Basolateral to Apical Transcytosis in Polarized Cells Is Indirect and Involves BFA and Trimeric G

18 Pages·2002·7.7 MB·English

Basolateral to Apical Transcytosis in Polarized Cells Is Indirect and
Involves BFA and Trimeric G Protein Sensitive Passage through the
Apical Endosome
Margarida Barroso and Elizabeth S. Sztul
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
Abstract. We have used temperature and nocodazole since transcytotic ligand can exit the compartment and
blocks in an in vivo basolateral to apical transcytosis be released into the apical medium when cells pre-
assay to dissociate the early transcytotic steps occur- loaded at 17°C are subsequently incubated at 37°C.
ring during the formation of transcytotic vesicles and We have used this system to examine the effect of Do
w
their microtubule-dependent translocation into the api- Brefeldin A (BFA) and the involvement of trimeric nlo
a
cal region, from the late steps when transcytotic cargo GTPases in the late (post apical transcytotic compart- de
d
is delivered into the apical media. We found that po- ment) steps of the transcytotic pathway. We found that fro
m
larized MDCK cells transfected with rabbit polymeric addition of BFA or cholera toxin, a known activator of http
IgA receptor (plgA-R) internalize basolaterally added Gsot, to cells preloaded with transcytotic ligand at ://ru
p
plgA-R ligand (Fab2 fragment of IgG against the 17°C significantly inhibits the exit of ligand from the re
receptor's ectodomain) at 17°C but do not deliver it apical transcytotic compartment. General structure and ss.org
to the apical PM. instead, the ligand accumulates in function of the apical endosome are not affected since /jcb
/a
an apically localized transcytotic compartment, distal neither BFA nor cholera toxin inhibit the recycling of rticle
to the basolateral endosome and the microtubule- apically internalized membrane markers (ricin and -p
d
requiring translocation step. We have characterized plgA-R ligand) from the same compartment. f/12
4
this compartment and show that it is distinct from The data suggest that transcytosis connects the /1/8
3
basolateral transferrin recycling endosomes, basolateral "membrane-sorting" sub-domain of the basolateral en- /1
2
6
early endosomes or late endosomes or lysosomes. The dosome with a homologous sub-domain of the apical 09
3
0
apical transcytotic compartment colocalizes with the endosome and that exit of transcytosing cargo from /8
3
compartment containing apically recycling membrane the apical endosome is controlled by a BFA and tri- .pd
f b
markers (ricin and apically internalized plgA-R ligand) meric G protein sensitive mechanism, distinct from y g
u
but is distinct from the compartment receiving apically that used for recycling of apically internalized proteins est o
internalized fluid phase marker (BSA). This compart- (ricin or plgA-R). n 0
1
ment is an intermediate station of the overall pathway Ja
n
u
a
ry 2
0
2
p 3
DEZIRALO epithelial cells possess two functionally, in liver; Sztul et al., 1983; Hoppe et al., 1985), from apical
morphologically, and biochemically distinct plasma to basolateral PM (e.g., IgG in enterocytes of neonatal rats;
membrane (PM) ~ domains (for review see Simons Rodewald and Abrahamson, 1980; Rodewald and Kraen-
and Fuller, 1985). Connection between the basolateral and buhl, 1984), or equally from both surfaces (e.g., fluid-phase
apical domain is mediated by ,sisotycsnart a multi-step vesic- marker in MDCK cells; Bomsel et al., 1989).
ular transport pathway. Depending on the cell type and the Basolateral to apical transcytosis has been extensively
marker protein followed, transcytosis can occur predomi- studied in rat liver and in polarized MDCK ceils by examin-
nantly from basolateral to apical PM (e.g., pIgA and pIgA-R ing the transcytotic traffic of a transmembrane protein, the
plgA-R (Mullock et al., 1979; Renston et al., 1980; Geuze
Address all correspondence to Dr. Elizabeth Sztul, Department of Molecu- et al., 1984; Hoppe et al., 1985; Limet et al., 1985; Mostov
lar Biology, Lewis Thomas Laboratory, Princeton University, Princeton, and Deitcher, 1986; Breitfeld et al., 1989). Transcytotic
NJ 08544. pathway can be subdivided into several spatially and/or tem-
porally distinct steps: (a) internalization of transcytotic
.1 Abbreviations used in this paper: ARF, ADP-ribosylation factor; BFA,
cargo from the basolateral surface via clathrin-coated pits
Brefeldin A; BSA-FITC, FITC-labeled BSA; plgA-R, polymeric IgA recep-
tor; PM, plasma membrane; TCVs, transcytotic vesicles. and vesicles; (b) delivery to the basolateral early/sorting en-
© The Rockefeller University Press, 0021-9525/94/01/83/18 $2.00
The Journal of Cell Biology, Volume 124, Numbers 1 & 2, January 1994 83-100 83
dosome; (c) active sorting from other transmembrane and BFA has been shown to dramatically modulate membrane
content proteins and segregation into a distinct endosomal traffic within the secretory and endocytic pathways (for re-
sub-domain; (d) budding of transcytotic vesicles (TCVs), view see Klausner et al., 1992). It inhibits the guanine
from that sub-domain; (e) translocation of IL'Vs into the sub- nucleotide exchange on ADP-ribosylation factor (ARF) and
apical region of cells via a microtubule-dependent mecha- thus prevents its binding to the membrane where ARF is re-
nism; and (f) subsequent delivery oftranscytotic cargo to the quired to promote the association of cytosolic coatomer and
apical PM (Geuze et al., 1984; Hoppe et al., 1985; Breitfeld other proteins (Waters et al., 1991; Narula et al., 1992;
et al., 1989, 1990; Casanova et al., 1990; Hunziker et al., Klausner et al., 1992; Donaldson et al., 1992; Randazzo et
1990). Although the initial stages of the transcytotic path- al., 1993; Palmer et al., 1993). Membrane "coats" might be
way, namely those before the formation of TCVs, are well involved in protein sorting or stabilizing proteins within a
documented, the later steps, i.e., those occurring within the specific membrane sub compartment in a manner analogous
apical region of cells, are poorly defined. One of the major to the action of adaptins during clathrin-mediated membrane
questions is whether sV'-LI deliver their cargo directly by fus- traffic or in preventing incorrect membrane fusion events
ing with the apical PM or whether the delivery is indirect (Pearse and Robinson, 1990; Rothman and Orci, 1992). The
and involves fusion of TCVs with other compartments, from effect of BFA on the transcytotic pathway in MDCK cells has
which transcytotic cargo is subsequently delivered to the ap- been examined previously and, as shown by Hunziker et al.
