Rab35 and Its GAP EPI64C in T Cells Regulate Receptor
Recycling and Immunological Synapse Formation*□
Genaro Patino-Lopez‡1, Xiaoyun Dong‡1, Khadija Ben-Aissa‡, Kelsie M. Bernot§, Takashi Itoh¶?, Mitsunori Fukuda¶?,
Michael J. Kruhlak‡, Lawrence E. Samelson§, and Stephen Shaw‡2
TohokuUniversity,Aobayama,Aoba-ku,Sendai,Miyagi 980-8578, Japan
Upon antigen recognition, T-cell receptor (TCR/CD3) and
tact site between the T cell and antigen-presenting cell, i.e. the
immunological synapse (IS). Enrichment occurs via mecha-
nisms that include polarized secretion from recycling endo-
somes, but the Rabs and RabGAPs that regulate this are
unknown. EPI64C (TBC1D10C) is an uncharacterized candi-
date RabGAP we identified by mass spectrometry as abundant
in human peripheral blood T cells that is preferentially
both on in vitro Rab35-specific GAP activity and findings in
constructs each impaired transferrin export from a recycling
transferrin receptor, TCR, and SNAREs implicated in TCR-po-
larized secretion. Rab35 localized to the plasma membrane and
to intracellular vesicles where it substantially colocalized with
TfR and with TCR. Rab35 was strongly recruited to the IS. Con-
jugate formation was impaired by transfection with Rab35-DN
or EPI64C and by EPI64C knock down. TCR enrichment at the
IS was impaired by Rab35-DN. Thus, EPI64C and Rab35 regu-
late a recycling pathway in T cells and contribute to IS forma-
tion, most likely by participating in TCR transport to the IS.
The immunological synapse (IS)3is a specialized contact
between T lymphocytes and antigen-presenting cells (APC)
tors, and signaling and cytoskeletal components (1–7). Enrich-
tion at the contact region (i.e.“mutual co-capping”) (8, 9).
Furthermore, submembranous flow of the actomyosin cortex
contributes to molecular concentration in the IS (10, 11).
Recent data indicate that polarized exocytosis from a rapid
recycling pathway is particularly important for the enrichment
of some synapse components, including TCR (12–16).
Rab proteins are major intracellular transport regulators in
eukaryotes; they function in vesicle formation, motility, dock-
ing, and fusion (17, 18). Over 60 mammalian Rab proteins have
been identified, and each is thought to regulate distinct intra-
ation with various interacting proteins. Guanine exchange fac-
between active GTP-bound and inactive GDP-bound Rab pro-
teins. Although the TBC (Tre/Bub2/Cdc16) domain is a hall-
mark of RabGAPs (19), few of the more than 50 putative TBC
domain-containing proteins present in the human genome
cess was identification of EPI64 (EBP50-PDZ interactor of
64kD) as a GAP specific for Rab27a (20).
EPI64 is a broadly expressed TBC domain-containing protein
first identified in placental microvilli (21). Recent studies have
two paralogs in mouse and human. One of these, EPI64B
EPI64C (mFLJ00332), is more divergent (only 60% identity in the
TBC domain) and lacks the capacity to regulate Rab27a in mela-
hematopoietic cells and especially primary T cells and demon-
strate that EPI64C is a GAP for Rab35. EPI64C and Rab35 both
tion analysis, transfection studies, and knock down show that
T cells and APC in a manner indicative of regulating polarized
Antibodies, Reagents, Cells, and Replication—Anti-Rab35
tut Curie, Paris), rabbit anti-EPI64C antibody raised against
* This work was supported, in whole or in part, by the National Institutes of
Health NCI Intramural Research Program. The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
supplemental Figs. S1–S8 and a movie.
1Both authors contributed equally to this work.
