From the Department of Molecular and Cell Biology, University of
California, Berkeley, California 94720-3200
The pH and trafficking of recycling endosomes
have previously been studied using transferrin. We have used another
approach, one in which the vesicle transport protein cellubrevin was
appended with a luminal IgG epitope to allow targeting of
fluorescein-5'-isothiocyanate (FITC)-labeled anti-IgG F(ab) antibodies
to the recycling endosomes in living cells. FITC-F(ab) was specifically
internalized by COS cells transfected with cellubrevin-Ig, which at
steady state accumulated in a pericentriolar region similar to
rhodamine-transferrin. Confocal microscopic analysis showed that
endosome labeling by these two markers was heterogeneous. This
differential distribution was not induced by the IgG tag, since
endogenous Cb and Tf were also partitioned into separate endosomal
populations. We used fluorescence ratio imaging of internalized
FITC-F(ab) to measure the pH of cellubrevin-enriched recycling
endosomes (pHCb) and FITC-transferrin to measure the
pH of transferrin-enriched recycling endosomes (pHTf). In
COS cells, cellubrevin endosomes (mean pHCb 6.1 ± 0.05; range, 5.2-6.6) were more acidic than transferrin endosomes
(mean pHTf 6.5 ± 0.05; range, 5.6-7.2). Similar
results were obtained in Chinese hamster ovary cells. Treatment with
the vacuolar H+-ATPase inhibitor bafilomycin A1
caused pHTf to increase (
pHTf = 1.2 pH
units) to a greater extent than pHCb (
pHCb = 0.5 pH units). Furthermore, inhibition of the
Na+/K+-ATPase by ouabain or
acetylstrophanthidin caused pHTf to decrease by 0.6 pH
units but had no effect on pHCb. Based on the combination of these morphological and functional data, we suggest that the recycling endosomes are heterogeneous in their biochemical
compositions, ion transport properties, and pH values.
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INTRODUCTION |
The cycling of molecules through the endocytic pathway has been
extensively studied by monitoring the trafficking of the
transferrin-transferrin receptor
(Tf·TfR)1 complex (reviewed
in Refs. 1 and 2). Surface-bound Tf is concentrated into coated pits
and internalized in endocytic vesicles, which rapidly fuse with sorting
endosomes, the population of early endosomes scattered throughout the
cell periphery (3-7). The low luminal pH (~6.0) of the sorting
endosomes promotes dissociation of Fe3+ from the bound Tf
(8, 9), and the Tf·TfR complex is then segregated into tubular
extensions that exclude the now soluble Fe3+ (10). These
tubular elements bud from the sorting endosomes and return the complex
to the cell surface, where TfR can reload with Fe3+-Tf to
repeat the cycle. On the recycling pathway, most of the Tf·TfR
complex clusters at a distinct perinuclear location in close apposition
to the microtubule organizing center. These perinuclear recycling
endosomes are distinguished from sorting endosomes by their distinct
intracellular location and by the lack of cargo destined for late
endosomes and lysosomes (3, 4, 6, 11).
Despite the consistent picture emerging from experiments investigating
Tf and TfR, recent biochemical and immunomicroscopic observations have
suggested that the endosomal pathway is more complex than was
originally perceived. Several membrane proteins that cycle through the
endosomal system have overlapping but distinct distributions,
suggesting that they may not always follow the same path (for example,
see Refs. 12-14). Many components of the vesicular traffic machinery
are also heterogeneously distributed among endosomes. The low molecular
weight GTPases Rab4 and Rab11 are both associated with subpopulations
of perinuclear recycling endosomes (15, 16). Cellubrevin (Cb), a
v-SNARE protein involved in TfR recycling, is associated with many
peripheral vesicles that do not contain TfR (17, 18). Likewise, in
neuroendocrine cells Cb is targeted to neurites that exclude the TfR
(19).
At present, the physiological properties and functional roles of the
putative endosome subpopulations are not known. Endosomal pH
measurements using dye-labeled Tf in conjunction with either cytofluorometry or cellular imaging have shown that peripheral sorting
endosomes have a pH in the range of 5.9-6.4 (20-24), while the
perinuclear recycling endosomes have a slightly higher pH of 6.4 (4,
22-24). As a first step to determine the physiological heterogeneity
of recycling endosomes, we have devised a "targeted fluorescence"
approach to observe the distribution and pH of Cb-containing recycling
vesicles. Cb was chosen because it is a constituent of the recycling
endosomes, but, as discussed above, previous experiments suggested that
it does not always co-distribute with Tf. Therefore, measurements made
with this marker could conceivably reveal physiological differences
within the recycling endosomes that may have been overlooked by the
Tf-based work. A fusion protein of Cb and the human IgG constant region
was created to allow targeting of pH-sensitive dyes to Cb-containing
recycling endosomes. We stably transfected COS-7 cells with the Cb-Ig
construct and performed a series of experiments to determine (i) the
cellular distribution of FITC-labeled anti-IgG F(ab) fragment added to
the extracellular media of live cells, (ii) the pH of the Cb-containing
recycling endosomes, and (iii) the roles of the H+-ATPase
and Na+/K+-ATPase in governing Cb-containing
recycling endosome pH. These results were compared with data obtained
from similar experiments using Tf as the recycling endosome marker. Our
results indicated that there are subpopulations of perinuclear
recycling endosomes that can be visualized by confocal microscopy and
are further distinguished by differences in pH and responses to ouabain
and bafilomycin A1. The potential roles of the recycling
endosome subpopulations are discussed.
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EXPERIMENTAL PROCEDURES |
Materials--
All salts, bafilomycin A1,
nocodazole, ouabain, chloroquine, paraformaldehyde, hygromycin B,
acetylstrophanthidin, fetal calf serum, DEAE-dextran and DABCO were
purchased from Sigma. Glycerol, methanol, and acetic acid were from
Fisher, and restriction enzymes were from Boehringer Mannheim and New
England Biolabs (Beverly, MA).
