Isolation and biochemical characterization of septate junctions:

differences between the proteins in smooth and pleated varieties

NANCY J. LANE

AFRC Unit of Insect Neurophysiology and Pharmacology, Department of Zoology, Dotvning Street, Cambridge CB2 3EJ, UK

and STEPHEN M. DILWORTH

CRC Molecular Embryology Croup, Department of Zoology, Dotoning Street, Cambridge CB2 3EJ, UK

Summary

Septate junctions are found only in invertebratetissues, and are almost ubiquitous within them. Inarthropods, the two major types are the 'pleated'and the 'smooth' varieties. Using tissues from dif-ferent species, including the cockroach Periplanetaamericana, procedures have been established forobtaining membrane fractions selectively enrichedin septate junctions. The junctions have been ident-ified in pellets of these fractions by both thinsectioning and freeze-fracturing. SDS-PAGE ofthese membrane fractions reveals two major poly-peptide species with apparent molecular 'weights of

22000-24000 and 17000-18000. Consistent differ-ences in these apparent molecular weights areobserved between the pleated and smooth varietiesof septate junction. These polypeptides are prob-ably integral membrane components, as they re-main associated after treatment with high concen-trations of urea. Evidence suggests a plane ofweakness in the mid-line of the extracellular septalribbons.

Key words: septate junctions, arthropod tissues, insect gut,junctional membrane proteins.

Introduction

Septate junctions (SJs) are found between the lateralborders of epithelial cells in most invertebrates, but arenot present between those of vertebrates. Although thejunctional types commonly occurring between cells invertebrates have been extensively studied biochemically,the SJs of invertebrates have been neglected, with a singleexception (Green et al. 1983). In contrast, their fine-structural features have been examined in depth (seeNoirot-Timothe'e & Noirot, 1980; Lane & Skaer, 1980;Lane, 1986).

SJs occur almost universally between the cells of manydifferent kinds of tissues in most invertebrate groups, andso undoubtedly have an important function in theseorganisms. In the past they were considered to act asintercellular permeability barriers (Green et al. 1979;Noirot-Timothe'e et al. 1978), which might be onlypartial or selective (Lane, 1984). However, recent workby Maddrell and his colleagues (O'Donnell et al. 1984;Skaer et al. 1987) shows that they actually do not form abarrier, and explains (Skaer & Maddrell, 1987) howinvertebrate epithelia can produce effective permeabilitybarriers in spite of the fact that they possess 'leaky' SJs.The major role of these junctions may therefore be

Journal of Cell Science 93, 123-131 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

primarily as adhesive devices, particularly given thepresence of actin filaments along their cytoplasmic sur-face (Lane & Flores, 1988). The intercellular septalribbons may impart the correct degree of flexibilityessential for maintaining the cell-to-cell integrity of thehighly undulating, complex lateral surfaces characteristicof invertebrate epithelia.

The EM studies of these junctions have shown manysubtle variations on a theme of the same basic structure(Green, 1978). Frequently, the same organism may havetwo major varieties, the pleated (PSJ), and the smooth(SSJ) septate junctions. In general, PSjs occur primarilyin ectodermal tissues, and SSJs in endodermal ones.These two types differ both in the features of theirintercellular ribbons and as regards their intramembra-nous particle (IMP) distribution (Flower & Filshie,1975; Noirot-Timothe'e & Noirot, 1980; Lane & Skaer,1980).

This report describes an initial investigation into thebiochemical properties of SJs. Beginning with a mem-brane preparation, procedures have been developed toenrich selectively for these junctions and, as a result, acomparison of PSJs and SSJs has been possible. Ourmethods have allowed their polypeptide structure to beexamined for the first time and demonstrate that they

123

have a very similar composition but the componentpolypeptides have slightly different apparent molecularweights. The availability of enriched SJ preparations willallow precise comparisons to be made between thevarious categories of SJ, and immunocytochemical tech-niques to be used in a manner not previously possible. Apreliminary abstract report on these data has beenpublished (Lane & Dilworth, 1987).

