From the Department of Molecular Pharmacology and The
Albert Einstein Cancer Center, Albert Einstein College of Medicine,
Bronx, New York 10461, § Department of Cell Biology, Harvard
Medical School, Boston, Massachusetts 02115, and ** Department of
Neurosciences, The Lerner Research Institute, Cleveland Clinic
Foundation, Cleveland, Ohio 44195
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Caveolae are vesicular organelles that represent
a subcompartment of the plasma membrane. Caveolins and flotillins are
two families of mammalian caveolae-associated integral membrane
proteins. However, it remains unknown whether flotillins interact with
caveolin proteins to form a stable caveolar complex or if expression of flotillins can drive vesicle formation. Here, we examine the cell type
and tissue-specific expression of the flotillin gene family. For this
purpose, we generated a novel monoclonal antibody probe that recognizes
only flotillin-1. A survey of cell and tissue types demonstrates that
flotillins 1 and 2 have a complementary tissue distribution. At the
cellular level, flotillin-2 was ubiquitously expressed, whereas
flotillin-1 was most abundant in A498 kidney cells, muscle cell lines,
and fibroblasts. Using three different models of cellular
differentiation, we next examined the expression of flotillins 1 and 2. Taken together, our data suggest that the expression levels of
flotillins 1 and 2 are independently regulated and does not strictly
correlate with known expression patterns of caveolin family members.
However, when caveolins and flotillins are co-expressed within the same
cell, as in A498 cells, they form a stable hetero-oligomeric
"caveolar complex." In support of these observations, we show that
heterologous expression of murine flotillin-1 in Sf21 insect
cells using baculovirus-based vectors is sufficient to drive the
formation of caveolae-like vesicles. These results suggest that
flotillins may participate functionally in the formation of caveolae or
caveolae-like vesicles in vivo. Thus, flotillin-1
represents a new integral membrane protein marker for the slightly
larger caveolae-related domains (50-200 nm) that are observed in cell
types that fail to express caveolin-1. As a consequence of these
findings, we propose the term "cavatellins" be used (instead of
flotillins) to describe this gene family.
Caveolae are small omega-shaped indentations of the plasma
membrane that have been implicated in signal transduction and vesicular transport processes (1, 2). Caveolae are most abundant in terminally
differentiated cells such as adipocytes, endothelial cells, smooth
muscle cells, skeletal and cardiac myocytes, and fibroblasts
(3-8).
Caveolin, a 21-24-kDa integral membrane protein, is a principal
component of caveolae membranes in vivo (9-13).
Caveolin-rich membrane domains purified by either detergent-based or
detergent-free methods are enriched in a variety of lipid-modified
signaling molecules such as heterotrimeric G proteins, Src-family
tyrosine kinases, Ha-Ras and Rap GTPases, and endothelial cell
nitric-oxide synthase (1, 14-24). Many of these signaling molecules
interact in a regulated manner directly with caveolin (18, 25, 26).
However, caveolin is only the first member of a growing gene family of
caveolin proteins; caveolin has been re-termed caveolin-1. Three
different caveolin genes (Cav-1, Cav-2, and Cav-3) encoding four
different subtypes of caveolin have been described thus far (2). There
are two subtypes of caveolin-1 (Cav-1 Recently, we have identified another family of integral membrane
proteins that may contribute to the structural organization of caveolae
membranes (31, 32). Micro-sequence analysis of purified caveolin-rich
membrane domains isolated from lung tissue revealed a novel ~45-kDa
component of caveolae membranes termed flotillin (31). Molecular
cloning of flotillin and analysis of the cDNA for this protein has
provided new avenues by which to explore the structure and function of
caveolae organelles. Interestingly, flotillin is a close homologue of
ESA1 (epidermal
surface antigen (33)), and together they define a new "flotillin family" of caveolae-associated integral membrane proteins (flotillin-1 and flotillin-2/ESA)) (31). However, the study of
flotillin-1 has been hampered by the lack of a flotillin-1 specific
antibody probe. Previously, we generated a rabbit anti-peptide antibody
against murine flotillin-1. This anti-peptide antibody did not
recognize flotillin-1 well in crude extracts, precluding a detailed
analysis of the tissue distribution and expression patterns of the
flotillin protein (31). These antipeptide antibodies also recognized
another protein of ~27 kDa we termed Here, we examine the cell type and tissue-specific expression of the
flotillins using a novel monoclonal antibody probe that recognizes only
flotillin-1. A survey of cell and tissue types demonstrates that
flotillins 1 and 2 have a complementary tissue distribution. In
addition, co-immunoprecipitation experiments revealed that flotillins 1 and 2 are part of a stable hetero-oligomeric complex that contains both
caveolins 1 and 2. However, protein levels of flotillin-1 and
flotillin-2/ESA remain unchanged in response to oncogenic
transformation of NIH 3T3 cells. Our data suggest that the expression
of the flotillins can be independently regulated from that of
caveolin-1 and caveolae formation. Also, we show that flotillin-1
represents a new marker protein for the slightly larger
caveolae-related domains (50-200 nm) that are observed in cell types
that fail to express caveolin-1 and do not contain detectable caveolae.