Construction of Cellubrevin-Ig and Other Plasmids--
Cb was
polymerase chain reaction-amplified from a rat basophilic leukemia
cDNA library (courtesy of Dr. Brian Seed, Massachusetts General
Hospital) with primers
(5'-CGCGGGAAGCTTGCCGCCACCATGTCTACAGGGGTGCCT-3' and
5'-CGCGGGGGATCCGAGACACACCACACAAT-3'), which allowed isolation of
full-length Cb sequence with 5' HindIII and 3'
BamHI restriction sites for cloning into the pCD2B
1
expression vector (courtesy of Dr. Brian Seed). This created an
in-frame fusion of Cb to the CH2 and CH3 domains of human IgG after its
hinge region. The resulting Cb-Ig construct was then transferred to the
pCD43/hsfi
vector via HindIII and
HpaI sites for stable transfection in COS cells.
pCD43/hsfi
(from Dr. Brian Seed) is a pCDM8-derived
plasmid that contains CD43 in the stuffer region, confers resistance to
hygromycin B, and has the SV40 origin of replication inactivated at the
SfiI site. Untagged Cb was amplified similarly with an
additional stop codon in the antisense primer and cloned into the
pcDNA3 vector (Invitrogen, San Diego, CA).
Organelle markers were constructed by attaching epitope tags to marker
sequences containing the organelle targeting signals. The catalytic
domain of UDP-glucuronyltransferase (UDPGT) was replaced with the CD4
epitope to generate CD4-UDPGT (courtesy of Dr. Brian Seed); the
cytoplasmic dilysine motif of UDPGT targets this construct to the ER.
The full coding sequences of galactosyltransferase (GalT) and furin
have been appended with a Flag epitope tag at the COOH terminus to
generate GalT-Flag and furin-Flag. GalT sequence with 5'
HindIII and 3' XhoI sites was isolated by
polymerase chain reaction amplification from a human liver cDNA
library with the primers 5'-CGCGGGAAGCTTGCCACCATGAGGCTTCGGGAGCCG-3' and
5'-CGCGGGCTCGAGGCTCGGTGTCCCGATGTC-3'; the ends of a mouse furin
cDNA (pAGEFur, courtesy of Dr. K. Nakayama, University of Tsukuba,
Ibaraki, Japan) were similarly modified by the primers
5'-CGCGGGAAGCTTGCCACCATGGAGCTGAGATCCTGG-3' and 5'-CGCGGGCTCGAGAGGGGCGCTCTGGTCTTT-3'. Both products were inserted via
5' HindIII and 3' XhoI sites into the
pCDM8-C-Flag vector, which contained the Flag epitope sequence for
in-frame insertion of the tag at the C terminus.
Transfections and Generation of Cell Lines Stably Expressing
Cb-Ig--
COS-7 cells were grown in DMEM (BioWhittaker, Walkersville,
MD) supplemented with 10% fetal calf serum and under 5%
CO2 atmosphere. CHO TRVb1 cells stably transfected with
human Tf receptor (courtesy of Dr. T. E. McGraw, Cornell
University Medical School, New York, NY) were grown in Ham's F-12
medium (Life Technologies, Inc.) supplemented with 10% fetal calf
serum and under 5% CO2 atmosphere. To isolate stable
transfectants, semiconfluent cells in six-well plates were transfected
with 1 µg of Cb-Ig/hsfi
using the lipofectamine
procedure (Life Technologies), transferred to 10-cm dishes the next
day, and subjected to selection with 200 µg/ml hygromycin B at
48 h post-transfection. Clones were screened by indirect
immunofluorescence. Organelle markers were transiently transfected with
the DEAE-dextran protocol of Seed and Aruffo (25).
Immunofluorescence and Laser Scanning Confocal
Microscopy--
Cells were washed twice with PBS, fixed in 3.7%
paraformaldehyde for 20 min, and permeabilized in ice-cold 100%
methanol for 20 s. Incubation for 15 min in 1% BSA/PBS preceded
staining with the primary antibodies for 1 h and the secondary
antibodies for 30 min. Washed coverslips were mounted on slides with a
non-bleach reagent (KPL mounting media from Kirkegaard and Perry Labs,
Inc. (Gaithersburg, MD) or 2.5% DABCO in 80% glycerol/PBS). A Zeiss Axiophot (Oberkochen, Germany) microscope with a × 63 objective was used for indirect immunofluorescence. For laser scanning confocal microscopy, cells were analyzed using a krypton/argon laser coupled with a Bio-Rad MRC1000 attached to a Zeiss Axioplan (Oberkochen, Germany) microscope with a Leitz Plan Apo × 63 oil/NA 1.4 objective. Separate excitation lines and emission filters were used for
each fluorochrome (FITC, 488 nm (excitation) and 522DF32 (emission); Texas Red, 568 nm (excitation) and 605DF32 (emission)). Single optical
sections separated by 0.54 µm were collected sequentially for each
fluorochrome. Confocal images were background-subtracted, merged using
the Confocal Assistant software program, and processed with Adobe
Photoshop software. For quantitation, red, green, or yellow endosomes
from merged 0.5-µm optical sections were counted by visual inspection
of the enlarged image on a computer monitor. The following dilutions of
antibodies were used: FITC-conjugated goat anti-human IgG, 1:25
(Cappel, Durham, NC); mouse anti-CD4 (Ortho Diagnostic Systems,
Raritan, NJ), 1:500; mouse anti-Flag, 1:100 (Eastman Kodak Co.);
Rh-conjugated goat anti-mouse IgG, 1:50 (Kirkegaard and Perry Labs);
rabbit anti-Cb, 1:100 (courtesy of Dr. Reinhard Jahn, Max Planck
Institute for Biophysical Chemistry); FITC-conjugated goat anti-rabbit
IgG, 1:50 (Kirkegaard and Perry Labs).
