUTATIONS

Epidermolysis bullosa (EB): blistering disorders; 1 in 50,000 live births

JUNCTIONAL COMPLEXES

microvilli

tight junction zonula occludens belt desmosome zonula adherens junctional complex spot desmosome macula adherens

keratin filaments

gap junction

hemidesmosome basal lamina GAP JUNCTIONS

Ions

Ions cAMP

Protein OLIGOMERIZATION

“GATING” OF Cx43-Binding Proteins

Biochem. J. (2006) 394, 527-543 Molecular Cell Biology, Seventh Edition ©2013 W.H. Freeman and Company

Table 4. Connexin-associated diseases and corresponding connexin proteins and .

Eur. J. Clin. Invest. (2011) 41(1):103-116.

JUNCTIONAL COMPLEXES

microvilli

tight junction zonula occludens belt desmosome zonula adherens junctional complex spot desmosome macula adherens

keratin filaments

gap junction

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 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 (H- ATPase) 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. TRANSEPITHELIAL TRANSPORT

A B

+ Na+ Na glucose K+ 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 inner barrier barrier

Na Na Na K K

Na entry is by Na extrusion is by a diffusion Na,K exchange pump

Naout Kcell Vo = f Vi = f Nacell Kout VOLTAGE SCANNING GALL BLADDER EPITHELIUM

.

A ∆V X X X X X X X X X X X X + J J J CC J C J C C J C

- 0 epithelium A -1 V (mV) ∆ 0 50 100 150 200 Position of electrode (µm) Aquaporins

In mammals at least 13 isoforms present 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 Active Receptor Active complex HORMONE H H R H R H R D N A ANTAGONIST

A A R' A R' mRNA

Inactive Inactive Specific Receptor complex 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

influe nza vi rus buds only from ve si cu lar s tomatitis vi rus buds only th e apic al m em brane fr om th e b asolat eral membrane Na,K-ATPase Localization in MDCK Cells Direct and Indirect Sorting Pathways 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 Three different routing pathways have been identified in epithelial cells. Newly synthesized proteins can follow a direct route (red pathways for apical proteins and black pathways for basolateral proteins). In some polarized systems, apical proteins may also be sorted in a transcytotic route (green pathways). Alternatively, proteins can be randomly targeted to both membrane domains and achieve their asymmetric distribution by selective stabilization or retention at one cell surface following the so-called random route (blue pathways). Membrane fusion is the final and irreversible step of each trafficking route and is mediated by SNARE proteins. Under normal homoeo- static conditions, only the appropriate organelles fuse with the cognate membrane. SNARE proteins mediating fusion events at the apical (red) and basolateral (black) membrane are depicted. Svelto et al., Biol. Cell 102:75, 2010. J Cell Biol. (1990) 111(3):987-1000 Step 2: Step 3: Step 1: (HA - apical; VSVG - basolateral)

Selective labeling of post-TGN vesicles: pulse with 35S-met then chase with 20°C block (TGN) in cycloheximide 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 APICAL SORTING SIGNALS

Svelto et al., Biol. Cell 102:75, 2010 GLYCOSYLPHOSPHATIDYL INOSITOL ANCHOR N O C-NH Protein CH 2 ethanolamine

CH 2 P

GLYCAN-GlcNH 2

O O C=O C=O BASOLATERAL SORTING SIGNALS

Svelto et al., Biol. Cell 102:75, 2010 BASOLATERAL SORTING SIGNALS Tyrosine-Dependent Basolateral Sorting Signals LDLR 9NSINFDNPVYQKTTEDEVHICHN LAP 405RMQAQPPGYRHVADGEDHA HA Y543 538NGSLQYRICI ASGPR H1 1MTKEYQDLQML Igp120 1RKRSHAGYQTI TGN38 5VTRRPKASDYQRLNLKL

Di-Hydrophobic Basolateral Sorting Signals FcRII-B2 22NTITYSLLKH MHC II/Ii 1MSSQRDLISNNEQLPMLGRRPGAPESKCSR BASOLATERAL SIGNALS IN THE CYTOPLASMIC DOMAIN OF THE LDL RECEPTOR

PROXIMAL DISTAL SIGNAL SIGNAL KNWRLKNINSINFDNPVYQKTTEDEVHICHNQDGYSYPSRQMVSLEDDVA CLATHRIN-COATED PIT SIGNAL CLATHRIN ADAPTOR PROTEIN COMPLEXES APICAL AND BASOLATERIAL SORTING SIGNALS

Stoops EH, and Caplan MJ. (2014). J Am Soc Nephrol. 25:1375-1386. SORTING SIGNALS CONCLUSIONS

1. Proteins can be specifically delivered to either the basolateral or apical membrane surface using basolaterally or apically targeted vesicles.

2. Different epithelial cells emphasize different pathways in the sorting mechanism.

3. Anchoring of polypeptides to the membrane through glycosylphosphatidyl inositol may be an important sorting signal to send some polypeptides to the apical surface.

4. Protein-based basolateral sorting signals within the cytoplasmic domain of some proteins have been identified.

5. The sorting mechanisms for secreted and membrane proteins may be different.