Receptor-mediated Transcytosis of IgA in MDCK Cells Is via Apical Recycling Endosomes

20 Pages·2002·5.86 MB·English

Published April 1, 1994
Receptor-mediated Transcytosis of IgA in
MDCK CellsI s via Apical Recycling Endosomes
Gerard Apodaca, Leonid A. Katz, and Keith E. Mostov
Department of Anatomy, Department of Biochemistry and Biophysics, and Cardiovascular Research Institute, University of
California, San Francisco, California 94143
Abstract. Classically, the polymeric immunoglobulin estimate that approximately 80 % of basolaterally inter-
receptor and its ligand, IgA, are thought to be sorted nalized IgA resides in the apical endosomal compart-
from basolateral early endosomes into transcytotic ment. In addition, approximately 50% of basolaterally
vesicles that directly fuse with the apical plasma mem- internalized transferrin, a basolateral recycling protein,
brane. In contrast, we have found that in MDCK cells has access to this apical endosomal compartment and
IgA is delivered from basolateral endosomes to apical is efficiently recycled back to the basolateral surface.
endosomes and only then to the apical cell surface. Microtubules are required for the organization of the
When internalized from the basolateral surface of apical endosomal compartment and it is dispersed in
MDCK cells IgA is found to accumulate under the nocodazole-treated cells. Moreover, this compartment
D
apical plasma membrane in a compartment that is ac- is largely inaccessible to fluid-phase markers added to o
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cessible to two apically added membrane markers: either pole of the cell, and therefore seems analogous n
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anti-secretory component Fab fragments, and avidin to the recycling endosome described in nonpolarized a
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internalized from the biotinylated apical pole of the cells. We propose a model in which transcytosis is not e
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cell. This accumulation occurs in the presence of api- a specialized pathway that uses unique transcytotic fr
cal trypsin, which prevents internalization of the vesicles, but rather combines portions of pathways o
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ligand from the apical cell surface. Using a modifica- used by non-transcytosing molecules. jc
tion of the diaminobenzidine density-shift assay, we b
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Primary function of epithelial cells is to regulate the ex- two surfaces meets (Bomsel et al., 1989; Parton et al., 1989; rg
change of macromolecules between their external Bomsel et al., 1990; Fujita et al., 1990). In addition, some o
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environment and the underlying tissue. This selec- proteins are sorted in early endosomes into transcytotic vesi- M
tive exchange is made possible by the impermeability of the cles and delivered to the opposite plasma membrane surface. a
y
monolayer to both large and small molecules and specialized Transcytosis is a key membrane trafficking process in 2
endocytotic pathways that allow transit of macromolecules polarized cells because it allows for the exchange of macro- , 2
across these cells (Mostov et al., 1992). Epithelia charac- molecules from one cell surface to the opposite one (Apo- 0
1
teristically have discrete plasma membrane domains at the daca et al., 1991; Mostov et al., 1992). In addition, this is 6
apical and basolateral poles of the cell, that are separated by the only pathway for delivery of newly synthesized mem-
tight junctions (Simons and Wandinger-Ness, 1990). Each brane proteins to the apical cell surface that is universally
domain has a distinct protein and lipid composition which found in all epithelial cells examined, and in some epithelial
is maintained despite an enormous flux of membrane traffic cells, such as hepatocytes, it is virtually the only pathway for
to and from each surface. In MDCK cells, for instance, an membrane proteins to this surface (Bartles et al., 1987).
area of plasma membrane equivalent to 40 % of the cell sur- Transcytosis is also central to the establishment of cell polar-
face is endocytosed per hour (von Bonsdorff et al., 1985). ity (Zurzolo et al., 1992).
Fluid-phase material endocytosed from the apical and Much of our understanding of the transcytotic pathway
basolateral surfaces of these cells is delivered to correspond- comes from studies of the polymeric immunoglobulin recep-
ingly distinct apical and basolateral early endosomes that are tor (pIgR) ~ and its ligand dimeric IgA. The generally ac-
not thought to interchange their contents (Bomsel et al., cepted model of pIgR traffic (Fig. )1 is based on studies in
1989; Parton et al., 1989). Although much of the endocy- rat liver and MDCK cells transfected with the pIgR cDNA
tosed fluid, and many membrane components are recycled (Geuze et al., 1984; Hoppe et al., 1985; Apodaca et al.,
to the original plasma membrane surface, some are sent in 1991). The pIgR and its ligand are internalized, along with
a microtubule dependent process to a common late endo- the other receptors and fluid phase markers, in coated pits
some or prelysosome, where material endocytosed from the
Address all correspondence to Gerard Apodaca, Ph.D., Department of Abbreviations .1 used in this paper: DAB, diaminobanzidine; FSG, fish skin
Anatomy, Box 0452, University of California, San Francisco, CA 94143- gelatin; MTOC, microtubule organizing center; pIgR, polymeric-immuno-
0452. globulin receptor; SC, secretory component, Tf, transferrin.
© The Rockefeller University Press, 0021-9525194/04/67/20 $2.00
The Journal of Number 125, Volume Biology, Cell ,1 April 1994 67-86 67
Published April 1, 1994
at the basolateral (sinusoidal) surface (step 1). Subsequently, clonal antibody ascites to ZO-I, a protein associated with tight junctions,
the receptor is delivered to a tubulo-vesicular early en- was obtained from Chemicon (Temecula, CA) and was used at 1:100 dilu-
tion. Guinea pig anti-SC Fab fragments (derived from affinity purified
dosomal compartment shared with other receptors. Here the
guinea pig serum) were used at 10-20 #g/ml and were prepared as described
pIgR and its ligand are segregated from recycling receptors (Breitfeld et al., 1989b). Fab-HRP was prepared by conjugating reduced
into microdomains of endosomal tubules and packaged into YlaF to sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-l-cau'boxylate
structures, which at least in thin sections have the appear- (Pierce Chemical Co., Piscataway, NJ) derivatized HRP according to the
manufacturer's protocol and was used at 25 /zg/ml. Avidin-TRITC and
ance of vesicles (step 2) (Geuze et al., 1984). These "trans-
avidin-HRP were obtained from Vector Laboratories (Burlingame, CA) and
cytotic vesicles" are thought to contain a variety of trans-
used at 25 #g/ml. ExtrAvidin-10 nm gold was from Signm (St. Louis, MO)
cytosing molecules (Barr and Hubbard, 1993), but not and was washed with PBS and diluted 1:5. Lysine-fixable dextran-FITC
recycling molecules (Sztul et al., 1991). In both hepatocytes (10,000 tool wt) was from Molecular Probes (Eugene, OR) and used at
and pIgR-expressing MDCK cells transcytotic vesicles are 10-15 mg/ml. Rabbit antiserum to the tmns-Golgi network resident protein
TGN-38 (kindly provided by G. Banting, University of Bristol, Bristol, En-
found accumulated near and in continuity with the apical
gland) has been described (Wilde et al., 1992) and was used at 1:100 dilu-
(bile canalicular) cell surface (Geuze et al., 1984; Hoppe et tion. Canine apo-transferrin (Sigma) was loaded with iron as described