Uptake of Labeled F(ab) and Transferrin--
For continual
uptake experiments, cells were washed twice with PBS and incubated for
2 h at 37 °C in DMEM containing 100 µg/ml of FITC-conjugated
goat anti-human IgG F(ab) fragment (Cappel), 100 µg/ml Rh-transferrin
(Molecular Probes, Inc., Eugene, OR), or 100 µg/ml Texas Red
transferrin (Molecular Probes), and 1% BSA. Following this, the
coverslips were washed three times with 10 mM acetic acid
in PBS, fixed, and mounted. For pulse-chase experiments, cells were
washed twice and incubated on ice for 20 min in serum-free DMEM
supplemented with 1% BSA and 100 µg/ml of FITC-conjugated goat
anti-human IgG F(ab) fragment. Following three washes to remove unbound
antibody, the cells were either fixed immediately or returned to normal
growth conditions for a 4-h chase. 100 µg/ml Rh-transferrin was added
to the chase media to co-localize internalized antibody with endocytic
structures. Surface-bound transferrin was removed with three washes of
10 mM acetic acid in PBS before fixation and viewing. For
fluorescence ratio imaging experiments, the recycling endosomes were
labeled with FITC-F(ab) in DMEM containing 10% serum, chased for 2-12 h, and washed with Ringer's solution (described below). For imaging the transferrin compartment, cells were serum-starved for 30 min in
DMEM with 1% BSA and then loaded with FITC-Tf (50 µg/ml, Molecular Probes) in 1% BSA/DMEM for 2-5 h. Acetylstrophanthidin or bafilomycin was added to the media after the first hour. There was a nominal chase
(~10 min) in the absence of Tf at room temperature while the
coverslips were prepared for imaging.
Fluorescence Ratio Imaging of Cytosolic and Endosomal
pH--
Endosomal compartments labeled with F(ab)- or Tf-FITC and
cytosol labeled with 2 µM BCECF-AM (Molecular Probes)
were monitored in separate experiments using digitally processed
fluorescence ratio imaging. Coverslips (22 mm diameter) with dye-loaded
cells were placed in an open perfusion chamber on an inverted IM35
Zeiss microscope. Fluorescence measurements from up to 16 cells were made during each experiment. A × 40 or × 60 oil immersion
objective (Zeiss) and either a × 1 (cytosol) or × 6.3 field
objective (organelles) was used to magnify the images before
transmission to the camera. A low light level DAGE 68 SIT camera
collected (through a 530-nm band pass filter) emission images of the
cells during excitation at 490 and 440 ± 5 nm (Omega Optical,
Brattleboro VT). Filters were changed with a Lambda 10
2 filter wheel
(Sutter Instruments, Novato CA). Separate images for each wavelength
were averaged over eight frames by a digital image processor (Axon
Image Lightning, Axon Instruments, Foster City CA) and subsequently
converted pixel by pixel to a ratio image. Experimental parameters such
as data collection rate (one ratio image every 5-60 s), changing the
filter wheel and opening/closing the shutter were controlled by a
133-MHz Pentium computer (Gateway 2000) running Axon's Imaging
Workbench. The ratio images were displayed in pseudocolor. Data were
collected by electronically selecting regions of the image for
quantitation. Cytosolic measurements were made from entire cells. For
organelles, only the brightest perinuclear regions were selected. FITC
fluorescence at 490 nm increases with pH, while fluorescence at 440 nm
is nearly insensitive to pH. Problems due to photobleaching and dye
loss were minimized by reducing the intensity (with neutral density filters), duration of illumination, and number of images collected.
Perfusion of Solutions during pH Measurements--
Ringer's
solution contained 141 mM NaCl, 2 mM KCl, 1.5 mM K2HPO4, 1 mM
MgSO4, 10 mM glucose, 2 mM
CaCl2, 10 mM HEPES, pH 7.4. Calibration
solutions contained 70 mM KCl, 70 mM NaCl, 1 mM each K2HPO4 and
KH2PO4, 1.3 mM MgSO4,
10 mM glucose, 1 mM CaCl2, and 10 mM HEPES or 10 mM MES, adjusted to various pH
values with NaOH and KOH. HEPES was used for solutions with pH > 6.5, and MES was used for solutions with pH
6.5.
Calibration of BCECF and FITC Fluorescence in Terms of
pH--
An in situ calibration was performed following each
experiment to convert 490-/440-nm values to pH. Calibration solutions containing a 5-10 µM concentration of the
K+/H+ exchange ionophore nigericin and a 5-10
µM concentration of the Na+/H+
exchange ionophore monensin were perfused over cells. By using these
solutions, we made no assumptions about Na+ and
K+ concentrations within cytosol or organelles and allowed
the equilibration of Na+ and K+ to drive the
equilibration of H+. At least four solutions at various pH
values (8.0, 7.5, 7.0, 6.5, 6.0, 5.5, and/or 5.0) were used per
calibration. For each cell and organelle, a calibration curve was
generated, the data were fit to a sigmoidal curve with Graphpad (San
Diego, CA), and the resulting fit was used to convert the ratio to pH
values using the equation pH = pK + log((R
Rmin)/(Rmax
R)), where R is the ratio,
Rmin and Rmax are the
minimum and maximum values determined from the fit, and pK
is the pKa determined from the fit. Experimental
data were compared using unpaired Student's t test (two-tailed). All data are presented as mean ± S.E. Differences were considered significant if p < 0.05.
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RESULTS |
Experimental Strategy--
Our methodology for measuring the pH of
recycling endosomes in living cells is outlined in Fig.
1. We placed a luminal epitope tag on Cb
by extending its C terminus with the monomeric constant region of human
IgG heavy chain. When this fusion construct (Cb-Ig) appeared at the
cell surface, the IgG epitope was exposed to the external medium and
could therefore be tagged by the exogenous addition of FITC-conjugated
antibodies. The bound antibody subsequently hitch-hiked on the cycling
protein and at steady state resided in the intracellular location of
its escort protein. Organelle pH measurements were then made in single
living cells using the pH sensitivity of the fluorescein moiety and
digital imaging microscopy. Measurements were made over a sustained
time and under a variety of conditions without the temporal limitations
of using Tf-FITC, which is present only transiently in the recycling
endosomes.

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Fig. 1.
Schematic representation of Cb-Ig
construction and targeting in living cells. Top, a
full-length rat Cb was fused in frame to the CH2 and CH3 domains of
human IgG. The resulting Cb-Ig construct thus contained a luminal IgG
epitope tag following the transmembrane (TM) domain of Cb.