Materials and methods

MaterialsThe animals used were 5th instar larvae of the moth, Manducasexta, adult specimens of the cockroach, Periplaneta ameri-cana, and adults of the lobster, Nephrops norvegicus. Tissueswere rapidly dissected from the animals, pooled, and eitherused at once, or frozen and stored at —20cC for future use. WithM. sexta and the lobster, the endodermal midgut or hepatopan-creas, respectively, were used for the extraction of SSJs. WithP. americana, two distinct lots of tissue were removed and keptseparate: the ectodermal tissues of the gut tract, including theoesophagus, hind gut and rectum, were dissected out, theircontents removed, and then they were used for the isolation ofPSJs; the endodermal midgut and associated mesenteric caecawere also dissected out, their contents removed and their SSJsthen isolated. This allowed a comparison to be made of SSJsand PSJs within the same animal; additionally, a comparison ofSSJs from two different insects or an insect with a crustaceancould also be made.

MethodsExtraction procedure. All steps were carried out at 4°C or on

ice. Tissues were dissected out into Isolation Buffer (IB) (1 mM-NaHCO3 plus 0 5 mM-CaCl2) and transferred to 40 or 50 ml IBper 0-4g tissue; they were then chopped with a razor blade orscissors and were homogenized in a loose-fitting Dounce type Ahomogenizer, using about 10 strokes. The approximate amountof starting material per organism was 0-04-01g wet wt(cockroach), 0'4-0'6g wet wt (moth) and 2-5-4-0g wet wt(lobster), while 10-50 insects or 2-4 crustaceans were used perexperiment. Inhibitors of proteolysis were added: 2/igml~ ofaprotinin, plus phenylmethylsulphonyl fluoride (PMSF),1:100 dilution of stock (20mgml~1) in dimethyl sulphoxide(DMSO). The homogenate was then filtered through two singlelayers of gauze (12 ply) in a funnel and washed through with200 ml IB. The solution was centrifuged (in 2 tubes of ~300mleach) for 15min at 4°C in a Sorvall GSA rotor, at 5000 revsmin"1 (2560g). The pellet was resuspended in 30 ml IB andrecentrifuged (in 2 tubes of ~45 ml each) for 15 min at 4°C in aSorvall SS34 rotor at 5000 revs min~' (3000 #). This pellet wassuspended in 3 ml distilled water (DW), and 60% sucrose wasadded to it to make a 50% sucrose concentration. Sucrosedensity step gradients (2 gradients of 13 ml each) were then run,with the 50% (sucrose plus tissue sample) overlain by 32%sucrose. These were spun for 90 min in a Beckman SW27Tiswing rotor at 25 000 revs min"' (81 700#) at 4°C. The mem-brane fraction at the 32 %/50 % boundary was removed intobicarbonate to wash, and pelleted (4 tubes of about 5 ml each) at25 000 revs min"1 (78 9O0#) in a Beckman SW40Ti rotor for20 min at 4°C. The washing was repeated and the preparationsspun again; at this stage the pellets could be stored overnight at4°C. This material was resuspended in 7-5 ml of 1 mM-NaHCC>3and 81 % sucrose was added slowly to the 7-5 ml to give a finalconcentration of 60%. This was made into sucrose stepgradients (6 tubes of 13 ml each) of 60% ( + sample)/50%/

42%/36%/32% and spun for 90 min at 25 000 revs min"1

(81 700#) in a SW27Ti Beckman rotor at 4°C. The 32%/36%,36%/42% and 42%/50% interfaces were collected, pelleted(in ~5 ml tubes) in a Beckman SW40Ti rotor, and washed bythe same method as used after the first gradient. The fractionsobtained after this second set of sucrose gradients were dividedinto three portions and pelleted: one for study by electronmicroscopy (EM), one for polyacrylamide gel electrophoresis(PAGE) and one for urea-treatment studies. The latter involvedincubating the pellet in l-6M-urea for 15, 30 or 60min at roomtemperature after stirring, then re-pelleting, washing andpelleting again into two samples, one for study by EM and oneby PAGE. The yield of the final SJ pellets was variable, but wasabout 1 — 10 /̂ /g per g wet wt.