ical PM. (1991), the drug inhibits the transcytotic delivery of plgA-R
Relevant to such considerations is the finding that polar- physiological ligand, dimeric IgA (dlgA), into the apical me-
ized MDCK cells contain distinct endosomal systems: one dia. The BFA induced block is early (ligand is not translo-
set of early/sorting endosomes is localized close to the cated into the apical region of cells) and is presumably due
basolateral PM and receives cargo endocytosed only from to the inability of plgA-R to be correctly sorted from the D
that PM domain and another, close to the apical PM which basolateral endosome into the transcytotic pathway (Hun- ow
n
receives cargo only from the apical domain. Cargo internal- ziker et al., 1993). Effects of BFA on later steps of transcyto- loa
d
ized into these distinct sorting endosomes does not mix, sug- sis have not been examined. ed
gesting that fusion between the two sets of endosomal com- Trimeric GTPases were originally identified as members from
partments is inhibited (Bomsel et al., 1989; Parton et al., of the signal transduction cascade, functionally coupling PM http
1989). Both of the endosomal systems "empty" into a com- receptors (e.g., rhodopsin and /~-adrenergic receptor) to ://ru
p
mon late endosomal system in which cargo internalized from effectors (cGMP; phosphodiesterase and adenylyl cyclase, re
basolateral and apical surface mixes before delivery to lyso- respectively) (Kaziro et al., 1991). However, it is now clear ss.o
rg
somes (Bomsel et al., 1989; Hughson and Hopkins, 1990). that they perform additional functions, including the regula- /jcb
Tphoela rized presence cells ofra ises distinct an interesting early endosomal question: populatioinsf transcytosis in tion1991; of Stow the sete cretory al., 1991; and Colombo endocytic et al.pa,t hways 1992; (BarrL eyte et et aall..,, /article
-p
occurs indirectly, is it possible that TCVs act as the vesicular 1992; Carter et al., 1993). Trimeric GTPases appear to df/1
link connecting the two endocytic compartments? cross-talk with BFA-responsive elements since they also 24
/1
To address this question we examined the compartments modulate the membrane association of specific coat proteins, /83
involved in the late steps of the transcytotic pathway by for example, the coatomer (present in Golgi-derived non- /126
0
analyzing the basolateral-to-apical transcytosis of plgA-R clathrin-coated vesicles), ARF (a compartment of clathrin 93
0
pseudo-ligand (Fab2 fragments of IgG directed against the and non-clathrin-coated vesicles), and ),-adaptin (found in /83
plgA-R ectodomain) in polarized MDCK cells transfected I-IA1/AP1 adaptor complex of the TGN-derived clathrin- .pd
with rabbit plgA-R (Mostov and Deitcher, 1986). To focus coated vesicles) (Serafini et al., 1991; Donaldson et al., f by g
ewxhcilcuhs ively dissociates on late events stages, occurring we used before an in the vivo delivery treatment of Wong 1991; and Ktistakis Brodsky, et al., 1992). 1992; It has Robinson been shown and that Kreis, activation 1992; uest on
0
cway. argo Specifically,i nto the apical we region took advantage from the later of the steps observation of the path- by moefm branes specific tanrdi meric thus can GTPases influence decreases coat assembly ARF binding and mem- to 1 Janu
a
Hunziker et al. (1990) that incubation of MDCK cells at brane traffic (for review see Bomsel and Mostov, 1992). ry 2
17°C results in a partial block of the transcytotic pathway. We have shown previously that rat liver sV'-'LI" contain a 02
3
At this temperature, pIgA-R ligand is endocytosed, sorted major 108-kD protein associated with their cytosolic surface
from non-transcytotic ligands into TCVs which are then and that this protein exists in cytosolic and TCV membrane
translocated on microtubules into the apical region of cells associated pools as a hexameric complex of'~600 kD (Sztul
(Hunziker et al., 1990; Breitfeld et al., 1990). However, et al., 1991; Gow et al., 1993). More recently, we have found
during 17°C incubations, delivery of the pIgA-R ligand into that the association of this complex with the TCV membrane
the apical media is inhibited (Hunziker et al., 1990) and the is regulated by a cholera toxin responsive Gs (Barroso, M.,
ligand accumulates in an undefined apical compartment. The and E. S. Sztul, manuscript in preparation), suggesting that
release of apically accumulated pIgA-R ligand into the apical trimeric GTPases might control transcytotic traffic in a man-
medium can be induced by shifting the cells to 37°C and is ner analogous to their role in other membrane traffic, i.e.,
not microtubule-dependent. by modulating the membrane association of specific coat
We used this temperature block to functionally character- components. Involvement of trimeric Gs in early stages of
ize the 17°C apical transcytotic compartment and to study transcytosis has been recently documented (Bomsel and
the mechanism controlling the movement of transcytotic Mostov, 1993) but the role of trimeric GTPases during late
cargo from that compartment to the cell surface. Specifi- stages of transcytosis has not been reported.