Bottom, Cb-Ig can be visualized in living cells by the
addition of FITC-conjugated anti-hIgG F(ab) antibodies to the
incubation medium. The continual cycling of Cb-Ig between the endosomes
and plasma membrane results in the transient exposure of the IgG tag to
the extracellular medium. When this occurs, the FITC-F(ab) antibodies
bind to the epitope and travel with Cb-Ig through the endosomal system.
Since in the steady state Cb-Ig resides in the recycling endosomes, the
fluorescent signal arises predominantly from this location.
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Cb-Ig Localizes to the Recycling Endosomes--
A stable clone of
COS-7 cells expressing Cb-Ig was isolated. To confirm that the fusion
construct was properly targeted to the recycling endosomes, we used
indirect immunofluorescence microscopy to colocalize Cb-Ig with markers
for the ER, Golgi, trans-Golgi network (TGN), and endosomes.
The ER, Golgi, and TGN were visualized by transient transfection with
epitope-tagged constructs specifically targeted to these compartments:
CD4-UDPGT (ER), galactosyltransferase-Flag (trans-Golgi),
and furin-Flag (TGN) (see "Experimental Procedures"). Endosomes
were labeled by a 2-h incubation in media containing 100 µg/ml
Rh-Tf.
Cells were fixed, permeabilized, and doubly stained with antibodies
against both human IgG on Cb-Ig and the epitope tag on the transfected
organelle marker. Cb-Ig was found at the cell surface and in a
perinuclear area (Fig. 2, B,
D, F, and H), similar to that reported
for unmodified Cb in CV-1, CHO, and rat brain glial cells (17-19). The
distribution of Cb-Ig clearly did not coincide with the ER marker
CD4-UDPGT, which was found in a branching, tubular network that
extended throughout the cytoplasm but was most concentrated around the
nucleus (Fig. 2A). Cb-Ig staining also differed from the
Golgi (GalT-Flag, Fig. 2C) and TGN (furin-Flag, Fig.
2E), two well defined tubular compartments that often formed bulbous, circular, and semicircular patterns in the perinuclear region
of these cells. The overlapping but clearly distinct distributions of
Cb-Ig, GalT-Flag, and furin-Flag was expected, as many organelles are
positioned near the cell center (26). In contrast to the other markers,
Rh-Tf (Fig. 2G) labeled a distinct pericentriolar spot
which co-localized with Cb-Ig. This perinuclear localization of
Tf has been used to define the recycling endosomes (1, 4, 11). We
therefore concluded that, like native Cb, the Cb-Ig fusion
construct was targeted to the recycling endosomes.

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Fig. 2.
Localization of Cb-Ig in stably transfected
COS-7 cells. The steady-state distribution of Cb-Ig in a stably
transfected cell line was examined by comparing its distribution with
transiently transfected, epitope-tagged organelle markers.
A-F, Cb-Ig is targeted to perinuclear structures
distinguishable from compartments of the secretory pathway. Cells were
fixed and permeabilized 48 h post-transfection and doubly stained
for Cb-Ig (B, D, F) and the ER marker
CD4-UDPGT (A), the Golgi marker GalT-Flag (C), or
the TGN marker furin-Flag (E). FITC-goat anti-hIgG was used
to visualize Cb-Ig, and the co-transfected markers were visualized with
either mouse anti-CD4 or mouse anti-Flag followed by Rh-goat anti-mouse
IgG. G-H, Cb-Ig and Tf were localized to the same
perinuclear region characteristic of the recycling endosomes. Endosomes
were labeled for 2 h with 100 µg/ml Rh-Tf (G) before
fixation and staining with FITC goat anti-hIgG to localize Cb-Ig
(H). Bar, 10 µm.
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Cycling of Cb Allows Antibody Uptake in Live Cells--
We further
investigated the trafficking of Cb-Ig with a series of antibody uptake
studies. Untransfected (Fig. 3,
A and B) and Cb-Ig-transfected (Fig. 3,
C-F) COS-7 cells were incubated in medium containing Rh-Tf
and FITC-conjugated anti-IgG F(ab) fragment antibodies, and the
patterns of F(ab) and Tf fluorescence were compared. A monovalent F(ab)
fragment was used to ensure that the added antibody did not induce
cross-linking of Cb-Ig. After a 2-h incubation in the continual
presence of antibodies at 37 °C, Cb-Ig-transfected cells showed
clear labeling of an organelle that was similar to that observed in the
fixed and stained cells (compare Fig. 3C with Fig. 2). No
fluorescence was detected in untransfected COS-7 cells (Fig.
3A), demonstrating that F(ab) antibody uptake was an
epitope-mediated event and that "background" fluorescence resulting
from fluid phase endocytosis of the antibody was minimal. In
Cb-Ig-transfected cells, FITC-F(ab) and Rh-Tf were found together in
vesicles dispersed throughout the cytoplasm and in the perinuclear
recycling endosomes (Fig. 3, C and D). Some bound
antibody was also present at the cell surface (Fig. 3C). As
assessed by indirect immunofluorescence and cytofluorometry, the FITC
signal was attenuated when cells were preincubated with unconjugated
anti-hIgG F(ab) antibodies for 2 h at 37 °C before FITC-F(ab)
labeling (data not shown). These results demonstrated that, in addition
to proper targeting, the fusion construct continually cycled through
the endosomal system in a manner analogous to that presumed for native
Cb.

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Fig. 3.
Labeling the Cb-Ig compartment in live cells
by antibody uptake. A-D, antibody uptake is dependent
on the expressed Ig-epitope. Untransfected cells (A,
B) and cells stably transfected with Cb-Ig (C,
D) were incubated in medium containing 100 µg/ml FITC-goat
anti-hIgG F(ab) fragment (A, C) and Rh-Tf
(B, D). Cells were fixed after a 2 h
37 °C incubation in the continual presence of labels. Only the
transfected cells (C) take up FITC-F(ab). E and
F, pulse-chased antibodies accumulate in the perinuclear
recycling endosomes. Cells stably transfected with Cb-Ig were
pulse-labeled with 100 µg/ml FITC-goat anti-hIgG F(ab) on ice for 20 min. F(ab) was removed, and the cells were chased at 37 °C for
4 h in medium containing 100 µg/ml Rh-Tf. Endocytosed FITC-F(ab)
fragment (E) was concentrated in the recycling endosomes as
was Rh-Tf (F). Bar, 10 µm.