Materials--
The cDNAs for murine flotillin-1 and
Drosophila flotillin-1 were as we previously described (31,
32). Antibodies and their sources were as follows: anti-caveolin-1 IgG
(mAb 2297; gift of Dr. John R. Glenney, Transduction Laboratories,
Lexington, KY); anti-ESA IgG (mAb 29; Transduction Laboratories);
anti-myc epitope IgG (mAb 9E10; Santa Cruz Biotechnology);
anti-caveolin-1 (pAb; rabbit anti-peptide antibody directed against
caveolin-1 residues 2-21; Santa Cruz Biotechnology). A mAb directed
against caveolin-2 (clone 65) was as we described previously (34). A
variety of other reagents were purchased commercially: fetal bovine
serum (JRH Biosciences); pre-stained protein markers (Life
Technologies, Inc.); Slow-Fade anti-fade reagent (Molecular Probes,
Eugene, OR). Protein lysates from the following cells were the generous gift of Drs. Roberto Campos-Gonzalez and John R. Glenney, Jr. (Transduction Laboratories): human fibroblasts, human endothelial cells, A-498 kidney carcinoma (HTB-44), HeLa cells (CCL-2.1), human
smooth muscle cells, SK-N-SH neuroblastoma cells (HTB-11), Jurkat cells
(TIB-152), K562 cells (CCL-243), MDBK cells (CCL-22), bovine pulmonary
endothelial cells, Madin-Darby canine kidney cells (CCL-34), L6
myoblasts (CRL-1458), RPE J cells (CRL-2240), PC12 cells (CRL-1721),
CREF fibroblasts, BC3H1 myoblasts (CRL-1443), P19 teratocarcinoma cells
(CRL-1825), v-Src-transformed NIH 3T3 cells, MEL cells, and 3T3-L1
fibroblasts (CCL-92.1). Most of these cell lines can be obtained from
the ATCC, and their ATCC numbers are as we indicated above in parentheses.
Hybridoma Production--
A monoclonal antibody to murine
flotillin-1 was generated by multiple immunizations of Balb/c female
mice with a fusion protein encoding the full-length flotillin protein.
Mice showing the highest titer of anti-flotillin-1 immunoreactivity
were used to create fusions with myeloma cells using standard protocols
(35). Positive hybridomas were cloned twice by limiting dilution. These
hybridomas were also screened against flotillin-2/ESA to prevent the
selection of a cross-reacting monoclonal antibody. Positive hybridomas
recognizing only flotillin-1, but not flotillin-2, were then injected
into mice to produce ascites fluid. IgGs were purified by affinity chromatography on protein A-Sepharose. These antibodies were produced in collaboration with Drs. Roberto Campos-Gonzalez and John R. Glenney,
Jr. (Transduction Laboratories).
Cell Culture--
HEK-293T cells were the gift of Dr. Anthony J. Koleske (in Dr. David Baltimore's laboratory at MIT, Cambridge, MA)
and were propagated in t75 tissue-culture flasks in Dulbecco's
modified Eagle's medium supplemented with antibiotics and 10% fetal
bovine serum, as we described previously (34). v-Abl- and Ha-Ras
(G12V)-transformed NIH 3T3 cells were as we described previously and
were propagated in t75 tissue-culture flasks in Dulbecco's modified
Eagle's medium supplemented with antibiotics and 10% donor bovine
serum (34, 36, 37).
Transient Expression of Flotillin cDNAs in 293T
Cells--
Constructs encoding C-terminal myc-epitope-tagged
full-length forms of murine and Drosophila flotillin were
created essentially as we described previously for the caveolins 1, 2, and 3 (5, 27, 29). These constructs (~5-10 µg) were transiently
transfected into 293T cells using standard calcium phosphate
precipitation. Forty-eight h post-transfection, cells were scraped into
lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl,
1% Triton X-100, 60 mM octyl glucoside). Recombinant
expression was analyzed by SDS-PAGE (15% acrylamide) followed by
Western blotting. Epitope-tagged forms of flotillin-1 were detected
using the monoclonal antibody, 9E10, that recognizes the myc-epitope (EQKLISEEDLN).
