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Themajor transmembraneproteinsforming occludingjunctions are calledclaudins;difin dffirent tissues,conferringdffirent perferent membersof thefamily are expressedmeability properties on the uarious epithelial sheets.Intracellular scaffold proteins bind to the transmembranecomponentsat occluding junctions and coordinatethesejunctions with cadherin-basedanchoringjunctions, so as to createjunctional complexes.The junctional scaffold proteins haue atleasttwo other crucialfunctions. Theyplay a part in the control of epithelial cell proliferation; and, in conjunction with other regulatory moleculessuch as Racand Cdc42,they gouerncell polarity. Epithelial cellshauean intrinsic tendencyto deuelopa polarized apico-basal axis.
The orientation ofthis axis in relation to the cell'sneighbors inan epithelial sheet depends on protein complexesinuoluing scaffold proteins thatassembleat cell-cell junctions, as well as on cytoskeletalpolarization controlled byRac/Cdc42and on influencesfrom the basal lamina.Thecellsof someepitheliahaueanadditionalpolarity intheplane of theepithelium,at right anglesto the apico-basalaxis.A separatesetofconseruedproteins,operatingin asimilar way in uertebratesand in insects,gouernsthis planar cell polarity through poorlyunderstoodsignaling processesthat are likewisebasedon cell-celljunctions.PASSAGEWAYSFROMCELLTO CELL:GAPJUNCTIONSAND PLASMODESMATATight junctions block the passagewaysthrough the gaps between cells, preventing extracellular molecules from leaking from one side of an epithelium to theother.
Another type of junctional structure has a radically different function: itbridges gaps between adjacent cells so as to create direct passagewaysfrom thecltoplasm of one into that of the other. These passagewaystake quite differentforms in animal tissues, where they are called gap junctions, and in plants,where they are called plasmodesmata (singular plasmodesma). In both cases,however, the function is similar: the connections allow neighboring cells toexchange small molecules but not macromolecules (with some exceptions forplasmodesmata). Many of the implications of this cell coupling are only beginning to be understood.Gap JunctionsCoupleCellsBoth Electricallyand MetabolicallyGap junctions are present in most animal tissues,including connective tissuesas well as epithelia, allowing the cells to communicate with their neighbors.Each gap junction appears in conventional electron micrographs as a patchwhere the membranes of two adjacent cells are separated by a uniform narrowr 159PASSAGEWAYSFROMCELLTOCELL:GAPJUNCTIONSAND PLASMODESMATAgap of about 2-4 nm.
The gap is spanned by channel-forming proteins, of whichthere are two distinct families, called the connexins and the innexins. These areunrelated in sequence but similar in shape and function: in vertebrates, bothfamilies are present, but connexins predominate, with 2l members in humans.ln Drosophilaand C. elegans,only innexins are present, with 15 family membersin the fly and 25 in the worm.The channels formed by the gap-junction proteins allow inorganic ions andother small water-soluble molecules to pass directly from the cltoplasm of onecell to the cltoplasm of the other, thereby coupling the cells both electrically andmetabolically.
Thus, when a suitable dye is injected into one cell, it diffuses readily into the other, without escaping into the extracellular space. Similarly, anelectric current injected into one cell through a microelectrode causesan almostinstantaneous electrical disturbance in the neighboring cell, due to the flow ofions carrying electric charge through gap junctions. With microelectrodesinserted into both cells, one can easily monitor this effect and measure properties of the gap junctions, such as their electrical resistanceand the ways in whichthe coupling changes as conditions change.In fact, some of the earliest evidenceof gap-junctional communication came from electrophysiological studies thatdemonstrated this type of rapid, direct electrical coupling between some typesof neurons.
Similar methods were used to identify connexins as the proteins thatmediate the gap-junctional communication: when connexin mRNA is injectedinto either frog oocytes or gap-junction-deficient cultured cells, channels withthe properties expected of gap-junction channels can be demonstrated electrophysiologically where pairs of injected cells make contact.From experiments with injected dye molecules of different sizes, it seemsthat the largest functional pore size for gap-junctional channels is about 1.5nm.Thus, the coupled cells share their small molecules (such as inorganic ions, sugars, amino acids, nucleotides, vitamins, and the intracellular mediators cyclicAMP and inositol trisphosphate) but not their macromolecules (proteins,nucleic acids, and polysaccharides) (Figure f9-33).A Gap-JunctionConnexonls MadeUp of SixTransmembraneConnexinSubunitsConnexins are four-pass transmembrane proteins, six of which assemble toform a hemichannel, or connexon.
\dhen the connexons in the plasma membranes of two cells in contact are aligned, they form a continuous aqueous channel that connects the two cell interiors (Figure l9-34A and Figure f 9-35). A gapjunction consists of many such connexon pairs in parallel, forming a sort ofmolecular sieve.The connexons hold the interacting plasma membranes a fixeddistance apart-hence the gap.Gap junctions in different tissues can have different properties becausetheyare formed from different combinations of connexins, creating channels thatdiffer in permeability. Most cell q,pes expressmore than one type of connexin,and two different connexin proteins can assembleinto a heteromeric connexon,with its own distinct properties.