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The recycling endosomes were also labeled with a pulse-chase protocol.
A 20-min incubation on ice with FITC-conjugated goat anti-human IgG
F(ab) antibodies labeled the plasma membrane of transfected cells,
whereas no fluorescent signal could be detected in untransfected cells.
After a 4-h chase at 37 °C, the surface label had dissipated and was
replaced by a perinuclear stain that colocalized with Rh-Tf (Fig. 3,
E and F). Surface and peripheral endosome
staining was not visible, presumably because the majority of
FITC-labeled Cb-Ig accumulated in the recycling endosomes at steady
state. Labeling of the recycling endosomes was apparent within 45 min
of chase and was still visible after 24 h (data not shown).
Laser Scanning Confocal Microscopy Distinguishes Endosomes Labeled
by Tf and Cb--
The distributions of Cb-Ig and Rh-Tf were examined
in greater detail with the use of laser scanning confocal microscopy
(Fig. 4). After a 3-h incubation with 100 µg/ml of Texas Red Tf, cells were fixed, permeabilized, and stained
with FITC goat anti-hIgG antibodies. Representative merged images from
single optical sections are shown. In contrast to the results obtained
with indirect immunofluorescence, Cb-Ig and Tf could be visualized in
separate pericentriolar vesicle populations. These differences were
most obvious near the bottom or top of the cell, but differences could
also be visualized in the middle sections where extensive
colocalization was also present (Fig. 4A). The same result
was obtained when Cb-Ig was localized by FITC-F(ab) antibody uptake
(Fig. 4B) and when endogenous Cb was visualized in
untransfected COS-7 cells (Fig. 4C). These results indicated
that neither the luminal IgG epitope nor F(ab) internalization was
affecting the localization of Cb-Ig. Observation of cells stained with
a single fluorophore (either Texas Red Tf or FITC-conjugated anti-hIgG
antibodies) demonstrated that bleed-through was not occurring in the
optical sections (data not shown).

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Fig. 4.
Localization of Cb and Tf assessed by laser
scanning confocal microscopy. Cb and Tf are heterogeneously
distributed among the recycling endosomes. Representative merged images
from single 0.5-µm optical sections are shown. For all conditions, Tf
was visualized by a 3-h incubation with 100 µg/ml Texas Red Tf.
A-C, Cb-Ig was visualized in stably transfected COS-7 cells
by either postfixation staining with FITC goat anti-hIgG antibodies
(A) or by a 3-h incubation with 100 µg/ml FITC goat
anti-hIgG F(ab) antibodies (B). Endogenous Cb was visualized
in untransfected COS-7 cells by postfixation staining with rabbit
anti-Cb antibodies followed by FITC goat anti-rabbit IgG antibodies
(C). D-F, cells were treated with 20 µM nocodazole for 1 h prior to fixation. Cb-Ig was
visualized in stably transfected COS-7 cells by either postfixation
staining with FITC goat anti-hIgG antibodies (D) or by a 3-h
incubation with 100 µg/ml FITC goat anti-hIgG F(ab) antibodies
(E). Transiently transfected, untagged Cb was visualized in
COS-7 cells by postfixation staining with rabbit anti-Cb antibodies
followed by FITC goat anti-rabbit IgG antibodies (F).
Cb-enriched (green), Tf-enriched (red), and Cb/Tf
intermixed (yellow) endosomes could be observed in all
optical sections under any of the given conditions. Bar in
A-C, 5 µm; bar in D-F, 10 µm.
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Microtubule depolymerization leads to the dispersal of many organelles
throughout the cytoplasm (6, 15, 26, 27) and would thus allow for a
more definitive examination of Cb/Tf codistribution. We therefore
repeated our experiments in cells treated with the microtubule
depolymerizing agent nocodazole. As shown in Fig. 4, D-F,
some degree of colocalization persisted in nocodazole-treated cells.
This was observed when Cb-Ig was visualized by postfixation staining
(Fig. 4D) or by FITC-F(ab) antibody uptake (Fig.
4E). Partial colocalization was also seen in COS-7 cells
transiently transfected with an untagged Cb (Fig. 4F).
Transient transfection of untagged Cb was required, because the
distribution of endogenous Cb could not be detected over background
fluorescence in nocodazole-treated cells. Transfection did not alter
the steady state distribution of Cb (data not shown) and allowed
individual structures to be resolved in nocodazole-treated cells. The
partial codistribution of Cb and Tf in both untreated and
nocodazole-treated cells thus indicated that the proteins were targeted
to overlapping but distinct subpopulations of recycling endosomes.
The percentage of Cb-enriched (green), intermixed
(yellow), and Tf-enriched (red) endosomes was
determined by visual inspection of merged optical sections such as
those shown in Fig. 4 (Table I). Since
the degree of overlap varied with the cell section, values are given
for bottom, middle, and top regions of the cell. Only perinuclear
vesicles were counted in cells prepared without nocodazole treatment.
Several trends could be ascertained from Table I: (i) in all optical
sections, only 20-30% of endosomes showed intermixing of Cb and Tf,
the rest being composed of Cb- or Tf-enriched vesicles; (ii) the
percentage of Cb-enriched endosomes increased as confocal sectioning
progressed from the bottom to the top of the cell; (iii) the percentage
of Tf-enriched endosomes decreased as confocal sectioning progressed
from the bottom to the top of the cell; and (iv) Cb-enriched vesicles
were more predominant than Tf-enriched or intermixed endosomes in
almost all optical sections. These general trends applied to all tested
conditions and further documented the heterogeneous distributions of Cb
and Tf in the recycling endosomes.