Tissue Western--
Approximately 200 mg (wet weight) of various
mouse tissues were lysed in immunoprecipitation buffer and homogenized
on ice with a Polytron tissue grinder, as described (4). Equal amounts (100 µg of protein) were loaded on an SDS-PAGE gel (12% acrylamide). After transfer to nitrocellulose, the blot was probed with antibodies directed against flotillin-1 and flotillin-2/ESA.
Immunoblotting--
Samples were separated by SDS-PAGE (15%
acrylamide) and transferred to nitrocellulose. After transfer,
nitrocellulose sheets were stained with Ponceau S to visualize protein
bands and subjected to immunoblotting. For immunoblotting, incubation
conditions were as described by the manufacturer (Amersham Pharmacia
Biotech), except we supplemented our blocking solution with both 1%
bovine serum albumin and 1% nonfat dry milk (Carnation).
Synthesis of Immobilized Flotillin-derived Peptides for Epitope
Mapping--
Flotillin-derived polypeptides were synthesized directly
onto an activated polymeric membrane by Research Genetics. The peptide chemistry was standard Fmoc with coupling mediated through butyl alcohol/DIC and Fmoc removal with 1:3
piperidine/N,N-dimethylformamide. For final
peptide protecting group removal, the membrane was placed in a bath of
50:47.5:2.5:1.5:1 DCM/trifluoroacetic acid/thioanisole/EDT/anisole for
1 h and, finally, washed and dried. These sheets were probed by
immunoblotting as described above.
Immunoprecipitation--
Immunoprecipitations were carried out
using protein-A-Sepharose CL-4B (Amersham Pharmacia Biotech) as
described previously (38), with minor modifications. Briefly, cells
were lysed in a buffer containing 10 mM Tris, pH 8.0, 0.15 M NaCl, 5 mM EDTA, 1% Triton X-100, 60 mM octyl-glucoside and subjected to immunoprecipitation with anti-caveolin-1 pAb (directed against residues 2-21; Santa Cruz
Biotech, Inc.). After extensive washing, samples were separated by
SDS-PAGE (15% acrylamide) and transferred to nitrocellulose. Blots
were then probed with IgGs directed against caveolin-1 (mAb 2297),
caveolin-2 (mAb 65), flotillin-1 (mAb 18), and flotillin-2/ESA (mAb 29).
Cell Culture Models of Skeletal and Neuronal
Differentiation--
C2C12-3 cells (39) were derived from a single
colony of C2C12 cells (40) cultured at clonal density and display a
more stable phenotype than the parental cell line. C2C12-3 myoblasts were cultured as described elsewhere (39). Briefly, proliferating C2C12-3 cells were cultured in high mitogen medium (Dulbecco's modified Eagle's medium containing 15% fetal bovine serum and 1%
chicken embryo extract) and induced to differentiate at confluence in
low mitogen medium (Dulbecco's modified Eagle's medium containing 3%
horse serum). Overt differentiation was indicated by the assembly of
multinucleated syncytia, which commenced 36-48 h after the cells were
switched to low mitogen media. PC 12 cells were grown in RPMI 1640 medium with 5% fetal bovine serum and 10% horse serum. PC 12 cells
were differentiated for 1-4 days by culturing the cells in low serum
medium (RPMI 1640 with 1% horse serum) with nerve growth factor (100 ng/ml) (41).
Baculovirus-based Expression of Murine Flotillin-1 in Sf21
Insect Cells--
Spodoptera frugiperda (Sf21) cells
were provided by Dr. Takashi Okamoto (Cleveland Clinic Foundation).
Sf21 cells were grown in Ex-cell 401 medium containing 10%
fetal bovine serum and antibiotics (penicillin-streptomycin) at
27 °C. The cDNA encoding murine flotillin-1 was subcloned into
the multiple cloning site of a transfer plasmid vector, pBacPAK9. A
mixture of 2 µg of recombinant plasmid pBacPAK 9-flotillin-1 DNA and
1 µg of purified engineered baculoviral vector DNA BacPAK 6 (Bsu36I digest) (CLONTECH) were
transfected into insect Sf21 cells, as suggested by the
manufacturer (42). Four days later, culture supernatants were removed
and centrifuged at 1,000 rpm for 10 min. Clarified supernatants
containing wild-type and recombinant baculoviruses were plaque-assayed
on a monolayer of Sf21 cells. Occlusion negative plaques were
picked and seeded onto 2.5 × 106 cells. After a 3-day
incubation, cells and culture supernatants were removed and centrifuged
at 1,000 rpm for 10 min. The cell pellets were analyzed by
immunoblotting analysis using anti-flotillin-1 antibodies or anti-myc
tag mAb 9E10. Those plaques testing positive for the presence of
flotillin-1 were selected for three rounds of plaque purification. The
selected plaques with the highest yield of expression were used as
recombinant baculovirus stock for producing protein by infecting insect
Sf21 cells. Transmission electron microscopy was performed as
described previously by our laboratory. Samples were fixed with
glutaraldehyde, postfixed with osmium tetroxide, and stained with
uranyl acetate and lead citrate, as detailed in Sargiacomo et
al. (14) and Lisanti et al. (15). Samples were examined
under the Philips 410 TEM.