cally, we tested whether addition of BFA or activation of tri- In this paper, we show that basolateral to apical transcyto-
meric G proteins influences the traffic of pIgA-R ligand from sis of pIgA-R ligand occurs indirectly by transport from the
the apical transcytotic compartment to the apical PM. basolateral endosome to an apical endosomal compartment
The Journal of Cell Biology, Volume 124, 1994 84
and that the transcytotic compartment colocalizes with a tion period. For treatment with cholera toxin, MDCK cells were incubated
specific sub-domain of the apical endosome, namely that in- at 37°C with cholera toxin for I h at 10/zg/ml, and with media for the next
4 h. Pertussis toxin was used at a concentration of 1/zg/ml throughout the
volved in apical membrane recycling. Wc also show that ad-
37°C incubation period.
dition of BFA and activation of trimeric G proteins modulate Ricin-HRP internalization from the apical surface was performed as indi-
late steps of the transcytotic pathway, i.e., those occurring cated for the pIgA-R ligand, except that 01 mM lactose washes were used
after transcytosed cargo is delivered to the apical endosome to remove the bound ricin after the 17°C period. Incubation at 37°C was
also performed in the presence of 10 mM lactose, to recover the ricin-HRP
but before its exit from that compartment. Since neither BFA
recycling back to the apical surface. HRP activity was tested using as a sub-
nor activation of trimeric G proteins inhibits apical mem-
strate Turbo-TMB (Pierce, Rockford, IL) and optical density was deter-
brane recycling through the same apical endosome, it ap- mined at 450 rim.
pears that a sorting mechanism sensitive to BFA and involv-
ecnecseroulF Confocal ypocsorciM
ing G proteins controls the exit of transcytotic cargo from the
apical transcytotic compartment into the apical recycling The in vivo transcytotic assay was used as previously described except that
pathway. the ligand was labeled with fluorescent dyes, Cy3 or FITC, as described by
the manufacturer. Fab2 fragment-Cy3 or -FITC was added to the
basolateral (10/~g/mi) or apical media (20 ~g/mi) and the cells incubated
for 4 h at 17°C. Cells were then washed and incubated at 37°C for different
Materials and Methods periods of time, under microtubule-depolymerizing conditions (as de-
scribed earlier). Other markers, holo-dog transfcrrin-FITC (50 /~g/mi),
slairetaM ricin-FITC (10 #g/ml), or BSA-FITC (5-6 mg/mi) were added simultane-
ously with pIgA-R ligand-Cy3 to either the basolateral or the apical media.
Nocodazole, ricin-FITC, and ricin-HRP were purchased from Sigma Double-labeling fluorescence confocal microscopy was used to analyze the
Chem. Co. (St. Louis, MO). FITC was obtained from Molecular Probes, colocalization between their staining patterns, under different internaliza-
Inc. (Eugene, OR). 3HSodium borohydride with a specific activity of tion conditions. The cells were washed on ice five times with PBS ++ for 5 D
o
50.4 Ci/mmol was obtained from New England Nuclear (Boston, MA). rain, and then fixed for 01 rain in 3% paraformaldehyde in PBS at room wn
Cy3 fluorochrome was purchased from Biological Detections Systems. temperature. Cells were washed extensively with PBS, the filters removed loa
Affinity-purified anti-goat IgG was from Cappel Labs. (Cochranville, PA). and mounted in 50% glycerol in PBS. Confocal imaging was performed ded
Anti-goat Fab2 were purchased from Jackson ImmunoReseareh Labs., using an MRC 600 confocai microscope mounted on a optiphot II Nikkon fro
Inc. (West Grove, PA). with a 60x oil immersion lens BioRad Labs. (Hercules, CT). Differences m h
between the photomultiplier tube sensitivity of the two different fluoro- ttp
Culture of MDCK Cells chromes emission spectrum were compensated during the collection of the ://ru
data to obtain images of equivalent brightness. Black and white photo- pre
MUCDSCFK) were cells growenx pressing in polycarbonate rabbit pIgA-R filters (kindly (0.4 prov#imde d pore sibzey; Dr. 24-ram K. Mostov, diam) gKordaakp.h s were Color obtained photographs using a were Screen-Star obtained system using and the T-sMaaxm e system 400 films but fromwi th ss.org
fired within Transwell chambers (Costar Corp., Cambridge, MA) or in tis- EKTAR-100 film from Kodak. /jcb
/a
bsruaen es culture as a insecretlsl substrate. (Nunc, IncT.,h ese Naperville, membranes IL) have which the usaed vantage Anapore of membe-i ng rticle
Results -p
transparent when wet. No difference was observed in the results obtained d
from cells grown on either membranes. The cells were cultured at 37°C, f/12
4
in 5 % CO2 atmosphere and in DMEM containing 10% FBS, penicillin and C°71 Block sparT citotycsnarT ograC in an yUacipA /1
/8
streptomycin. One confluent 100-ram plate was used to plate a 6-well Trans- Localized Compartment 3/1
well and cells were used for transcytosis experiments after 3 d of growth. 26
0
Nunc tissue culture inserts were used in 24-well plates, under the same plat- To analyze the pathway traversed by basolaterally internal- 93
ing conditions. As shown by Fodbtlowicz and MeUman (1990), apical inter- ized transcytotic cargo after its sorting from the basolateral 0/8
3
nalization of radiotabeled dog holo-transferrin was <5 % of basolateral in- endosome and before its release into the apical medium, we .pd
ternalization, indicating that MDCK cells form polarized PM domains 3 d f b
after plating. used a 17°C temperature block which has been shown to al- y g
low transcytotic cargo sorting and its microtubule-dependent ue
In Vivo sisotycsnarT Assay movement from the basolateral to the apical region of cells, st on
0
but prevents its apical release. To examine the compartment 1
Fab2 fragments of antibodies raised against the extracellular domain in which transcytotic cargo accumulates during the 17°C Jan
(secretory component) of the rabbit plgA-R were used as a reporter mole- ua
cule (Breiffeld et al., 1989). Affinity-purified immunoglobulins were sub- block, Cy3-1abeled pseudo-ligand was internalized at 17°C ry 2
jected to pepsin digestion to generate Fabh fragments. The ll0-kD frag- for 4 h from the basolateral surface of MDCK cells trans- 02
3
ments were then delebaloidar using detaitirt sodium borohydride (Rice and fected with rabbit pIgA-R and grown on filter supports.
