LC3-dependent Intracellular Membrane Tubules Induced by γ-Protocadherins A3 and B2

Abstract

Clustered protocadherins (Pcdhs) are a family of cadherin-like molecules arranged in gene clusters (α, β, and γ). γ-Protocadherins
(Pcdh-γs) are involved in cell-cell interactions, but their prominent intracellular distribution in vivo and different knock-out phenotypes suggest that these molecules participate in still unidentified processes. We found using
correlative light and electron microscopy that Pcdh-γA3 and -γB2, but not -γC4, -α1, or N-cadherin, generate intracellular
juxtanuclear membrane tubules when expressed in cells. These tubules recruit the autophagy marker MAP1A/1B LC3 (LC3) but are
not associated with autophagic vesicles. Lipidation of LC3 is required for its coclustering with Pcdh-γ tubules, suggesting
the involvement of an autophagic-like molecular cascade. Expression of wild-type LC3 with Pcdh-γA3 increased tubule length
whereas expression of lipidation-defective LC3 decreased tubule length relative to Pcdh-γA3 expressed alone. The tubules were
found to emanate from lysosomes. Deletion of the luminal/extracellular domain of Pcdh-γA3 preserved lysosomal targeting but
eliminated tubule formation whereas cytoplasmic deletion eliminated both lysosomal targeting and tubule formation. Deletion
of the membrane-proximal three cadherin repeats resulted in tubes that were narrower than those produced by full-length molecules.
These results suggest that Pcdh-γA and -γB families can influence the shape of intracellular membranes by mediating intraluminal
interactions within organelles.

Full text

Rachel B. Hazan and Greg R. Phillips
Yildirim, Rebecca Lee, Kimita Suyama,
Fernández-Monreal, Twethida Oung, Murat
Hugo H. Hanson, Semie Kang, Mónica
INTERACTIONS
and B2: A ROLE FOR INTRALUMINAL
-Protocadherins A3γTubules Induced by
LC3-dependent Intracellular Membrane
Cell Biology:
doi: 10.1074/jbc.M109.092031 originally published online May 3, 2010
2010, 285:20982-20992.J. Biol. Chem.
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LC3-dependent Intracellular Membrane Tubules Induced by
␥
-Protocadherins A3 and B2
A ROLE FOR INTRALUMINAL INTERACTIONS
*
□
S
Received for publication, December 4, 2009, and in revised form, April 6, 2010 Published, JBC Papers in Press, May 3, 2010, DOI 10.1074/jbc.M109.092031
Hugo H. Hanson
‡1
, Semie Kang
‡1
,Mo´ nica Ferna´ ndez-Monreal
‡
, Twethida Oung
‡
, Murat Yildirim
‡
, Rebecca Lee
‡
,
Kimita Suyama
§
, Rachel B. Hazan
§
, and Greg R. Phillips
‡2
From the
‡
Department of Neuroscience, Mount Sinai School of Medicine, New York, New York 10029 and the
§
Department of
Pathology, Albert Einstein College of Medicine, Bronx, New York 10461
Clustered protocadherins (Pcdhs) are a family of cadherin-
like molecules arranged in gene clusters (
␣
,
␤
, and
␥
).
␥
-Pro-
tocadherins (Pcdh-
␥
s) are involved in cell-cell interactions, but
their prominent intracellular distribution in vivo and different
knock-out phenotypes suggest that these molecules participate
in still unidentified processes. We found using correlative light
and electron microscopy that Pcdh-
␥
A3 and -
␥
B2, but not -
␥
C4,
-
␣
1, or N-cadherin, generate intracellular juxtanuclear mem-
brane tubules when expressed in cells. These tubules recruit the
autophagy marker MAP1A/1B LC3 (LC3) but are not associated
with autophagic vesicles. Lipidation of LC3 is required for its
coclustering with Pcdh-
␥
tubules, suggesting the involvement of
an autophagic-like molecular cascade. Expression of wild-type
LC3 with Pcdh-
␥
A3 increased tubule length whereas expression
of lipidation-defective LC3 decreased tubule length relative to
Pcdh-
␥
A3 expressed alone. The tubules were found to emanate
from lysosomes. Deletion of the luminal/extracellular domain
of Pcdh-
␥
A3 preserved lysosomal targeting but eliminated
tubule formation whereas cytoplasmic deletion eliminated both
lysosomal targeting and tubule formation. Deletion of the mem-
brane-proximal three cadherin repeats resulted in tubes that
were narrower than those produced by full-length molecules.
These results suggest that Pcdh-
␥
A and -
␥
B families can influ-
ence the shape of intracellular membranes by mediating intralu-
minal interactions within organelles.
Clustered protocadherins (Pcdhs)
3
comprise ⬃50 different
cadherin-like transmembrane proteins arranged into three
genomic clusters, termed
␣
,
␤
, and
␥
(1, 2). The Pcdh genes
within the
␣
and
␥
clusters, numbering 14 and 22, respectively,
share constant cytoplasmic domains within their respective
clusters by alternative splicing (3). The Pcdh-
␥
s participate in
cell-cell interactions (4, 5) with homophilic properties (6) and
have been localized at synapses (6–8), but also have a promi-
nent intracellular distribution in cultured cells and in vivo (6, 8).
For Pcdh-
␣
s, conflicting evidence has been presented as to
whether they mediate cell-cell interactions (9–11), but a syn-
aptic localization has been reported (1).
Complete genetic deletion of Pcdh-
␥
s reduced the number of
synaptic specializations in spinal cord but also caused substan-
tial apoptosis of spinal cord interneurons (7, 12). Conditional
knock out in retina also increased apoptosis of some retinal cell
types but, unlike in spinal cord, did not affect synaptic connec-
tivity (13). Conditional knock out in astrocytes did not cause
apoptosis but delayed synaptic development (14). The multiple
effects of Pcdh-
␥
deletion may indicate still unidentified cellu-
lar roles for these molecules.