Generation and Epitope-mapping of a mAb Probe Specific for
Flotillin-1--
A fusion protein carrying the full-length flotillin-1
protein was used to generate a flotillin-1-specific monoclonal antibody probe. This antibody does not recognize flotillin-2/ESA (see
"Experimental Procedures"). Thus, this antibody can be used in
conjunction with other published antibodies to study the function and
differential expression of distinct flotillin and caveolin family members.
Fig. 1 demonstrates that this novel mAb
probe recognizes both mammalian flotillin-1 (murine) and
Drosophila flotillin-1 proteins. Immunoblotting with mAb
9E10 was also used in parallel to detect these C-terminal myc-tagged
proteins.
As this flotillin-1 mAb reacts with both murine and
Drosophila forms of flotillin-1, it must recognize an
evolutionarily conserved amino acid epitope. Thus, we performed epitope
mapping by generating a series of 48 overlapping peptides that are
derived from the sequence of murine flotillin-1 (listed in Table
I). These peptides were synthesized as an
immobilized array that can be probed by immunoblotting. Fig.
2 shows the results of this
epitope-mapping strategy. The flotillin-1 mAb only reacted with a
single peptide (number 38 in Table I). As expected based on its
reactivity with both mammalian and Drosophila flotillin-1,
this epitope is highly conserved but is divergent in flotillin-2/ESA.
An alignment of this region of flotillin-1 and other flotillin-related
proteins is shown in Fig. 2C.
Cell Type and Tissue-specific Expression of Flotillins 1 and
2--
To establish the tissue distributions of flotillins 1 and 2, we
prepared tissue extracts from a variety of different murine tissues
(Fig. 3). Flotillin-1 is detected mainly
in striated muscle tissues (heart, diaphragm, and psoas muscle),
adipose tissue, and lung. In striking contrast, flotillin-2/ESA shows a
much wider tissue distribution but is virtually absent in skeletal
muscle (diaphragm and psoas muscle). Thus, flotillins 1 and 2 show a relative complementary tissue distribution.
To identify model cell systems to study flotillins 1 and 2, we examined
the expression of flotillins in a variety of commonly used cell lines
and primary cultured cells (Fig. 4). Note
that flotillin-2/ESA is most widely expressed, whereas flotillin-1 shows a more restricted distribution. More specifically, flotillin-1 was most abundant in A498 kidney cells, muscle cell lines (smooth muscle cells, L6 skeletal myoblasts, and BC3H1 myoblasts) and fibroblasts.
Flotillins 1 and 2 Form a Stable Hetero-oligomeric Complex with
Caveolins 1 and 2--
Given that flotillins have been shown to
co-fractionate with caveolin-1 using three independent fractionation
schemes used to purify caveolae membranes, we wondered whether they
form a complex with caveolins (31). To address this issue, we performed a series of co-immunoprecipitation experiments using A498 cells; these
cells co-express both flotillin 1 and 2 (See Fig. 4).
Lysates from A498 cells were prepared and subjected to
immunoprecipitation with an anti-peptide antibody that recognizes the unique N terminus of caveolin-1 that is not found in other caveolin family members. These immunoprecipitates were then probed by Western blot analysis using monoclonal antibodies directed against caveolin-1 (mAb 2297), caveolin-2 (mAb 65), flotillin-1 (mAb 18), and
flotillin-2/ESA (mAb 29).
Fig. 5 demonstrates that the anti-peptide
antibody directed against the unique N terminus of caveolin-1 can be
used to co-immunoprecipitate caveolin-2, flotillin-1, and flotillin-2.
Thus, it appears that caveolins 1 and 2 and flotillins 1 and 2 form a
stable complex in vivo.
Differential Expression of Flotillins and Caveolins in
Oncogenically Transformed NIH 3T3 Cells--
Caveolin-1 mRNA and
protein expression are reduced or absent in NIH 3T3 cells transformed
by a variety of activated oncogenes (such as v-Abl and Ha-Ras (G12V));
caveolae organelles are also missing from these transformed cells (36).