Means, .)1791 A lacipyt noitaraperp had a cificeps ytivitca of 2-I x 60I
MDCK cells were polarized and formed a tight monolayer
cpm/~g.
as assayed by polarized transferrin internalization and by
Radiolabeled Fab2 fragments were added (5/~g/ml) to the basolaterai
media of the Transwell chambers in DMEM containing 20 mM Hepes, pH impermeability of the monolayer to WGA (data not shown).
7.3, and 0.5 % BSA. After internaiization at 17°C for 4 h, cells were washed After internalization, the cells were washed, processed for
extensively with PBS ++ (containing Mg +2 and Ca )+2 and further incubated
fluorescence microscopy, and examined for distribution
with nocodazole (33/~M) during I h on ice, to depolymerize the microtubu-
of ligand-Cy3 using a confocal microscope. 18 horizontal
lar network (Hunzikcr et ai., 1990). Cells were then incubated at 37°C for
different periods of time in the presence or absence of nocodazole. planes (1/~m apart) spanning the total height of the cells were
Immunoprecipitatiun with affinity-purified anti-goat IgG and protein obtained. The average height of the filter-grown cells was of
A-Scpharose was used to recover the Fabh fragments released into the ap- 15-20/~m, which is within the range reported by Bacalao
icai media. The immunoprecipitates and the remaining filters were counted,
and Stelzer (1989) for a fully polarized cell monolayer. The
and the amount of label recovered represents the ligand intcrnaiized into the
transcytotic pathway. To show that the transport of the Fabh fragments pIgA-R ligand panels in Fig. 1 A show 9 of the original 18
across the cells was a specific citotycsnart event catalyzed by the pIgA-R, focal planes, each "~2/zm apart, scanning the cells from the
we performed an analogous internalization experiment with MDCK cells apical 1( #m from the apical PM) to the basolateral side 71(
not transfected with pIgA-R. Non-transfected cells internalized low amount m~/ from the apical PM). pIgA-R ligand internalized into
of ligand (<2%) and did not transport the Fab2 fragments to the apical
cells at 17°C is present in punctate structures distributed
surface of the cells.
BFA was used at a concentration of 10/~M throughout the 37°C incuba- throughout the cell, but concentrated in the apical (-1 to -5
Barroso and Sztul Transcytosis in Polarized Epithelia 85
erugiF .1 plgA-R~ligand and
transferrin are distinct in com-
- the after partments 17°Cinter
nalization period. )A( MDCK
cells internalized plgA-R li-
gand-Cy3 dna CTIF-nirrefsnart
for 4 h at 17°C from the baso-
lateral surface. The cells were
for processed then lebal-elbuod
Con- microscopy. fluorescence
focal microscope was used
to scan the MDCK cell layer
between axis, vertical its along
the apical PM and the baso-
lateral PM, generating 81
apart) hori- consecutive (1/~m D
o
zontal focal planes. 9 of the w
n
original 81 focal planes are loa
d
shown representing the top/ ed
apical (-I to -5 #m which fro
m
correspond to 1-5 m~t from h
ttp
PM), medial the apical the to -11 the and pan) -osab/rewol (-7 ://rup
re
lateral gions of (-13 the ceils. A double to -17 /~m) re- ss.org
filter was used to scan simul- /jcb
dnagtaneously il panels) for the dna Cy3 Flrrc (plgA-R (trans- /article
-p
ferrin panels) signals. Bar, 01 df/1
#m. (B) Enlargements of two 24
/1
planes focal are shown, repre- /8
3
the top/apical senting (-2 #m) /1
2
6
and the medial (-7 #m) re- 0
9
3
gion of the cells. The yellow 0
/8
color in the merge panels (ar- 3.p
rows) indicates overlap be- df b
tween the pIgA-R ligand-Cy3 y g
u
rin-FITC (red) signal and the transfer- (green) signal. Bar, est on
0
01 #m. 1
Ja
n
u
a
ry 2
/zm) region of the cells and less common in the medial (-7 riod, no ligand is released into the apical media (data not 02
3
to -11 #m) and in the basolateral (-13 to -17 t,m) regions shown). The samea mount of ligand is transcytosed when the
of cells. The presence of ligand within the apical regions of internalization was performed at 37°C for 10 min but the re-
the cells indicates that translocation from the basolateral to lease shows a slight lag time in comparison to the 17°C inter-
the apical region is not inhibited by the 17°C block (although nalization. Basolateral recycling of ligand internalized at
it might be slowed) and is in agreement with the findings of 17°C occurs faster than transcytosis, with close to 30% of
Hunziker et al. (1990) showing that in cells loaded under the the internalized plgA-R ligand reaching the basolateral me-
same conditions with dlgA, the majority of dlgA-containing dium after 60 min chase at 37°C (Fig. 2 B). Similar recy-
structures accumulate in the upper part of the cell. cling kinetics are observed when ligand internalized at 37°C
To examine whether ligand accumulated at 17°C is capable is analyzed: slight increase in the overall recycling (29% at
of reaching the apical PM, we compared the release of radio- h 1 and 38 % at 5 h) is observed. The slow rate oftranscytosis
