Loss of Rab27 function results in abnormal lung
epithelium structure in mice
Giulia Bolasco, Dhani C. Tracey-White, Tanya Tolmachova, Andrew J. Thorley,
Teresa D. Tetley, Miguel C. Seabra and Alistair N. Hume
Am J Physiol Cell Physiol 300:C466-C476, 2011. First published 15 December 2010;
doi:10.1152/ajpcell.00446.2010
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Am J Physiol Cell Physiol 300: C466 –C476, 2011.
First published December 15, 2010; doi:10.1152/ajpcell.00446.2010.
Loss of Rab27 function results in abnormal lung epithelium structure in mice
Giulia Bolasco,1 Dhani C. Tracey-White,1 Tanya Tolmachova,1 Andrew J. Thorley,2 Teresa D. Tetley,2
Miguel C. Seabra,1,3,4 and Alistair N. Hume1
1
Molecular Medicine, 2Pharmacology and Toxicology, National Heart and Lung Institute, Imperial College London, London,
United Kingdom; 3Instituto Gulbenkian de Ciência, Oeiras; and 4Faculdade de Ciências Médicas, Universidade Nova de
Lisboa, Lisboa, Portugal
Submitted 3 November 2010; accepted in final form 13 December 2010
Rab27 proteins; intracellular transport; alveolar epithelium type II
cell; Clara cell
of the Rab family regulate intracellular transport
in eukaryotic cells (5, 22, 24). Analyses of mammalian genomes indicate that this family comprises ⬃60 members that
have intracellular compartment-specific localization and function. Rabs act as molecular switches cycling between an active
GTP and inactive GDP bound states. Active Rabs recruit
effectors to the membrane upon which they reside, and these
transduce functions in vesicle formation, transport, docking, or
fusion. After nucleotide hydrolysis, inactive Rab is then retrieved to the cytosol in complex with Rab GDP dissociation
inhibitor (GDI) in readiness for reutilization in further rounds
of transport. Nascent Rabs are posttranslationally modified by
covalent attachment of geranylgeranyl groups to carboxylterminus cysteine residues, which is essential for their targeting
to specific intracellular membranes.
SMALL GTPASES
Address for reprint requests and other correspondence: A. N. Hume, School
of Biomedical Sciences, Queen’s Medical Centre, Nottingham NG7 2UH, UK
(e-mail: [email protected]).
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While a subset of “housekeeping” Rabs are expressed ubiquitously, Rab27 GTPases (Rab27a and Rab27b) are expressed
in cell types with specialized secretory functions (6, 29). In
these cells, Rab27 localizes to, and regulates, the motility and
exocytosis of lysosome-related organelles (LROs) and secretory granules; e.g., melanosomes in melanocytes, lytic granules
in cytotoxic T cells (CTL), and dense granules in platelets (1,
8, 11, 25, 28, 32). To facilitate these diverse functions Rab27
interacts with eleven known downstream effectors (5, 13). For
instance, in melanocytes, we and others (9, 21) have shown
that Rab27a-GTP sequentially recruits effector Melanphilin/
Slac2-a and molecular motor myosin 5a to melanosomes
thereby allowing their retention in actin-rich peripheral dendrites. Meanwhile, in CTLs, Rab27a mediates granule docking
and exocytosis via effector Munc13– 4 (17). Differential engagement of effectors is in part due to their cell type-specific
pattern of expression (10). In some cell types, Rab27a and
Rab27b isoforms are expressed together, and recent data suggest that they may perform sequential roles in granule docking
and exocytosis via engagement of different effectors (14, 20).
Previous analysis revealed that Rab27 proteins are expressed
in the murine lung, suggesting that they play an important role
in lung cell types with specialized secretory functions (6, 29).
Within the conducting airways (bronchioles), Clara cells perform an important role in production and release of lung lining
fluid components; e.g., surfactant protein A, Clara cell-specific
protein (CCSP). While in the gas exchange, (alveolar) epithelium type II (AEII) cells perform a similar function producing
surfactant protein and phospholipid components (3, 15). Lining
fluid proteins and lipids perform an essential role in the innate
immune response to invading pathogens; e.g., defensins and
collectins, and reduction of surface tension that maintains the
alveolar structure; e.g., surfactant proteins and lipids. Interestingly, within AEII cells a subset of surfactant components are
stored in lysosome-related organelles (LROs), known as lamellar bodies (LB), that release their cargo into the airspace upon
stimulation (4).