We show here that Pcdh-
␥
A3 and -
␥
B2 generate intracellu-
lar membrane tubules that recruit the autophagic vesicle pro-
tein LC3, the mammalian homologue of yeast Atg8. LC3 is lipi-
dated via a reaction similar to the ubiquitin ligase cascade and
targets to nascent autophagosomes where it is thought to sta-
bilize the phagophore (15, 16) or promote its closure (17). How
LC3 carries out this function(s) is completely unknown. LC3 is
a member of a family of proteins including GABARAP, GEC-1,
and GATE-16 (18), all of which are implicated in protein or
vesicle trafficking. We found that LC3 recruitment to Pcdh-
␥
-
induced tubules was dependent on LC3 lipidation. Coexpres-
sion of Pcdh-
␥
A3 with lipidation-defective LC3 reduced tubule
length whereas expression of wild-type LC3 increased tubule
length compared with Pcdh-
␥
A3 expressed alone. The tubules
emanated from lysosomes. The luminal/extracellular domain
of Pcdh-
␥
A3 was found to be required for tubule formation,
and shortening its length resulted in narrower tubules. Thus,
Pcdh-
␥
s, in concert with LC3 family members, could partici-
pate in the biogenesis of their own trafficking organelles via
intraluminal homophilic interactions. The effect of LC3 pertur-
bation on tubule length is consistent with a role for LC3 in
promoting or stabilizing membrane expansion.
EXPERIMENTAL PROCEDURES
Plasmid Constructs—Pcdh-
␥
A3-GFP has been described (8).
Pcdh-
␥
B2-YFP and Pcdh-
␥
C4-YFP were provided by Dr.
Joshua Weiner (University of Iowa). Luminal/extracellular
deleted Pcdh-
␥
A3-GFP was provided by Drs. Marcus Frank and
Ingrid Haas (Max Planck Institute). Pcdh-
␣
1-GFP was pro-
vided by Dr. Qiang Wu (Shanghai Jiao Tong University).
*This work was supported by National Institutes of Health Grant NS051238
and an Irma T. Hirschl award (to G. R. P.).
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1– 4.
1
Both authors contributed equally to this work.
2
To whom correspondence should be addressed: Dept. of Neuroscience,
Mount Sinai School of Medicine, Box 1065, One Gustave L. Levy Place, New
York, NY 10029. Fax: 212-659-8574; E-mail: greg.phillips@mssm.edu.
3
The abbreviations used are: Pcdh, protocadherin; GFP, green fluorescent
protein; YFP, yellow fluorescent protein; RFP, red fluorescent protein;
N-cad, N-cadherin; LC3, MAP 1A/1B LC3; CLEM, correlative light and elec-
tron microscopy; wt, wild type; ER, endoplasmic reticulum; CCD, cytoplas-
mic domain; ECD, extracellular domain.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 27, pp. 20982–20992, July 2, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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N-cadherin (N-cad)-Venus was provided by Dr. Hidekazu
Tanaka (Osaka University School of Medicine). RFP-LC3 was
provided by Dr. Zhenyue Yue (Mount Sinai School of Medi-
cine). hcRed-LC3wt and hcRed-LC3⌬G were provided by Dr.
Isei Tanida (National Institute of Infectious Diseases, Tokyo,
Japan). Cytoplasmic domain-deleted Pcdh-
␥
A3-GFP (⌬CCD-
GFP) has been described (6). Pcdh-
␥
A3-GFP with the three
membrane-proximal cadherin repeats deleted was generated
by PCR amplification of the distal three cadherin repeats and
ligating to the cytoplasmic, transmembrane, and extracellular
membrane-proximal segment just after the end of the sixth
cadherin repeat (corresponding to amino acid 667) using an
introduced Sal1 site to fuse the segments.
Cell Cultures, Transfection, and Immunostaining—Human
embryonic kidney (HEK293) cells were grown in Dulbecco’s
modified Eagle’s medium with 10% fetal bovine serum. Cells
were plated on 35-mm live imaging dishes with gridded glass
bottoms (Mattek) for analysis by correlative light and electron
microscopy (CLEM). Cells were plated on 25-mm coverslips in
6-well plates for light microscopy alone. Cells were transfected
with 4
␮
g of each plasmid using Lipofectamine 2000 (Invitro-
gen) according to the manufacturer’s instructions. For lysoso-
mal inhibition, after transfection cells were treated overnight
with 20 mMNH
4
Cl. Primary neurons were grown and trans-
fected as described (6). Cells were fixed and immunostained
exactly as described (6) except that the concentration of Triton
X-100 was 0.2%.
Antibodies—Antibodies to the constant cytoplasmic domain
of mouse Pcdh-
␥
s and to the cytoplasmic domain of N-cad have
been described (8, 19, 20). Antibodies to the constant cytoplas-
mic domain of mouse Pcdh-
␣
s were generated using GST fused
to the entire Pcdh-
␣
constant cytoplasmic domain. Polyclonal
anti-GFP was from Clontech. Chicken antibodies to MAP2
were from Covance. Monoclonal anti-KDEL was from Stress-
gen. Monoclonal anti-LAMP-2 (H4B4, developed by J. T.
August and J. E. K. Hildreth) was from the Developmental Stud-
ies Hybridoma Bank developed under the auspices of the
NICHD and maintained by the University of Iowa, Department
of Biology.