labeled plgA-R ligand into the apical and basolateral media observed in our system is probably due to technical reasons,
after internalization either at 17°C for 4 h or at 37°C for 10 most likely a result of prolonged washing on ice which we
min and subsequent chase at 37°C. As shown in Fig. 2 A, use to remove unbound ligand after the internalization pe-
~48 % of plgA-R ligand internalized at 17°C is transcytosed riod. We also need to stress that direct comparison between
and released into the apical media when the cells are incu- rates of transcytosis obtained in different studies is difficult
bated at 37°C for 5 h. During the 17°C internalization pe- since transcytosis has been studied using different types of
ehT lanruoJ of lleC ,ygoloiB emuloV ,421 4991 68
The Apical Transcytotic Compartment Is Not a
Basolateral Endosome
A 5o Since the basolateral surface of MDCK cells extends up to
37°C 10mln ~5/~m from the apical PM (where tight junctions are local-
C°71 4h ized when assayed by WGA apical binding at low tem-
"~,E 40 perature-data not shown), it was possible that the pIgA-R
=_.~°l
ligand trapped at 17°C was in the basolateral endosomes
localized above and around the nucleus. We therefore deter-
mined the accessibility of the apical transcytotic compart-
e- o ment to transferrin, a protein shown to be endocytosed and
~,o recycled through the basolateral endosome in MDCK cells
(Fuller and Simons, 1986; Podbilewicz and Mellman,
~.c_ 1
o 1990). pIgA-R ligand and canine holo-transferrin, labeled
~o with different fluorochromes (Cy3 and FITC, respectively),
were cointernalized from the basolateral surface of MDCK
I I I I
100 200 300 400 cells for 4 h at 17°C. The cells were then washed, processed
for double-label fluorescent microscopy, and analyzed using
Time at 37°C (rain)
a confocal microscope (Fig. 1 A, transferrin panels). Trans-
ferrin was mainly localized in the apical/medial (-3 to -9
)m~/ part of the cells, consistent with its published distribu-
B
tion within polarized cells (Hughson and Hopkins, 1990). To D
o
w
examine more closely the pIgA-R ligand containing- and the n
lo
50 ~ transferrin containing-compartments, all focal planes were ade
d
examined at higher magnification and the level of colocaliza- fro
_~s 4o tion analyzed by merging the Cy3 (pIgA-R ligand) and the m h
C0370C 71 4h lOmln FfIrToCm either (transferrin) the top/apical signals. (-2/~mTwo representative panel) or the focal medial planes (-7 ttp://rup
!::_• ,o"~ 30 #m panel) regions of the cells are shown in Fig. 1 B. pIgA-R ress.o
ligand and transferrin can be detected in both regions of the rg
~mO 20 cells but the amount of transferrin in the top/apical area is /jcb
/a
- "~~-Qo 10 ethxattr emely region low. does More not colocalizes ignificantly, with transferrin the pIgA-R present ligand within (Fig. rticle-pd
o<. = - 1 B, -2/zm panel, .)sworra Similarly, no colocalization was f/12
4
seen in planes taken between 1 and 4 #m from the apical PM /1
/8
I I i i (data not shown). Transferrin is more abundant in the medial 3/1
100 200 300 400 2
portion of the cell (5 #m-ll mx/ from apical PM), and as 60
9
Time at 37°C (min) shown in Fig. 1 B, -7/zm panel, a proportion of it colocal- 30/8
izes with the plgA-R ligand (arrows). A similar level of 3.p
erugiF .2 Quantitation of the transcytotic and recycling syawhtap colocalization was observed in planes taken between 5 and df b
of plgA-R ligand. Radiolabeled plgA-R ligand saw internalized 11/zm from the apical PM (data not shown). The colocaliza- y gu
)selcricfrom the or basolateral for 01 min surface at 37°C of MDCK nepo( cells .)selcric for 4 Cells h at 17°C were krad( then tainodn tmraonsstf errin likely in reflects the basolateral the presence endosomal of both plgA-R compartment ligand est on 0
washed and transferred to 37°C for different periods of time. The 1
apical and basolateral medium were collected and -ipicerponummi before they are sorted out into different pathways. This is Jan
tated or basolateral with anti-ligand medium antibodies. saw plotted Radioactivity as % of total released label into present apical in cfornascitstieonnt pepared with the from recent rat liver finding contains that an both early the endosomal plgA and uary 20
2
both media and in cells on filters. )A( Basolateral-to-apical transcy- the transferrin receptors (Casciola-Rosen and Hubbard, 3
tosis (ligand released into the apical media). )B( Basolateral recy- 1992). The data presented in Fig. ,1 A and B show that the
cling (ligand released into the basolateral media). majority, if not all, of apical (within 4 #m from the apical
PM) structures containing plgA-R ligand internalized at
17°C are distinct from the transferrin containing basolateral
endosome.
plgA-R ligands i.e., dIgA, monomeric Fab or dimeric Fab
and it appears that different ligands might be handled differ-
The Apical Transcytotic Compartment
ently by MDCK cells (Hirt et al., 1993; Bomsel, M., per-
Is Not Accessible to ApicaUy Internalized
sonal communication).