Several studies provide evidence for an important role for
Rab GTPases in the physiological function of the lung. For
example, Rab3D has been found to localize to a population of
LBs in AEII cells, although its precise function in this context
remains unknown (30). Rab14, meanwhile, was found partially
localized to LBs, and knockdown resulted in partial inhibition
of their evoked release suggesting a role for Rab14 in this
process (7). Most recently, analysis of the chocolate mutant
mouse and the ruby mutant rat that contain Rab38 gene
mutations revealed a role for this protein in surfactant exocytosis and LB structure (18, 19). In this study, we investigated
the cell-type specific and intracellular localization of Rab27
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Bolasco G, Tracey-White DC, Tolmachova T, Thorley AJ,
Tetley TD, Seabra MC, Hume AN. Loss of Rab27 function results
in abnormal lung epithelium structure in mice. Am J Physiol Cell
Physiol 300: C466 –C476, 2011. First published December 15, 2010;
doi:10.1152/ajpcell.00446.2010.—Rab27 small GTPases regulate secretion and movement of lysosome-related organelles such as T cell
cytolytic granules and platelet-dense granules. Previous studies indicated
that Rab27a and Rab27b are expressed in the murine lung suggesting that
they regulate secretory processes in the lung. Consistent with those
studies, we found that Rab27a and Rab27b are expressed in cell types that
contain secretory granules: alveolar epithelial type II (AEII) and Clara
cells. We then used Rab27a/Rab27b double knockout (DKO) mice to
examine the functional consequence of loss of Rab27 proteins in the
murine lung. Light and electron microscopy revealed a number of
morphological changes in lungs from DKO mice when compared with
those in control animals. In aged DKO mice we observed atrophy of the
bronchiolar and alveolar epithelium with reduction of cells numbers,
thinning of the bronchiolar epithelium and alveolar walls, and enlargement of alveolar airspaces. In these samples we also observed increased
numbers of activated foamy alveolar macrophages and granulocyte containing infiltrates together with reduction in the numbers of Clara cells
and AEII cells compared with control. At the ultrastructural level we
observed accumulation of cytoplasmic membranes and vesicles in Clara
cells. Meanwhile, AEII cells in DKO accumulated large mature lamellar
bodies and lacked immature/precursor lamellar bodies. We hypothesize
that the morphological changes observed at the ultrastructural level in
DKO samples result from secretory defects in AEII and Clara cells and
that over time these defects lead to atrophy of the epithelium.
RAB27 FUNCTION IN LUNG EPITHELIUM
proteins in the murine lung as well as the effects of loss of
Rab27 upon morphology of the pulmonary epithelium.
MATERIALS AND METHODS
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and 12 mg/kg, respectively) and heparin (300 U/ml). Animals were
perfused with PBS through the right ventricle of the heart until the
lungs were visually free of blood. The trachea was then exposed, and
a Luer cannula (BD Insyte; 20 gauge 1.1 ⫻ 30 mm) was inserted and
secured with surgical thread. The lungs and heart were then removed
and fixed by careful inflation with 10% formalin neutral buffer
solution via the trachea at a constant hydrostatic pressure of 30
cmH2O at the height of the carina in the upright position for 15 min.
The lungs were further incubated overnight in fixative, and the right
lung was embedded in paraffin. After deparaffinization and rehydration, 4-␮m sections were stained using hematoxylin and eosin.
Stained sections were then observed using a Zeiss Axiovert 200
inverted microscope and images captured using a Hamamatsu Orca
ER CCD. For mean linear intercept analysis of the integrity of
alveolar epithelium, 10 randomly acquired low-magnification (20⫻)
images for each of 3 age-matched animals were overlaid with a grid
comprising 10 equally spaced horizontal lines, and the number of
times that the epithelium intersected the grid was recorded for each
image. The mean was determined and plotted as a percentage of
age-matched control (wild-type, WT) samples. For measurement of
bronchiolar thickness, bronchiolar epithelium areas were identified
on the basis of intensity; the area was then calculated by subtraction of the luminal-airway area from the total bronchiolar area. The
bronchiolar perimeter was then determined by taking the average
of the inner and outer perimeter measurements (determined with
area measurements). The area was then divided by the perimeter to
give a thickness measurement (area/length of perimeter). This
measurement was performed on 10 randomly selected bronchioles
Fig. 1. Rab27a and Rab27b proteins are expressed in the lungs of mice and
humans. A: lung lysates were immunoblotted using Rab27a and Rab27b
isoform-specific antibodies. Lysates from control animals (Rab27aash/ash and
Rab27BKO) lacking Rab27a and Rab27b, respectively, confirm the specificity
of the antibody staining. Additionally, lysates were blotted with calnexinspecific antibodies as a control for equal loading. B: real-time PCR was
performed to measure the expression of Rab27a- and Rab27b-specific transcripts in total cDNA prepared from samples of resected human lung. Signal
was normalized using 18s rRNA as control and expression level is displayed
as ⌬Ct value (inversely related to expression level).