2To whom correspondence should be addressed: National Institutes of
Health, Bldg. 10, Rm. 4B36, 10 Center Dr., MSC 1360, Bethesda, MD 20892-
1360. Tel.: 301-435-6499; Fax: 301-496-0887; E-mail: firstname.lastname@example.org.
3The abbreviations used are: IS, immunological synapse; APC, antigen-pre-
green fluorescent protein; SEE, staphylococcus enterotoxin E; SNARE, sol-
uble NSF attachment protein receptor; TBC, Tre/Bub2/Cdc16; TCR, T-cell
receptor; TfR, transferrin receptor; mRFP, monomeric red fluorescent
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anti-EEA1, anti-LAMP-2, anti-calnexin, and anti-TfR were
Bethesda, MD), anti-?-tubulin antibody was from Sigma-Al-
antibodies were Alexa Fluor 546 phalloidin, goat anti-mouse
IRDye 680 and goat anti-rabbit IRDye 800CW (LI-COR, Lin-
coln, NE). Other reagents were lucifer yellow (Sigma), staphy-
kat Tag cells were kindly provided by Dr. Gerald Crabtree
(Stanford University); Raji B-lymphoma cells and human
embryonic kidney 293 cells were from ATCC (Manassas, VA).
Primary human T lymphocytes were isolated from the blood of
healthy human volunteers by leukapheresis and elutriation or
immunomagnetic negative selection (24, 25). Unless otherwise
indicated, all results shown are representative of at least three
8a, 8b, 9b, 10, 11a, 11b, 14, 21, 27a, 35, TCR-?, Vamp3, SNAP3,
and syntaxin4 were amplified from Jurkat Tag RNA (RNAeasy
Mini kit; Qiagen, Inc.) by reverse transcription PCR (Super-
Script One-Step RT-PCR for Long Templates; Invitrogen) and
cloned into pENTR vectors by recombination (pENTR/D-
TOPO cloning kit; Invitrogen). EPI64C (R141K), Rab35 (S22N
or Q67L), and Rab27a (T23N or Q78L) were generated using
QuikChange site-directed mutagenesis (Stratagene); mutants
into Gateway destination vectors from the Protein Expression
Laboratory (NCI-Frederick, National Institutes of Health, Fred-
erick, MD) (pDest732 (N-terminal GFP tag), pDest733 (N-termi-
nal monomeric red fluorescent protein (mRFP) tag), pDest472
(C-terminal GFP tag for TCR-?)) by an LR reaction according to
the manufacturer’s recommendations (Invitrogen) to construct
mammalian expression vectors. The TCR-? chain was also sub-
Western Blotting and Analysis—Cells were lysed in 1?
NuPAGE LDS sample buffer (Invitrogen) containing 20 mM
dithiothreitol (Sigma), sonicated three times for 10 s (Ultra-
sonic Processor GE130; Sonics & Materials, Inc., Newtown,
CT), and heated at 70 °C for 7 min. Equal amounts of protein
(45 ?g) from each preparation were resolved by 4–12% SDS-
NuPAGE gels, transferred to nitrocellulose membranes, and
analyzed by Western blot using an Odyssey Infrared Imaging
System (LI-COR Biosciences).
Conjugate Formation—Conjugate formation was deter-
mined by flow cytometry. Human Jurkat cells were transfected
with plasmids encoding tagged proteins as indicated in each
figure legend. The next day, Raji cells were loaded with 0.5 ?M
Cell Tracker carboxyfluorescein succinimidyl ester (Invitro-
with 5 ?g/ml SEE, and washed. Jurkat cells and Raji APCs were
then mixed at a ratio of 1:1 (1.0 ? 106cells), centrifuged at
?18 ? g for 10 s, and incubated for 30 min at 37 °C in 5 ml of
polystyrene round-bottom tubes. Cells were fixed for 10 min
with 2% paraformaldehyde. After gentle resuspension, cells
lyzed with Flowjo 7.2 (Tree Star, Inc., Ashland, OR). Conjugate
formation was determined as the fraction of RFP? events that
were also carboxyfluorescein succinimidyl ester-positive. Sta-
tistical significance was assessed by one-tailed paired Student’s
t test. For imaging studies, Raji cells were stained with Cell-
Tracker Blue, and conjugates were formed in similar manner
and analyzed by confocal microscopy as below.