Fluid-Phase Marker
To ensure that the internalized ligand was not degraded,
we measured the amount of label recovered during immuno- Since MDCK cells contain distinct basolateral and apical
precipitation of ligand (intact ligand) from media and cells endosomal compartments, we used BSA-FITC as a fluid-
and compared it to the amount of label recovered in the su- phase marker to analyze the relationship between the apical
pernatant after the immunoprecipitation (degraded ligand). transcytotic compartment and the apical endocytic pathway.
Less than 5 % of the label was recovered in the supernatant plgA-R ligand labeled with Cy3 was internalized from the
and might represent degradation products. basolateral surface of the cells for 4 h at 17°C. During the
Barroso and Sztul Transcytosis in Polarized Epithelia 87
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The Journal of Cell Biology, Volume 124, 4991 88
last 2 h of the internalization period, BSA-FITC was added and the apical endosomal compartment involved in mem-
to the apical surface of the same cells. As shown previously, brane traffic, we used ricin, a 60-kD lectin which binds
fluid-phase endocytosis and recycling occurs at 17°C but de- to galactose residues of membrane glycoproteins and glyco-
livery of internalized material to late compartments of the lipids and thereby acts as a non-specific probe for mem-
endosomal/lysosomal pathway is inhibited (Dunn et al., brane flow. Ricin can be used as a general marker for en-
1980; Bomsel et al., 1989). Consequently, BSA-FITC inter- docytosis and recycling of apically internalized membrane
nalized at this reduced temperature will fill only the early en- proteins since van Deurs and co-workers (van Deurs et al.,
dosomal compartment. After internalization, the cells were 1990; Prydz et al., 1992) have previously demonstrated that
washed and processed for double-label fluorescence micros- the majority of apically internalized ricin recycles back to the
copy. The apical regions (-1 to -4/~m from the apical PM) apical PM, while a small percentage is transcytosed to the
of the cells were scanned by confocal microscopy to obtain basolateral PM or transported to the lysosomes and the Golgi
four focal planes. complex.
As shown in Fig. 3 A, both plgA-R ligand-Cy3 and BSA- MDCK cells internalized plgA-R ligand-Cy3 from the
FITC are present in the top/apical region of the cells, in basolateral surface for 4 h at 17°C. During the last 2 h, ricin-
compartments similar in morphology and localized in close FITC was internalized from the apical surface of the cells.
proximity. In general, BSA-FITC-containing structures are The cells were processed for double-label fluorescence mi-
found in the periphery of the cells while plgA-R ligand-Cy3 croscopy and the apical (1-4/~m from the apical PM) region
containing compartments accumulate in a more central re- of the cells was then analyzed using a confocal microscope.
gion. The peripheral localization of the fluid-phase marker A representative focal plane is presented. As shown in Fig.
is consistent with its inability to reach late endocytic com- 3 C, both plgA-R ligand and ricin are detected in the apical
partments at 17°C (Dunn et al., 1980; Bomsel et al., 1989). region of the cells in morphologically similar structures. A
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When the signals from the two fluorochromes were merged, merge of the Cy3 and FITC signals results in marked w
n
the level of colocalization between the compartments was colocalization between the ricin-FITC and plgA-R ligand- loa
d
very low indicating that the transcytotic compartment does Cy3 containing compartments (arrows). Although the co- ed
not colocalize with fluid phase markers present within the localization is not complete, it is significantly higher than the from
h
apical early endosome. colocalization of BSA-FITC and plgA-R ligand-Cy3 inter- ttp
To examine the relationship between the transcytotic com- nalized under the same conditions (compare Fig. 2, A and ://ru
p
partment and the late endosomal and lysosomal systems, we B, merge panels). These results indicate that the apical trans- re
compared the localization of the transcytotic compartment cytotic compartment overlaps with an apical membrane- ss.o
rg
(marked by plgA-R ligand internalized for 4 h at 17°C) to recycling compartment and that apically internalized fluid- /jcb
tnahlaitz ed of late during endosomes subsequent and lysoscohmasees for 1 (containing h at 37°C). BSA The inter- fluid pahpaicsael anedn dosomal membrane-bound system. markers segregate within the /article
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phase BSA was internalized from the apical surface since it df/1
has been shown that cargo endocytosed for more than 30 min The Apical Transcytotic Compartment Is Accessible to 24
/1
at 37°C from either surface of MDCK cells enters a shared Apically Internalized plgA-R Ligand /83
/1
late endosomal and lysosomal system and thus the internal- pIgA-R ligand is internalized from both the basolateral and 26
0
ized BSA will label the entire cellular late endosomal- the apical PM domains in polarized MDCK cells transfected 930
lysosomal system (Bomsel et al., 1989; Patton et al., 1989). with pIgA-R (Breitfeld et al., 1989). However, after endocy- /83
.p
As shown in Fig. 3 B, no colocalization was observed, indi- tosis, the ligand follows distinct transport pathways: while df b
cating that the transcytotic compartment is distinct from late basolaterally internalized pIgA-R ligand is predominantly y g
u
endosomes or lysosomes. transcytosed, the majority of apically endocytosed pIgA-R est o
The Apical Transcytotic Compartment Is Accessible to ligand recycles back to the apical PM (Breitfeld et al., 1989). n 01
Apically Internalized Ricin Therefore, pIgA-R ligand internalized from the apical sur- Jan
face of MDCK cells can be used as a specific marker for the ua