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Chemicals and antibodies. Unless otherwise stated all chemicals
were obtained from Sigma-Aldrich (Poole, UK). The following antibodies were used for immunoblotting: rabbit polyclonals anti-Rab27a
and anti-Rab27b raised against the COOH-terminus hypervariable
region of each protein at 1:1,000 (Davids Biotechnologie, Regensberg, Germany) and rabbit anti-Calnexin Stressgen (SPA-860D, BioWhittaker; Wokingham, UK) at 1:10,000. For immunohistochemistry,
rabbit anti-pro-surfactant protein C (SPC) antibody (Abcam ab28744,
Cambridge, UK) was used at 1:1,000, rabbit anti-Clara cell-specific
protein (CCSP) (UK cat no. 07-623, Upstate, Dundee) was at 1:1,000,
goat anti-SPC (Research Diagnostics RTSURFCCabG) was at 1:100,
and rabbit polyclonal anti-Rab27a and anti-Rab27b antibodies (as
above) were each at 1:100.
Mouse strains. All mice were treated humanely and in accordance
with the UK Home Office regulations under project license PPL
70/7078 at the Central Biomedical Services of Imperial College,
London, UK. C57BL/6J wild-type mice were purchased from B&K
Universal Limited (Hull, UK). Rab27a-deficient mice, ashen
(Rab27aash/ash) mice were described previously (31) and the generation of Rab27b KO mice and double Rab27KO was described elsewhere (27). All strains were maintained on a C57/Bl/6 background.
Immunoblotting. Immunoblotting was performed as described previously (12). Perfused lung lysates were prepared using a Polytron
homogenizer and an appropriate volume (10⫻ vol/wt) of lysis buffer
(150 mM NaCl, 20 mM Tris·HCl, pH 7.5, 1 mM DTT, 1% CHAPS,
and 1⫻ PI cocktail) followed by incubation on ice for 15 min. Nuclei
and debris were then harvested by centrifugation (3,000 g at 4°C for
10 min), and the protein content of the PNS was quantified using BCA
protein assay kit (Pierce, UK).
Real-time PCR analysis. Total RNA from resected human lung
tissue obtained from 19 transplant donors was reverse transcribed
using a reaction mix of 1⫻ RT buffer (500 ␮M each dNTP, 3 mM
MgCl2, 75 mM KCl, 50 mM Tris·HCl, pH 8.3), 20 units of RNasin
Rnase inhibitor (Promega, Madison, WI), 10 mM dichloro-diphenyltrichloroethane (DDT), 100 units of Superscript II RNase H-reverse
transcriptase (Invitrogen, Uppsala, Sweden), and 250 ng of random
hexamers (Promega). First-strand cDNA synthesis was carried out in
a final volume of 20 ␮l, incubating at 20°C for 10 min and 42°C for
30 min, and inactivating reverse transcriptase by heating at 99°C for
5 min and cooling at 5°C for 5 min.
Real-time PCR were performed using the 7000 Abi Prism (Applied
Biosystems, Foster City, CA) with optimized PCR conditions. The
reaction was carried out in a 96-well plate adding 3 ␮l of diluted
template cDNA to a final reaction volume of 25 ␮l. The PCR master
mix was assembled with TaqMan Universal Master Mix Reagents
(Applied Biosystems) and each Taqman Gene Expression Assay,
Hs00608302 (Applied Biosystems) for human Rab27a, Hs01072206
(Applied Biosystems) for Rab27b. Each target assay was replicated
three times and performed in multiplex reaction with the 18S rRNA
endogenous control gene (4310893E, Applied Biosystems). The thermal cycling conditions comprised an initial denaturation step at 95°C
for 10 min, and 50 cycles at 95°C for 15 s and 65°C for 1 min.
Real-time quantitative values were obtained from the Ct number at
which the increase in signal associated with exponential growth of
PCR products starts to be detected. Results, expressed as amount in
target genes (Rab27a, Rab27b) expression relative to the reference
gene (18s rRNA), were calculated with the ⌬Ct method. Briefly, the
⌬Ct value of the samples was determined by subtracting the average
Ct value of the target gene from the average Ct value of the 18s rRNA
gene.