Microscopy—Detailed methods for CLEM after confocal
microscopy have been described (21–23). For Pcdhs or N-cad
transfected alone, cells were fixed 24 h after transfection with
4% glutaraldehyde in 0.1 Msodium cacodylate buffer with 1 mM
CaCl
2
. For cotransfection with hcRedLC3wt or ⌬G constructs,
cells were imaged live prior to fixation with glutaraldehyde
because we found hcRed fluorescence to be incompatible with
glutaraldehyde fixation. Confocal stacks of transfected cells
were acquired on a Zeiss LSM 510 META microscope. Differ-
ential interference contrast and fluorescent images were
acquired. The location of the cell was documented with respect
to the coverslip grid using brightfield illumination. The cells
were then fixed if imaged live and processed for transmission
electron microscopy. The material was washed in sodium caco-
dylate buffer and treated with 1% osmium tetroxide, 1.5%
potassium ferracyanide in 0.1 Mcacodylate buffer for1hat4°C.
The cells were then dehydrated in solutions of ethanol at
increasing concentrations of 50%, 60%, and 70%, kept in 2 ml of
3% uranyl acetate in 70% ethanol for 12 h at 4 °C, washed in 70%
ethanol, and further dehydrated with increasing concentra-
tions of 80%, 90%, and 100% ethanol. After dehydration, cells
were infiltrated with a 1:1 solution of resin (Embed 812 kit;
Electron Microscopy Sciences) and 100% ethanol for 24 h at
room temperature. After infiltration, the resin/ethanol was
replaced with a 1-ml layer of pure resin, and an open ended
embedding capsule was placed on the dish surrounding the cell
of interest. The resin was then hardened in a vacuum oven at
65 °C for 8 –12 h. After the first layer was solidified, the capsule
was topped off with more resin and put back in the oven for
another 8 –12 h. To separate the block from the dish, a hot plate
was heated to 60 °C, and the dish was placed on a preheated hot
plate for exactly 3 min. The dish was removed from the hot
plate and the capsule carefully peeled free from the dish.
The imaged cell was relocated in the block face and sectioned
through. Sections were contrasted with lead citrate and uranyl
acetate, and serial sections of the cell of interest were docu-
mented at magnifications of ⫻10,000, ⫻15,000, and ⫻30,000.
Confocal, differential interference contrast, and transmission
electron microscopy images were realigned and oriented using
nuclear and other morphological landmarks. Immunoelectron
microscopy was performed as described (8).
FIGURE 1. Pcdh-
␥
s generate electron-dense membrane profiles when
expressed in cells. A, three cells are labeled in the images. Cells 1 and 2 have
been transfected with Pcdh-
␥
B2-YFP, whereas cell 3 remains untransfected.
Pcdh-
␥
B2-YFP accumulates in a line at the junction between cells 1 and 2
(arrowheads) but does not accumulate at the junction between cells 1 and 3
(arrows), consistent with its participation in a transcellular interaction
between cells 1 and 2. Band C, interfaces between cells 1 and 2 and cells 1 and
3 were examined by CLEM. The membranes at the junction between cells 1
and 2 were significantly more electron-dense (B) relative to the membranes at
the junction between cells 1 and 3 (C).
Pcdh-
␥
Intracellular Tubules
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Pcdh-
␥
Intracellular Tubules
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Quantification—Induction of LC3 clustering by Pcdh-
␥
s,
-
␣
1, or N-cad was quantified as follows. Confocal sections of
random cells doubly transfected with RFP-LC3 and Pcdh or
N-cad GFP/YFP/Venus constructs were acquired on a Zeiss
LSM 410 microscope. It was then determined whether RFP-
LC3 in each cell was punctate or diffuse. At least 75 cells were
scored for each condition over three independent experiments,
and the number of punctate LC3 cells was averaged. All condi-
tions were counted blindly. ImageJ was used on imported trans-
mission electron microscopy images to determine tubule
length and diameter and averaged from 20–30 tubules from
two cells/condition. A ttest was used to determine significance.
For immunoelectron microscopic quantification of anti-Pcdh-
␥
-labeled organelles, the overall number of gold particles/or-
ganelle was determined, and for each organelle, the orientation
of particles was designated as “paired,” representing an adhe-
sive-like conformation, or “unpaired.” The ratio of unpaired to
paired labeled organelles was determined from 51 labeled
organelles.
RESULTS
Pcdh-
␥
s Generate Electron-dense Membrane Appositions—
Pcdh-
␥
-GFP fusions are functional in vivo (7, 12–14, 24). When
expressed in cultured hippocampal
neurons and cell lines, Pcdh-
␥
-
GFPs exhibited an intracellular dis-
tribution that shifted to the cell sur-
face at cell-cell contacts upon deletion
of the cytoplasmic region (6), indicat-
ing specific retention signals within
the cytoplasmic domain. To deter-
mine the identity of the compart-
ments that harbor full-length intra-
cellular Pcdh-
␥
s, we performed
CLEM (21, 23) of transiently trans-
fected cells. This allowed the precise
relocalization of GFP-tagged Pcdh-
␥
s under the electron microscope.
Although largely intracellular, in
some cases
␥
B2-YFP (Fig. 1A,
arrowheads) was found at the inter-
face between two transfected cells
(Fig. 1A, cells 1 and 2). Pcdh-
␥
A3-
GFP was previously observed to tar-
get to cell-cell interfaces (5, 6).
␥
B2-
YFP never accumulated at the
interface between a transfected cell
and a nontransfected cell (Fig. 1A,
cells 1 and 3). Both Pcdh-
␥
-positive
and -negative cell-cell interfaces
were relocated under the electron microscope by CLEM. The
␥
B2-YFP positive interface between cell 1 and cell 2 (Fig. 1B)
was more electron-dense with rigid alignment of plasma mem-
branes compared with the
␥
B2-YFP-negative interface between
cell 1 and the nonexpressing cell 3 (Fig. 1C).