Electrophoresis. Polyacrylamide slab gel electrophoresis wascarried out according to the method of Laemmli (1970), using4% stacking and 11% running gels. The pelleted junctionswere dissolved in 100-150/il of SDS sample buffer (SDS-mer-captoethanol) before loading onto the gel. Protein detection wasby staining with Coomassie Brilliant Blue, and two sets ofmolecular weight (/Wr) markers (Sigma) were used (66K, 45K,36K, 29K, 24K, 20K and 14K, and 205K, 116K, 97K, 66K,45Kand29K (K=10 3 M r )) .

Electron microscopy. Tissue samples or pellets were fixed in2-5% glutaraldehyde in 0-1 M-phosphate buffer plus 6% su-crose. Samples for thin sectioning were postfixed for 1 h in 1 %OSO4, treated with 0 5 % tannic acid, and stained en bloc for30-60min in 2% aqueous uranyl acetate (UA). They wereembedded in Araldite after dehydration in an ascending seriesof ethanol; ultrathin sections were double stained in UA andlead citrate. Elastase treatment involved 40 min of incubation oftissue in a 4mgml~1 solution of elastase in Ringer prior tofixation; after this time the cells had fallen apart (Satmary &Bradley, 1984). Fixed samples for freeze-fracture were placed in25 % glycerol in buffer before fracturing in a Balzers BA36Oapparatus at -100°C at 2xlO~6 Torr (1 Torr<=> 133-3 Pa). Allmicrographs were taken on a Philips EM420 or 300 electronmicroscope at 80 kV.

Results

Intact tissues

The lateral borders of the oesophagus, hind gut andrectum of the cockroach feature PSJs (as in Fig. 1A),while the midgut of larval Manduca, adult Periplanetaand lobster hepatopancreas are characterized by typicalSSJs (as in Fig. IB). Lateral borders of arthropodepithelial cells tend to be joined near the apical surface byzonular SJs, intercalated with and followed by maculargap junctions (as in Fig. 1A). Freeze-fracture replicasshow that these SJ membranes are characterized byparticle rows aligned in parallel; in PSJs the IMPs areseparated one from another (Fig. 1C) while in SSJs theyare often fused into ridges (as in Fig. ID).

Septate junction isolation

In developing isolation procedures for SJs, it was necess-ary to assay by EM, as no physiological test for theirpresence exists. After sucrose density centrifugation,each interface had therefore to be assayed ultrastructur-ally for the presence and degree of enrichment with SJs.Negative staining yields inadequate information as to therelative proportion of junctions to non-junctional mem-brane isolated, so thin sections of pellets had to be

124 N. J. Lane and S. M. Dilworth

PF

\EF

\

Fig. 1. Thin sections of lanthanum-impregnated insect tissue, to show PSJs in foregut (A) and SSJs in midgut (B). Notedifferences in septal ribbons when cut en face (arrows). Clusters of gap-junctional connexons (gc) may be intercalated betweenseptal ribbons or lie below them. Freeze-fracture replicas of arthropod PSJs (C) and SSJs (D), showing the differentarrangement of IMPs in the two types. In unfixed tissues, IMPs cleave onto the P face (PF) in PSJs (C) and onto the E face(EF) in SSJs (D). In both cases, complementary grooves occur on the opposite fracture face. A, X 47 000; inset, X 148000;B, X86000; inset, X191000; C, X58 0O0; D, X61000.

Isolation of septate junctions 125

Fig. 2. Low-power view (A) of the SSJ pellet taken from the 36%/42% (w/v) interface of the sucrose step-gradient, shown inlane 5 in Fig. 7. The arrows indicate some of the numerous SJs that are present. At higher power (B), it can be seen that theSSJs (small arrows) often open out into single membranes (curved arrows) or sometimes run into gap junctions (large arrows).A, X8000; B, X47 000.Fig. 3. Freeze-fracture replicas of pellets taken from the 36%/42% (w/v) (A) and 42%/50% (B) interface of a SSJpreparation. Note the characteristic ridge-like alignments of IMPs and the way the junctional area may be abruptly transformedinto non-junctional membrane (* in A). The outer membrane half-leaflet (EF in B) shows grooves complementary to the IMProws. A X90000; inset in A X76000; B X65 000.