It has been shown previously that fluid-phase markers are apical receptor-mediated recycling pathway. To examine the ry 2
0
sorted from cointernalized membrane-bound markers within relationship between the transcytotic and the apical pIgA-R 23
the early endosome (Maxfield and Yamashiro, 1991). Since recycling compartments, MDCK cells internalized pIgA-R
pIgA-R ligand remains bound to its receptor during transcy- ligand-Cy3 basolaterally and pIgA-R ligand-FITC apically
tosis it is, in effect, a membrane-bound marker. Thus, we for 3 h at 17°C. The ceils were then washed and processed
posited that plgA-R ligand might be present within an api- for double-label fluorescence microscopy. Four focal planes
cal endosomal compartment containing membrane-bound ,1( 2, ,3 and 4 #m from the apical surface) were obtained and
markers sorted away from fluid-phase material. oT examine are shown in Fig. 4. Basolaterally internalized pIgA-R ligand
the relationship between the apical transcytotic compartment is detected predominantly 3 and 4 #m below the apical PM
erugiF .3 The apical transcytotic compartment si selectively accessible to apically added markers, plgA-R ligand-Cy3 saw internalized
for 4 h at 17°C from the basolateral surface of the MDCK cells. CTIF-ASB saw added to the apical surface of the cells during the last
2 h of the internalization period )A( or during 1 h at 37°C chase after the internalization period (B). In ,C ricin-FITC saw added to the
apical surface of the cells during the last 2 h of the internalization period. The ceils were then processed for double-label fluorescence
.ypocsorcim Confocal microscope saw used to scan the top/apical (1-4 #m from the apical PM) regions of the cells and a evitatneserper
focal plane is shown. Merge panels were produced by overlapping the Cy3 and the FITC signals. Bar, 01 #m.
osorraB dna lutzS sisotycsnarT ni deziraloP ailehtipE 98
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.4 erugiF(added ligand-Cy3 Basolaterally to (added ligand-FITC the basolateral surface) and pIgA-R dna apically internalized ligand plgA-R colocalize in an apical compartment. MDCK ceils internalized plgA-R to the apical surface) for 3 h at 17°C. The cells then were uary 20
2
processed for microscope Confocal microscopy. fluorescence double-label saw used to the scan top/apical (1-4 mzt from the apical PM) 3
regions FITC the and Cy3 the overlapping ofby produced were panels Merge shown. are obtained planes focal four the and cells the .slangis
Bar, 01 #m.
while apically internalized pIgA-R ligand is concentrated in the basolateral surface at 17°C is delivered to an apical
more apical regions. Merging the Cy3 and the FITC signals transcytotic compartment which is not accessible to fluid-
shows significant colocalization of the two markers )sworra( phase marker internalized from the apical PM but which
in all except the most apical (-1 /~m) focal plane .)sworra( receives apically endocytosed membrane-associated mark-
It appears, therefore, that the apical transcytotic compart- ers. These results suggest that the apical endosomal system
ment and the apical receptor-mediated recycling compart- is divided into distinct compartments, one handling fluid
ment overlap within the apical region of the cells. phase cargo and another involved in membrane traffic. To
test this hypothesis, plgA-R ligand-Cy3 and BSA-FITC were
cointernalized from the apical surface of MDCK cells at
Apically Internalized Fluid-Phase Marker and
17°C for 2 h. The cells were then processed as above and
Apically Internalized plgA-R Ligand Are Present in
the apical region of cells examinedb y confocal microscopy.
Distinct Apical Endosomal Compartments
As shown in Fig. 5 A, both plgA-R ligand and BSA are pres-
We have shown above that pIgA-R ligand internalized from ent within the apical region of thec ells but the compartments
The Journal of Cell Biology, Volume 124, 1994 90
erugiF .5 plgA-R ligand and
a fluid-phase marker coin-
ternalized from the sur- apical
face are present in distinct
apical compartments, plgA-R
ligand-Cy3 and BSA-FITC
were added to the apical sur- D
o
face of inter- and cells MDCK wn
nalized for 2 h at 17"C )A( or loa
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for 01 min at 37"C (B). The ed
cells were then processed for from
mi- double-label fluorescence h
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croscopy. Confocal micro- ://ru
scope saw used to scan the p
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top/apical region (1--4 #rn ss.o
from the apical PM) of the rg
cells and a representative fo- /jcb
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over- by produced were panels cal plane is shown. Merge rticle-p
FITC the and Cy3 the lapping df/1
signals. Bar, 01 .m~t 24
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containing the two ligands, although in close proximity, do into Entry the Apical citotycsnarT Does Compa:tment 0/8
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not show significant colocalization. The results suggest that Not Involve gnilcyceR from Apical the PM .pd
the separation of markers occurs within the early endosome f by g
since the internalization is carried out at 17°C, the tempera- ue
ture at which endocytosed proteins should be prevented from The data presented above suggest that the basolateral to api- st o
n
moving into late endosomes. However, to ensure that the cal transcytotic pathway includes movement of transcytotic 01
separation is not due to BSA-FITC partially bypassing the cargo through an apical endosome before the cargo is deliv- Jan
u
a
temperature block and gaining entrance into the late endo- ered to the apical PM. However, a major concern in inter- ry 2
somes, we repeated the experiment at 37°C. pIgA-R ligand- preting such studies is to distinguish between a situation in 02
3
Cy3 and BSA-FITC were internalized at 37°C for 10 min, which transcytotic cargo enters the apical recycling endo-
