* Division of Hematology/Oncology, Children's Hospital and the Dana Farber Cancer Institute, Division of Medical Science,
Program in Biological and Biomedical Sciences, Harvard University, Boston, Massachusetts 02115
We have recently cloned and characterized ankyrin-3 (also called ankyrinG), a new ankyrin that is widely distributed, especially in epithelial tissues, muscle, and neuronal axons (Peters, L.L., K.M. John, F.M. Lu, E.M. Eicher, A. Higgins, M. Yialamas, L.C. Turtzo, A.J. Otsuka, and S.E. Lux. 1995. J. Cell Biol. 130: 313-330). Here we show that in mouse macrophages, ankyrin-3 is expressed exclusively as two small isoforms (120 and 100 kD) that lack the NH2-terminal repeats. Sequence analysis of isolated Ank3 cDNA clones, obtained by reverse transcription and amplification of mouse macrophage RNA (GenBank Nos. U89274 and U89275), reveals spectrin-binding and regulatory domains identical to those in kidney ankyrin-3 (GenBank No. L40631) preceded by a 29-amino acid segment of the membrane ("repeat") domain, beginning near the end of the last repeat. Antibodies specific for the regulatory and spectrin-binding domains of ankyrin-3 localize the protein to the surface of intracellular vesicles throughout the macrophage cytoplasm. It is not found on the plasma membrane. Also, epitope-tagged mouse macrophage ankyrin-3, transiently expressed in COS cells, associates with intracellular, not plasma, membranes. In contrast, ankyrin-1 (erythrocyte ankyrin, ankyrinR), which is also expressed in mouse macrophages, is located exclusively on the plasma membrane. The ankyrin-3-positive vesicles appear dark on phasecontrast microscopy. Two observations suggest that they are lysosomes. First, they are a late compartment in the endocytic pathway. They are only accessible to a fluorescent endocytic tracer (FITC-dextran) after a 24-h incubation, at which time all of the FITC-dextran- containing vesicles contain ankyrin-3 and vice versa. Second, the ankyrin-3-positive vesicles contain lysosomal-associated membrane glycoprotein (LAMP-1), a recognized lysosomal marker. This is the first evidence for the association of an ankyrin with lysosomes and is an example of two ankyrins present in the same cell that segregate to different locations.
The ankyrins are a family of plasma membrane-associated proteins that link integral membrane proteins
to the underlying membrane skeleton. There are now
three family members: erythrocyte ankyrin (ankyrin-1,
Ank1, AnkR) (42, 48), brain ankyrin (ankyrin-2, Ank2,
AnkB) (58), and epithelial or general ankyrin (ankyrin-3, Ank3, AnkG) (37, 60).
In the RBC, ankyrin-1 links the transmembrane anion
exchanger, band 3, to Ankyrin-2 is the major form of ankyrin in the nervous
system. The 220-kD protein is found in most neuron cell
bodies and dendrites, as well as in glia (58). A 440-kD isoform is expressed in the fetal brain, targeted specifically to
unmyelinated axons and dendrites (8, 40).
Ankyrin-3 is much more widely distributed than its sister genes and is the major ankyrin in epithelia, myocytes,
hepatocytes, melanocytes, megakaryocytes, Leydig cells,
and neuronal axons (60). In the brain, ankyrin-3 is expressed as isoforms of 270- and 480-kD and localizes to the
nodes of Ranvier and to the initial segments of axons (34,
37, 60).
All three ankyrins have a similar, three-domain structure. The NH2-terminal, plasma membrane-binding or
"repeat" domain contains 24 tandem 33-amino acid repeats and binds many of the integral membrane protein
ligands of ankyrins (12, 50). The central, spectrin-binding domain binds spectrin and fodrin (nonerythroid spectrin) (2, 3, 14, 33, 62) and is critical for attaching the underlying membrane skeleton to the lipid bilayer. The COOHterminal, regulatory domain influences the affinity of ankyrin
for its ligands via an acidic, alternately spliced segment in
the middle of the domain (14).
The kidney contains multiple isoforms of ankyrin-3: 215, 200, 170, 120, and 105 kD (60). The two smallest isoforms
lack the repeat domain and differ only by the presence or
absence of the acidic regulatory domain insert (60). These
two small ankyrin-3 isoforms are the only forms observed
in intestine, testis, and liver. Immunohistochemical staining suggests they reside in the cytoplasm instead of in the
plasma membrane (60).