Pcdh-
␥
A3 and -
␥
B2 Induce Intracellular Tubules—We took
advantage of the property that Pcdh-
␥
s render membranes
electron-dense to identify the intracellular organelles that har-
bor the A, B, and C subclasses of Pcdh-
␥
s and compare these
with a Pcdh-
␣
family member (Pcdh-
␣
1) and the classical cad-
herin, N-cad. Three representatives of the Pcdh-
␥
cluster,
␥
A3-
GFP,
␥
B2-YFP, and
␥
C4-YFP, were compared with each other
and with a member of the
␣
cluster, Pcdh-
␣
1-GFP (
␣
1-GFP), as
well as the classical N-cad-Venus. Intracellular juxtanuclear
accumulations of
␥
A3-GFP were present in most transfected
cells (Fig. 2A,box). The area corresponding to
␥
A3-GFP fluo-
rescence (Fig. 2A,boxes) was analyzed by CLEM in individual
cells (Fig. 2, A,right panel, and B). In all cells, precisely at
the area of
␥
A3-GFP fluorescence, electron-dense membrane
tubules were observed that were completely absent in untrans-
fected cells. High magnification of the
␥
A3-GFP induced
tubules (Fig. 2B) revealed a diameter of ⬃60–70 nm. These
FIGURE 2. Pcdh-
␥
A3 and -
␥
B2 but not -
␥
C4 generate intracellular tubules. Aand B,
␥
A3-GFP accumulates mostly intracellularly in HEK293 cells (6). A, cell
expressing
␥
A3-GFP was identified by confocal microscopy (left and center) and processed for CLEM. Boxed region in confocal micrographs was visualized by
electron microscopy (right). This region contained part of the nucleus, several mitochondria, and other tubulovesicular organelles resembling ER, which were
also found in nontransfected cells. Darker, electron-dense tubule-shaped organelles, that were never found in untransfected cells, were found in the region of
␥
A3-GFP expression. B, high magnification images are shown of electron-dense organelles generated by
␥
A3-GFP. The tubules measured ⬃60 –70 nm across
and were in some cases observed to emanate from other round organelles of ⬃100 –250 nm in diameter, which contained smaller structures inside (arrow-
head). C,
␥
B2-YFP also generated tubules at sites of intracellular accumulation and was also sometimes associated with other organelles (arrowhead). D,in
contrast,
␥
C4-YFP did not generate tubules at sites of intracellular accumulation but rather produced membrane sheets adjacent to the nucleus. N, nucleus; M,
mitochondria.
FIGURE 3. Pcdh-
␣
1-GFP and N-cad-Venus do not produce intracellular tubules. A,
␣
1-GFP, a representative
of the
␣
cluster, exhibited almost exclusive intracellular accumulation (left). Low magnification electron
micrograph of boxed region (middle). When visualized by electron microscopy,
␣
1-GFP produced elon-
gated rough ER-like organelles that were found associated with ribosomes (right,arrowheads). B, although
found more frequently at cell-cell interfaces, N-cad-Venus could be found intracellularly in some cells
(box,left). Low magnification electron micrograph of boxed region (middle). Examination of intracellular
N-cad-Venus regions revealed membrane whorls and numerous smaller vesicles that lacked ribosomes
(right). N, nucleus; M, mitochondria.
Pcdh-
␥
Intracellular Tubules
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tubules were distinct from rough and smooth ER-like profiles
that were oftentimes found in the vicinity of the tubules but
were also observed in untransfected cells. In some cases the
tubules were observed to emanate from 100–250 nm vesicular
organelles that also contained smaller dot-like profiles (Fig. 2B,
right panel,arrowheads).
Intracellular accumulations were also observed for
␥
B2-YFP
and
␥
C4-YFP (Fig. 2, Cand D, respectively) and were examined
by CLEM. Tubules similar to those found in
␥
A3-GFP-trans-
fected cells were found in
␥
B2-YFP-transfected cells (Fig. 2C).
In contrast,
␥
C4-YFP did not induce
the formation of tubules (Fig. 2D)
but instead accumulated in layered
membrane sheets similar to the type
of accumulations observed for
some ER proteins (25, 26). Unlike
␥
A3-GFP-induced tubules, the
␥
C4-YFP-induced sheets were con-
tinuous through several serial sec-
tions, demonstrating that
␥
C4-YFP
induces accumulation of sheets
rather than tubules (supple-
mental Fig. 1). In addition,
␥
C4-YFP
sheets were found to be continuous
with the nuclear envelope, suggest-
ing an ER origin (supplemental
Fig. 1).
By comparison,
␣
1-GFP was al-
most exclusively intracellular and
absent at cell-cell contacts (Fig. 3A)
in agreement with its apparent
lack of adhesive activity (9, 10). By
CLEM,
␣
1-GFP showed ER-like
accumulations that, at higher mag-
nification, appeared to be associated
with ribosomes (Fig. 3A,right panel),
suggesting an expansion of rough
ER. N-cad-Venus was more promi-
nent at cell junctions (not shown)
but also could be found intracellu-
larly (Fig. 3B). CLEM of N-cad-
Venus intracellular accumulations
showed a different ER-like profile
with wrapped sheets and vacuoles
(Fig. 3B,right panel) that, unlike
␣
1-GFP, did not associate with ribo-
somes. These whorled membranes
have also been shown to be associ-
ated with accumulations due to ER
expansion (25, 26). Thus,
␥
A3-GFP
and
␥
B2-YFP produce a specific
type of intracellular tubule versus
the ER and sheet-like accumula-
tions observed for the other
constructs.