al., 1985; Hunziker et al., 1990). Microtubules are required (Podbilewicz and Mellman, 1990) and used at 25/zg/hal. In addition this
for this accumulation of transcytotic vesicles to occur. In protein was coupled to keyhole limpet hemocyanin and the conjugate used
to produce rabbit antibodies against this protein (coupling and immuniza-
nocodazole-treated MDCK cells IgA is internalized into
tion of rabbits was performed by Caltag, South San Francisco, CA). Anti-Tf
basolateral endosomes but translocation of IgA from the
antibodies were affinity purified on a canine-Tf sepharose coltmm and used
basolateral to apical pole of the cells is prevented (Hunziker at 10-20 #g/ml. The mouse monoclonal antibody, H68.4, was kindly
et al., 1990). Upon reaching the apical surface, the pIgR is provided by I. Trowbridge (Scripps Research Institute, La Jolla, CA) and
cleaved to SC, which is released with IgA into secretions recognizes the Tf receptorf rom a number of different species (White et al.,
1992). Expression of the Tf receptor in pIgR-expressing MDCK was
(step 3). A vesicular fraction highly enriched in pIgR and
confirmed by immunoprecipitating the 95,000-Mr protein from radiola-
IgA has been isolated from rat liver and termed transcytotic
beled cell lysates. For immunofluorescence studies the ascites was diluted
carrier vesicles (Sztul et al., 1991). These vesicles have been 1:100. AC17 is a mouse monoclonal antibody (kindly provided by E. Rod-
shown to fuse with isolated bile canalicular membranes in riguez-Boulain, Cornell University Medical College, New York, NY) to a D
95,000-Mr lysosomal membrane glycoprotein and was used at 1:100 dilu- o
a cell-free fusion assay that reconstitutes the fusion of trans- w
tion (Nabi et al., 1991). The mouse monoclonal antibody DM5~ specific n
cytotic vesicles with the apical surface (Sztul et al., 1993). for cc-tubulin, was obtained from Sigma and used at 1:250 dilution. A rabbit lo
We were interested in investigating the nature of the sub- antiserum specific for 7-tubulin (kindly provided by T. Stearns, University ad
apical IgA-containing vesicles/tubules found in hepatocytes of California, San Francisco, CA) has been described (Stearns et al., 1991) e
d
atrnadn scytotic in pIgR-expressing vesicles that aMreD CK waiting cells. to fuse with Do they the represent apical aaIngndG ti-human antiwabso dies used IgA, waetr e goat a 1:100 obtained anti-rat dilution. from IgG, Jackson goatF ITC- anti-mouse ImmunoorRe search Texas IgG, red-conjugated goat Laboratories anti-rabbit goat from
surface, as has been suggested by several investigators (WestCn'ove, PA) and used at 10-20 t~g/ml. The anti-rat, anti-mouse, and jc
(Geuze et al., 1984; Hoppe et al., 1985; Hunziker et al., anti-rabbit antibodies had minimal cross-reactivity with each other and b
1990)? Alternatively does the apical accumulation of IgA with human serum proteins. .ru
p
represent ligand that has been internalized from the apical r
Cell erutluC e
cell surface into apical endosomes, or IgA delivered from ss
basolateral endosomes directly to apical endosomes? In ad- MDCK strain II cells expressing the wild-type rabbit pIgR have been de- .o
r
dition, we wished to determine at what point in the trans- scribed (Breitfeld et al., 1989a). Although the results presented in this pa- g
cytotic pathway IgA is segregated away from macro- per are from one clone of MDCK strain II cellst ransfected with the plgR on
molecules that are either delivered to prelysosomes, (e.g., eDNA, we have obtained very similar results using two other independent M
clones of pIgR-expressing MDCK cells (not shown). Cells were maintained a
fluid-phase markers), or recycled back to the basolateral cell in Minimal Essential Medium (MEM; obtained from the UCSF Cell Cul- y
hasvue rface analyzed (e.g., transferrin). the transcytotic oT address pathway these of IgA questions in pIgR- we tUur/em l Facility) penicillin, supplemented and 100/~g/ml with streptomycin 10% FBS in (Hyclone, 5% CO2/95 % air. Logan, UT), In order 100 2, 2
0
expressing MDCK cells. Using these cells it is possible to to maintain a high level of receptor expression, new cells were thawed every 1
3--4 wk, and were split I:10 and pessnged once weekly. For all experiments, 6
follow the distribution of IgA in relationship to ligands and
cells were cultured on 12-ram diam, 0.4-t~m pore size Transwells (Costar;
fluid-phase markers that define the major endocytotic path- Cambridge, MA) as described (Breitfeld et al., 1989a). The cells were fed
ways, and to analyze events occurring at both the apical and everyday and used 3-4-d postculture.
basolateral poles of the cells. In contrast to the model for
noitazilanretnI of sdnagiL dna esahp-diulF
transcytosis described above, we find that in MDCK cells
,srekraM ,tnemtaerT elozadocoN gnippirtS of
IgA is delivered from basolateral endosomes to apical endo-
Cell-surface Ligands, and Biotinylation
somes and only then to the apical cell surface. In addition,
we find that transferrin (Tf), a basolateral recycling protein, Ligands and fluid phase markers were internalized from the apical and/or
has access to this apical endosomal compartment, where it basolateral surface of filter-grown MDCK cells. Priort o "If internalization,
is recycled back to the basolateral surface. it was necessary to incubate the cells for a minimum of 4 h at 37°C in
MEM/BSA (MEM, Hanks Balanced Salts, 0.6% wt/vol BSA, 20 mM
Hepes, pH 7.4) to deplete intraccllular stores of Tf. Otherwise, a Tf-
dependent signal could not be obtained in our immunottuorescenco analy-
Materials and Methods sis. All incubations in MEM/BSA were performed in a circulating water
bath. It was also possible to deplete the ceils of'IT by incubating them over-
Antibodies and Proteins night in MEM, Earle's balanced salts, 0.6% BSA, 20 mM Hepes, pH 7.4
in the tissue culture incubator. No obvious harmful effects were observed
Purified human dimeric IgA was kindly provided by J.-P. Vaerman (Catholic in serum-starved cells incubated in either fashion, as the distribution of
University of Louvaln, Brussels, Belgium) and was used at a concentration pIgR andT f-receptor were unaltered and the appropriate ligands were faith-
of 50/zg/ml. IgA-HRP was custom prepared from human dimeric IgA and fully recycled or transcytosed. For basolateral uptake of ligands or fluid-
HRP by Zymed (South San Francisco, CA) and used a2t5 /~g/ml. Rat mono- phase markers the cells were rinsed with MEM/BSA at either 81 ° or 37°C
The Journal of Cell Biology, Volume 125, 1994 68
Published April 1, 1994
and the edge of the filter on the side opposite the blotted carefully ceils was capped a syringe at -20°C. The were slides stored at -20°C until viewing
to excess medium. remove The unit Transweil placed was on a 1~-52 drop with the conf~al microscope.
of MEM/BSA containing the ligand or fluid-phase marker. rOF apical up- The specificity of the immunoflnorescence staining was confirmed as
take, the cells rinsed were with MEM/BSA at the appropriate temperature, follows:
excess fluid was aspirated fromt he cell-side of the Transwell and 051 gl /gA. No IgA signal was detected in cells in which this ligand was not
ofligend or marker, diluted fluid-phase in added. MEM/BSA, incu- All was internalized, or in MDCK cells not expressing the pIgR. The anti-rat,
bations were performed in a humid chamber. At theen d of the experiment anti-mouse, and anti-rabbit secondary antibodies didn ot cross-react with
cells were two typically washed to ttihmreese equilibrated MEM/BSA with the IgA, and the anti-human IgA secondary antibody did not cross-react
at the appropriate temperature, and either rapidly cooled down to 4°C or with any of the other markers, antibodies, ligands, fluid-pbase or secondary
if appropriate fixed immediately. antibodies.