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Table I
Distribution of endosomes containing Cb, Cb and Tf, and Tf
The degree of overlap between Cb and Tf is shown. Images such as those
in Fig. 4 were visually screened for green (Cb-enriched), yellow (Cb-
and Tf-intermixed), or red (Tf-enriched) vesicles. At least 650 endosomes from five or more cells were quantitated for each condition
and section. Results are presented as the percentage of endosomes that
are Cb-enriched, Cb and Tf intermixed, and Tf-enriched. Comparisons
were made between Tf and endogenous Cb visualized by postfixation
staining (Cb fix), untagged transfected Cb visualized by postfixation
staining after nocodazole treatment (nocodazole/Cb fix), Cb-Ig at
steady state with or without nocodazole treatment (Cb-Ig fix and
nocodazole/Cb-Ig fix), or internalized antibodies bound to Cb-Ig with
or without nocodazole treatment (F(ab) and nocodazole/F(ab)). In cells
prepared without nocodazole treatment, only perinuclear vesicles were
considered. Middle sections were defined as those images in which the
nucleus and cell body were clearly defined.
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pHCb Is Lower Than pHTf--
The epitope
specificity of the F(ab) antibody uptake in conjunction with the
4 °C pulse labeling and 37 °C chase resulted in specific labeling
of the Cb-Ig-containing recycling endosomes (Fig. 3E). We
used this protocol for in vivo pH measurements. Briefly,
cells chased for 2-12 h were set into an open perfusion chamber and
alternately illuminated with 490- and 440-nm light. An example is shown
in Fig. 5, A and B.
The resulting ratio image (490/440 nm) from the bright perinuclear spot
estimated organelle pH (Fig. 5C). Raw ratio data from three
of the cells in Fig. 5, A-C, are shown in Fig.
6A. Organelle viability was
demonstrated by the instantaneous alkalization resulting from perfusion
with Ringer's solution containing 30 mM NH4Cl.
Following treatment with NH4Cl, membranes were
permeabilized with nigericin and monensin in equimolar Na+
and K+ with varying pH values. The calibration data from
this experiment are presented in Fig. 6B. The ratio values
at a given pH were stable for many minutes and provided a consistent
reading; multiple exposures to the pH 6.5 solution produced the same
organelle ratio value. Using this calibration curve, we estimated an
average pHCb of 6.2 for the three cells in Fig. 5. An
apparent pKa of 6.6 for FITC-F(ab) was derived from
the calibration data obtained from all experiments performed in both
COS-7 cells (Fig. 6C) and CHO cells stably transfected with
Cb-Ig (not shown). The pHCb distribution from all
experiments performed in COS-7 cells (Fig. 6D,
solid bars) showed a wide range of recorded
values, from 5.2 to 6.6 with an average of 6.1 ± 0.05 (n = 35 cells; 12 experiments). An identical
pHCb of 6.1 ± 0.05 (n = 16 cells;
four experiments) was measured in CHO cells stably transfected with
Cb-Ig (Fig. 6E, solid bars).

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Fig. 5.
Fluorescent ratio imaging of
pHCb. Cb-Ig cells were incubated with 100 µg/ml of
FITC-goat anti-hIgG F(ab) for 30 min at 4 °C and then chased for
5 h at 37 °C. Cells were illuminated with either 490- (A) or 440-nm (B) wavelength light, and images
were captured with a low light level DAGE 68 SIT camera. A pixel by
pixel ratio image was created and displayed in pseudocolor
(C). "Hotter" colors in the ratio image indicate
increasing pH.
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Fig. 6.
Quantitation of pHCb
and comparison with pHTf.
A and B, numerical data from three of the four
cells in Fig. 5 are plotted versus time. pHCb
increased during a 30 mM
NH4+ pulse, showing that membranes of
the recycling endosomes were intact (A). Calibration data of
cells shown in A indicated that the signals were stable for
>20 min. Cells were pulsed with solutions at various pH values
containing 10 µM nigericin and monensin(B).
C, summary of all calibrations (35 cells). The 490-/440-nm
fluorescence ratio for each cell was plotted versus pH of
the calibration solution, and the maximum values from individual fits
were set to 1. The raw calibration data were then expressed relative to
the fitted maximum to allow comparison among experiments. These data
indicated that the apparent pKa was ~6.6.
D and E, frequency histogram of organelle pH in
COS-7 (D) and CHO (E) cells. pHCb was
measured as in A and B, and pHTf was
measured as in Table II. Data represent a summary of all
pHCb and pHTf measurements made and are
expressed as a percentage of the total number of cells measured for
each marker. For COS-7 cells, there were 35 measurements for the Cb
compartment and 46 measurements for the Tf compartment; for CHO cells,
there were 16 measurements for both the Cb and Tf compartment.
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Since the values obtained using Cb-Ig were considerably lower than
previous data obtained with FITC-Tf, we repeated the pH measurements
using FITC-Tf loaded into the same cell lines. Cells were incubated
with FITC-Tf for 2-5 h and then chased for 10 min in Ringer's
solution at room temperature. Imaging of the perinuclear recycling
endosomes again produced an array of values (Fig. 6D, hatched bars), although both the range (5.6-7.2)
and average pHTf of 6.5 ± 0.05 (n = 46 cells; six experiments) were significantly more alkaline
(p < 0.05) in the Tf-labeled compartment. A similar pHTf of 6.6 ± 0.06 (n = 16 cells;
five experiments) was obtained in CHO cells stably transfected with
human Tf receptor and Cb-Ig (Fig. 6E, hatched
bars). A broad pHTf distribution has previously been observed by others recording the pHTf of individual
endosomes (22). The average pH obtained with either Cb-Ig or Tf thus
represented a range of values that varied from cell to cell and, most
likely, from endosome to endosome.
Bafilomycin and Ouabain Exert Different Effects on pHCb
and pHTf--
Previous Tf-based studies have reported that
endosomal pH is maintained by a H+-ATPase and regulated by
Na+/K+-ATPase activity (21-24, 28, 29). In
order to test whether these mechanisms were active in the
Cb-Ig-containing recycling endosomes, we applied bafilomycin and either
ouabain or acetylstrophanthidin (a membrane-permeant ouabain analog) to
COS-7 cells and determined the effect on endosomal pH. Treatment with
100 nM bafilomycin A1, an inhibitor of the
vacuolar-type H+-ATPase, elicited an increase in
pHCb. In the experiment shown in Fig.