Lung histology and morphometry. Male mice were terminally
anesthetized by intraperitoneal injection of ketamine-xylazine (100
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RAB27 FUNCTION IN LUNG EPITHELIUM
lungs of 3 age-matched, sex-matched mice stained with pro-SPC
antibody were imported into Volocity 5.4 image analysis software,
and AEII cells were identified on the basis of staining intensity above
background as well as area measurements. P values were derived
using the Student’s t-test.
Conventional electron microscopy. Lungs of C57BL/6 mice were
infused and fixed with a mixture of 2% (wt/vol) PFA, 2% (wt/vol)
glutaraldehyde (TAAB) in 0.1 M sodium cacodylate buffer (Agar),
pH 7.4, postfixed with 1% (wt/vol) OsO4 supplemented with 1.5%
(wt/vol) potassium ferrocyanide, dehydrated in ethanol, and infiltrated
with propylene oxide (Agar)/Epon (Agar) (1:1) followed by Epon
embedding. Ultrathin sections were cut with an Ultracut S microtome
(Leica), counter-stained with lead citrate, and observed with a transmission electron microscope (TEM) Jeol 1010. Images were obtained
using a Gatan ORIUS CCD camera. For measurement of lamellar
body area, electron microscope images were imported into Image J
software, the perimeter of each organelle was defined manually, and
the area was measured using the measure tool within the software.
Ultracryotomy and immunogold labeling. Lungs of C57BL/6 mice
were infused and fixed with 2% (wt/vol) PFA and 0.1% (wt/vol)
glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.1). Samples
were cut in 0.5-mm3 blocks, embedded in 12% gelatin, and infused in
2.3 M sucrose (26). Mounted gelatin blocks were frozen in N2, and
ultrathin (50 nm) cryosections were cut at ⫺120°C with an Ultracryomicrotome (Leica). Sections were retrieved in 1.15 M (wt/vol) sucrose-2% (vol/vol) methylcellulose solution and processed for immunolabeling. After the sections were blocked with 0.5% (wt/vol) BSA,
single immunolabeling was performed on the sections in a humid
chamber with primary antibodies, and protein A was coupled to
10-nm diameter gold particles (PAG-10 nm).
Fig. 2. Immunohistochemistry of frozen sections
of murine lung reveals that Rab27 proteins are
expressed in the bronchiolar and alveolar epithelium. Frozen sections of C57BL/6 murine lungs
were fixed and stained with antibodies specific
for Rab27a (A and B) or Rab27b (C) (green in
overlay images). In A and C, arrows indicate
localization of Rab27a and Rab27b to alveolar
epithelial type II (AEII) cells, whereas arrowhead indicate localization of Rab27a and Rab27b
to the bronchiolar epithelium. In B, arrow shows
localization of Rab27a to granules in the apical
projections of Clara cells, whereas arrowhead
highlights the expression of Rab27a in ciliated
cells. Scale bars A and C ⫽ 100 ␮m and B ⫽ 5 ␮m.
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from each of 3 age-matched animals. P values were derived using
the Student’s t-test.
Immunohistochemistry. Immunofluorescence was performed on either deparaffinized, formalin-fixed paraffin sections or frozen sections. Sections were deparaffinized using Histoclear and then taken
through reducing alcohols to water, and rinsed in 1⫻ PBS. For antigen
retrieval, samples were boiled for 9 min in 10 mM sodium citrate
buffer (pH 6.0) and cooled for 5–10 min in water. Slides were washed
in 1⫻ PBS and incubated with blocking buffer (1⫻ PBS, 0.025%
Triton X-100, 1% BSA, 10% donkey serum) for 1 h and then
incubated overnight at 4°C with primary antibody diluted in blocking
buffer. The following day slides were washed three times with
washing buffer (1⫻ PBS, 0.1% Triton X-100), incubated with Alexa488- or Alexa568-conjugated secondary antibodies, and finally
washed as before and mounted using Immunofluor mountant (MP
Biomedicals, Cambridge, UK). Nuclei were visualized using 4,6diamidino-2-phenylindole (DAPI) stain. For immunofluorescence of
frozen sections, perfused lungs were manually inflated via the trachea
using a 1:1 mixture of OCT (Cellpath, Powys, UK) and PBS, and
lungs were then embedded in OCT, frozen using liquid N2, and stored
at ⫺80°C. Semithin sections (5 ␮m) were cut at ⫺20°C using a
cryostat (Bright Instruments, Huntingdon, UK) and fixed with 4%
paraformaldehyde (PFA) (wt/vol), 1⫻ PBS for 20 min at ambient
temperature and then processed for blocking and immunolabeling as
described for the deparaffinized sections. Images of fluorescent antibody-stained sections were obtained using either wide-field Zeiss
Axiovert 200 controlled by Simple PCI acquisition software with
images recorded using a Hamamatsu EM-CCD or a Zeiss LSM-510
inverted confocal microscope (Zeiss, Welwyn Garden City, UK).