Microscopy—Jurkat cells transfected with mRFP- and/or
GFP-tagged plasmids were allowed to interact for 10 min with
poly-lysine-precoated glass coverslips (MatTek Corporation,
Ashland, MA) and then were either fixed for 10 min with 4%
paraformaldehyde or permeabilized for 10 min with 0.1% Tri-
ton X-100; in some studies cells were also stained with mouse
anti-?-tubulin antibody, followed by goat anti-mouse IgG-Al-
exa 647 (Invitrogen) at room temperature for 1 h or the culture
medium was replaced with imaging medium (RPMI without
imaged with a Zeiss LSM 510 META confocal microscope
using a ?100 (N.A. 1.4) oil immersion objective lens (Zeiss,
with a Perkin-Elmer Ultraview spinning disc confocal system.
Images were captured with an Orca-ERII CCD camera
(Hamamatsu). A hot air blower and an objective warmer were
used to maintain live samples at 37 °C. Multicolor imaging was
done using multitrack configuration (filters, laser power, and
detector gain) that gives no detectable bleed-through between
channels. For live imaging during IS formation Raji cells were
pulsed with 2 ?g/ml SEE for 15 min at 37 °C and plated on Lab
Tek chambers in RPMI. For detection and quantification of
TCR accumulation at the synapse, conjugates were fixed with
anti-mouse IgG-Alexa 488 without detergent to detect surface
each field with conjugates and quantitation performed using
selection of the optical slice with highest green fluorescence in
the contact area, the mean green intensity was measured along
the plasma membrane at the contact site and at a comparable
area along the plasma membrane elsewhere in the cell (where
results was done “blind” in the sense that the scoring observer
did not know the identity of the constructs transfected. Statis-
tical comparisons were carried out by the nonparametric
Purification of Glutathione S-Transferase-Rabs and in Vitro
GAP Assay—Purification of glutathione S-transferase-Rab and
glutathione S-transferase-EPI64C proteins and thrombin
digestion were performed as described previously (27, 28). The
GTP- loading protocol and the in vitro GAP assay were per-
formed as described previously (29). In this study 200 pmol of
Rab proteins and 5 pmol of EPI64C protein were used for the
RNA Interference—The following 20-nucleotide sequences
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GGACTTA-3?; B, 5?-GTACCAAAGCCAGCACCAAA-3?).
cells/sample along with 30–40 ?g of plasmid DNA. Trans-
fected Jurkat cells were used after 48–60 h for suppression.
Measurement of Transferrin Recycling—Transferrin recy-
transferrin and determining the amount retained in cells after
ences were that: Jurkat cells in suspension were used rather
the medium used was RPMI rather than Dulbecco’s modified
Eagle’s medium; and the mRFP tag was integral to the trans-
fected constructs rather than co-transfected.
EPI64C Induces Vacuoles—After transfection into Jurkat cells,
the nucleus (Fig. 1A). Many transfected cells (51% of 100 cells
scored blind) developed one or more large (?1 ?m) vacuoles vis-
vacuoles were not observed in Jurkat cells transfected with a con-
(R141K). As controls, other TBC domain proteins (TBC1D15,
AS160) were similarly transfected but did not cause formation of
an endocytic/recycling pathway, imaging was performed after
lucifer yellow was readily detectable in the large vacuoles (Fig.
1B). To characterize the vacuoles we stained with markers of
key intracellular compartments. TfR receptor was strikingly
localized to the vacuole membranes, indicating their involve-
ment in a recycling pathway (Fig. 1C). In contrast, the markers
mic reticulum (calnexin) were not localized on the vacuole
membrane but rather retained their normal pattern (Fig. 1C
and supplemental Fig. S2).
In T cells there is a recycling pathway that mediates TCR
be fully characterized (13). We conjectured that our observa-
tion of EPI64C-mediated vacuole formation might be con-
nected to that process. As an initial screen for this possibility,
in synaptic TCR exocytosis would be present on these EPI64C-
induced vacuoles (Fig. 1D). The results demonstrated that
of the vacuoles (Fig. 1D and supplemental Fig. S3).