Nocodazole (Calbiochem-Behring Corp., dissolved was CA) Diego, San ZO-I, ~-TfR, TGN-38, ~-tubuUn, and "~-tubuUn. The specificity of
in DMSO at 33 mM and stored at -20°C. In all experiments in which this staining for these molecules based was on their established distribution in
drug was used, cells were pretreated 60 rain at 4°C in the presence of 33 these and other cells. None of antibodies these cross-reacted with IgA, and
Mt~ nocodazole. The drug was included in subsequent incubations. when omitted from the protocol staining no detected. was signal The distri-
In many of the experiments ceil-surface receptors and their were ligands bution of these proteins identical was whether orn ot internalized. was IgA
stripped from the cell surface as follows: ceils were treated for 30 min at .fT No signal for Tfwas detected in starved cells in which the was ligand
C04 with 50-100 im/gt~ of TPCK-treated trypsin or 50/~g/rni of proteinase not internalized or if added was "If to the apical surface of the cells for 30
K diluted in MEM/BSA. Subsequently, the ceils were washed twice with 37°C. at rain In addition, in cells that had internalized Tfbut incubated were
ice cold MEM/BSA, one time 01 rain with either soybean 125/~g/ml trypsin with preimmune senrou m detected. was Labeling was labeling not detected
inhibitor or 2 mM fluoride dissolved phenylmethylsuifonyl in MEM/BSA. in cells in which the purified affinity anti-Tf antibodies were omitted and
For morpholngicai analysis the cells were subsequently rinsed with PBS these antibodies did not cross-react with IgA. The distribution of Tf was
containing 0.5 mM and MgCI2 0.9 mM 21CaC (PBS+), and immediately identical whether or not IgA was internalized.
fixed.
Before internaliTation of avidin-TRITC, avidin-gold, or avidin-HRP it gninnacS Laser lacofnoC sisylanA of yltnecseroulF
was necessary to biotinylate the apical surface of the cells. All manipula-
delebaL slleC
tions were performed at 4°C. Cells were washed three times with HBSS
containing Ca +2 and Mg +2 salts and 20 mM Hepes, pH 7.4, SSBI-I( )+ and
The Bio- a krypton-argon with coupled a laser using analyzed were samples
treated twice for 51 rain with 0.5 mg/ml of NHS-LC-biotin (Pierce, Rock-
Pad MRC600 head, confocal attached to an Nikon microscope Optiphot H
ford, IL) dissolved in the same buffer. The cells weret hen washed twice D
quickly and one time for 01 rain with MEM/BSA. In the immunofluores- with a Plan Ape 60X 4.1 NA objective lens. The samples were scanned o
simultaneously for and F1TC Texas red (or propidium iodide or TRITC) w
cence analysis NHS-SS-biotin was used, and apical following internaliza- emission using the 1K and K2 filter blocks. In each figure the left panel n
tion of avidin-TRITC, the apical surface of the stripped cells was of biotin- lo
avidin complexes by incubating the cells twice for 20 rain in reducing shows FITC fluorescence while the right shows Texas red, propidium io- a
dide, or parameters TRITC Collection fluorescence. were as follows: zoom d
solution. This reagent was prepared by dissolving 551 mg of reduced e
= 0.3 or 0.5 s/scan, 3.5, 5 frames/imnge, filter, Kaiman motor step size -- d
cgintathione wailthf serum+ PBS and and in 0.960 immediately fixed. 1~ml of of added. 50% NaOH washed (wt/vol) was were Ceils 38 mM NaCI. Immediately prior to use 1 ml of 5.0Comos ,mt~ sofh~tre diaphragm closed and regions or set of at colocaiization were identified using 3/1 open. The data analyzed using was the from
format merge (TIFF) side function. and the The contrast levels converted were images of the images adjusted to tagged-information-file- in the Pho- jc
Fixation and Labeling Fluorescent of sUeC b
toshop (Adobe program Co., CA) View, Mountain on Macintosh a llci (Ap- .r
u
fixed were Samples with a using paraformaidehyde pH-shifl protocol (Bom- ple, Cupertino, CA). The contrast-corrected images were imported into p
sel et al., 1989) or glutaraidehyde. with In the protocol, pH-shift cells erew. Pngemaker (Aldns Corporation, Seattle, )AW and printed from an Agfa re
9,800 imngesetter at 2,400 dots per inch, using a line screen of 051 s
fixed 5 rain at room temperature with 4% paraformaldehyde, 80 mM s
Pipes/KOH, pH 6.5, 5 mM EGTA, 2.0 mM MgCl2, and then transferred lines/inch. .o
r
to 4% dissolved paraformatdehyde in 001 mM NaBorate, pH 1L0, anidn - g
cubated 01 rain at room temperature. Cells were washed 2 x 3 rain with Analysis Ultrastructural of lgA-HRP sisotycsnarT on
PBS, pH 8.0, and nonmacted paraformaidehyde was quenched 01 min at M
room temperature with 57 mM ,1Ch-IN 20 mM Glycine, pH 0.8 (both dis- Following internalization of IgA-HRP and avidin-gold, the cells were a
solved in PBS, pH 8.0). Following two 5-rain washes with PBS, pH ,0.8 washed three times quickly with MEM/BSA and one time with ice cold y
2
nonspecific (FSG), sites and 0.025% (wt/vol) saponin (Sigma). were blocked with PBS, 0.7% In (wt/vol) experiments fish skin gelatin in which the PBS glutaraldehyde .+ % (vol/vol) The by ceils ice-cold adding 0.5 were immediately fixed in 200 mM Na cncodylate, pH 7.4, 1 mM CaCI2, 5.0 mM , 2
0
nucleus was stained with propidium iodide 001 lm/g~t of boiled A RNAse MgCI2, and incubating the cells for 30 rain at room texture. Cells 1
was added to the blocking solution. In some experiments coils were fixed were rinsed three times with 200 mM Na cacudylate buffer, pH 7.4, and 6
with dissolved glutaraldehyde (vol/vol) % 0.2 in 80 mM Pipes/KOH, pH ,8.6 %1.0 (wt/vol) diaminobenzidine (DAB), dissolved in 200 mM cacodylate
5 mM EGTA, 2.0 mM MgCI2, for 01 rain at room temperature. Non- buffer, was added for 2 rain at room temperature. The BAD solution was
reacted glutaraldehyde quenched was by incubating the cell thrteiem es 51 aspirated and replaced with fresh BAD solution containing %10.0 (vol/vol)
rain with freshly prepared mg/mi 1 4I-IBaN dissolved in PBS, pH .0.8 The rain at incubated 30 and H202 room temperature tihne dark. were Samples
cells washed were twice with blocked and PBS in PBS-FSG-sapouln as de- rinsed with 200 mM Na cacodylate, pH 7.4, and osmicated with %1 4OsO
scribed above. (wt/vol), 200 mM Na cacodylate, pH 7.4, %1 (wt/vol) IGFe(CN)6, for 90
The cells were incubated fixed with the appropriate primary antibodies, rain 4°C. at After several rinses with 0241 the samples were block-stained
diluted in PBS-FSG-saponin, for 45 min at 37°C in a humid chamber, and overnight with 0.5% (wt/vol) uranyl acetate in H20. Filters were de-
then washed 3 x 3 rain with PBS-FSG-sapouin, 1 x 3 rain with PBS- hydrated in a graded series of ethanol, embedded in the resin epoxy 211-XL
sapouln, and 1 × 3 rain with PBS-FSG-saponin. Subsequently, the cells (Ladd Res. Inds., Inc., Burlington, VT), and sectioned with a diamond
were mill 45 incubated at 37°C with the appropriate combination of fluores- (Diatome, knife Fort Sections, Washington, PA). 200-225-nm (as thick de-
diluted propidium labeled (2/~g/ml) secondary iodide cently antibodies and by termined their mounted were colors) interference on nickel butvar-coated
in PBS-FSG-sapouin. The cells were washed 3 × 3 rain with PBS-FSG- grids viewed and at 80 Vk in micro- electron EM-10 Germany) (West Zeiss a
saponin, 1 × 3 rain with PBS-saponin, and 2 × 3 rain with PBS, pH ,0.8 scope without further contrasting.
