Defective CFTR apical endocytosis and enterocyte brush border in myosin VI-deficient mice.

Abstract

In polarized epithelial cells such as those that line the inner ear, kidney and gut, myosin VI has been localized to the intermicrovillar domains where it is proposed to regulate clathrin-dependent endocytosis; however, a direct role for myosin VI in apical endocytosis has not been shown. We examined the apical membrane distribution and endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in myosin VI-deficient Snell's Waltzer Myo6((sv/sv)) mice. Confocal microscopy and cell-surface biotinylation confirmed that surface levels of CFTR in the intestine of Myo6((sv/sv)) mice were markedly higher, and CFTR internalization from the apical plasma membrane was reduced compared with heterozygous controls. Consistent with a defect in CFTR endocytosis and accumulation at the cell surface, exaggerated CFTR-mediated fluid secretion was observed in Myo6((sv/sv)) mice following treatment of isolated jejunum with the cyclic GMP-activated heat stable enterotoxin. These data establish that myosin VI modulates apical endocytosis and may be an important physiological modulator of CFTR function and CFTR-associated secretory diarrhea in the gut.

Full text

Page 1

# 2007 The Authors
Journal compilation#2007 Blackwell Publishing Ltd
doi: 10.1111/j.1600-0854.2007.00587.x
Traffic 2007; 8: 998–1006
Blackwell Munksgaard
Defective CFTR Apical Endocytosis and Enterocyte
Brush Border in Myosin VI-Deficient Mice
Nadia Ameen1,2,* and Gerard Apodaca2,3
1Department of Pediatrics, University of Pittsburgh
School of Medicine, Pittsburgh, PA 15261, USA
2Department of Cell Biology and Physiology, University
of Pittsburgh School of Medicine, Pittsburgh,
PA 15261, USA
3Laboratory of Epithelial Cell Biology, Renal-Electrolyte
Division, Department of Medicine, University of
Pittsburgh School of Medicine, Pittsburgh,
PA 15261, USA
*Corresponding author: Nadia Ameen, nameen@pitt.edu
In polarized epithelial cells such as those that line the
inner ear, kidney and gut, myosin VI has been localized
to the intermicrovillar domains where it is proposed
to regulate clathrin-dependent endocytosis; however,
a direct role for myosin VI in apical endocytosis has not
been shown. We examined the apical membrane distri-
bution and endocytosis of cystic fibrosis transmembrane
conductance regulator (CFTR) in myosin VI-deficient
Snell’s Waltzer Myo6(sv/sv)mice. Confocal microscopy
and cell-surface biotinylation confirmed that surface
levels of CFTR in the intestine of Myo6(sv/sv)mice were
markedly higher, and CFTR internalization from the apical
plasma membrane was reduced compared with hetero-
zygous controls. Consistent with a defect in CFTR endo-
cytosis and accumulation at the cell surface, exaggerated
CFTR-mediated fluid secretion was observed in Myo6(sv/sv)
mice following treatment of isolated jejunum with the
cyclic GMP-activated heat stable enterotoxin. These data
establish that myosin VI modulates apical endocytosis
and may be an important physiological modulator of
CFTR function and CFTR-associated secretory diarrhea
in the gut.
Key words: CFTR, diarrhea, endocytosis, intestine, myo-
sin VI, Snell’s Waltzer mice
Received 13 December 2006, revised and accepted for
publication 23 April 2007, uncorrected manuscript pub-
lished online 26 April 2007, published online 6 June 2007