␥
A3-GFP and
␥
B2-YFP Cocluster
with LC3—To determine the origin
of the tubules within the secretory
pathway, we searched for organelle markers that might colocal-
ize with
␥
A3-GFP- and
␥
B2-YFP-induced tubules (see
supplemental Fig. 2). The autophagy protein LC3, when fused
to either GFP or RFP, has been shown to be a reliable marker for
autophagosomes in cultured cells and in vivo (18, 27). We found
that both
␥
A3-GFP and
␥
B2-YFP induced the clustering of, and
colocalized with, RFP-LC3 (Fig. 4A). In contrast, neither
␥
C4-
YFP,
␣
1-GFP, nor N-cad-Venus affected the clustering of RFP-
LC3, which remained diffuse in transfected cells (Fig. 4A).
Quantification showed that ⬃70% of cells transfected with
FIGURE 4. Pcdh-
␥
A3 and -
␥
B2 induce clustering of, and colocalize with, LC3. A, the indicated constructs
(top panels) were transfected together with RFP-LC3 (middle panels). All Pcdh-
␥
constructs generated clear
intracellular accumulations (arrows and arrowheads). Only
␥
A3-GFP and
␥
B2-YFP induced clustering of, and
colocalized with, RFP-LC3 (arrowheads).
␥
C4-YFP did not induce clustering of RFP-LC3 despite its intracellular
location (arrows).
␣
1-GFP and N-cad-Venus also did not affect the distribution of RFP-LC3. B, the percentage of
cells with punctate RFP-LC3 was determined from three independent experiments. Error bars, S.E. C, colocal-
ization of Pcdh-
␥
A3-GFP and RFP-LC3 in neurons transfected with the two constructs at 12 days in vitro is
shown. Blue channel is MAP2 immunostaining. Arrowheads indicate colocalized profiles. Inset shows codistri-
bution of the two constructs in an organelle-like profile.
Pcdh-
␥
Intracellular Tubules
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␥
A3-GFP or
␥
B2-YFP and RFP-LC3 exhibited clustered LC3
(Fig. 4B). All constructs evaluated were expressed at compara-
ble levels in cells (see supplemental Fig. 3). Furthermore, no
accumulation of autophagic vesicles or autolysosomes, which
have been visualized using the same electron microscopic prep-
aration method (22, 23), could account for the high levels of
clustered LC3 in
␥
A3-GFP- or
␥
B2-YFP-transfected cells (see
Fig. 2).
␥
A3-GFP, when cotransfected with RFP-LC3 into cul-
tured hippocampal neurons at 12 days in vitro, also produced
intracellular clusters, some of which were found to cocluster
with RFP-LC3 (Fig. 4C,arrowheads and inset).
It is known that LC3 clustering artifacts due to protein aggre-
gation can lead to misidentification of LC3 puncta (28, 29). LC3
is lipidated at the glycine residue near its carboxyl terminus via
a reaction similar to the ubiquitin ligase pathway, and this lipi-
dation is required for LC3 targeting in autophagy (29). Nonspe-
cific clustering of LC3 at protein aggregates lacks the require-
ment for lipidation of LC3 (29). We sought to determine
whether LC3 lipidation is required for clustering with
␥
A3-
GFP. Wild-type hcRed-LC3 or lipidation-defective hcRed-
LC3-⌬G (29) was transfected with
␥
A3-GFP, and the extent of
LC3 clustering was evaluated quantitatively (Fig. 5). Wild-type
hcRed-LC3 was found to cocluster
with
␥
A3-GFP (Fig. 5A,left panels)
similar to RFP-LC3 (see Fig. 4). In
contrast, hcRed-LC3-⌬G did not
cocluster with
␥
A3-GFP (Fig. 5A,
right panels). Quantitative analysis
demonstrated that
␥
A3-GFP in-
duced the formation of wild-type
hcRed-LC3 clusters but not that of
hcRed-LC3-⌬G. Thus, lipidation of
LC3 is required for the colocaliza-
tion of LC3 with Pcdh-
␥
-induced
intracellular tubules.
LC3 Mediates Pcdh-
␥
Intracellu-
lar Tubule Elongation from Ly-
sosomes—We suspected that wild-
type and lipidation-defective LC3
might differentially affect tubule
formation or elongation when coex-
pressed with
␥
A3-GFP. To test this,
we compared the tubules produced
by
␥
A3-GFP alone or in the pres-
ence of wild-type hcRed-LC3 or
hcRed-LC3-⌬G. In contrast to the
relatively long electron-dense tu-
bules formed by
␥
A3-GFP in the
presence of wild-type hcRed-LC3
(Fig. 6A), the electron-dense tubules
were significantly shorter in the
presence of hcRed-LC3-⌬G (Fig.
6B). Instead of tubules, most of the
electron-dense organelles in hcRed-
LC3-⌬G/
␥
A3-GFP-cotransfected
cells consisted of vesicles similar to
the type that were often connected
to the tubules in RFP-LC3 and
␥
A3-
FIGURE 5. Lipidation-defective LC3 does not colocalize with
␥
A3-GFP.
A,
␥
A3-GFP was cotransfected with wild-type hcRed tagged LC3 (hcRed-
LC3wt) or similarly tagged LC3 truncated at the glycine residue near the car-
boxyl terminus (hcRed-LC3⌬G), the site for lipid conjugation. hcRed-LC3wt
was clustered and colocalized with
␥
A3-GFP, whereas hcRed-LC3⌬G was not.
B, results were quantified as the percentage of cells with punctate LC3 from
three independent experiments. Error bars, S.E.
FIGURE 6. hcRed-LC3⌬G shortens
␥
A3-GFP-induced tubule length. A,
␥
A3-GFP was cotransfected with
hcRed-LC3wt and processed for CLEM. B, cotransfection of hcRed-LC3⌬G with
␥
A3-GFP and CLEM. Intracellular
accumulation of
␥
A3-GFP (green channel) did not recruit hcRed-LC3⌬G(red channel), and CLEM of this site
revealed fewer tubules that were shorter in length than those observed when hcRred-LC3wt was cotrans-
fected. C, quantification of tubule length for
␥
A3-GFP cotransfected with hcRed-LC3wt or ⌬Gversus
␥
A3-GFP
alone. Results were averaged from 20 –30 tubules from two different cells/condition. A ttest was used to
determine significance. **, p⬍0.001; *, p⬍0.05. Error bars, S.D. N, nucleus.