We find that two similar, small, NH2-terminal truncated
proteins are also the only isoforms of ankyrin-3 in bone
marrow-derived macrophages, where they associate with
intracellular membranes of phase-dense lysosomes (i.e.,
lysosomes that appear dense in the phase-contrast microscope). Judging from when they are labeled by soluble,
fluid-phase tracer molecules (25, 68, 71), these vesicles occur very late in the endocytic pathway. Specifically, 24 h
after an initial 15-min pulse, fluorescence from endocytically incorporated dextrans completely coincides with
staining from ankyrin-3 antibodies. Taken together with immunofluorescent experiments that show both ankyrin-3 and
lysosomal-associated membrane glycoprotein 1 (LAMP-1)1
on the same late endocytic vesicles, we identify these vesicles as lysosomes.
Macrophages
Murine bone marrow-derived macrophages were obtained as previously
described (70). Briefly, femurs from C3H-HeJ +/+ mice (The Jackson
Laboratory, Bar Harbor, ME) were removed and their marrow was extruded and cultured in bone marrow macrophage medium (BMM) for 6-7 d.
BMM comprises DME (GIBCO BRL, Gaithersburg, MD) containing
30% L cell-conditioned medium, a source of macrophage colony stimulating factor, and 20% heat-inactivated FBS (Biocell Laboratories, Rancho
Dominguez, CA). Cells were harvested and either plated onto glass coverslips and incubated overnight to allow cells to adhere for immunofluorescence or replated at one-third density and allowed to grow for an additional 2 d before harvesting protein or RNA.
Antibodies
Ankyrin-3 antibodies were generated and characterized previously in our
laboratory (60). Briefly, a unique portion of the regulatory domain of
mouse ankyrin-3 was fused to glutathione-S-transferase (GST) and used
as an antigen (Ank3-R1 antibody). Also, antibodies were prepared to the
"B" insert (DKCTWFKIPKVQEVL), which lies between the repeat and
spectrin-binding domains, and to the NH2-terminal end of the kidney isoform lacking the repeat domain (MALPHSEDAITGDTD). These are referred to as Ank3-B and 5 Anti-human erythrocyte protein 4.1 and eight different anti-human
spectrin antisera were provided by Dr. Orah Platt (Children's Hospital,
Boston, MA). Affinity-purified antibodies against Preparation of Proteins
Kidney membrane proteins were prepared as previously described (60).
Macrophage protein extracts were prepared by lysing dense but not confluent macrophages from one well of a six-well dish into 1 ml of extraction
buffer (16) containing 0.5% SDS, 0.1% Triton X-100, 40 mM Hepes, pH
7.15, 50 mM Pipes, pH 6.9, 75 mM NaCl, 1 mM MgCl2, 0.5 mM EGTA, 0.1 mg/ml leupeptin, 0.1 mg/ml pepstatinA, and 0.1 mg/ml PMSF (first dissolved in 100% ethanol). Extracts were immediately added to one-third
volume of 4× Laemmli sample buffer (41), boiled for 5 min, and applied to SDS polyacrylamide gels.
Immunoblotting
SDS-PAGE was performed using the Laemmli buffer system (41) with
5% stacking and 10% running gels. 30 µl (50-100 µg) of macrophage extracts or kidney membrane proteins and 30 µl (~200 µg) of prestained
molecular weight markers (Bio Rad Laboratories, Hercules, CA) were run
overnight at a constant 30 V. The proteins were transferred to ImmobilonP membranes (Millipore Corp., Bedford, MA) in 48 mM Tris-HCl, 39 mM
glycine, 20% methanol, and 0.0375% SDS, pH 8.3, using a BioRad semidry transfer apparatus. Filters were blocked for a minimum of 1 h at room
temperature in TTBS (10 mM Tris HCl, pH 7.0, 0.05% Tween-20, and 150 mM NaCl) containing 5% BSA. Filters were washed three times for 5 min
each in TTBS, and then incubated for 1 h at room temperature in primary
antibody diluted 1:500 in TTBS. Washing was repeated as above, and then filters were incubated at room temperature with goat anti-rabbit IgG conjugated with alkaline phosphatase (Bio Rad Laboratories) according to
the manufacturer's instructions. Filters were washed again as above, and
bound antibody was visualized with nitroblue tetrazolium and 5-bromo-4chloro-3-indolyl phosphate (BioRad kit).
Preparation of RNA
Medium was removed from macrophage cultures by aspiration, and each
monolayer was washed twice with ice-cold PBS lacking calcium and magnesium. For 150-mm dishes, 4.5 ml of 10 mM EDTA, pH 8.0, containing
0.5% SDS was added, and the lysate was scraped off the dish and collected
into a glass centrifuge tube. Then 4.5 ml of 0.1 M sodium acetate, pH 5.2, containing 10 mM EDTA, pH 8.0, was used to rinse the plate; the eluate
was collected and added to the lysate. Water saturated phenol (9 ml) was
added to the lysate and the mixture was shaken vigorously at room temperature for 2 min. Aqueous and organic phases were separated by a 10min centrifuge spin at 4°C at 3,000 g. The aqueous phase was reextracted twice more, once in a 24:24:1 mixture of phenol/chloroform/isoamyl alcohol and once in a 24:1 mixture of chloroform/isoamyl alcohol. Ice-cold 1 M
Tris-HCl (pH 8.0, 990 µl) and 5 M NaCl (405 µl) were added to the aqueous phase and mixed. 2 vol of ice-cold ethanol were then added, and the
precipitated RNA was centrifuged for 15 min at 4°C at 12,000 g. The RNA
was redissolved and reprecipitated as above, and then dissolved in icecold 10 mM Tris and 1 mM EDTA, pH 8.0.