alone, 5 rain with 0.1% Triton dissolved X-100, in PBS, pH followed 8.0, oT confirm that IgA-HRP was transcytosed, efficiently this ligend was
wash a 5-rain by in PBS, pH ,0.S alone. Cells were posttixed in 4% parafor- basolaterally internalized for 01 rain at ceils. Fol- pIgR-expressing by 37°C
dissolved maldehyde in 001 mM Na-cucodylate, pH 7.4, for 30 rain at room a lowing 2-h chase at 37°C approximately %08 of IgA-HRP was found in
tempera~ue, washed twice with PBS, and mounted inp-phenylene diamine the apical medium (l~'mcytosed), % was released basolaterally, 5 and the
(Sigma) (Johnson, )1891 which prepared was as 2 follows: ml of 200 mM remainder was cell associated, IgA-HRP was not intemulized significantly
Tris, pH added 8.2, was to 20 mi of glycerol and nitrogen bubbling by mixed in the presence of IgA unlabeled excess 100-fold or by MDCK ex- cells not
gas through the solution for 20 min at room temperature. 200 milligrams pressing the plgR. Similarly, no BAD detected reaction was at tEhMe level
of p-phenylene diamine added was and the mixing continued for two addi- if an excess of (2.5 IgA during mg/ml) added was the internalization of the
tional hours. The mounting medium was stable for 7-10 d when stored in IgA-HRP to the cell surface, if the IgA-HRP during omitted was the inter-
Apodaca et .la Transcytosis of IgA Is via Apical Endosomes 69
Published April 1, 1994
nalization step, or if IgA-HRP was internalized by non-transfected MDCK cally these values welreets hsa n % 5 of the total counts). Finally these values
cells. were normalized to those obtained when AgI]152~[ and Avidin-HRP or
Fab-HRP were cointernalized for 01 rain at 37°C or 120 rain at 18°C.
sisylanA of fT]IW[ gnilcyceR
Results
Iron-saturated "If was iodinated to a specific activity of 5.0-9.0 × 601
cpm/~tg using ICI as described (Breitfeld et al., 1989a). The cells were
depleted of endogenous "If by incubating for 4 h in MEM/BSA and fT]I521[ noitalumuccA of Compartment Subapical a lgA in of
(5 ~tg/mi) was internalized from the basolateral surface of the cells for 2 h gnisserpxe-Rglp MDCK Cells
at 18°C. The cells ware washed two times quickly and one time 3 rain with
MEM/BSA at 18°C before incubation at 37°C in the presence of 50 ~tg/ml In Fig. 2 we have analyzed the time and temperature require-
of cold "Ft. At the appointed times the cells were rapidly cooled on ice and ments for basolaterally internalized IgA to accumulate at the
the apical and basolateral media were collected. [12Sl]Tf was stripped from
apical pole of pIgR-expressing MDCK cells. Individual sec-
the cell surface by incubating the cellsf or 60 rain at 4°C with 750 mM Gly-
tions, obtained with a confocal microscope, are shown from
cine, pH 2.5, diluted 5:1 with PBS +. The Transwells were rinsed with
PBS + and the filters were cut out of their holders. The total fTJ-1521[ ini- theb asal portion of the cellb elow the nucleus (level 4), from
tially boundt o the cells includes ligand recycled to the basolateral surface, the lateral surface of the cell at the level of the nucleus (level
ligand transcytosed into the apical medium, ligand stripped from the cell 3), from the apical region of the cell above the nucleus
surface with acid, and cell associated ligand not sensitive to glycine strip-
(level 2), and from the apical pole of the cell at or above the
ping (endocytosed), and was quantitated in a gamma counter (Beckman In-
struments, Palo Alto, CA). [125I]Tf uptake was inhibited >95% when the level of the tight junctions (level 1 ).