a time period sufficient for BSA-FITC to fill the early endo- some before being delivered to the apical PM from one
somes butn ot the late endosomes (definaesd a compartment where pIgA-lRi gand first moves to the apical PM and is then
containing mannose 6-P receptor) (Bomsel et al., 1989). As reendocytosed from the apical surface and delivered to the
shown in Fig. 5 B, plgA-R ligand and BSA were present in apical early endosome. To examine whether entry into the
neighboring compartments but a merge of the Cy3 and FITC apical endosome occurs directly or via reendocytosis, we
signals did not show coloealization. The data suggest that added Cy3 labeled plgA-R ligand basolaterally and FITC-
markers internalized from the apical surface are rapidly labeled anti-ligand ([Fab]2 fragment of rabbit IgG raised
sorted within the apical early endosome and are restricted against goaItg G)a pically and incubated the cells for various
to eithetrh e fluid-phase or the membrane recycling compart- times and at different temperatures. Anti-ligand-FITC will
ments. This segregation phenomenon has been observed in be internalized only if it can bind to ligand exposed on the
electron microscopic analyses of receptor-ligand dissocia- apical PM and be endocytosed along with it; we have previ-
tion after endocytosis in nonpolarized cells or uptake from ously determined that anti-ligand is not internalized when
the basolateral PM (Geuze et al., 1984; Mueller and Hub- added to the apical surface of cells in the absence of ligand
bard, 1986; Dunn eta l., 1989). It appears that the apical en- (data not shown). As shown in Fig. 6 A, when the incubation
dosome behaves the same way and effectively segregates with basolaterally added ligand and apically added anti-
markers into specific sub-domains. ligand was done at 17°C for 4 h, no significant FITC staining
Barroso and Sztul Transcytosis in Polarized Epithelia 91
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erugiF .7 Incubation of presence the in at ceils 37°C of nocodazole ad
e
allows exit of transcytotic cargo from the apical compartment. d fro
ligand-Cy3 MDCK ceils plgA-R internalized from the basolateral m
h
surface for 4 h at .C071 The cells were then supplemented with ttp
shifted and nocodazole to 37"C for (,4) 1 or h 5 (B). The were cells ://ru
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processed for described as microscopy fluorescence confocal in the re
legend to Fig. .1 Bar, 10 #m. ss.o
rg
/jcb
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rticle
To ensure that endocytosis of the anti-ligand is totally de- -pd
f/1
pendent on the availability of the ligand, we repeated the 2
4
erugiF .6 Entry of into ligand the transcytotic apical plgA-R com- above experiments but added the anti-ligand to the basolat- /1/8
3
partment does not reendocytosis involve from the apical surface. eral side of MDCK cells and the ligand to the apical side. /1
2
plgA-R ligand-Cy3 00 #g/ml) saw added to the basolateral sur- Since pIgA-R ligand added to the apical side recycles only 609
face and anti-ligand-FITC (20 #g/ml) saw added to the apical to the apical surface, it should never be exposed to the anti- 30/8
surface of MDCK cells. The either cells were incubated for 4 h at 3
ligand added to the basolateral media. As shown in Fig. 6, .p
saw C071 added )4,( or ftoor ligand-Cy3 h 2 at the 37°C plgA-R (B). Alternatively, apical surface and anti-ligand-F1TC to the C and D, after incubation at either 71 or 37°C, respectively, df by g
basolateral for surface at h 17°C 4 (C) or of for MDCK ceils and h 2 at 37"C (D). the The cells incubated processed were ceils either ninod icate Frrc that staining pIgA-Rl igand was detected internalized inside the from cells. the Tbhaessolea teral data uest on
for double-label fluorescence microscopy. Confocal microscope surface at 17°C enters the apical endosome without prior de- 01
saw used to the scan top/apical region 4--1( #m for the apical PM) livery to the apical PM. Jan
u
oconsists f the ceils and a representative plane focal is shown. Each panel of a double image showing the ligand-Cy3 staining (/eft Analysis of Transcytotic Ligand Movement from the ary 20
2
)lenap staining anti-ligand-FITC the and thgir( .)lenap Bar, 01 #In. Apical Compartment to tAhpei cal Medium 3
To examine only the late steps of transcytosis, we dissected
was detected inside the cells suggesting that the ligand does the overall pathway into the late stages (exit of transcytotic
not reach the apical PM. cargo from the apical transcytotic compartment and its deliv-
To show that the ligand and anti-ligand can interact with ery to the apical PM) and the early stages (delivery into the
each other, we incubated ceils with basolaterally added apical transcytotic compartment). We preloaded the apical
ligand and apically added anti-ligand for 2 h at 37°C. As compartment with ligand internalized from the basolateral
shown previously in Fig. 2 A, ,,020% of transcytotic ligand surface at ,21°71 and then chased the ligand from that com-
reaches the apical PM after a 2-h 37°C incubation. Since api- partment by shifting the cells to 370C in the presence of a
cal recycling of ligand has been shown to occur (Breitfeld et micmtubule-disrupting drug, nocaxiazole. In the presence of
al., 1989; Hunziker et al., 1990), we expected that the anti- nocodazole,t ranscytotic cargo within the apical transcytotic
ligand would interact with the ligand at the apical PM and compartment (this cargo is already past the microtubule-
be endocytosed together. As shown in Fig. 6 B, significant requiring step) will be able to move to the apical PM (this
amounts of anti-ligand are present within the apical region step does not require microtubules; Hunziker et al., 1990;
of the cells and show complete colocalization with the ligand Breitfeld et al., 1990) but cargo present in proximal com-
.)sworra( partments will not enter the apical transcytotic compartment
The Journal of Cell Biology, Volume 124, 1994 92