Material from Mercer (Epithelial lecture) and Nichols MCB 5068 – Exam 2 Review Material from Mercer (Epithelial lecture) and Nichols

EPITHELIAL CELLS Bob Mercer rmercer@wustl.edu October 18, 2016

EPITHELIAL CELL Molecular Biology of the Cell, Alberts

EPITHELIAL CELL

EPITHELIAL CELL Molecular Biology of the Cell, Alberts

LIMITING JUNCTION/TIGHT JUNCTION

TJ PLASMA MEMBRANE PROTEINS Tetra span proteins: TJ-associated marvel protein (TAMP): Occludin (marvelD1; 65 kDa; 522 aa; 4 splice isoforms identified) marvel: myelin and lymphocyte protein and ṟelated proteins for vesicle trafficking and membrane ḻink Claudins (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

EXPRESSION OF DIFFERENT CLAUDINS IN THE HUMAN INTESTINE AND HEPATOBILIARY TRACT Barmeyer, Schulzke, and Fromm. (2015). Seminars in Cell & Developmental Biology, 42:30-38

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

PROTEIN INTERACTIONS AT THE ZO

PLASMA MEMBRANE ADHEREN JUNCTION PROTEINS E-Cadherin- Ca-dependent binding Nectin- Ig Subfamily; Ca-Independent binding Vezatin- Myosin binding

CATENIN COMPLEXES

SPOT AND HEMI- DESMOSOMES

JUNCTIONAL COMPLEXES

GAP JUNCTIONS

CONNEXIN OLIGOMERIZATION

“GATING” OF CONNEXONS

Cx43-Binding Proteins Biochem. J. (2006) 394, 527-543

TRANSEPITHELIAL TRANSPORT

Epithelial Cell Transport Mechanisms Absorptive Cell Secretory Cell

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 (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.

ADH Stimulated Water Permeability

AQP2 Movement to the Apical Membrane of the Collecting Duct Principal Cells in Response to ADH

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

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.

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.

APICAL SORTING SIGNALS Svelto et al., Biol. Cell 102:75, 2010

BASOLATERAL SORTING SIGNALS Svelto et al., Biol. Cell 102:75, 2010

CONCLUSIONS 1. Proteins can be specifically delivered to either the basolateral or apical membrane surface. 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.