Images were edited using Adobe Photoshop CS software. For AEII
cell counting 10 randomly collected images of alveolar areas of the
RAB27 FUNCTION IN LUNG EPITHELIUM
RESULTS
present in both ciliated and Clara cells (Fig. 2B, arrowhead and
arrow, respectively). In high-magnification images of bronchiolar epithelium, Rab27a was found to be particularly apparent in apical vesicles in Clara cells that may represent secretory
vesicles (Fig. 2B, arrow). Within the alveolar epithelium,
Rab27a was found to be highly expressed within a subset of
cells (Fig. 2A, arrow). Double immunolabeling of surfactant
protein C (SPC), which is specifically expressed in AEII cells,
revealed that Rab27a-positive cells are AEII cells (Fig. 3A).
High-magnification images of AEII cells showed that while
SPC is present in punctate structures (presumably LBs),
Rab27a is present on small vesicles throughout the cytoplasm
that do not significantly colocalize with LBs (Fig. 3B). Similar
analysis using specific antibodies indicated that Rab27b is also
expressed in ciliated and Clara cells of the bronchiolar epithelium (Fig. 2C, arrowhead), as well as a subset of cells in the
alveolar epithelium (Fig. 2C, arrow). Double immunolabeling
of frozen sections with SPC-specific antibodies confirmed that
a subset of these Rab27b-positive cells are AEII cells (Fig. 3C,
Fig. 3. Rab27 proteins are localized to cytoplasmic vesicles in AEII cells. Frozen sections of C57BL/6 murine lungs were fixed and stained with antibodies
specific for Rab27a (A, B) or Rab27b (C, D) (green in overlay images), surfactant protein C (SPC, red in overlay images), and DAPI (blue in overlay images)
and analyzed using confocal immunofluorescence microscopy. Low-magnification images show Rab27a (A) and Rab27b (C) expression in AEII cells (arrows).
High-magnification images show the intracellular distribution of Rab27a (B) and Rab27b (D) in AEII. Scale bars A and C ⫽ 25 ␮m, B and D ⫽ 5 ␮m.
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Rab27A and Rab27B are expressed in the murine lung. As a
first step to investigate the role of Rab27 proteins in the
function of the pulmonary epithelium, we used isoform-specific Rab27 antibodies to test the expression of Rab27a and
Rab27b proteins in murine lung lysates. In control (WT) lung
lysate we observed that both isoforms are expressed (Fig. 1A).
The specificity of Rab27a and Rab27b signal was confirmed by
blotting of lysate derived from mutant mice lacking individually Rab27a (Rab27aash/ash) or Rab27b (Rab27bKO), respectively. Moreover, quantitative real-time PCR analysis of
Rab27a and Rab27b mRNA levels in resected human lung
tissue samples showed that both genes are significantly expressed in the lung (Fig. 1B).
Rab27 proteins are expressed in Clara cells and AEII cells.
We then used immunofluorescence microscopy to reveal the
distribution of Rab27 isoforms in frozen sections of C57Bl/6
mouse lung. This showed that Rab27a is highly expressed in
the bronchiolar epithelium (Fig. 2A, arrowhead) where it is
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Fig. 4. Electron microscopy of ultrathin cryosections of murine lungs reveals Rab27 proteins localization to cytoplasmic vesicles, lamellar bodies, and
multivesicular bodies in AEII cells. Ultrathin cryosections of C57Bl/6 murine lungs were labeled with antibodies specific for surfactant protein C (SPC; A and
B), Rab27a (C and D) or Rab27b (E and F) and detected using second antibodies conjugated to 10-nm protein A gold (PAG). Electron microscopy analysis of
labeled sections revealed strong expression of SPC (A and B) in the internal vesicular membranes of MVBs as well as LBs and cytoplasmic vesicles in AEII
cells. Similarly Rab27a (C and D) and Rab27b (E and F) were localized to the membranes of cytoplasmic vesicles, LBs, and multivesicular bodies (MVBs) in
AEII cells. Scale bars ⫽ 100 nm.