However, it remains unknown whether flotillins 1 and 2 are
down-regulated in response to oncogenic transformation.
Fig. 6 shows that although caveolin-1
expression is dramatically down-regulated in v-Abl and Ha-Ras
(G12V)-transformed NIH 3T3 cells, the expression of flotillins 1 and 2 remains virtually unaffected in v-Abl and Ha-Ras (G12V)-transformed NIH
3T3 cells. The expression of caveolin-2 is shown for comparison. As we
have previously demonstrated that these transformed cells do not
contain detectable caveolae (36), it appears that co-expression of
caveolin-2, flotillin-1, and flotillin-2/ESA is not sufficient to drive
caveolae formation. Thus, flotillins 1 and 2 can be expressed within
cells that lack morphologically distinguishable caveolae.
However, this does not preclude a role for the flotillins in the
formation of caveolae (50-100 nm) or the slightly larger caveolae-related domains (50-200 nm) that are observed in cells that
fail to express caveolin-1 and do not contain detectable caveolae (See below).
Flotillin-2/ESA Is Up-regulated during the Differentiation of C2C12
Skeletal Myoblasts in Vitro--
As flotillins 1 and 2 were
co-expressed in a variety of muscle cell lines (smooth muscle cells, L6
myoblasts, and BC3H1 myoblasts) and are expressed in muscle tissue
types (heart, diaphragm, and psoas muscle), we assessed whether
flotillins are induced during differentiation of C2C12 cells from
myoblasts to myotubes. Fig. 7 shows that
although flotillin-1 is undetectable in myoblasts and myotubes,
flotillin-2/ESA is dramatically induced during this process of
differentiation.
Expression of Flotillins 1 and 2 during PC12 Cell
Differentiation--
As flotillin-2/ESA was particularly abundant in
brain, we next assessed the expression of flotillins during neuronal
differentiation using PC12 cells as a model system. Although PC12 cells
underwent morphological differentiation in response to treatment with
nerve growth factor, little or no change in the expression of
flotillins 1 or 2 was observed (Fig.
8).
Recombinant Expression of Flotillin-1 in Insect Cells Is Sufficient
to Drive the Formation of Caveolae-sized Vesicles--
Full-length
flotillin-1 was integrated into an engineered baculovirus genome via a
recombinant transfer plasmid, as detailed under "Experimental
Procedures." For these constructions, a myc epitope tag was placed at
the C terminus with a polyhistidine tag following the myc tag (Fig.
9A).
Murine flotillin-1 was expressed very well in Sf21 insect cells
using the baculovirus system. Fig. 9B shows that flotillin-1 migrated at its expected molecular weight, including the myc and polyhistidine tags. We and others have previously shown that these tags
do not interfere with the targeting of recombinant flotillin-1 and
caveolins or the ability of caveolin-1 expression to drive vesicle
formation (5, 13, 18, 27, 29, 31, 32, 43-45). However, it remains
unknown whether the expression of recombinant flotillin-1 is sufficient
to drive vesicle formation.
Electron microscopic analysis of uninfected Sf 21 insect cells did not
show the appearance of caveolae, as we have reported previously (44)
(Fig. 9C). However, insect cells infected with murine
flotillin-1 accumulated a uniform population of caveolae-like structures within their cytoplasm. This population of vesicles was
relatively homogeneous in size and were the same size as expected for
mammalian caveolae or slightly larger, ~50-200 nm in diameter (Fig.
9C).
Similarly, we have previously reported that insect cells infected with
either caveolin-1 or caveolin-3 accumulated a uniform population of
caveolae-like structures within their cytoplasm. However, this
population of vesicles was more homogeneous in size, with a diameter of
~50-100 nm (44, 46). In contrast, expression of caveolin-2 did not
drive vesicle formation, despite evidence of infection such as viral
particles (46). These results indicate that a transmembrane domain
alone is not sufficient to drive vesicle formation in Sf21
insect cells.
Thus, in this functional assay system, flotillin-1 behaved more like
caveolins 1 and 3 than another member of the caveolin gene family,
namely caveolin-2. These results suggest that flotillin-1 may
participate in the formation of caveolae-related vesicles that are of a
larger diameter than caveolae.
Flotillins are a new family of caveolae-associated integral
membrane proteins. They were first identified by microsequence analysis
of purified caveolae. Subsequently, molecular cloning and data base
searches revealed two flotillins, flotillin-1 and flotillin-2/ESA (31).