radioactive llgand was internalized in the presence of a 100-fold excess of Following 5-rain a pulse at 37°C basolaterally internalized
cold ligand. IgA was found in discrete vesicles below, at the level of, and
above the nucleus (Fig. 2 A). At the level of the tight junc-
Analysis of p3I]IgA sisotycodnE and tions the majority of the IgA remained at the cell margins,
citotycodnetsoP etaF however, some IgA was also found in apical elements at the
apex of the cell. When the 5-rain pulse was followed by a 10-
AgI]1521[ was iodinated using the ICI method to a specific activity of D
1.0-2.0 x 701 cpm//~g. Endocytosis of AgI]1521[ was measured as de- rain chase, IgA was found throughout the cell including a o
scribed (Breitfeld et al., 1989b). When specified 25/~g/mi of trypsin was large accumulation of ligand apically in both clusters and as wn
included in the apical medium. At the conclusion of the experiment small individual vesicles that were typically centrally dis- lo
AgI]I521[ was stripped from the cell surface and the results quantitated as tributed and radiated towards the margins of the cell (Fig. 2 ad
described above for the analysis of f'1']I521[ recycling. The postendocytotic e
f(Barteei tfeld of a perte internalized al., 1989b). When cohort specified of [IzsI]IgA 25/tg/mi was of analyzed trypsin was as describedi ncluded Bthe) . basal Following and lateral a 25-rain portions chase of little the ceil IgA (Fig. could be 2 C). found Instead, in d fro
in the apical medium. the IgA was largely distributed in vesicular elements present m
in the apical cytoplasm above the nucleus and at the apex of jc
BAD tfihs-ytisneD yassA the cell. Following the 25-rain chase the intensity of IgA b.r
staining had decreased and by 90 rain of chase little IgA up
In the original DAB density-shift assay, cells were homogenized following could be detectedi n the cell (Fig. 2 D). These results demon- re
internalization of markers (one of which was conjugated to HRP and the s
other radiolabeled) and a fraction rich in endosomal markers was purified strate that IgA moves rapidly from peripheral basolateral en- s.o
by centrifugation (Courtoy et al., 1984). During homogenization there was dosomes and subsequently accumulates at the apical pole of r
g
always some breakage of endosomes. This resulted in loss of radiolabeled the cell as it is being transcytosed. The distribution of the o
signal and the release of HRP conjugate that could non-specifically cross- pIgR was almost identical to its ligand; it was found through- n
link vesicles when these fractions were reacted with DAB and H202. Fol- M
out the cell but was especially concentrated at the apical pole
lowing the advice ofJ. Kaplan (University of Utah, Salt Lake City, UT), a
we have modified this original protocol, by omitting the homogenization of the cell. y 2
ostfep fT]1521[ and performing (5 /~g/mi), [125I]IgA (5 the DAB reaction #ogn/ mi), whole Fab--HRP cells. After (25 internalization #g/ml), or The distribution of IgA in cells incubated at 18°C was also , 2
assessed. At this temperature IgA can be recycled basolater- 0
avidin-HRP (25/~g/ml) the cells were washed with ice-cold MEM/BSA and 1
ally, however, release of the ligand from the apical pole of 6
radiolabeled ligands stripped from the cell surface with 100 ~tg/ml trypsin
(in the case of [125I]IgA) or with 50/zg/ml proteinase K (in the case of the cell is inhibited (Hunziker et al., 1990). Following a 30-
)fT]I521[ for 3 x 10 rain at 4°C. The cells were then washed twice with rain incubation at 18°C the IgA was found in a peripheral en-
ice-cold HBSS +. DAB reaction buffer (0.5 ml) was added to both apical dosomal compartment that lies close to the basolateral cell
and basal compartments of the Transwell. This reagent was prepared by add-
surface (Fig. 2 e). Although similar to the distribution seen
ing 3.3 ml of 3 mg/mi of DAB (dissolved in HBSS +, pH adjusted to 7.4
after 5 rain at 37°C, at 18°C there was little IgA located
with NaOH, and filtered), and 20/zl of 30% (vol/vol) H202 to 20 mi of
HBSS +. In control reactions H202 was omitted from the DAB reaction above the nucleus. After 2 h of internalization, IgA was also
buffer. Following a 45-rain incubation at 4°C the cells were washed twice founndo t only tihpnee ripheral endosomes but also through-
with HBSS +, the filters were carefully excised from their holders, boiled out the cytoplasm (Fig. 2f). tAhte level of the tight junctions
for 2 min in 0.4 mi of SDS lysis buffer (0.5% [w/v] SDS, 100 mM
some IgA was also found concentrated in a centralized spot
triethanolamine, pH 8.6, 5 mM EDTA, 0.02% [wt/vol] NAN3), and vortex
shaken for 51 rain at 4°C. Under these conditions <5 % of the total counts of bright fluorescence. This cluster of IgA was often more
were associated with the filter. The supernatants were then centrifuged at compact and condensed than observed at 37°C and may
100,000 g in a Sorvall TAOTFR rotor for 25 rain at 20°C. Radioactivity was reflect alterations in the interaction of this compartment with
quantitated in a gamma counter.
the cytoskeleton at 18°C. This distribution is similar to that
In this assay radiolabeled ligand present in the avidin-HRPo r Fab-HRP
reported by Hunziker et al. (Hunziker et al., 1990). The dis-
filled apical endosomal compartment is cross-linked by the DAB reaction
into a dense, detergent-insoluble complex that is recovered in the pellet fol- tribution of IgA 2 h at wasn ot altered if thei ncubation period
lowing centrifugation. The absolute value for the amount of ligand present was extended to 4 h (not shown). Like at 37°C, IgA internal-
in the DAB cross-linked endosomes was obtained by dividing the amount ized for 30 rain at 18°C was translocated to the apical pole
of ligand present in the pellet by the total amount of ligand present in both
of the cell when the cells were chased in ligand freem edium
the pellet and supernatant. The percent of ligand that pelleted when H202
was d omitw, from the DAB reaction was subtracted from this value (typi- for 90 rain at 18°C (not shown).
The Journal of Cell Biology, Volume 125, 1994 70
Published April 1, 1994
Transcytosing IgA Accumulates in Apical Endosomes accessible to apically internalized ligands. A prediction of
the classical model for transcytosis presented in Fig. 1 is that
We weren ext interested in determining the nature of the api-
transcytotic vesicles wouldb e inaccessible to apically inter-
cal compartment in which the IgA was accumulating. We
nalized ligands. If, however, the apically distributed IgA was
considered three possibilities: the apically distributed IgA
accessible to apically internalized ligands it might suggest
could represent transcytotic vesicles that had not yet fused
that one of the latter possibilities was true.
with the apical plasma membrane, or IgA that had reached
In the first part of our analysis we have labeled apical endo-
the apical surface and had been re-internalized into apical
somes with monovalent anti-SC Fab fragments (derived
endosomes, or IgA that was delivered directly from baso-
from affinity purified anti-SC antibodies). When added api-
lateral to apical endosomes. To distinguish among these
cally the anti-SC Fab fragments act as a pseudoligand for the
three possibilities we determined if the basolaterally inter-
pIgR and are efficiently internalized and recycled from the
nalized IgA that accumulates in the apical compartment is
apical cell surface (>95 % of this ligand is recycled apically)
(Breitfeld et al., 1989b). Little of this ligand is transcytosed
in the apical-basolateral direction. As demonstrated in Fig.
3 a there was a significant degree of colocalization of
basolaterally internalized IgA with Fab fragments internal-
ized apically for 10 min at 37°C. Colocalization of the two
ligands was also apparent in endosomal structures that lay
below the level of the tight junctions and above the nucleus
(not shown). Of course at the light level it is not possible to
rule out that IgA and Fab fragmenatrse segregated into sepa-
rate subdomains of an apically distributed compartment.