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RAB27 FUNCTION IN LUNG EPITHELIUM
Loss of Rab27 protein results in atrophy of the pulmonary
epithelium. Based on the previous observation that Rab27
proteins are expressed in the murine lung, we next aimed to
investigate the function of Rab27 in this context. As a first step
we examined the morphology of lungs from mice lacking both
Rab27 isoforms (DKO). As shown in Fig. 6A, we observed
thinning of the bronchiolar epithelium as well as marked
enlargement of the alveolar air spaces in lungs from the DKO
compared with control (WT) samples. These morphological
alterations were clearly observed in high-magnification images
of bronchioles (Fig. 6B), which show disorganization and
shortening of bronchiolar cilia, while alveolar areas clearly
show enlargement of alveolar spaces and thinning of the
alveolar epithelium (Fig. 6C). Quantification using mean linear
intercept measurement (Fig. 6D) indicated that airspace enlargement in the DKO reaches significance in the lungs of 12
wk- and 18-mo-old mice. This also indicated that airspace
enlargement becomes more evident with increasing age. Meanwhile, measurement of the thickness of the epithelium in
multiple bronchioles confirmed that there is significant thinning of the epithelium in DKO compared with age-matched,
sex-matched control (WT) samples (Fig. 6E). In addition to
these changes, we also observed increased incidence of activated foamy macrophages (see online supplementary Fig. S3A,
arrows) and cellular infiltrates containing granulocytes (supplementary Fig. S3, B and C, arrows) in 12-mo-old DKO mice
compared with those of age-matched, sex-matched controls
(WT) (4/5 DKO vs. 2/9 control samples). Morphological analyses of the lungs of mice lacking individual Rab27 isoforms
indicate that Rab27b plays a more significant role in maintenance of the integrity of the pulmonary epithelium than does
Rab27a (data not shown).
The numbers of cells expressing AEII and Clara marker
proteins are reduced in the lungs of DKO mice. Given that loss
of Rab27 proteins resulted in generalized atrophy of the pulmonary epithelium, we investigated the effect of loss of Rab27
Fig. 5. Cryoimmunoelectron microscopy of murine lungs reveals Rab27a localization to secretory/CCSP granules within Clara cells. Ultrathin cryosections of
C57Bl/6 murine lungs were labeled with antibody specific for Rab27a (A and B) and detected using protein A conjugated to 10-nm gold particles (PAG). Electron
microscopy analysis at higher magnification (B) of labeled sections revealed expression of Rab27a (A and B) in membrane of Clara cell granules. Mito,
mitochondria; CCSP, Clara cell secreted protein granule. Scale bars ⫽ 500 nm.
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arrows). High-magnification images of double-labeled sections
reveal little colocalization of Rab27b with SPC-positive LBs
(Fig. 3D). This analysis also showed that Rab27b is strongly
expressed on cytoplasmic granules in an additional cell type
present throughout the alveolar epithelium (Fig. 3C, arrowhead
and see online supplementary Fig. S1A at AJP-Cell Physiol
website). The specificity of antibody staining was confirmed by
staining frozen sections of lung from double knockout (DKO)
mice (see online supplementary Fig. S2).
Rab27 proteins associate with cytoplasmic vesicles, LBs,
and multivesicular bodies in AETII cells and apical secretory
granules in Clara cells. We then used cryoimmunoelectron
microscopy to investigate further the identity of Rab27-positive structures within AEII cells in ultrathin cryosections of the
lungs of C57BL/6 mice. Staining with SPC-specific antibodies
(Fig. 4, A and B) showed that this protein is present in the
internal membranes of multivesicular bodies (MVBs), LB
precursor vesicles, and mature LBs. Similar analysis showed
that Rab27a is associated with MVBs and vesicles present
throughout the cytoplasm (Fig. 4, C and D). Furthermore,
Rab27a did not show strong colocalization with LBs. Meanwhile, Rab27b was localized to cytoplasmic vesicles and in
some cases to the limiting membrane of LBs (Fig. 4, E and F).
Consistent with immunofluorescence, immunoelectron microscopy analysis confirmed the existence of a second class of
Rab27b-expressing cell type within the capilliary network of
the alveolar wall. High-magnification images revealed that
these cells are eosinophils, based on the presence of cytoplasmic crystalloid granules that were labeled with anti-Rab27b
antibodies (see online supplementary Fig. S1, B–E). Meanwhile, in the bronchiolar epithelium, examination of immunogold-labeled cryosections revealed that Rab27a is present on the
limiting membrane of secretory CCSP granules in Clara cells (Fig.