Pcdh-
␥
Intracellular Tubules
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GFP-cotransfected cells (see Fig. 2B,right panel). Quantifica-
tion revealed an almost 50% reduction in tubule length in cells
transfected with
␥
A3-GFP plus hcRed-LC3-⌬G relative to
␥
gA3-GFP plus wild-type hcRed-LC3. Cells transfected with
␥
A3-GFP alone had a tubule length intermediate to the two
(Fig. 6C). Thus, LC3 promotes
␥
A3-GFP tubule elongation in a
lipidation-dependent fashion.
In cells cotransfected with hcRed-LC3-⌬G and
␥
A3-GFP,
the predominant electron-dense organelles were 100–250-nm
vesicles (Fig. 7A,right panel) rather than tubules (Fig. 7A,left
panel), suggesting that the initial substrates for tubule forma-
tion may be a subclass of vesicular organelles that sprout
tubules when Pcdh-
␥
s accumulate in them. We sought to deter-
mine the types of organelles that harbor Pcdh-
␥
s by immuno-
staining of
␥
A3-GFP-transfected cells with subcellular markers
including those for ER, Golgi, early endosomes, and late endo-
somes/lysosomes (supplemental Fig. 2). We observed a signifi-
cant colocalization of Pcdh-
␥
s with anti-LAMP-2 immuno-
staining (Fig. 7Band supplemental Fig. 2). Both
␥
A3-GFP and
␥
B2-YFP, constructs that induced tubules, exhibited significant
colocalization with LAMP-2 (supplemental Fig. 4). Thus, in
these cells, the combined data sug-
gest that Pcdh-
␥
A and B, but not C,
families, together with LC3, induce
the sprouting of tubules as they
accumulate in late endosomes/lyso-
somes in a manner dependent on
LC3 lipidation.
To verify the lysosomal origin
of
␥
A3-GFP-induced tubules, we
treated
␥
A3-GFP-transfected cells
with NH
4
Cl, an inhibitor of lysoso-
mal acidification. In this case, large
vacuoles (Fig. 7C) were induced
with a reduction in tubules, indi-
cating that tubulogenesis is tied to
lysosomal function. A few tubules
(Fig. 7C,arrowhead) were observed,
however, to emanate from the
enlarged vacuoles (Fig. 7C,arrow).
Differential Effect of Luminal/
Extracellular and Cytoplasmic Do-
main Deletion on Lysosomal Tar-
geting and Intracellular Tubule
Biogenesis—The luminal/extracellu-
lar domains of Pcdh-
␥
A3 and Pcdh-
␥
B2, but not Pcdh-
␥
C4, were shown
to participate in cell-cell interactions
with homophilic properties, whereas
the cytoplasmic domains promoted
retention (6). We sought to deter-
mine whether the luminal/extracel-
lular and/or cytoplasmic domains
mediate tubule formation. Cyto-
plasmic-deleted
␥
A3-GFP (⌬CCD-
GFP) was mostly found at the cell
surface (6), but intracellular accu-
mulations could be found in a few
transfected cells (Fig. 8A,arrows). These never induced cluster-
ing of RFP-LC3, nor did they colocalize with LAMP-2 (Fig. 8A,
arrows). In contrast, when the luminal/extracellular domain
was deleted (⌬ECD-GFP; Fig. 8A,arrowheads), both RFP-LC3
clustering and LAMP-2 colocalization were observed. CLEM
showed that ⌬ECD-GFP accumulated in distorted electron-
dense organelles that contained smaller profiles similar to those
found in lysosomes (Fig. 8B,bottom,arrows). In this case, no
tubules were observed. In contrast, ⌬CCD-GFP generated
membrane whorls (Fig. 8B,top) similar to the type (25, 26)
produced upon expression of some ER-targeted proteins.
⌬CCD-GFP-induced intracellular membrane whorls were pos-
itively immunolabeled with anti-KDEL antibodies (Fig. 8C,
arrowheads), confirming their ER origin.
Shortening the Luminal/Extracellular Domain Decreases
Tubule Width—The above results suggested that the cytoplas-
mic domain promotes lysosomal targeting whereas the lumi-
nal/extracellular domain is required for tubulogenesis, poten-
tially by mediating intraluminal interactions. It is known that
cadherins mediate their adhesive interactions via the most dis-
tal extracellular moiety, the first cadherin repeat (30–32). We
FIGURE 7. Lysosomes are the substrates for tubulogenesis. A, electron-dense organelles in
␥
A3-GFP and
hcRed-LC3⌬G-cotransfected cells resemble lysosomes (right panel,arrow)versus organelles from
␥
A3-GFP-
tranfected cells in which the tubules (left panel,arrow) retained electron-dense properties whereas lysosomal-
like organelles were less electron-dense. B, colocalization of
␥
A3-GFP with lysosomal marker LAMP-2 indicates
Pcdh-
␥
s target to lysosomes, implicating them as the substrate for tubule growth. C,NH
4
Cl inhibition of
lysosomal acidification reduces tubulogenesis. Enlarged vacuoles (arrow) only occasionally sprouted tubules
(arrowhead). N, nucleus; M, mitochondria.