studied. After the initial extraction procedures, thesucrose step density gradient interfaces yielding thehighest concentration of SJs were at 36%/42%; theseusually consisted of circular profiles or short lengths of SJ(Figs 2A and 3A). Rather longer lengths were sometimes

found at other interfaces (e.g. 42 %/50 % in Fig. 3B). SJswere identifiable both in thin sections of pellets whereintercellular septa could be seen (Figs 2A,B and 4) and infreeze-fracture replicas (Fig. 3A and B). The lattershowed that the isolated junctions had the intramembra-

126 N. J. Lane and S. M. Dilworth

Fig. 4. Both PSJ (A) and SSJ (C) may split open (at small arrows), and here half-septal ribbon combs can be seen. There alsomay be a fuzz (large arrows in A) on the cytoplasmic face of the junctions. The isolated SJs may form circles (C) or may branch(A). The septal ribbons, viewed en face (arrows in D), prove that these structures are truly SJs. Collapsed gap junctions igjs.)are to be seen in some preparations (B). A. Cockroach PSJ, X99000. B. Lobster SSJs, X85 000. C. Cockroach SSJ, X54000;inset, Nephrops SSJ, X168 000. D. Lobster SSJs, X83 000.

nous features characteristic of intact SJs (for example,compare Fig. 3A with Fig. ID). Although the bulk of thegap junctions accumulate at a lighter interface (32 %/36 % sucrose) than the SJs, some gap-junctional mem-branes occasionally remained attached to the SJs andmight be found in the same interfaces. PSJ (Fig. 4A) aswell as SSJ preparations (Figs 2A,B, 4C) were obtainedthat often appeared to have 90 % or more of the totalmembrane profiles occupied by unequivocal SJs. Theisolated junctions sometimes have a 'fuzz' on theircytoplasmic surface (Fig. 4A). Apparent non-junctionalmembrane in these samples may be derived from junc-tional membranes that have split open during preparation(as in Fig. 4A). This has also been observed with SSJsleaving half-septal 'combs' on either side of the mem-brane (Fig. 4C). Often the junction-enriched membrane

fuses into a complete circle (Figs 4A,C and 5A) andfrequently en face views can also be seen where tangentialsections reveal the intercellular septal ribbons in surfaceview (Figs 4D, 5A,B) similar to those in intact tissues. Insuch preparations, the septal ribbon spacing may appar-ently 'slip' and the septa become much more closelypacked (Fig. 5A) than is the case in situ. There are alsodifferences in the cleft width and intercellular cleftdensity in the isolated SJs (e.g. see Fig. 5D), which maybe very striking.

Polypeptide composition of isolated junctionsSDS-PAGE of the enriched membrane preparationsdemonstrates the presence of two major polypeptides inboth SSJ and PSJ preparations. These have molecular

Isolation of septate junctions 127

Fig. 5. Isolated cockroach (A) and lobster (B) SSJs, showing the close packing of the interseptal ribbons (arrows) when vieweden face. Pellets of cockroach PSJ, incubated in 6M-urea for 30min (C), show that the SJs are still intact although junctionalmembrane separation (small arrows) or junctional collapse (large arrows) may occur. Intact gap junctions (see inset) may stilloccasionally be present. Under normal isolation procedures, SJs may undergo collapse or appear denser, as with these cockroachSSJs (D), the protein profile of which is shown in Fig. 7 (lane 5). A, X65 000; B, X56000; C, X93 000, inset, X 173000; D,X125 000.Fig. 6. Free membrane surface of intact insect tissue after elastase incubation. The separated cells exhibit unstained hemi-septalribbons (arrows) that can be seen in en face views as they project from the stained plasma membrane (arrows), after the other,previously associated, cells have fallen away, x 107 000.

weights in the region of 22-24K and 17-18K (Fig. 7) andsimilar results have been obtained with M. sexta as withthe cockroach. However, both polypeptides show a smallvariation between the two junctional types. The largerspecies is 1-2K larger in the SSJs than in the PSJs, whilstthe smaller polypeptide is larger in the PSJ preparationsthan the SSJs. Other polypeptide species are sometimesobserved in the membrane preparations, but always insmaller quantities (as judged by staining intensity withCoomassie Blue). It is not clear whether these are derivedfrom contaminating membranes in the preparation or areadditional components of the septate junctions. It isfeasible that the 17-18K polypeptides are similar to themajor gap-junctional polypeptide present in arthropods

(Finbow et al. 1984; Ryerse, 1986). However, as yet noreactions with antibodies against this gap-junctional pro-tein have been observed (data not shown).