Moreover, adjacent cells expressing differentni nteractingp l a S m am e m D r a n e s100,'1000 i..lsoooa-;Figure19-33 Determiningthe sizeof agap-junctionchannel.Whenfluorescentmoleculesof varioussizesareinjectedintoone of two cellscoupledby gapjunctions,moleculeswith a massof lessthan about1000daltonscan passinto the othercell,cannot.but largermoleculesFigure19-34 Gapjunctions.(A)A threedrawingshowingthedimensionalof twointeractingplasmamembranesadjacentcellsconnectedby gapjunctions.Eachlipid bilayeris shownasaoair of redsheets.Proteinassembliescalledconnexons(green),eachof whichisformedby sixconnexinsubunits,penetratethe apposedlipid bilayers(red).join acrosstheTwo connexonsgap to form a continuousintercellularaqueouschannelconnectingthe twoof connexinscells.(B)Theorganizationinto connexonsand connexonsintocanTheconnexonschannels.intercellularand thebe homomericor heteromeric,intercellularchannelscan be homotypicor heterotypic.homomenc&4KsfErI W OC O n n e X OtnnSr e g i s t e rf o r m i n go p e n c h a n n e lb e t w e e na d j a c e n tc e l l s(A)connexoncomposedofs i xs u b u n i t s!Eheteromericc o n n e xnr s(B)connexonshomotypiccheterotypici n t e r c e l l u l acr h a n n e l sI 160Chapter19:CellJunctions,CellAdhesion,and the ExtracellularMatrixr ar g egapJu nctronFigure19-35Gapjunctionsas seeninthe electron microscope.(A)Thin-sectionand (B)freeze-fractureelectronmicrographsof a largeand a smallgapjunctionbetweenfibroblastsin culture.In(B),eachgapjunctionis seenasa clusterof homogeneousintramembraneparticles.particleEachintramembranecorrespondsto a connexon.(FromN.B.Gilula,in CellCommunicationlR.P.Cox,ed.l,pp.
1-29. New York:Wiley,1974.\memoranesconnexins can form intercellular channels in which the two aligned half-channels are different (Figure l9-348).Each gap-junctional plaque is a dyramic structure that can readily assemble, disassemble,or be remodelled, and it can contain a cluster of a few to manythousands of connexons (seeFigure l9-358). Studieswith fluorescently labeledconnexins in living cells show that new connexons are continually added aroundthe periphery of an existing junctional plaque, while old connexons are removedfrom the middle of it and destroyed (Figure f 9-36). This turnover is rapid: theconnexin molecules have a half-life of a few hours.The mechanism of removal of old connexons from the middle of the plaqueis not known, but the route of delivery of new connexons to its periphery seemsclear: they are inserted into the plasma membrane by exocytosis,like other integral membrane proteins, and then diffuse in the plane of the membrane untilthey bump into the periphery of a plaque and become trapped.
This has a corolIary: the plasma membrane away from the gap junction should contain connexons-hemichannels-thathave not yet paired with their counterparts onanother cell. It is thought that these unpaired hemichannels are normally heldcRoSs SECTtONSFigure19-36 Connexinturnoverat a gap junction.Cellsweretransfectedwith a slightlymodifiedconnexingene,codingfor a connexinwith a shortamino-acidtag containingfour cysteinesin the sequence...Cys-Cys-X-X(whereX denotesan arbitraryamino acid).ThistetracysteineCys-Cystogcan bind strongly,and in effectirreversibly,to certainsmallfluorescentdyemoleculesthat can be addedto the culturemediumand will readilyentercellsby diffusingacrossthe plasmamembrane.In the experimentshown,agreendye wasaddedfirst,and the cellswerethen washedand incubatedfor 4 or t hours.At the end of thistime,a red dye wasaddedto themediumand the cellswerewashedagainand fixed.Connexinmoleculesalreadypresentat the beginningof the experimentare labeledgreen(andtake up no red dye becausetheirtetracysteinetagsarealreadysaturatedwith greendye),whileconnexinssynthesizedsubsequently,duringthe4- or 8-hourincubation,arelabeledred.Thefluorescenceimagesshowopticalsectionsof gap junctionsbetweenpairsof cellspreparedin thisway.Thecentralpart of the gap-junctionplaqueis green,indicatingthat itconsistsof old connexinmolecules,whilethe peripheryis red indicatingthat it consistsof connexinssynthesizedduringthe past4 or 8 hours.Thelongerthe time of incubation,the smallerthe greencentralpatchof oldmolecules,and the largerthe peripheralring of new moleculesthat havebeen recruitedto replacethem.
(FromG. Gaiettaet al.,Sclence296:503-507,2002.With permissionfrom AAAS.)4 h incubation8 h incubationE N - F A CVEI E W8 h incubation2pmPASSAGEWAYSFROMCELLTOCELL:GAPJUNCTIONSANDPLASMODESMATAin a closedconformation,preventingthe cell from losingits smallmoleculesbyleakagethroughthem. But thereis alsoevidencethat in somephysiologicalcircumstancesthey can open and serve as channelsfor the releaseof smallmolecules,such as the neurotransmitterglutamate,to the exteriol or for theentry of smallmoleculesinto the cell.GapJunctionsHaveDiverseFunctionsIn tissues containing electrically excitable cells, cell-cell coupling via gap junctions serves an obvious purpose.
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