Reverse Transcription-PCR
Macrophage RNA (5 µg) was heated to 65°C for 5 min and transferred
to ice for an additional 5 min. Denatured RNA was then added to a buffer
containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT,
0.5 mM each of dGTP, dATP, dCTP, and dTTP, 10-50 µg/ml oligo(dT), 1 µl
(40 U) of RNAsin (Promega, Madison, WI), and 4 µl (800 U) of Moloney
murine leukemia virus reverse transcriptase (GIBCO BRL) and incubated at 42°C for 60 min. RNA was then hydrolyzed by adding NaOH to a
final concentration of 0.375 N at 65°C for 30 min. The base was neutralized with an equal molar amount of acetic acid. cDNA was amplified using
AmpliTAQ (Boehringer Mannheim Biochemicals) with isoform-specific
ankyrin-3 primers (see Fig. 1) (15 ng/µl) in a buffer containing 100 mM Tris-HCl, pH 8.3, 15 mM MgCl2, 500 mM KCl, and 0.2 mM dNTP. The PCR program was 94°C (30 s), 60°C (30 s), and 72°C (5 min) for 30 cycles
followed by extension at 72°C (7 min).
Primer sequences were: REGR1; 5 5 Amplification of the 5 Primer sequences were: MEMR1; 5 DNA Sequencing
PCR products were isolated and subcloned into the pCR II vector using
the TA cloning kit (Invitrogen, San Diego, CA). Plasmid DNA was prepared by the cetyltrimethylammonium bromide method (15) and sequenced on both strands by the dideoxynucleotide chain termination
method (65) using Sequenase version 2.0 (United States Biochemical
Corp., Cleveland, OH) and synthetic oligonucleotide primers.
Immunofluorescence Microscopy
For ankyrin-3 localization, cells plated onto glass coverslips were fixed in
4% paraformaldehyde at room temperature for 5 min, and then lysed in a
0.1% Triton X-100 buffer containing 40 mM Hepes, pH 7.15, 50 mM
Pipes, pH 6.9, 75 mM NaCl, 1 mM MgCl2, and 0.5 mM EGTA as described
previously (16). For LAMP-1 localization, cells plated onto glass coverslips were fixed in 50 mM phosphate buffer, pH 7.8, containing 2% paraformaldehyde, 9 mg/ml lysine, and 2 mg/ml NaIO4 for 1-2 h. The fixed
cells were permeabilized in 0.01% saponin.
For double immunofluorescence experiments, cells were fixed as described for LAMP-1 but were lysed in 0.25% saponin. Both LAMP-1 and
ankyrin-3 were detectable under these conditions, although staining of
each protein was less intense than it was under the optimal conditions of
cell lysis described above (LAMP-1 = 0.01% saponin; ankyrin-3 = 0.1%
Triton X-100). This was particularly true for ankyrin-3, which was barely
detectable in cells lysed with saponin. In a survey experiment, 0.25% saponin was the only concentration where both ankyrin-3 and LAMP-1
could be detected. The number of LAMP-1-positive or dextran-filled vesicles observed was reduced when higher concentrations of saponin were
used for lysis or completely ablated when Triton X-100 was used instead of saponin.
All cells were then washed three times (5 min each) in 2 ml of PBS containing 0.02% NaN3 at room temperature, and then incubated at room
temperature for 1 h with primary antibodies diluted in PBS. Cells were
washed in PBS again and incubated for 1 h at room temperature with a 1:800
dilution of a goat anti-rabbit or goat anti-mouse secondary antibody conjugated with the fluorochrome CY-3 (Jackson ImmunoResearch Laboratories, West Grove, PA). Stained cells were washed a final time, mounted
in 9:1 glycerol/PBS, and visualized on an Axiochrom microscope (Carl
Zeiss Inc., Thornwood, NY). Images were captured on T-Max 400 film
(Eastman Kodak Co., Rochester, NY).
Labeling of Endocytic Organelles
Dextran was used as an endocytic tracer. Macrophages were incubated at
37°C for 15 min in BMM containing 10 mg/ml of an anionic, fluoresceinconjugated dextran (Mr 10,000) that could be fixed with paraformaldehyde (Molecular Probes, Eugene, OR). The cells were then washed and
chased with BMM (not containing dextran) for periods up to 24 h. The
dextran-treated cells were processed at specific time points for immunofluorescence microscopy as described above.