Flotillin-2/ESA was independently identified as a epidermal surface
antigen using a mAb probe directed against epidermal cell plasma
membrane fractions. This antibody probe also was sufficient to detach
cultured keratinocytes, suggesting that ESA may function in
cell-cell or cell substrate attachments. In collaboration with Lodish,
Schnitzer, and colleagues (31), we have shown that both flotillins 1 and 2 are dramatically enriched within caveolae purified from
adipocytes and endothelial cells (31). However, the rabbit anti-peptide
antibodies that we generated against murine flotillin-1 did not
recognize flotillin-1 well in crude extracts, precluding an analysis of
its true tissue distribution and expression patterns. These
anti-peptide antibodies also recognized another unknown protein of
~27 kDa, which we termed flotillin cross-reacting determinant.
Here, we have generated and characterized a novel monoclonal antibody
probe that selectively recognizes murine flotillin-1. As this antibody
is directed against an evolutionarily conserved epitope, this mAb probe
also recognizes Drosophila flotillin-1. A survey of
mammalian cell lines and murine tissue types reveals that flotillins 1 and 2 have distinct tissue distributions, and flotillin-2 appears to be
more ubiquitously expressed than flotillin-1. Using A498 cells, we show
that caveolins (Cav-1 and Cav-2) and flotillins form a stable
hetero-oligomeric complex as seen by co-immunoprecipitation studies
using an antibody directed against the unique N terminus of caveolin-1.
This suggests that flotillins 1 and 2 may play some unknown structural
role within caveolae membranes or caveolae-related domains.
Caveolin-1 mRNA and protein expression are down-regulated in
response to transformation of NIH 3T3 cells by activated oncogenes such
as v-Abl and Ha-Ras; caveolae organelles are also missing from these
transformed cells (36). Conversely, recombinant expression of
caveolin-1 in these cell lines is sufficient to restore caveolae formation. Interestingly, we find here that these transformed cells
that lack morphologically detectable caveolae continue to express
flotillins 1 and 2 and caveolin-2. These results indicate that
expression of flotillins is not sufficient to generate mature invaginated caveolae.
Using two independent model cell systems, we also show that the
expression of flotillins is independently regulated during myoblast
differentiation and neuronal differentiation. This suggests that
flotillins 1 and 2 may be functionally redundant or that their
individual functions may be tailored to a given tissue and cell type.
Taken together, our data suggest that the expression levels of
flotillins 1 and 2 do not strictly correlate with known expression
patterns of caveolin family members. However, when caveolins and
flotillins are co-expressed within the same cell, as in A498 cells,
they form a stable hetero-oligomeric caveolar complex. In this regard,
the antibodies we have generated and characterized should provide an
invaluable tool to elucidate the function of flotillin-1.
A number of investigators have purified caveolae from cells and tissues
that lack apparent expression of caveolin (47-51). These membranes
were purified based on their Triton insolubility and light buoyant
density in sucrose gradients; they have also been purified based simply
on their light buoyant density in the absence of detergents. These
domains are ~50-200 nm in diameter and have been termed
Triton-insoluble complexes, detergent-resistant membranes, low-density
membranes, and caveolae-related domains. Like caveolae, these
caveolae-related microdomains are dramatically enriched in cholesterol,
sphingolipids, and lipid-modified signaling molecules (47, 48).
The existence of "caveolae-related domains" or caveolae-related
domains that fail to contain caveolin has caused considerable confusion
(47). However, this was at a time when only one caveolin gene was known
to exist, i.e. caveolin (now termed caveolin-1). In
addition, it has been shown that caveolin-1 and caveolae are down-regulated in response to cell transformation, whereas caveolin-2 levels remain constant (34). As a consequence, many commonly used cell
lines lack caveolin-1 protein expression and visible caveolae, as they
are immortalized or transformed. This suggests that caveolae may be
more versatile structures than we have previously imagined. In
addition, expression of caveolin-2 in the absence of caveolin-1 is
apparently not sufficient to generate invaginated caveolae that are
visible by transmission electron microscopy (34).
A similar situation also existed for caveolae purified from striated
muscle, as cardiac myocytes and skeletal muscle myocytes fail to
express caveolin-1; we now know that muscle cell caveolin contain
caveolin-3 (28, 29, 52). Thus, if a cell does not express detectable
amounts of caveolins-1, -2, or -3, this does not mean that they do not
express a protein that fulfills an equivalent function, a functional
homologue rather than a structural homologue. Other detergent-insoluble
membrane proteins have recently been cloned such as VIP-36, VIP-17/MAL,
and the flotillin gene family (flotillin-1 and flotillin-2/ESA), and
one or more of them may represent functional homologues of the
caveolins (31, 47, 53).