However, using a density-shift assay (described below) we
estimate that approximately % 75 of the IgA is present in this D
o
Fab-labeled apical endosomal compartment (see Fig. 6 b for w
quantitation). In this and many of the subsequent experi- nlo
ments IgA was continuously internalized for 30 rain at 37°C a
d
to completely label all of the IgA-accessible compartments e
d
brightly. The distribution of IgA internalized for 30 min at fr
37°C is like that showinn Fig. 2 b, with the majority of IgA o
m
present in the apical region of the cell and some present in jc
peripheral basolateral structures. Similar results were ob- b
tained if cells were pulsel abeled with IgA for 5 min and sub- .ru
p
sequently chased prior to the addition of apical ligand. r
e
Identical results were observed if Fab fragments were in- s
s
ternalized from the basolateral surface and IgA was internal- .o
r
ized apically (not shown). Similarly, the meeting of apically g
o
added Fab fragments with basolaterally internalized IgA oc- n
curred in cells incubated at 18°C (Fig. 3 b), a temperature M
a
at which transcytosis is inhibited. The significant degree of y
colocalization observed at 37 ° and 18°C suggests that the 2
apically accumulated IgA did not represent unfused trans- , 2
0
cytotic vesicles, but rather an endosomal compartment that 1
6
was accessible to both basolaterally internalized IgA and
apically internalized ligands.
lgA Is Delivered to Apical Endosomes Under
Conditions Where Apical Endocytosis of the Ligand
Is Prevented
The apical accumulation of IgwAe observed coulbde the re-
erugiF .1 model Classical for transcytosis of the plgR in MDCK sult of ligand that was first delivered to the apical plasma
cells. In the classical model for transcytosis AgI is internalized, membrane and then internalized into an apical endosomal
along with and molecules, recycling fluid-phase into a common compartment. It is known that cleavage of the pIgR to SC is
). 1 (step early endosome basolateral Within com- endosomal this slow relative to apical endocytosis; consequently, the pIgR
de- are markers Fluid-phase occur. events sorting several partment
and its bound ligand can be endocytosed from the apical cell
livered to (e.g., receptors many while endosomes, late )rotpecer-fT
surface (Breitfeld et al., 1989b). If this is the case it should
is IgA pack- membrane. back plasma basolateral the are recycled
be possible to preventI gA from being internalized from the
with fuse ultimately 2), which (step vesicles transcytotic into aged
apical cell surface by including trypsin in the apical medium.
proteinase a surface cell apical the At membrane. plasma apical the
sevaelc the receptor to ,CS releasing it into secre- IgA bound and We have previously used trypsin to cleave pIgR molecules
tions (step .)3 BEE, basolateral early LE, endosome; late endo- as theya rrive at the basolateral surface of the cell, thereby,
some/prelysosome; ,JT junction. tight preventing their subsequent endocytosis and transcytosis
Apodaea et al. Transcytosis of lsA Is via Apical Endosomes 17
Published April 1, 1994
Figure .2 Time and tempera-
ture dependence of IgA accu-
mulation in a subapical com-
partment of pIgR-expressing
MDCK cells. IgA was inter-
nalized from the basolateral
D
surface of MDCK cells ex- o
w
pressing the pIgR for 5 rain at
n
37°C (a). Following the 5-rain lo
pulse of IgA the cells were a
d
rapidly washed and chased in e
d
the absence of ligand for 01 fr
(b), 25 (c), or 90 min at 370C o
m
b(ads)o.la terally IgA was also for internalized30 (e) or jcb
021 rain at 18°C (f). At the .r
u
end of the internalization p
r
period the cells were washed e
s
and ligand bound to cell sur- s
face receptors stripped with .o
r
g
trypsin at 4°C. The cells were o
fixed with paraformaldehyde n
and stained for IgA, for the M
tight junction protein ZO-1, ay
and for the nucleus. Individ- 2
ual sections, obtained with a , 2
scanning laser confocal mi- 0
1
croscope, are shown from the 6
basal portion of the cell below
the nucleus (level 4), from the
lateral surface of the cell at the
level of the nucleus (level 3),
from the apical region of the
cell above the nucleus (level
2), and from the apical pole of
the cell at or above the level of
the tight junctions (level 1 ).
The samples were scanned
simultaneously for FITC and
Texas red (or propidium io-
dide) emission which are dis-
played in the left and right
halves of each panel, re-
spectively. All images are
at the same magnification.
Bar, I0/zm.
ehT lanruoJ of Biology, Cell emuloV 125, 4991 27
Published April 1, 1994
(Casanova et al., 1991; Aroeti et al., 1993). In contrast, if lateral surface of the cell while at the same time trypsin was
IgA is delivered directly from basolateral to apical endo- included in the apical medium. As shown in Fig. 4 (d and
somes then addition of apical trypsin should have littleef fect e) the delivery of IgA to the apical compartment of these
on the ability of the IgA to enter the apical endosomal com- trypsin-treated cells occurred largely undiminished at either
partment. 37* or 18°C, respectively. These results suggested that under
To distinguish between the two possibilities described these conditions much of the IgA was being delivered
abovwee first needed to confirm that trypsin treatment could directly to this apical compartment. We have also performed
be used to prevent internalization of IgA from the apical cell the analysis described in Fig. 2 (a-d) in the presence of api-
surface. [l~I]IgA was prebound to pIgR molecules at the cal trypsin. Similar results were obtained, although as ex-
apical cell surface at 40C, and then the cells were warmed pected there was a more rapid decrease in IgA signal (data
up for 1-5 min at 37°C in the absence or presence of 25 not shown). As an additional control for the effectiveness of
#g/ml of trypsin. As shown in Fig. 4 a, in the absence of the apical trypsin treatment in preventing internalization of
trypsin, the [t2sI]IgA was internalized, albeit inefficiently, plgR present at the apical plasma membrane, Fab fragments
during the 37°C incubation. However, in the presence of tryp- were added in conjunction with the trypsin to the apical sur-
sin no internalized AgI]I521[ was detected during the same face of the cellss howinn Fig. 4 (d and e). Under these condi-
5-min internalization period (Fig. 4 b). In addition, the vast tions internalization of Fab fragments was completely
majority of IgA (>95 %) was rapidly released into the apical prevented. Compare untreated cells in the left panel of Fig.
medium even after a 1-min incubation period in the presence 3 a with trypsin-treated cells in the left panel of Fig. 4 d.