Actin-based transport within cells is a function of myosins,
a large family of molecular motors that use energy derived
from ATP hydrolysis to generate force. This force is used
by myosins to move cargo over short distances along actin
filaments. Myosin VI is unique among the myosin motors
as it moves toward the minus end of polymerized actin
fibers. Myosin VI has been variously localized to the Golgi
complex, to areas of cell ruffling, to clathrin-coated pits and
to uncoated transport vesicles, and it may regulate diverse
cellular processes including membrane ruffling, mainte-
nance of the Golgi complex, brush border assembly,
development of stereocilia on the hair cells of the inner
ear and endocytosis (1).
Myosin VI may regulate multiple steps of receptor-
mediated endocytosis. It is associated with clathrin-coated
pits and purified clathrin-coated vesicles, and it interacts
biochemically with clathrin, adaptor protein 2 (AP2) and a
proline-rich 122 amino acid region at the C-terminus of
disabled 2 (Dab2) (2,3). The latter is a clathrin-associated
sorting protein that promotes endocytosis of a subset of
receptors including the low-density lipoprotein receptor
(LDLR) (2,3). Expression of the C-terminal globular tail
domain of myosin VI in NRK cells, which interacts with
clathrin-coated pits but lacks a motor domain, inhibits
receptor-mediated endocytosis of transferrin in a dominant-
negative manner (2). While clathrin-mediated endocytosis
of a-amino3-hydroxy-5-methyl-4-isoxazole propionic acid-
type glutamate receptors is defective in the neurons of
myosin VI-deficient Snell’s Waltzer Myo6(sv/sv)mice, no
deficit in constitutive transferrin receptor uptake is ob-
served (4). These results indicate that in vivo myosin VI
may differentially regulate the endocytosis of a subset of
receptors, possibly in a cell-type-specific manner. Other
studies indicate that the association of myosin VI with
clathrin-coated pits may depend on the expression of
both Dab2 and an alternatively spliced variant of myosin
VI that includes a relatively large (?32 amino acid) insert
at the interface between a coiled-coil region and the tail
of the protein (2,5). In the absence of Dab2 expression, or
in splice variants lacking the large insert, myosin VI may
associate with uncoated vesicles where it promotes their
transit through the actin-rich cortical cytoskeleton (5,6).
In polarized epithelial cells such as Caco-2 and tissues such
as the kidney and intestine, myosin VI has been localized to
the terminal web region, intermicrovillar space and along
the microvilli, in clathrin-coated vesicles and tubules
beneath the apical membrane (2,3,7,8). This distribution
for myosin VI in polarized cells and tissues, and its move-
ment away from the plasma membrane and toward the
minus ends of actin filaments (9), suggests an important
role for myosin VI in regulating apical endocytosis, possibly
by driving coated pit invagination and vesiculation in the
milieu of the actin-rich terminal web. However, direct
evidence for myosin VI in apical endocytosis is lacking.
The cystic fibrosis transmembrane conductance regulator
(CFTR) is an apical membrane chloride channel implicated
in cystic fibrosis and secretory diarrhea (10). In the
intestine, CFTR provides the major exit pathway for fluid
secretion elicited by protein kinase A and protein kinase G
998
www.traffic.dk