Pcdh-
␥
Intracellular Tubules
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deleted the three membrane-proximal cadherin repeats (cad-
herin repeats 4–6, ⌬EC4–6-GFP; see supplemental Fig. 3)to
determine whether the distal part of the extracellular domain
might be involved in tubulogenesis. We found that this con-
struct induced clustering of RFP-LC3 and colocalized with
LAMP-2 (Fig. 9A). CLEM of intracellular ⌬EC4–6-GFP re-
vealed that juxtanuclear tubules
were generated, but with an appar-
ent narrower width. We quantified
the width of tubules generated by
␥
A3-GFP,
␥
B2-YFP, and ⌬EC4–6-
GFP (Fig. 9B) and found that
although both
␥
A3-GFP and
␥
B2-
YFP generated tubules of identical
width (⬃65 nm), ⌬EC4–6-GFP-
generated tubules were significantly
narrower (⬃40 nm) (Fig. 9C). Thus,
the molecular length of the luminal/
extracellular domain is correlated
with the diameter of the intracellu-
lar tubules, suggesting an intralumi-
nal interaction between Pcdh-
␥
molecules on apposing membranes
stabilizing tubule structure.
Pcdh-
␥
s Delinate Subdomains of
Tubulovesciular Organelles in Vivo—
By immunoelectron microscopy,
Pcdh-
␥
s were shown to have a pro-
minent intracellular component in
vivo (6, 8). In many cases, Pcdh-
␥
labeled constrictions of, or emana-
tions from, organelle profiles (Fig.
10A,arrowheads), in a paired arrange-
ment on opposing membranes (Fig.
10B,left panel). When quantified,
Pcdh-
␥
-labeled organelles had this
arrangement 55% of the time versus
45% of organelles with unpaired
gold particles (Fig. 10B,right panel).
These results are consistent with a
role for Pcdh-
␥
-mediated intralu-
minal interactions in organelle
dynamics in vivo (Fig. 10C).
DISCUSSION
The cellular function of all of the
Pcdhs has been poorly defined (33),
unlike the well characterized classi-
cal cadherins. The Pcdh-
␥
s can
mediate transcellular interactions
with homophilic properties (5, 6)
and, for the Pcdh-
␣
s, less is known
about their functions at the cellular
level, but these most likely do not
mediate transcellular interactions
(9, 10, 34). Knock-out phenotypes
for Pcdh-
␥
s are consistent with
transcellular interactions as one fea-
ture but also point to other modes of action (7, 12–14, 24, 35).
Pcdh-
␥
s exhibit a prominent intracellular distribution in cul-
tured neurons and in neural tissue (6, 8). Here, we show that
Pcdh-
␥
A3 and -
␥
B2 generate, from substrate organelles, intra-
cellular tubules that require the autophagic molecule LC3 for
their elongation. Based on the accumulated data, we speculate
FIGURE 8. Deletion of the luminal/extracellular and cytoplasmic domains of Pcdh-
␥
A3 differentially
affect lysosomal localization, LC3 clustering, and tubule formation. A, intracellular accumulations of
⌬CCD-GFP, when found, did not induce clustering of RFP-LC3 and did not colocalize with LAMP-2. In contrast,
the luminal/extracellular deletion (⌬ECD-GFP) did induce clustering of RFP-LC3 and colocalized with LAMP-2.
B, electron micrographs of ⌬ECD-GFP and ⌬CCD-GFP intracellular accumulations are shown. ⌬ECD-GFP was
found in clustered juxtanuclear organelles that contained smaller particles in the lumen (arrows), suggestive of
distorted lysosomes whereas ⌬CCD-GFP generated larger membrane whorls that resembled ER distortion as
has been observed previously (25, 26). C, intracellular ⌬CCD-GFP induced membrane whorls labeled positive
with anti-KDEL antibodies (arrowhead). N, nucleus.
Pcdh-
␥
Intracellular Tubules
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that Pcdh-
␥
s have the ability to generate their own trafficking
organelles at intracellular sites where they accumulate by alter-
ing membrane shape or curvature and promoting intraluminal
interactions.
Nonconventional Roles for Pcdh-
␥
s—The predominant phe-
notype when the Pcdh-
␥
cluster is inactivated in neurons is cell
death (7, 12, 13, 24), and this could be either cell-autonomous
or non-cell-autonomous (13, 24). Prevention of neuron death
in Pcdh-
␥
-deleted animals by dou-
ble knock out with Bax revealed syn-
aptic defects in spinal cord (12) pre-
sumably due to disrupted Pcdh-
␥
transcellular interactions. However,
completely normal retinal connec-
tivity was restored by rescue from
apoptosis in Pcdh-
␥
retinal knock
outs (13), indicating that retinal
connectivity has no requirement for
Pcdh-
␥
s despite the high expression
of the molecules there. The exis-
tence of two, apparently separable,
phenotypes, neuronal death and
synapse loss, indicates the potential
for multiple functions for Pcdh-
␥
s
in various neuronal systems. Either
or both synapse development and
cell survival could be dependent on
intracellular trafficking of Pcdh-
␥
s.
There is a growing list of mole-
cules that influence intracellular
membrane shape, and multiple ER-
resident transmembrane proteins
that drive the structural dynamics of
ER are now beginning to be elabo-
rated (36), including the reticulon
family which can transform the ER
from sheets into tubules (37). It is
possible that Pcdh-
␥
s could define
a subdomain of membranes in
organelles in which they accumu-
late and modify these membranes in
a manner similar to the reticulons
(37, 38). Pcdh-
␥
s lack a cleavable
prodomain (39), that for classical
cadherins blocks adhesion during
intracellular transport (40), and
therefore its membrane-membrane
adhesive activity is likely to be active
inside the lumen of intracellular
compartments where it could par-
ticipate in the stabilization of
organelle shape (Fig. 10C). An adhe-
sive-like mechanism for mainte-
nance of ER shape has previously
been proposed (36) and could also
participate in the stabilization of the
tubule morphology. Such Pcdh-
␥
-
generated tubules might emanate
from any organelle that harbors them in neurons.