5, A and B). We also observed gold particles in Clara cells in
cryosections labeled with Rab27b-specific antibodies (data not
shown).
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Fig. 6. Histological and morphometric analysis
of the lungs of double knockout (DKO) mice
reveals airspace enlargement and atrophy of the
epithelium. The lungs of DKO and control mice
were removed, inflation-fixed in formalin (as
described in MATERIALS AND METHODS), and embedded in paraffin. Sections of 5 ␮m were then
cut and stained with hemotoxylin and eosin. A–C
show images of these stained sections from 12mo-old DKO and age-matched, sex-matched
control (WT) mice. A: low-power overview of lung
structure (scale bar ⫽ 60 ␮m); B and C: highmagnification images showing details of morphology of bronchiolar and alveolar epithelium
(scale bars ⫽ 20 ␮m). D: mean linear intercept
analysis of the integrity of the alveolar epithelium in the lungs of age-matched, sex-matched
DKO and control (WT) (*P ⬍ 0.05; **P ⬍
0.002). E: analysis of the thickness of randomly
selected areas of the bronchiolar epithelium of
DKO and control (WT) (***P ⬍ 0.0005).
specifically in Clara and AEII cells. To do this we stained
deparaffinized sections of mutant and control lungs with proSPC and CCSP-specific antibodies that detect AEII and Clara
cells, respectively, and then used immunofluorescence microscopy to investigate the effects of loss of Rab27 on the number
and distribution of these cell types. By this approach we
observed a significant reduction in the number of SP-C immune-positive cells in DKO compared with control samples
from age-matched, sex-matched WT mice (mean AEII cells/
20⫻ field 32 vs. 49.3, respectively, Fig. 7A). Similar analysis
of the number of Clara cells in both large and small bronchioles
of DKO mice revealed a similar reduction in the number of CCSP
immune-positive cells compared with control (WT) bronchioles
(Fig. 7B). In addition, we frequently observed that, in contrast to
the control, the coverage of Clara cells in DKO bronchioles was
patchy with large areas entirely devoid of Clara cells. These
observations suggest that the number of AEII and Clara cells is
reduced in the lungs of DKO versus WT mice.
DKO AEII cells accumulate large, mature LBs and contain
few LB precursors. Finally, we used electron microscopy to
investigate the ultrastructural alterations in Clara and AEII
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cells resulting from loss of Rab27 protein. Consistent with
immunofluoresence data, this indicated that the bronchiolar
epithelium in DKO mice is thinner than that of controls (Fig. 8A).
While in Clara cells we observed a dramatic accumulation of
cytoplasmic vesicles that most likely represent CCSP-containing secretory granules, together with a reduction in the abundance of early secretory pathway organelles, such as endoplasmic reticulum, that was commonly observed in control samples
(Fig. 8A, high magnification image). Similarly, using electron
microscopy to study AEII cells, we observed a marked increase
in both the number and size of mature LBs and a concomitant
reduction in the number of LB precursor structures and mitochondria in DKO compared with control samples (Fig. 8B).
This was confirmed by quantification (Fig. 8, C and D). This
analysis also confirms that the changes in morphology are
progressive and more apparent with aging (Fig. 8C). Parallel
electron microscopy analysis of AEII cells in mice deficient in
individual Rab27 isoforms revealed similar accumulation of
mature LB in AEII cells of mice lacking Rab27b alone,
indicating that Rab27b, and not Rab27a, plays a significant role
in the regulation of LB homeostasis and AEII cell function
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Fig. 7. Immunohistochemistry reveals a reduction in the number of cells positive for AEII and Clara marker proteins in the lungs of DKO mice. Paraffin sections
of lungs from 18-mo-old control (WT) and DKO mice were dewaxed and stained with ProSPC- (A) or CCSP (B)-specific antibodies to detect AEII and Clara
cells and DAPI to detect cell nuclei. A: left, fluorescence and phase contrast images of the number and distribution of Pro-SPC-positive AEII cells and the
morphology of lung parenchyma in mutant and control samples. Right, quantification of the Pro-SPC data showing the number of AEII cells present in 10
randomly selected low-magnification fields from mutant and control samples. *P value ⬍0.0001. B: fluorescence micrographs show the typical distribution of
Clara cells in the large and small bronchiolar airways in the lungs of 18-mo-old control (WT) and DKO mice. Scale bars ⫽ 20 ␮m.