In support of this hypothesis, we now show here that heterologous
expression of murine flotillin-1 is sufficient to drive the formation of caveolae-like vesicles. These vesicles have a relatively uniform diameter of ~50-200 nm but are slightly larger than caveolae (~50-100 nm). These results suggest that flotillins may participate functionally in the formation of caveolae or
caveolae-like vesicles in vivo. Thus, flotillin-1 represents
a new integral membrane protein marker for the slightly larger
detergent-resistant caveolae-related domains (50-200 nm) that are
observed in cell types which fail to express caveolin-1 and do not
contain detectable caveolae. In light of this new functional data, we
propose the more descriptive term "cavatellins2" be
used to refer to this gene family
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and Cav-1
) that differ in
their respective translation initiation sites (27). The tissue
distribution of caveolin-2 mRNA is extremely similar to caveolin-1
mRNA (5). In striking contrast, caveolin-3 mRNA and protein is
expressed mainly in muscle tissue types (skeletal, cardiac, and
smooth) (28-30).
lotillin
ross-
eacting
eterminant (FCRD) (31). The identity
and significance of flotillin cross-reacting determinant remains unknown.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
Characterization of a mAb probe specific for
flotillin-1. C-terminal myc-tagged forms of mammalian
(m, murine) and Drosophila (Dr)
flotillin-1 were transiently expressed in 293T cells. A,
murine flotillin-1; B, Drosophila flotillin-1.
Lysates were generated and probed with mAb 9E10 that recognizes the
myc-epitope or a novel mAb probe directed against murine flotillin-1
(mAb 18). Note that mAb 18 recognizes both mammalian and
Drosophila flotillin-1.
Peptides derived from murine flotillin-1 used for epitope-mapping
View larger version (30K):
[in a new window]
Fig. 2.
Epitope mapping of a flotillin-1 specific
mAb. A, a series of 48 overlapping flotillin-1-derived
peptides were synthesized as discrete spots on a membranous support.
Their sequences are listed in Table I. FLO, flotillin.
B, these immobilized peptides were then incubated with mAb
18 directed against murine flotillin-1. Note that a single spot was
immunoreactive with the antibody probe. This corresponds to peptide 38 with the following sequence, RARAEAEQMAKKAE. C, an alignment
of the relevant sequences of a number of flotillin family members is
shown. These include murine and Drosophila flotillin-1,
murine and human flotillin-2, and two flotillin-related open reading
frames (ORF) in Synechococcus, a cyanobacterium.
The relevant immunoreactive peptide is over-lined in
bold. Overlapping regions found in adjacent nonreactive
peptides are also over-lined. Note that this sequence is
remarkably conserved between murine and Drosophila
flotillin-1. The accession numbers are as follows: AF044734; U90435;
M60922; U07890; U30252 (for open reading frames 1 and 2).
View larger version (19K):
[in a new window]
Fig. 3.
Western blot analysis of the tissue
distribution of flotillin-1 and flotillin-2/ESA. Extracts of mouse
tissues were prepared as described under "Experimental Procedures."
In addition, a lane containing differentiated 3T3-L1
adipocytes was included as a comparison with adipose tissue.
Upper panel, anti-flotillin-1 (mAb 18); lower
panel, anti-flotillin-2/ESA (mAb 29). Equal amounts of protein
were loaded in each lane. In the upper panel, we
do not yet know the significance of the faint flotillin band in the
heart lane; this may represent a degradation product or a
product of alternate translation initiation, as flotillin-1 has three
closely spaced methionines at its extreme N terminus (Met at amino acid
residues 1, 11, and 23).
View larger version (46K):
[in a new window]
Fig. 4.
Expression of flotillins 1 and 2 in
established cell lines and primary cultured cells. 10 µg of
total cellular protein extracted from a given cell line (as indicated)
was separated by SDS-PAGE, transferred to nitrocellulose, and probed
with anti-flotillin-1 (mAb 18) and anti-flotillin-2/ESA (mAb 29). Note
that both flotillins 1 and 2 are co-expressed in A498 kidney cells,
smooth muscle cells, L6 myoblasts, BC3H1 myoblasts, and 3T3-L1
fibrobasts. H, human; B, bovine; C,
canine; R, rat; M, mouse.
View larger version (26K):
[in a new window]
Fig. 5.
Caveolins 1 and 2 and flotillins 1 and 2 form
a stable hetero-oligomeric complex in vivo. A498
cells were lysed and subjected to immunoprecipitations with
anti-caveolin-1 pAb (N-2-21), which recognizes the unique extreme N
terminus of caveolin-1. These immunoprecipitates were then probed by
Western analysis with monoclonal antibodies directed against caveolin-1
(mAb 2297), caveolin-2 (mAb 65), flotillin-1 (mAb 18), and
flotillin-2/ESA (mAb 29). Protein A-Sepharose beads alone were also
incubated with lysates and processed in parallel as a negative control
for nonspecific binding. Note that anti-caveolin-1 IgG can be used to
specifically co-immunoprecipitate caveolin-2 and flotillins 1 and 2. Thus, it appears that flotillins and caveolins form a stable
hetero-oligomeric complex. WB, Western blot.