7A, bafilomycin caused
pHCb to increase from 6.3 to 7.0. On average, bafilomycin
treatment shifted pHCb from 6.2 ± 0.08 to 6.7 ± 0.09 (n = 10 cells; three experiments). We also
measured the effect of bafilomycin on pHTf (Table
II); pretreatment of cells with 100 nM bafilomycin caused a more pronounced shift in pHTf from 6.5 ± 0.05 to 7.7 ± 0.05 (n = 18 cells; three experiments). The
bafilomycin-induced alkalization to a pHTf greater than 7.0 has been observed by others (23, 24, 29). These results demonstrated
that both classes of recycling endosomes maintained a steady state pH
by the continual action of a H+-ATPase operating to counter
a leak of proton equivalents. Similar conclusions about pH regulation
in the endosomes, phagosomes, Golgi, and TGN have been published
(30-33). It was possible that the recycling endosome pH might have
been affected only indirectly by bafilomycin, due to an effect of the
drug on cytosolic pH (pHc). We therefore measured
pHc in COS-7 cells using BCECF-AM. The average pHc (7.5 ± 0.03, n = 53 cells; six
experiments) in COS-7 cells was unaffected by bafilomycin (data not
shown), so alkalization of the recycling endosomes was not due to a
secondary effect arising from a change in pHc. We concluded
that recycling endosomes maintained their acidity due to the activity
of an H+-ATPase, while pHc was regulated by
other mechanisms.

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Fig. 7.
Effects of bafilomycin and ouabain on
pHCb. pHCb is alkalinized by bafilomycin
and is unaffected by ouabain. A, pHCb was
measured in COS-7 cells as described in the legend to Fig. 6. A
representative trace is shown from a cell over which 100 nM
bafilomycin was perfused. The cell rapidly alkalinized due to
inhibition of the H+-ATPase but remained intact as
indicated by its continued responsiveness to
NH4+ (data not shown). On average,
bafilomycin treatment shifted pHCb from 6.2 ± 0.08 to
6.7 ± 0.09 (n = 10 cells; three experiments).
B, there was no pH change when 100 µM ouabain
was applied to the cells. Traces from two cells within the same dish
are shown; they had different pHCb values, but neither
responded to ouabain treatment. R, Ringer's solution.
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Table II
Drug effects on pHTf
The effect of bafilomycin and acetylstrophanthidin on pHTf is
shown. COS-7/Cb-Ig cells were incubated in 50 µg/ml FITC-Tf for 2-5
h. Drugs were added for 1-4 h prior to pH measurements.
Acetylstrophanthidin (1 µM) acidified the compartment,
whereas bafilomycin (100 nM) dramatically alkalinized the
compartment. As FITC-Tf is rapidly transported out of the recycling
endosomes, drug pretreatment was necessary in order to ensure that an
effect could be seen before FITC-Tf exited the compartment. These
measurements therefore represented an effect on the average
pHTf but did not show the real-time changes induced by
inhibition of the Na+/K+-ATPase or H+-ATPase.
The control Tf data from Fig. 6D are presented for
comparison.
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In contrast to bafilomycin, ouabain had no effect on pHCb
(Fig. 7B). Based on the speed with which Tf and bulk
membrane enter the recycling endosomes (4, 10, 34), perfusion of
labeled cells with ouabain should have inhibited the recycling endosome Na+/K+-ATPase within 5 min. Yet, as shown in
Fig. 7B, ouabain had no effect on pHCb over this
time course. Treatment with 1 µM of the membrane-permeant
Na+/K+-ATPase inhibitor acetylstrophanthidin
also had no effect over 30-60 min (three experiments; data not shown).
However, as shown in Table II, inhibition of the
Na+/K+-ATPase by pretreatment with
acetylstrophanthidin caused pHTf to decrease; the average
pHTf of 6.5 obtained from FITC-Tf-loaded cells acidified to
pHTf 5.9 in cells pretreated with 1 µM
acetylstrophanthidin. It thus appeared that Tf and Cb-Ig were targeted
to overlapping perinuclear compartments that differed in both average
pH and responses to inhibitors of Na+/K+-ATPase
and H+-ATPase activity.
We also examined the effects of altering endosomal pH on the cycling of
Cb-Ig and Tf to the cell surface by quantitative assays using
125I-protein A and 125I-Tf. These studies
showed that bafilomycin slowed trafficking of both Cb-Ig and Tf from
the recycling endosomes to the plasma membrane, while ouabain and
acetylstrophanthidin had no effect on either marker (data not
shown).
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DISCUSSION |
"Targeted Fluorescence" Method to Study pH of the Recycling
Endosomes--
The targeted fluorescence method is based on binding
exogenously added fluorescent antibodies to specific "resident"
proteins that cycle between the plasma membrane and their home
organelle. We chose Cb because it is thought to cycle between recycling
endosomes and the plasma membrane (17-19). Proper targeting of Cb-Ig
to recycling endosomes was confirmed by colocalization with Rh-Tf but
not with markers for the ER, Golgi, or TGN. Uptake of FITC-F(ab)
antibodies was mediated by binding to Cb-Ig exposed at the surface (and
not by bulk endocytosis), probably due to the fact that very low
concentrations of FITC-F(ab) allowed specific labeling of the recycling
endosomes (e.g. 100 µg/ml as opposed to 5-10 mg/ml
required for fluid phase endocytosis (22, 24)). Preincubation with
unlabeled F(ab) significantly reduced subsequent uptake of FITC-F(ab),
demonstrating that Cb indeed cycled between endosomes and the plasma
membrane. The "targeted fluorescence" strategy has also been useful
for examining pH of the TGN and may be a general strategy for studying organelles containing proteins that cycle between the cytosol and the
plasma membrane (33, 35).
We found that pHCb ranged from 5.2 to 6.6 (mean
pHCb 6.1). FITC-Tf also showed a wide range of values,
although both the range (pHTf 5.6-7.2) and average (mean
pHTf 6.5) were significantly more alkaline than
pHCb. A broad distribution (pH 5.5-7.2) has also been
observed in endosomes labeled with fluorescein/rhodamine-Tf, although
the majority fell within a pH range of 6.0-6.5 (22, 24, 29). Rybak
et al. (36) have proposed that variations in endosome size,
shape, buffering capacity, and ion transport activity could account for
this broad pH distribution. A wide pH range may not be limited to the
endosomal system, since highly variable pH values in both the Golgi
(32) and immature secretory vesicles (37) have also been reported.