of the proteinase. These observations demonstrate that tryp- We have performed several additional controls to verify
sin is able to rapidlyr emove IgA bound to plgR from the cell the effectiveness of the apical trypsin in preventing IgA-pIgR
surface before it has a chance tob e internalized, and that the complexes from being internalized from the apical plasma
binding of IgA to the pIgRd oes not render the plgR insensi- membrane. First, identical results were obtained using as lit-
tive to trypsin treatment. tle as 5 t~g/ml trypsin, indicating that we were working in
In addition, we have analyzed the effect of apical trypsin considerable excess of the trypsin needed to cleave the pIgR. D
o
on the fate of a 5-min pulse of basolaterally internalized Second, Fab fragments bind to pIgR-IgA complexes (Le- w
n
[mI]IgA. As is shown in Fig. 4 c the addition of trypsin in- maitre-Coelho et al., 1981; and Bomsel, M., and K. E. lo
creases the initial rate of ligand transcytosis over that ob- Mostov, unpublished results), so if pIgR-IgA complexes a
d
served in the absence of proteinase. This observation sug- transiently appeared at the apical surface before reinternal- e
d
gests that in the absence of trypsin a fraction of the ization, the Fab fragments could still bind. Third, when ana- fr
transcytosing IgA is being reinternalized from the apicalc ell lyzed by PAGE, trypsin-treated baF]I~21[ fragments re- om
surfacea nd presumably recycling while in transit to the api- mained intact and were not degraded (not shown). In jc
cal secretions of the cell. It also points out the importance addition, if soybean trypsin inhibitor was added to the tryp- b
.r
of using apical trypsin to prevent reinternalization of the sin-Fab medium after the incubation at 37 ° or 180C, the Fab u
p
pIgR from the apical cell surface. fragments were rapidly internalized to the same extent as re
Having confirmed that trypsinc ould be effectively used to non-trypsin-treated Fabs (not shown). This indicatetsh at the ss
prevent ligand internalization we determined the effect of effect of trypsin is to inactivate the ability of the plgR to bind .o
r
apical trypsin on IgA accumulation in the apical compart- Fab fragments, and not to inactivate the Fab fragments. og
ment. IgA was internalized continuously from the baso- To confirm that IgA was being delivered to an apical en- n
M
a
y
2
, 2
0
1
6
Figure .3 AgI delivered is to an compartment. endosomal apical AgI saw internalized from the basolateral surface of the cells for 30 min
at and 370C anti-SC last during the apically internalized simultaneously were fragments Fab 01 rain of period incubation the (a). Alterna-
tively, AgI saw basolaterally and internalized baF apically fragments for 021 rain at (b). the end 18°C At of experiment the the were cells
and washed cell surface ligand stripped from the apical cell surface with trypsin. Cells with were paraformaldehyde, stained fixed with
the appropriate antibodies, and scanned simultaneously for red emission which FITC and Texas are in displayed the and left right halves
of each panel, Sections are respectively. at or the above level of junctions. the tight Images are at the same magnification. Bar, 10/zm.
acadopA te .la sisotycsnarT of AgI Is aiv Apical semosodnE 37
Published April 1, 1994
dosomal compartment under conditions where apical inter- al., 1989). Under these conditions there was significant
nalization of the ligand is prevented, we have performed the colocalization of the two ligands in the apical region of the
following experiments. IgA was internalized basolaterally cell, both at the level of the tight junctions (Fig. 5 a), and
for 20 min at 370C. The apical surface of the cell was bi- in the apical cytoplasm below the level of the tight junctions
otinylated and avidin-TRITC was added apically while IgA (Fig. 5 b). Avidin-TRITC was not detected below this level
was internalized basolaterally for an additional 10 min. (data not shown). In addition, colocalization of the two
Trypsin was included in the apical medium throughout the markers was observed if the period of internalization of the
30-rain internalization period. The internalized avidin-TRITC two markers was limited to just 10 min at 37°C (data not
serves as a general membrane marker for the apical en- shown), confirming that a large fraction of the IgA was rap-
dosomal compartment and unlike Fab fragments internaliza- idly being delivered directly to an apical early endosomal
tion of avidin-TRITC is not prevented by the action of tryp- compartment.
sin, as the pool of biotinylated proteins (and lipids) is largely To demonstrate that colocalization of IgA and avidin was
trypsin resistant. The 10-min internalization period is long occurring at the ultrastructural level, IgA-HRP was internal-
enough to fill the apical early endosomal compartment but ized for a total period of 30 min at 37°C, while avidin-gold
short enough to prevent entry of the avidin-TRITC into the was internalized for the final 10 min from the biotinylated
prelysosomal compartment (Bomsel et al., 1989; Parton et apical pole of the cell. Apical trypsin was included through-
D
o
w
n
lo
a
d
e
d
fr
o
m
jc
b
.r
u
p
r
e
s
s
.o
r
g
o
n
M
a
y
2
, 2
0
1
6
Figure .4 Trypsin prevents internalization of IgA from the apical cell surface. In a and b [125I]IgA was bound to the apical surface of cells
for 90 min at 4°C, the cells were washed, and then reincubated at 37°C in the absence (a) or presence (b) of 25 #g/rrd of trypsin added
to the apical chamber of the Transwell. At the indicated times the cells were rapidly cooled on ice and the apical media was collected.
[nsI]IgA was stripped from the cell surface by using acid treatment at 4°C and the filters were cut out of their holders. The total AgI]Is2~[
initially bound to the cells included ligand released into the apical medium, ligand stripped from the cell surface with acid, and cell-
associated ligand not sensitive to stripping (endocytosed), and was quantitated in a gamma counter. Values are from duplicate filters and
varied <10%. At time 0, prior to warming the cells up to 37°C, virtually 100% of the ligand was at the cell surface. In c AgI]I52~[ was
internalized from the basolateral surface of the cells for 5 rain at 37°C, the cells were washed, and then chased for 120 rain in the absence
(-) or presence of (+) apical trypsin. The percent of total ligand released apically (tmnscytosed) and basolaterally (recycled) are shown.
The remainder of the counts were intracellular. Values are from duplicate filters and varied <10 %. In d-e IgA was internalized basolaterally
for 30 rain at 37°C or 120 rain at 18°C, respectively, while Fab fragments plus 25 #g/ml of trypsin were added apically. Cells were fixed
with paraformaldehyde, stained with the appropriate antibodies, and scanned simultaneously for F1TC and Texas red emission which are
displayed in the left and right halves of each panel, respectively, d and e are projections (sums) of four sections from the apical pole of
the cell. Use of projections gives one an overview of the distribution of a particular marker in a single image. Images are at the same
magnification. Bar, 01 #m.
ehT lanruoJ of Cell ,ygoloiB emuloV 125, 4991 47
Published April 1, 1994
D
o
w
n
lo
a
d
e
d
fr
o
m
jc
b
.r
u
p
r
e
s
s
.o
r
g
o
n
M
a
y
2
, 2
0
1
6
Figure .5 IgA is delivered to an apical endosomal compartment under conditions that prevent internalization of the ligand from the apical
cell surface. In a and b IgA was internalized basolaterally for 30 min at 37°C while avidin-TRYIC was added during the last 10 min of
the incubation periotdo the biotinylated apical pole of the cell. Trypsin was included in the apical pole of the cell throughout the internaliza-
tion period. At the end of the experiment cell surface avidin-SS-biotin complexes were stripped from the cell surface with reduced
glutathione. Cells were fixed with paraformaldehyde, stained with the appropriate antibodies, and scanned simultaneously for FITC and
TRITC emission which are displayed in the left and fight halves of each panel, respectively, a is a section at the level of the tight junctions.
b is a section above the nucleus, approximately 2-gm below the level of the tight junctions. Arrows are intended as landmarks to guide
the reader in identifying regions of colocalization. All images are at the same magnification. In c IgA-HRP was internalized from the
basolateral cell surface for 30 rain at 37°C. Avidin-gold, bound to the biotinylated apical pole of the cell, was internalized apically during
the last 01 rain. Trypsin was included in the apical medium throughout the internalization period. Meeting was observed in tubulovesicular
elements close to the apical cell surface. Cells were processed for electron microscopy as described in the Materials and Methods. Solid
arrows, examples of structures in which IgA-HRP and avidin-gold colocalize; open arrows, examples of structures in which only IgA-HRP
is found; arrowheads, examples of areas where avidin-gold is found in the absence of IgA-HRP. Bars: (a and b) 01 #m; (c) 0.5 #m.