(data not shown). Ultrastructural analysis of AEI cells confirmed that there is marked thinning of the alveolar epithelium
in DKO compared with control lungs (Fig. 8B).
DISCUSSION
In this study we used light and electron microscopy to
investigate the localization and function of Rab27 proteins in
the lung. Our main findings are that Rab27a and Rab27b are
expressed in Clara and ciliated cells in bronchioles as well as
AEII cells in the alveolar region. Rab27b is additionally
strongly expressed in cells in the parenchyma that we have
defined as eosinophils based on our finding that they contain
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eosinophil-specific crystalloid granules. In particular, we find
that Rab27b is associated with cytoplasmic face of these
granules. These observations are largely consistent with previous analysis of expression of GFP-Rab27a transgenic protein
and Rab27b-driven lacZ expression in the lungs of mice (6,
29). However, our finding of high Rab27b expression in
eosinophils is surprising given that previous studies indicated
that Rab27a, not Rab27b, is expressed in circulating eosinophils in humans (2). Meanwhile, our analysis of the structure of
the lungs of DKO mice revealed a generalized atrophy of both
the alveolar and bronchiolar epithelium together with mild
airspace enlargement that was particularly apparent in mice
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Fig. 8. Electron microscopy reveals an accumulation of mature secretory organelles in
AEII and Clara cells in DKO mice. Ultrathin
sections of lungs from control (WT) and
DKO mice at 18 mo of age were observed
by transmission electron microscopy. A and
B: typical appearance of Clara cells in the
bronchiolar epithelium (A) and alveolar epithelium type II and type I cells (B) in DKO and
control mice. A, right: high magnifications
of regions of the left image. B, insets: high
magnification images of typical lamellar
bodies from each sample. This analysis reveals a marked accumulation of enlarged
secretory granules in Clara cells and lamellar
bodies in AEII cells as well as thinning of
the AEI cells. Mlb, mithocondria-like bodies; prec, precursors; cv, caveolae; s, surfactant; LB, lamellar body; nu, nucleus. Scale
bars ⫽ 0.5 ␮m. C: quantification of the
differences in organelle content (lamellar
body, lamellar body precursor, and mitochondria) in AEII cells in control (WT) and
DKO lung samples for mice of indicated
ages. For WT and DKO 5-wk-old samples,
WT 12-wk-old, and 18-mo-old samples: n ⫽
18; whereas for DKO 12-wk-old and 18-moold samples: n ⫽ 11 and 15, respectively.
*P ⬍ 0.05; **P ⬍ 0.005. D: quantification
of the differences in lamellar body size in
AEII of 18-mo-old WT (n ⫽ 231 LBs in 18
different AEII cells) and DKO (n ⫽ 121 LBs
in 14 different AEII cells) mice (ns, observed differences did not reach significance).
over 12 mo old. Moreover, we observed a reduction in the
abundance of SP-C/CCSP-positive cells in DKO mice that was
more apparent after 12 mo of age. This suggests that Rab27expressing AEII and Clara cell numbers may be reduced in the
DKO mutant compared with WT control. At the subcellular
level, we observed that Rab27 was localized to apical granules
in Clara cells that may represent secretory granules, whereas in
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AEII cells, Rab27 was located on cytoplasmic vesicles and
MVBs. In DKO samples, we observed a striking accumulation
of mature secretory organelles; i.e., Clara cell granules and
LBs in AEII cells, and a reciprocal reduction in the numbers of
mitochondria, granule precursors, and ER membranes.
Based on these findings, we hypothesize that the function of
Rab27 proteins in the lung is to regulate the release of exocytic
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RAB27 FUNCTION IN LUNG EPITHELIUM
ACKNOWLEDGMENTS
We thank Lorraine Lawrence for technical assistance in the preparation of
paraffin-embedded and frozen sections and Dr. Martin Spitaler for assistance
with microscopy.
The current address for A. N. Hume: School of Biomedical Sciences,
Queen’s Medical Centre, Nottingham NG7 2UH, UK; and current address for
G. Bolasco: EMBL Mouse Biology Unit, Campus A. Buzzati-Traverso, Via E.
Ramarini, 32, 00015 Monterotondo (RM), Italy.
GRANTS
This work was supported by Wellcome Trust Programme Grant (Reference
075498/Z/04/Z) to M. C. Seabra.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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granules in specialized secretory cell types such as Clara and
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