Page 2

(11). The latter is stimulated by the Escherichia coli heat
stable enterotoxin (STa) (12). In the native intestine, CFTR
is present on the apical plasma membrane, in coated pits,
and clathrin-coated vesicles (13); and CFTR is internalized
by clathrin- and AP2-mediated endocytosis (14–16). Cell-
surface CFTR can be isolated in a complex with clathrin,
Dab2 and myosin VI. Furthermore, endocytosis of CFTR is
blocked by expression of a dominant-negative myosin VI
tail construct when expressed in nonpolarized human
embryonic kidney 293 (HEK293) cells (17). However, it is
unknown whether myosin VI plays a physiological role
in regulating CFTR endocytosis in chloride-transporting
epithelial tissues and contributes to the pathogenesis of
diseases such as secretory diarrhea.
In the current study, we examined the distribution of
surface CFTR in Myo6(sv/sv)mice and sought direct
evidence for defective endocytosis of CFTR from the
apical plasma membrane in vivo. We observed that
steady-state levels of surface CFTR were markedly higher
and endocytosis from the apical membrane was reduced
in Myo6(sv/sv)intestines compared with Myo6(sv/wt)hetero-
zygous controls. Consistent with the increased levels of
surface CFTR and defective endocytosis, we observed
increased fluid accumulation and secretion in intestinal
loops following in vivo STa treatment in Myo6(sv/sv)mice.
To our knowledge, these are the first observations doc-
umenting (1) a physiological role for myosin VI in regulating
apical endocytosis of a transmembrane protein and (2)
a physiological role for CFTR endocytosis in regulating fluid
secretion in vivo.
Results
Myosin VI determines microvillar length in the
small intestine
TheSnell’swaltzerMyo6(sv/sv)mouse exhibits head tossing,
circling,hyperactivity anddeafness. Subcellular examination
of the inner ear reveals structural abnormalities in stereo-
cilia of the hair cells, and analysis of brain tissue indicates
that myosin VI is important for synapse formation and
glutamate receptor endocytosis (4,18). Other preliminary
data, that was not shown, indicate that the enterocytes of
Myo6(sv/sv)mice have reduced microvillar height (18). We
initially examined intestinal tissues from Myo6(sv/sv)and
Myo6(sv/wt)mice using light and electron microscopy to
confirm previous observations and identify any histologic
and structural abnormalities that would provide insight into
the importance of myosin VI to intestinal development and
function. Macroscopic examination revealed that the in-
testines from Myo6(sv/sv)mice were smaller in caliber and
more fragile than heterozygote Myo6(sv/wt)controls. While
the Myo6(sv/sv)mice were considerably smaller in size
(mean weight: 22 g, n ¼ 10) compared with heterozygotes
(mean weight: 35 g, n ¼ 11, p < 0.05), there was no
evidence of intestinal obstruction or diarrhea under steady-
state conditions.
Examination of hematoxylin- and eosin-stained sections
revealed mild reduction in the thickness of the muscularis
layer, but no obvious differences in crypt and villus orienta-
tion, size and number between Myo6(sv/sv)and Myo6(sv/wt)
mice (Figure 1). Furthermore, villus height and surface
morphology appeared similar between Myo6(sv/sv)and
Myo6(sv/wt)micewhenthetissuewasexaminedbyscanning
electron microscopy (Figure 2A,B versus 2D,E). Consistent
with previous observations (18), closer examination of villus
enterocytesin transmission electron microscopic (TEM)sec-
tions of the jejunum revealed that the microvilli of Myo6(sv/sv)
mice were somewhat disordered and shorter (mean length
0.8 mm ? 0.02 SEM) than Myo6(sv/wt)mice (mean length
1.2 mm ? 0.03 SEM). The difference between the two was
statistically different, p < 0.005 (Figure 2C,F,G), indicating
that myosin VI may regulate microvillar height. Examination
by TEM did not reveal structural differences between
Myo6(sv/sv)and Myo6(sv/wt)in actin rootlets or the level of
apical membranes in relation to the microvilli. The promi-
nence of actin rootlets extending into the terminal web in
TEM sections from Myo6(sv/sv)(Figure 2F) compared with
Myo6(sv/wt)(Figure 2C) reflects differences in uranyl con-
trast of the TEM sections.
CFTR surface distribution is increased in the
intestine of Myo6(sv/sv)mice
If Myosin VI is a physiologically important regulator of
apical endocytosis, we hypothesized that its absence
would result in the accumulation of normally endocytosed
proteins at the apical cell surface. As a marker of the apical
endocytic pathway, we used CFTR, an endogenous pro-
tein known to be internalized from the apical cell surface
through clathrin-dependent endocytosis (14,16). Immuno-
fluorescence was used to gain insight into the distribution
of CFTR in the intestine of Myo6(sv/sv)mice. We also
examined the distribution of myosin VI because its local-
ization in mouse intestine has not been described. Similar
to its distribution in wild-type mouse intestine (19), CFTR
was found on the apical pole of Myo6(sv/sv)enterocytes
(Figure 3A–D). However, the fluorescence intensity of CFTR
staining was more prominent (two-fold higher, p < 0.05) in
Myo6(sv/sv)jejunum compared with that of Myo6(sv/wt)con-
trols (Figure 3C,D,I). The distribution of myosin VI in the
mouse jejunum was consistent with previous reports of
Figure 1: Light microscopic appearance of hematoxylin- and
eosin-stained sections from jejunum. A) Myo6(sv/wt)and B)
Myo6(sv/sv)mice. Scale bar ¼ 50 mm.
Traffic 2007; 8: 998–1006
999
Defective CFTR Endocytosis in Myosin VI-Deficient Mice