acatxxlA et al. sisotycsnarT of lgA Is aiv Apical semosodnE 57
Published April 1, 1994
out the 30-rain internalization period. The IgA-HRP was expect maximum colocalization of the two markers, and in-
detected by DAB cytochemistry. IgA-HRP was found in deed observe a high degree of colocalization of ttwhoe mark-
vesicular and tubular elements distributed throughout the ers in apical endosomes (Fig. 6 a). Our maximal levels for
cytoplasm but concentrated in the apical region of the ceils colocalization, as determined by the HRP cross-linking as-
when semi-thick sections (200-225 urn) of epon-embedded say, are comparable to or even better than those obtained in
cells were examined. Particularly in the apical region of the many similar studies (Ajioka, 1986; Ward et al., 1990).
ceil, just under the apical plasma membrane, there were When we normalize to the maximum amount of [mI]IgA
large accumulations of short tubules that contained both found in the apical endosomal compartment, we estimate
avidin-gold and IgA-HRP (Fig. c, 5 solid arrows). This ob- that 80.5 + 2.39% of AgI]I~zp internalized for 30 min at
servation confirms the IgA-HRP is entering an apical early 37r"eCs iaidnne s apical endosomal compartment (Fig. 6 b).
endosomal compartment. Colocalization also occurred in This result is consistent with our morphological analysis in
more vesicular organeiles close to the plasma membrane which the majority of transcytosing IgA resideisn the apical
(Fig. 5 c) and occasionally in perinuelear multivesicular region of the cell in a compartment that overlaps with apical
bodies. endosomes. Moreover, we obtain a very similar result when
Although it was possible to find regions of IgA-HRP that AgI]Is2~[ is internalized basolateraliy and avidin-HRP api-
had no gold (Fig. 5 c, open arrows), it was less common to cally for only 01 rain at 37"C (>80%). In addition, AgI]q~l[
find avidin-gold in the absence of the other marker (Fig. 5 was also delivered to apical endosomes a following 2-h inter-
,c arrowheads). Wbee lieve this reflects the inefficient inter- nalization period at 180C. Undert hese particular conditions
nalization of the avidin-gold particles. In addition, some of we estimate that approximately 34.4 + 2.0% of the ligand
the IgA-HRP structures could represent tubules or vesicles had been delivered to the apical endosomes (Fig. 6 b). This
derived from basolateral early endosomes not yet fused with observation is consistent with our morphological evidence
the apical endosomal compartment, recycling vesicles de- (see Fig. 2 f) that although IgA is delivered to apical endo-
rived from basolateral endosomes, or some other uniden- somes at this reduced temperature, a large fraction of the
tiffed compartment. In these examples >90% of the avidin- ligand remains bians olateraclo mpartments that are presum- D
o
gold particles are found to colocalize with the IgA-HRP. ably inaccessible to apicaily internalized ligand. The lower w
Avidin-gold was not detected in the basal portions of the level of colocalization at 18"C may also a reflect less efficient nlo
call. Similar results were obtained if the two markers were internalization of ligand that occurs at 18"C. a
d
internalized for 120 min at 18"C (data not shown). In addi- As a control for the validity of our assay we have also per- e
d
tion, we observe significant colocalization between IgA- formed both a morphological analysis of IgA transcytosis fr
HRP and apically internalized ricin-gold (data not shown) and the HRP density-shift assay in nocodazole-treated cells. o
m
which is a non-specific membrane marker that is efficiently As demonstrated in Fig. 6 (c-d) meeting of basolaterally in- jc
internalized anrde cycled at the apical celslu rface (van Deurs ternalized IgA and apically internalized Fab fragments was b
et al., 1990). prevented in nocodazole-treated ceils. As expected, the IgA .ru
p
remained along the basolateracle lslu rface (Fig. 6 c) and was r
e
not delivered to the cytoplasm at the apical pole of the cell s
Biochemical Estimation of the Amount of lgA (Fig. 6 d). The Fab fragments were found dispersed across s.o
Delivered to the Apical Endosomai Compartment the apical region of the cell in small vesicles (Fig. 6 d, left). rg
o
oT quantitate the amount of IgA delivered directly to apical These results confirpmr evious observations that the translo- n
endosomes we have used modification a of the density- DAB cation of IgA from the basolateral to apical pole of the cell M
a
shift protocol (Courtoy et al., 1984). In our assay p2q]IgA requires microtubules, and is largely inhibited in nocoda- y
was internalized basolaterally for a total period of 30 rain at zole-treated ceils (Breitfeld et al., 1990; Hunziker et al., 2
37"C to completely fill all of the IgA-accessible compart- 1990). Similar results were observed if the experiments were , 2
0
ments. Avidin-HRP was internalized during the last 01 rain performed at 37"C (data not shown). 1
6
of the incubation period from the biotinylated apical pole of As predicted, entry of pzq]IgA into apical endosomes
the cell. Apical trypsin was included throughout the inter- was inhibited sevenfold at both *73 and 18"C when the DAB-
nalization period. At the end of the experiment, ligand was density shift assay was performed in nocodazole-treated cells
removed from the ceil surface, and the cells were treated (Fig. 6 b). Again, these estimates were normalized to the
with DAB and .2021-1 When ceils are treated in this manner, maximum amount of [mI]IgA found in the apical en-
[tzq]IgA present in the avidin-HRP filled apical endosomal dosomal compartment. This observation confirms that the
compartment is cross-linked by the DAB reaction into a majority of IgA was being delivered from basolateral endo-
dense, detergent-insoluble complex. When tcheeal rles solu- somes to apical endosomes, and that the process was
bilized in SDS the p~I]IgA present in DAB cross-linked microtubule dependent. In addition, the sevenfold inhibition
apical endosomes is recovered by centrifugation. AgI]qz~[ of meeting in drug-treated cells confirms that [tzq]IgA was
present in other compartments remains soluble and is found only density shifted when both the pzq]IgA and avidin-
in the supernatant following centrifugation. The percent of HRP were in the same compartment. Note that endocytosis
IgA itnh e pellet represents that fraction of IgA present in the from both poles of the cell is largely unaffected by drug treat-
apical endosomal compartment. It is well established that ment and the amount of [t~I]IgA internalized by the
DAB cross-linking is not 100% efficient, even for markers nocedozole-treated ceils actually increased (data not shown)
that are completely colocalized (Courtoy, 1984; Ajioka, (Breitfeld et al., 1990; Hunziker et al., 1990).
1986; Ward et al., 1990). As such our values were normal- As additional controls for the specificity of the DAB
ized to reactions in which IgA and avidin-HRP were co- density-shift assay we report that there was no shift when
internalized from the biotinylated apical pole of the cell( see avidin-tLRP was not internalized, and when [t2q]IgA and
legend to Fig. 6 for these values). Under these conditions we avidin-HRP were bound to opposite poles of thec ei4la" tC ,
ehT lanruoJ of lleC ,ygoloiB emuloV ,521 4991 67