Nonautophagic Role for LC3—LC3 is the mammalian homo-
logue of yeast Atg8 which was identified as a gene required for
proper formation of autophagic vesicles or vacuoles in yeast.
LC3 was first identified as an ⬃18-kDa protein that copurified
with microtubules and with MAP1A/1B (41). LC3 was found to
translocate to autophagosomes and can be present on the cyto-
solic and luminal sides (27). In mammals, LC3 has three other
FIGURE 9. Shortening of the extracellular domain decreases tubule width. Cadherin repeats 4 –6 were
removed, leaving only cadherin repeats 1–3 in the extracellular domain (⌬EC4 – 6-GFP; see supple-
mental Fig. 3). A,⌬EC4 –6-GFP induces clustering of RFP-LC3 and colocalizes with LAMP-2. B, low (left) and high
(center) magnification electron micrographs of intracellular tubules induced by ⌬EC4 – 6-GFP are shown. Right,
widths of tubules generated by
␥
A3-GFP,
␥
B2-GFP and ⌬EC4 –6-GFP constructs were quantified. Decrease in
width of ⌬EC4 –6-GFP tubules was statistically significant (ttest, p⬍0.0001). Widths of tubules induced by
␥
A3-GFP and
␥
B2-GFP were not different (p⬎0.2). Error bars, S.D. C, high magnification of images illustrates
widths of
␥
A3-GFP (left),
␥
B2-GFP (center), and ⌬EC4 –6-GFP induced tubules (right). N, nucleus.
Pcdh-
␥
Intracellular Tubules
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homologues, GABARAP, GEC-1, and GATE-16, which also
translocate to autophagosomes upon lipidation (18). The exact
role of LC3/Atg8 and related proteins in autophagy is still not
clear. One study showed that the size of autophagic vacuoles
depends on Atg8 levels in yeast (15). Knock outs of different
genes of the autophagic cascade, which eliminate LC3 family
lipidation, were shown to alter the shape or size of autophagic
vesicles, or prevent their closure, but not eliminate them (17).
We found that Pcdh-
␥
-induced intracellular tubules are
shorter in the presence of the lipidation-defective LC3. Our
results support the idea, previously suggested, that LC3 con-
trols the rate of intracellular membrane expansion (15), which
may indicate a generalized role for LC3 in nonautophagic pro-
cesses, some of which have recently begun to be elaborated (42)
and are consistent with a suggested role for LC3 family mem-
bers in membrane and receptor trafficking (43–48). It is possi-
ble that intracellular Pcdh-
␥
s may
be involved in regulating LC3 fami-
ly-mediated organelle trafficking in
addition to their roles in cell-cell
interactions.
Whether or not the Pcdh-
␥
-in-
duced tubules represent a precur-
sor to autophagic vesicles or
another type of organelle that uti-
lizes LC3 for its biogenesis remains
to be conclusively determined, but
autophagy is unlikely given that
autophagic vesicles were never
found in the vicinity of intracellular
Pcdh-
␥
s and that the tubules origi-
nate from lysosomes. The tubules
observed in the present study do
bear a resemblance, tubules con-
nected to vesicular profiles, to those
found using biochemical purifica-
tion of LC3-containing membranes
(49). Because no increases in auto-
phagic vesicles were observed in
Pcdh-
␥
-transfected cells it is likely
that the tubules observed are one of
the few examples of nonautophagic
organelles that utilize LC3 for
its biogenesis. Alternatively, the
tubules could be an arrested stage
of autophagic organelle biogenesis
prior to the generation of the phago-
phore. Regardless of whether the
tubules are autophagy-related or
-unrelated, the observation that cer-
tain Pcdh-
␥
s can induce organelle
formation in an LC3-dependent
fashion points to a potential novel
intracellular role for the molecules
in trafficking in addition to cell-cell
interactions.
Mechanism for Trafficking of Rec-
ognition Units—The diversity of
Pcdh-
␥
s in the nervous system generated by alternative splicing
(3) strongly suggests a role in cell surface recognition. It is
possible that through intracellular homophilic interactions,
Pcdh-
␥
trafficking might be directed into “packets” (Fig. 10C)
that when inserted into the plasma membrane contain a homo-
geneous population of a single Pcdh-
␥
isoform. The addition of
such recognition packets to the surface may have more imme-
diate consequences on synaptic development versus slow accu-
mulation of individual Pcdh-
␥
molecules at cell-cell interac-
tions sites in neurons via conventional secretory mechanisms.
Acknowledgments—We thank Drs. Qiang Wu, Isei Tanida, Joshua
Weiner, Hidekazu Tanaka, Marcus Frank, Ingrid Haas, and Zhenyu
Yue for reagents; Drs. Ana Maria Cuervo and Zhenyu Yue for helpful
discussions; and William G. Janssen for technical advice.
FIGURE 10. Arrangement of Pcdh-
␥
s within organelle profiles. A, immunoelectron microscopy of adult
hippocampal CA1 region with antibodies to Pcdh-
␥
constant intracellular domain. Pcdh-
␥
s have a prominent
intracellular distribution (8) and label tubulovesicular organelles in axons and dendrites. Pairing of gold parti-
cles (arrowheads) at apparent constriction sites suggests the possibility of an adhesive conformation within
organelles. Such paired labeling of gold particles has also been observed at sites of transcellular interactions
(8). Pcdh-
␥
s were also observed in lysosomal-like organelles (bottom left). B, example of organelle labeled in
paired (left,arrowheads) or unpaired (right) configuration. C, model for intraluminal interactions mediated by
Pcdh-
␥
s. Interactions could be stabilized by LC3 (squares). Homophilic intraluminal interactions may facilitate
the budding of new organelles containing high concentrations of a single Pcdh-
␥
isoform.
Pcdh-
␥
Intracellular Tubules
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