Page 3

myosin VI protein localization in the chicken intestine (8); it
was localized just below the actin-rich microvilli in the
terminal web region. The staining was somewhat punctate,
consistent with myosin VI localization to coated pits (Fig-
ure 3E,F). Lack of myosin VI expression in Myo6(sv/sv)
intestinal tissues was verified by immunofluorescence (Fig-
ure 3H). Double-label immunofluorescence using anti-CFTR
and myosin VI antibodies in sections of jejunum from
Myo6(sv/wt)mice (Figure 4) confirmed colocalization of CFTR
and myosin VI in the apical pole of enterocytes just beneath
the brush border, a distribution consistent with their localiza-
tion in endocytic structures (13).
To confirm that surface CFTR expression was increased in
the Myo6(sv/sv)intestine, we performed cell-surface bio-
tinylation of apical proteins exposed on the mucosal
surface of intestinal loops of jejunum (20). Levels of
surface-biotinylated CFTR were at least fivefold higher in
Myo6(sv/sv)jejunum compared with that of heterozygote
controls under steady-state conditions (Figure 5A,C, p <
0.05). The increase in surface-biotinylated CFTR detected
in Myo6(sv/sv)jejunum was not because of increased levels
of CFTR expression, as immunoblots confirmed that CFTR
expression was similar in the jejunum of Myo6(sv/sv)and
Myo6(sv/wt)mice (Figure 5B).
Endocytosis of CFTR from the apical plasma
membrane is defective in Myo6(sv/sv)enterocytes
The increased levels of CFTR staining at the apical pole of
Myo6(sv/sv)enterocytes suggested that surface expression
Figure 2: Ultrastructual examination of
jejunal villi and enterocyte microvilli in
Myo6(sv/sv)and Myo6(sv/wt)mice. A and B,
D and E) SEM analysis of tissues at low
(A and D) and high (B and E) magnification.
C and F) Thin-section analysis of enterocyte
brush border. G) Morphometric analysis of
microvillar length in villus enterocytes from
TEM sections of Myo6(sv/wt)and Myo6(sv/sv)
jejunum. Data are shown as mean ? SEM
(n ? 20).
1000
Traffic 2007; 8: 998–1006
Ameen and Apodaca

Page 4

of CFTR may be increased because of defective myosin
VI-dependent internalization. To test this hypothesis, we
measured endocytosis of CFTR from the apical surface of
the jejunum in Myo6(sv/sv)and Myo6(sv/wt)mice. In control
tissues, CFTR was efficiently internalized with ?15% of
cell-surface-biotinylated CFTR internalized after 1 minute,
and ?45% of cell-surface CFTR internalized by 5 minutes
(Figure 6). In contrast, in Myo6(sv/sv)jejunum, CFTR inter-
nalization was markedly impaired. Only ?5% of surface-
biotinylated CFTR was internalized by 1 minute and ?15%
after 5 minutes. The difference in CFTR internalization at all
time-points examined was statistically significant, p <
0.05. We confirmed by Western blot analysis that the
expression of proteins known to interact with myosin VI,
Figure 3: Distribution of CFTR, fila-
mentous actin and myosin VI in the
small intestine of Myo6(sv/sv)and
Myo6(sv/wt)mice. A–D) Distribution of
CFTR in villus (A and B) and crypt (C
and D) sections of jejunum. A) Staining
for CFTR alone in Myo6(sv/sv)jejunum
is shown in green. B) CFTR (green)
mergedwithrhodamine–phalloidin
(red) and Hoechst nuclear stain (blue)
is shown in Myo6(sv/sv)villus section.
The CFTR merged with rhodamine–
phalloidin in crypt of C) Myo6(sv/wt)and
D) Myo6(sv/sv)jejunum. Hoechst stain
labels nuclei blue. E and F) Staining
for myosin VI in villus sections from
Myo6(sv/wt)jejunum. E) Myosin VI alone
shown in green is marked by arrow. F)
Myosin VI merged with rhodamine–
phalloidin. Hoechst stain labels nuclei
blue. G) Control staining in Myo6(sv/wt)
jejunum without CFTR antibody and
labeled with rhodamine–phalloidin and
Hoechst nuclear stain. H) Staining for
myosin VI alone in Myo6(sv/sv)tissue. I)
Quantification of CFTR apical fluores-
cence after subtraction of values for
secondary alone
Myo6(sv/sv)andMyo6(sv/wt)jejunum.Data
are shown as mean ? SEM (n ? 40
crypts). FI, fluorescence intensity.
insections from
Figure 4: CFTR
myosin VI in the apical pole of crypt
enterocytes in Myo6(sv/wt)jejunum.
A–C) Low magnification images showing
distribution of CFTR (A, green), myosin VI
(B, red) and merged image (C, yellow)
(D, differential interference contrast (DIC))
in the apical pole of crypts. E–G) Higher
magnification images of a single crypt
showing distribution of CFTR (A, green),
myosin VI (B, red) and (C, merge) in apical
pole (H, DIC image). Scale bar ¼ 10 mm.
colocalization with
Traffic 2007; 8: 998–1006
1001
Defective CFTR Endocytosis in Myosin VI-Deficient Mice