D'Souza et al. (35) have also used Cb-targeted fluorescence
to measure pHCb in CHO cells lacking the
Na+/H+ exchanger. Their reported value of
pHCb 6.7 contrasts with the pHCb 6.1 we
observed in both COS-7 and CHO cells. This discrepancy may be due to
differences in wild-type versus
Na+/H+ exchange-deficient CHO cells or could
reflect an unexpected physiological effect on pHCb induced
by the "H+ suicide" method for selecting
Na+/H+ exchange-deficient cells. This
difference deserves further attention.
Differential Distribution of Cb and Tf among Recycling
Endosomes--
Although Cb apparently co-localized with Rh-Tf, it
became obvious that the two labels were in different subpopulations of endosomes. First, confocal microscopy demonstrated that the majority of
labeled endosomes contained differing amounts of Cb-Ig and Tf (Fig. 4
and Table I). This difference was also observed with native Cb (Fig. 4
and Table I), thereby demonstrating the similar localization of native
Cb and Cb-Ig. Second, pH measurements yielded distinct results
depending on whether Cb-Ig or Tf was used as the organelle marker.
Previous work using Tf as a recycling endosome marker recorded an
average pHTf of 6.4 (4, 22-24). We confirmed these
findings in COS-7 and CHO cells but consistently found pHCb < pHTf. Furthermore, bafilomycin A1 caused
pHCb to increase by 0.5 pH units to 6.7, while
pHTf increased 1.2 pH units to 7.7. Finally,
acetylstrophanthidin caused pHTf to decrease (also see Refs. 21-24), but neither ouabain nor acetylstrophanthidin affected pHCb, regardless of the initial pHCb value.
A model summarizing our data (Fig. 8)
proposes that recycling endosomes are heterogeneous, ranging between
the two extremes of a highly enriched Tf subpopulation and a highly
enriched Cb subpopulation. All occupy a perinuclear location, thereby
leading to the gross colocalization of Tf and Cb (15, 17, 18). These endosomes utilize a H+-ATPase to generate luminal acidity
but have different pH values and Na+/K+-ATPase
activity and can be segregated morphologically using nocodazole. The Cb
population was more acidic and was not affected by
Na+/K+-ATPase inhibitors, while the Tf
population was somewhat more alkaline and was acidified by inhibitors
of the Na+/K+-pump. The differential
distribution of Na+/K+-ATPase activity between
Tf- versus Cb-containing recycling endosomes may also be
responsible for the difference in pHCb and pHTf
after bafilomycin treatment; Na+/K+-pump
activity could generate a positive membrane potential that would drive
H+ out of the Tf compartment, leading to a more alkaline pH
than that found in the Cb-containing recycling endosomes, which appear to lack Na+/K+-ATPase activity. Either absence
or regulation of the Na+/K+-ATPase activity
could account for our pHCb observations. Other channels and
pumps are likely to play a role in pH homeostasis as well.

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Fig. 8.
Model for the heterogeneous composition of
the recycling endosomes. Endocytosed molecules are transported
from the plasma membrane to sorting endosomes (pH 6.0), whereupon
ligands and receptors are dissociated. Soluble ligands continue to late
endosomes (pH 5.5), while membrane proteins are sorted to the
perinuclear recycling endosomes. Cb and Tf are partitioned into
subpopulations of this compartment. The Tf-enriched subclass has an
average pH of 6.5, which is generated by a H+-ATPase and
regulated by a Na+/K+-ATPase. In contrast, the
Cb-enriched subclass has an average pH of 6.1, which is similarly
generated by a H+-ATPase but lacks detectable
Na+/K+-ATPase activity. Inhibition of the
H+-ATPase also has a differential effect on the
compartmental pH of the two subpopulations.
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Previous data are also consistent with the proposal that recycling
endosomes are heterogeneous. Daro et al. (15) showed that
recycling endosomes contained both Tf and Cb; but one subpopulation contained Rab4, while another did not. In addition, Rab11 was found on
both Tf-positive and Tf-negative recycling vesicles (16). Finally,
cleavage and inactivation of Cb by tetanus toxin reduced Tf release by
only 20-33% (18), although Cb's proposed role as a v-SNARE for
trafficking of endosomal vesicles to the plasma membrane (17, 18, 38)
would have predicted a complete block (39). Our model predicts that the
20-33% of tetanus toxin-sensitive Tf was present in the pool of
recycling endosomes that contained both Tf and Cb (e.g. see
Table I), while the 67-80% of Tf present in endosomes that did not
contain Cb were insensitive to the toxin.
Possible Function of Recycling Endosome
Subpopulations--
Although we do not know the exact functions and
trafficking patterns of the Tf and Cb endosome subpopulations, an
intriguing possibility is that recycling endosomes act as a sorting
station for targeting internalized proteins to the TGN. This idea could explain the apparent structural reorganization of the endosomal system
that has been proposed for cells expressing a chimeric TGN38/TfR
protein (TfR internalization motif replaced with the TGN38 YQRL
targeting signal; see Ref. 40). The TGN38/TfR construct localized not
to the TGN, but instead to a juxtanuclear structure that was
morphologically distinct and significantly more acidic (pH 6.0) than
the wild-type TfR-containing recycling endosomes (pH 6.5). In light of
the work presented here, we propose an alternative explanation that the
TGN38/TfR was targeted to the Cb-containing subpopulation of recycling
endosomes that is present at all times. Further work will be required
to elucidate the role of the Cb-containing recycling endosomes as a
possible sorting station for targeting internalized proteins to the
TGN.
We thank members of the Moore and Machen labs
for critical reading of the manuscript, Amy Robinson for the figures,
Dr. Brian Seed for providing the cDNA libraries and expression
vectors used in this study, Dr. Kazuhisa Nakayama for supplying the
furin plasmid, Dr. Robert Murphy for sharing his preprint with us, Dr.
Duncan Stuart for technical assistance with the confocal microscope, and Axon Instruments for providing the imaging hardware and
software.