View larger version (18K):
[in a new window]
Fig. 6.
Expression of flotillins and caveolins in
normal and transformed NIH 3T3 cells. Lysates were prepared from
normal and transformed (Ha-Ras (G12V) and v-Abl) NIH 3T3 cells and
subjected to immunoblot analysis with antibodies directed against
caveolin-1 (mAb 2297), caveolin-2 (mAb 65), flotillin-1 (mAb 18), and
flotillin-2/ESA (mAb 29). In contrast to caveolin-1, flotillins 1 and 2 as well as caveolin-2 are still well expressed in these transformed
cell lines. Caveolin-2 expression in v-Abl-transformed NIH 3T3 cells is
slightly reduced, as observed previously (34).
View larger version (29K):
[in a new window]
Fig. 7.
Expression of flotillins 1 and 2 in C2C12
skeletal myoblasts and myotubes. Cell lysates were prepared from
C2C12 myoblasts (day 0) and differentiating myotubes (days 3 and 6).
Note that flotillin-2/ESA expression is induced during myoblast
differentiation (~7-10-fold), whereas flotillin-1 remains
undetectable. Each lane contain an equal amount of
protein.
View larger version (33K):
[in a new window]
Fig. 8.
Expression of flotillins 1 and 2 in
differentiating PC12 cells. Cell lysates were prepared from
undifferentiated (day 0) and differentiating (days 2 and 4) PC12 cells.
Note that the expression of flotillin-2/ESA remains relatively
constant, whereas the expression of flotillin-1 declines slightly. Each
lane contain an equal amount of protein.
View larger version (31K):
[in a new window]
Fig. 9.
Recombinant expression of flotillin-1 in
insect cells is sufficient to drive the formation of caveolae-like
vesicles, but of a larger diameter than caveolae. A,
construction of epitope-tagged flotillin-1 for expression in
Sf21 insect cells. Flotillin-1 contains two hydrophobic domains
of 27 and 18 amino acids, respectively. The first of these domains
(from position 10 to 36) may represent an atypical signal peptide or
may be a transmembrane domain. The second hydrophobic region (from
position 134 to 151) is another potential transmembrane domain. Note
that the construct contains a C-terminal myc tag followed by a
polyhistidine tag. B, expression of flotillin-1 in
Sf21 insect cells. Lysates from insect cells infected with the
flotillin-1 baculovirus vector were prepared and subjected to
immunoblot analysis with a monoclonal antibody probe that recognizes
the myc epitope (mAb 9E10). Uninfected Sf 21 cells served as a negative
control. Each lane contains equal amounts of total protein.
C, morphological analysis of flotillin-1-induced vesicle
formation. Sf21 insect cells were infected with flotillin-1 and
cells were fixed and processed for transmission electron microscopy.
Upper panel, uninfected control cells lacking flotillin-1
induced vesicles. The black-dotted structures are ribosomes.
Lower panel, infected Sf21 cells expressing
flotillin-1 contain a relatively uniform population of vesicles of
~50-200 nm in diameter. The black bar-like structures are
baculoviruses. Bar = 100 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
FOOTNOTES |
---|
* This work was supported by a National Institutes of Health Grant (NCI) R01-CA-80250 (to M. P. L.) and grants from the Charles E. Culpeper Foundation (to M. P. L.), G. Harold and Leila Y. Mathers Charitable Foundation (to M. P. L.), and the Sidney Kimmel Foundation for Cancer Research (to M. P. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of National Institutes of Health (NCI) post-doctoral fellowship CA-71326.
Present address: Allergan, Inc., 2525 Dupont Dr., Irvine, CA 92713.
Supported by National Institutes of Health FIRST Award MH-56036
(to T. O.).
§§ To whom correspondence should be addressed: Dept. of Molecular Pharmacology and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-8828; Fax: 718-430-8830; E-mail: lisanti{at}aecom.yu.edu.
2
Cavatellin, is derived from the word
cavatellia shell-shaped Italian pasta with a ruffled or scalloped
outside that originated in the Puglia region of Italy.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ESA, epidermal surface antigen; mAb, monclonal antibody; PAGE, polyacrylamide gel electrophoresis; Fmoc, N-(9-fluorenyl)methoxycarbonyl.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|