Page 5

including AP2, clathrin and Dab2, was not altered by lack of
myosin VI expression in intestinal tissues (Figure 7), thus
ruling out the trivial explanation that impaired CFTR endo-
cytosis in Myo6(sv/sv)mice was a result of alterations in the
expression of myosin VI-associated endocytosis machin-
ery. Taken together, these observations indicate that
surface accumulation of CFTR in the Myo6(sv/sv)jejunum
can be explained in part by a defect in endocytosis.
STa-elicited fluid secretion is increased in Myo6(sv/sv)
jejunal loops
As further evidence that myosin VI regulated CFTR surface
expression and function in vivo, we measured fluid accu-
mulation in jejunal loops from Myo6(sv/sv)mice following
treatment with STa. This cyclic GMP-activated entero-
toxin, produced by enteropathogenic E. coli, binds to
enterocytes in the jejunum and elicits massive fluid
secretion by activating CFTR and stimulating its traffic to
the cell surface (20). The STa treatment resulted in
a statistically significant (p < 0.05) fourfold higher fluid
accumulation in Myo6(sv/sv)jejunum than controls (Fig-
ure 8) consistent with an increase in surface expression
of functional CFTR in Myo6(sv/sv)jejunum. These results
indicate that myosin VI plays a physiological role in
regulating the number of functional CFTR transporters on
the cell surface in vivo.
Discussion
A number of surface receptors, integral membrane pro-
teins and ion channels implicated in human disease reside
on the apical plasma membrane of epithelial cells. For ion
channels, such as CFTR, accumulating evidence suggest
that chloride efflux islinked to its trafficking to andfrom the
apical plasma membrane of ion transporting epithelial
tissues, but the mechanisms of its internalization and
relationship to ion transport is not clear (21–23). The
availability of the Snell’s Waltzer Myo6(sv/sv)mice and the
abundance of CFTR in the intestine, an epithelial chloride
transporting tissue, allowed us to directly examine myosin
VI’s role in CFTR apical endocytosis in vivo and address its
relevance to disease pathogenesis. The study was also
Figure 5: Cell surface expression of CFTR in jejunum of
Myo6(sv/sv)and Myo6(sv/wt)mice. A) Surface proteins were
labeled in the cold with sulfo-NHS-SS-biotin and either left
untreated (Bþ) or treated with MESNA (B?). Following MESNA
treatment, enterocytes were collected by gently scraping the
mucosa, the cells were lysed, biotinylated proteins were recov-
ered by streptavidin–agarose precipitation and CFTR detected by
Western blot analysis. B) Western blot analysis of total CFTR
in Myo6(sv/sv)and Myo6(sv/wt)lysates. Equal amounts of protein
(15 mg) were loaded in each well. C) Quantification of biotinylated
CFTR at the cell surface. Data are shown as mean ? SEM (n ? 4
mice from each group).
Figure 6: Apical endocytosis of CFTR in Myo6(sv/sv)and
Myo6(sv/wt)intestine. A) Surface proteins were labeled at 48C
usingsulfo-NHS-SS-biotinandallowedtointernalizefor1–5minutes
at 378C. Following MESNA treatment at 48C, the enterocytes were
lysed, biotinylated proteins were recovered with streptavidin–
agarose and endocytosed CFTR was detected by Western blot.
B) The fraction (percent) of biotinylated surface CFTR internalized
over time is shown. Data are shown as mean ? SEM (n ? 4 mice
from each group).
1002
Traffic 2007; 8: 998–1006
Ameen and Apodaca