To shed light on the strength of association of thesepolypeptides with the membranes, the preparations weretreated with high concentrations of urea. No diminutionin the amount of either of the major polypeptide specieswas observed (Fig. 7) although some of the minorcomponents did decrease in staining intensity. EM analy-sis after urea treatment showed that the basic septatejunctional structure was essentially intact but that some-times partial disruption of the septal ribbons in both SSJand PSJ (Fig. 5C) occurred; the component junctionalmembranes can then be seen to be in the process of

128 N. y. Lane and S. M. Dilworth

1 2 3 4 5 6 S

Fig. 7. SDS-PAGE separation of the major junctionalproteins in SJ preparations from cockroach gut. Gels stainedwith 12% Coomassie Blue; S shows the Mr standards: 66K,45K, 36K, 29K, 24K, 20K and 14K. The initial crude pelletsat 32%/50% (w/v) sucrose are shown in lanes 1 (PSJ) and 2(SSJ), followed by a similar pellet treated with urea in lane 3(PSJ). Pellets after further junctional enrichment at36%/42% (w/v) sucrose interfaces are shown in lanes 4(PSJ) and 5 (SSJ) and at 42%/S0% (w/v) sucrose interfacesin lane 6 (PSJ). Note the two bands at about 23K and 17Kfor the SSJs, and 22K and 18K for the PSJs. Sections ofpellets from another sample from the same preparations (lane5) are shown (see Fig. 2A,B) to indicate the relative purity ofSJs at these interfaces.

separating, again leaving single membranes with half-septal 'combs'. These can be assumed to be junctional,again as long as they are in continuity with junctionalmembranes.

After elastase digestion, the free plasma membranereveals en face sections of septal ribbons (Fig. 6); this ispresumably the half-septum remaining after the othercell's half-septal contribution has been pulled away.

Discussion

As encountered in zonulae occludentes (Stevenson et al.

1986) and zonulae adhaerentes (Geiger et al. 1987),there are difficulties in isolating pure zonular junctionalstructures, as belt-like structures cannot be isolated intactin the same way as macular structures (such as gapjunctions (see Buultjens et al. 1988)). Preparations ofzonular SJs are therefore also difficult to obtain ascompletely pure samples. Here we have developed aprocedure that yields highly enriched preparations of SJmembranes, however. In situ, gap junctions may beintermingled with the SJs, and two-thirds down thelateral epithelial cell border in arthropods, the SJs areterminated and membrane areas are encountered thatcontain gap junctions. Hence the isolated SJs containsome, but not many, gap junctions, which chiefly separ-ate into a different interface. The same result wasobtained in the earlier study on the isolation of SSJs fromthe midgut of larval Tenebrio (Green et al. 1983), wheregap junctions were also obtained, but there they wereinterpreted to have a MT of 36000.

An examination by PAGE of the polypeptides presentin these purified SJ preparations shows two majorspecies. These have Mr values in the region of 22-24Kand 17-18K. Both polypeptides are intimately associatedwith the membrane as they do not elute with highconcentrations of urea. This suggests they may beintegral membrane proteins, and could be the proteincomponent seen in freeze-fracture studies as IMPs. The17-18K polypeptides could possibly be due to contami-nation of the preparation with gap junctions, which inarthropods have a major component of approximatelythat sameMr (Finbow et al. 1984; Ryerse, 1986; Buult-jens et al. 1988; Lane & Finbow, 1988). As the stainingratios of the 22-24K polypeptide to the 17-18K arealways approximately the same, we feel this is unlikely.Moreover, so far we have not observed any cross-reactionwith antibodies to 17-18K gap-junctional proteins.Alternatively, it is a possibility (albeit probably remote)that the major component of the gap junctions is also amajor component of SJs and vice versa. A final possi-bility is that this SJ-associated polypeptide is unrelated tothat in gap junctions and the size similarity is fortuitous.These possibilities are now open to investigation.