EPI64C Vacuole Induction Is Mediated by Rab35 GAP
Activity—To identify the Rab that EPI64C regulates to induce
vacuole formation, we screened Rabs identified by proteomic
target Rab would localize to the EPI64C-induced vacuoles and
therefore screened by co-transfection of the GFP-tagged Rab
4J. J.Hao,G.Wang,T.Pisitkun,G.Patino-Lopez,M. A.Knepper,R. F.Shen,and
S. Shaw (2007) submitted for publication.
min cells were washed and imaged. C, Jurkat cells were transfected with
mRFP-EPI64C and stained with anti-EEA1, anti-LAMP2, anti-calnexin, or anti-
TfR, respectively. D, Jurkat cells were cotransfected with mRFP-EPI64C and
syntaxin4 (Stx-4), VAMP-3, or TCR-?. (Note that Stx-4 localizes not only to
FIGURE 2. EPI64C and Rab35 localization and expression. A, Jurkat cells
teins. Rab35 was unique in the extent of enrichment on the EPI64C-induced
vacuoles compared, for example, to Rab5a and Rab27a. Scale bar, 5 ?m.
topoietic cells and human embryonic kidney (HEK) 293 cells. PBT (peripheral
blood T cells) and monocytes are from human blood; Raji is a human Burkitt
lymphoma B-cell line.
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was the one most strongly enriched on the vacuoles (Fig. 2A).
Minimal enrichment on the vacuoles was seen with other Rabs
(e.g. Fig. 2A, Rab5a and Rab27a).
Systematic microarray analysis available from SymAtlas had
indicated that EPI64C expression occurs preferentially in
hematopoietic cells whereas Rab35 is broadly expressed (32).
Our analysis confirmed expression of EPI64C in the human
hematopoietic cells tested and enrichment particularly in pri-
mary human peripheral blood T lymphocytes (Fig. 2B). Similar
analysis of Rab35 expression confirmed its co-expression in
lymphoid cells with EPI64C as well as in a non-hematopoietic
kidney epithelial cell line.
measured its in vitro GAP activity for Rab35 (Fig. 3, A and B).
GAP activity of EPI64C is specific for Rab35 as it does not
controls, Rab3a or Rab27a.
EPI64C causes vacuoles in T cells by its Rab35-GAP activity
that decreases intracellular Rab35-
GTP. This hypothesis makes two
testable predictions, both of which
Rab35 (S22N) caused vacuoles (of
50 cells scored, 70% had vacuoles
?1.0 ?m in diameter), but the con-
did not (0% of 50 cells) (Fig. 3C).
Second, constitutive active Rab35
(Q67L) blocked vacuole formation
induced by EPI64C (0% of 50 cells
had vacuoles), but the control con-
stitutive active (Rab27a-Q78L) con-
struct did not (Fig. 3D, and data not
Furthermore, Rab35-DN impaired
recycling of TfR back to the plasma
membrane in Jurkat cells similar to
its function in HeLa cells (23) (Fig.
4A). Notably, EPI64C overexpres-
sion also impaired this process.
Thus, functional studies of Rab35
confirmed the two key predictions
vacuole formation through its
Rab35 GAP activity and analysis of
Rab35-dependent events. In addi-
tion to inducing large vacuoles
(Fig. 4B), transfection of GFP-
Rab35-DN into Jurkat cells prom-
inently highlighted smaller intra-
cellular vesicles, reminding us of
vesicles that transport both TCR
and TfR (13). Staining of the trans-
fected cells revealed extensive colocalization of Rab35-DN
not only with TfR (Fig. 4B) but also with CD3 (Fig. 4C). This
result suggested a role for Rab35 in trafficking of TCR as well
as of TfR.