Another preliminary isolation of SJs has been reportedby Green et al. (1983) but was on SSJs only. They wereunable to isolate PSJs from the tissues of insects,molluscs or annelids. The SSJs isolated from Tenebriowere found to contain major proteins of 31K and 32Kwith a 47K glycoprotein deemed to be a septal com-ponent. These authors employed a different technique,using variations of methods devised for gap-junctionalisolation, and did not include protease inhibitors that wefound to be important. The gels shown of Tenebrio tissue(Green et al. 1983) have a large number of bands that runtogether and so are difficult to interpret. Their gap-junctional pellet shows a faint band at 16K as well asothers at 28 and 31-36K; although the authors do notcomment on these, they could be dimers of the 16Kprotein monomers. Their SSJ gel contains numerousbands, including ones at 16, 31, 32 and 47K. After urea,there is still a 16-18K band, plus several other bands,similar to those in our cockroach protein profiles. Before

Isolation of septate junctions 129

urea treatment, the Tenebrio SSJs also have bands in the22-24K region, which could correspond to the proteinswe have found to be present in our preparations, notsurprisingly, given the close relationship of Tenebrio andthe cockroach. The response to urea, in both studies, is toremove many proteins that are much more apparent inthe initial crude Tenebrio preparations, but some ofwhich still remain; in the cockroach and moth, however,fewer are left, suggesting that greater purity has beenachieved. This is paralleled by a greater apparent purityin the low-power micrographs of our SJ pellets comparedwith those of Tenebrio.

The presence of half-septal ribbons, or combs inisolated SJs that are splitting open, suggests that the half-ribbons may be firmly anchored into the membranes.Such a theory is corroborated by studies of insect tissuesafter elastase treatment, which reveal that cells, normallyheld together by SSJs, fall apart after incubation inelastase solutions (Satmary & Bradley, 1984). At thatstage we have found, with the EM, that the lateralmembranes still reveal half-septal ribbons on their freedlateral membranes (see Fig. 6; and Bradley & Lane,unpublished observations), suggesting a plane of weak-ness at the central mid-line of the intercellular septa, alsoindicated by the effects of urea treatment. Already-anchored half-septal ribbons may be involved in septalrecognition during the assembly of SJs.

When isolated SJ pellets are sectioned tangentially, thepositively stained septal ribbons can be seen in en faceviews. In some cases, the spacing between adjacentribbons as well as the cleft width are different from thosein the in situ preparations. This suggests junctionalcollapse and that the anchoring of the septal ribbons hadslipped, which could be due to the severing, duringisolation, of the cytoskeletal attachments to the junctionalIMPs that normally hold the septal ribbons in place(Lane & Flores, 1988); a comparable slipping was alsoobserved by Green et al. (1983). Isolated SJs often showa 'fuzz' on the cytoplasmic face of the membrane, whichcould be actin and/or an associated protein; the minorpolypeptides observed on the gels migrating in thevicinity of 42-45K in crude membrane preparationscould arise from these cytoskeletal components.

Perhaps the most unexpected result of this study is thatthere appears to be a different major (apparently integralmembrane) protein in the PSJ compared with the SSJ.That in the SSJ is always relatively larger (23-24K) thanthat in the PSJ (22-23K), even though the apparent MT

values may vary slightly in different PAGE runs. It isinteresting to speculate whether this might be consistentwith the different appearance of their component IMPpopulations in freeze-fracture replicas; the IMPs in theSSJ are larger than those making up the PSJ (compareFig. ID with C). It is also intriguing that the other majorprotein in SSJs is always smaller (i.e. 17K) than those(i.e. 18K) in the PSJs. It would be useful to determinewhat the true significance of these apparent differences inMr may be, and how the two polypeptide species relate tothe structures observed in replicas by EM. The avail-ability of purified preparations makes such a studypossible.

We thank Mr William Lee for his unfailing support andassistance with the sectioning and replication of pellets, and inthe preparation of the photographic montages. We are indebtedto Mr J. B. Harrison for assistance with sectioning in the earlystages of the project. We are also grateful to Drs MalcolmFinbow and Eldridge Buultjens for providing us with the anti-18K Nepluvps gap junctional antibody, both crude and affinity-purified, and to Dr Jan Ryerse for providing us with anti-18KDrosophila gap junctional antibody. We thank the AFRC andthe CRC for their continuing support during this researchproject.

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