Rab35 and TCR Colocalize in Resting T Cells and at the
IS—Localization of wild-type GFP-Rab35 in transfected Jurkat
cells was analyzed. It localized both to plasma membrane and to
intracellular vesicles, including the pericentriolar region (Fig. 5A
and supplemental Fig. S4). Colocalization analysis showed TCR
and Rab35 on solitary vesicles (Fig. 5B and supplemental Fig. S5)
resembling those seen with anti-CD3 on Rab35-DN-transfected
lar region (Fig. 5B). The Rab35-positive vesicular compartment
in synaptic exocytosis (Fig. 5C). Thus, Rab35 marks a recycling
compartment including both small solitary vacuoles and a major
We assessed Rab35 distribution during SEE superantigen-
induced synapse formation between Jurkat T cells and Raji B
FIGURE 3. EPI64C is a Rab35 GAP, in vitro and in-cell evidence. EPI64C was analyzed for in vitro GAP
activity on bacterially produced Rab35 and in-cell GAP activity. A, kinetic analysis. B, results of densito-
metric analysis expressed as the amount of the GTP-bound form of the indicated Rab after the reaction
(Rab3a, 24 min; Rab27a, 48 min; and Rab35, 48 min) as a percentage of the amount before the reaction.
Error bars represent the mean ? S.D. of data from three independent experiments. C, Jurkat cells were
transfected with dominant negative constructs Rab35 (S22N) and Rab27a (T23N) and constitutive active
constructs Rab35 (Q67L) and Rab27a (Q78L). Only Rab35 dominant negative induced large vacuoles.
D, Jurkat cells were transfected with mRFP-EPI64C and Rab35-constitutive active (Q67L) or dominant
negative (S22N) constructs. Only the constitutive active prevented EPI64C-induced vacuoles. N indicates
nucleus. Scale bars, 2 ?m.
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cell APCs (Fig. 6A). In the absence of SEE, Rab35 was evenly
APCs. However, in the presence of SEE an expanded region of
contact formed between T cell and APC into which Rab35 was
revealed that Rab35 and TCR-? were highly colocalized at the
synapse (Fig. 6B). Among cell duplexes of Jurkat cells and Raji
plemental movie). In contrast, TCR-? and Rab35 were never
observed enriched at the contact region (0%) in the absence of
antigen. Moreover, other Rabs did not accumulate at the synapse
(data not shown). In addition, the pericentrosomal accumulation
of Rab35 and TCR was often observed close to the synapse as
expected based on the reorientation of the microtubule-organiz-
Rab35 and EPI64C Regulate T-cell Conjugate Formation—To
define the functional relevance for EPI64C and Rab35, a flow
quantitatively whether Rab35 or EPI64C constructs perturbed
significantly inhibited conjugate formation, but the GAP-de-
fective EPI64C mutant R141K did not. Transfection with
Rab35-S22N (dominant negative DN) blocked conjugates to
the same extent as EPI64C, but transfection with the Rab35-
constitutive active did not. This alteration was not due to
decreased surface CD3 in the transfected cells (supplemental
Fig. S6) or to a general impairment in adhesion (supplemental
strong enrichment of Rab35-DN at the interface. In contrast,
Jurkat transfected with Rab35-Q67L (constitutive active “CA”)
consistently showed enrichment of Rab35 CA at the contact
(similar to that observed with Rab35 wild type, Fig. 7B). Rab35
FIGURE 4. EPI64C and Rab35 control receptor recycling. A, EPI64-C and
Rab35 DN control transferrin recycling. Jurkat cells transfected with mRFP-
tagged EPI64-C, Rab35 DN, or mFRP were loaded with fluorescently labeled
transferrin, and Tf recycling to the cell surface was quantified. Cells were
immunofluorescence with anti-TfR (B) or anti-CD3-? (C) (green). Rab35DN
colocalized with both TfR and CD3 on vesicles (arrowheads) and vacuoles
is not associated with TfR (38).
FIGURE 5. Rab35 localization and colocalization with TCR. Confocal
images of Jurkat cells transfected with Rab35 alone or with other constructs.
A, localization of GFP-Rab35 in transfected Jurkat cells compared with two
other Rabs. Arrow highlights Rab35 localization in the pericentriolar region.
White arrowheads highlight small vesicles enriched in Rab35, many of which
Red arrowhead highlights peripheral processes. N indicates nucleus. B, colo-
in the pericentriolar region; white arrowheads highlight small vesicles
enriched in Rab35 and TCR-?. C, GFP-VAMP3 is localized on many of the
Rab35-positive granules (arrowheads) in co-transfected Jurkat cells. N indi-
cates nucleus. Scale bars, 2 ?m.
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