Expression of Epitope-tagged Ankyrin-3 in COS Cells
The HA epitope was fused to the COOH terminus of mouse macrophage
ankyrin-3 and ligated into the pcDNA3 vector (Invitrogen). The vector
used in transfection experiments was purified by alkaline lysis and cesium
chloride density gradient centrifugation as described previously (64). Purified vector was transfected into COS cells using the ProFection CaPO4
transfection kit (Promega, Madison, WI) according to manufacturer's recommendations. Cells were allowed to take up the DNA for 16 h, washed,
and incubated for an additional 2 d before HA-tagged ankyrin-3 was visualized via immunofluorescence using the monoclonal 12CA5 anti-HA antibody (Boehringer Mannheim Biochemicals) as described above.
Macrophage Ankyrin-3 Lacks the
NH2-terminal Repeats
In the mouse kidney, ankyrin-3 is expressed as multiple
isoforms (see Fig. 2) that are detected by Western blotting
with the Ank3-R1 antibody (60) (Fig. 1). Antibody staining is specific since it is successfully competed by the immunizing antigen (Fig. 2, lane C). The calculated molecular masses, based on mobility in SDS polyacrylamide gels,
are 215, 200, 170, 120, and 105 kD (60). We have previously shown that the two smallest isoforms (105 and 120 kD)
lack the NH2-terminal repeat domain (60). Bone marrow-
derived macrophages also express small (120 and 100 kD) isoforms of ankyrin-3 (Fig. 2, lane D, arrows). These isoforms are specific (Fig. 2, lane E) and are the only forms of
ankyrin-3 consistently observed in macrophages. Some
gels contain other, lower molecular mass bands (<80 kD)
that stain specifically with Ank3-R1 (Fig. 2, lanes D and
E), but these are probably proteolytic fragments of the
larger bands because they vary in size and intensity from experiment to experiment.
To further characterize the macrophage forms of ankyrin-3
and the analogous kidney isoforms, we recloned ankyrin-3
from mouse bone marrow-derived macrophage RNA by
reverse transcription (RT)-PCR. Primers that flank the regulatory domain (REGF1 and REGR1; Fig. 1) produce two
PCR products of ~1,025 and 1,600 bp (Fig. 3, lane A, arrows). Subcloning (Fig. 3, lanes G and H) and sequencing show these represent the regulatory domain with and
without the acidic, alternatively spliced exon that is located in the center of the domain (for review see Fig. 1).
The spectrin-binding domain is also present in macrophage
RNA as shown by the 1,800-bp product (Fig. 3, lane C, arrow) obtained using primers SPINTF1 and SPR1 (Fig. 1).
The identity of this domain was also confirmed by subcloning (Fig. 3, lane F) and sequencing. Interestingly, primers
MEMF1 and MEMR1 (Fig. 1) did not yield a product.
This result supports the protein analysis and confirms that
the NH2-terminal repeats of ankyrin-3 are not present in
macrophage RNA. All macrophage ankyrin-3 cDNAs
were completely sequenced and compared with ankyrin-3 cDNA clones from kidney. This analysis revealed that
macrophage ankyrin-3 contains all of the sequence of the
spectrin-binding (amino acids [aa] 874-1,455) and regulatory (aa 1,456-1,960) domains previously reported from
mouse kidney (60) (GenBank No. L40631). The two isoforms of ankyrin-3 expressed in macrophages differ from
each other only by the presence or absence of an insert in the regulatory domain (aa 1,588-1,783) that is also found
in kidney ankyrin-3 (60) and is analogous in size and
charge to the "2.1 insert" in the regulatory domain of
ankyrin-1 (42, 48).
It is important to note that the primer SP5F1, which hybridizes to the 5
Ankyrin-3 Associates with Intracellular Vesicles in
the Macrophage
Immunofluorescence microscopy using the Ank3-R1 antibody, which recognizes both isoforms of ankyrin-3 expressed in macrophages, reveals that ankyrin-3 surrounds
intracellular vesicles throughout the macrophage cytoplasm
(Fig. 5, A and B) and is not associated with the plasma
membrane. This surprising result was confirmed with a
second antibody, 5
Macrophage Ankyrin-3 cDNA Expression in COS Cells
The vesicular location of ankyrin-3 was further substantiated by transient overexpression of HA-tagged ankyrin-3
in COS cells. HA was fused to the COOH terminus of
mouse macrophage ankyrin-3 and ligated into the pcDNA3
mammalian expression vector. The fusion protein was visualized using the 12CA5 mAb, which is directed against
the HA epitope. This experiment shows that macrophage ankyrin-3 localizes to intracellular vesicle membranes and
not plasma membranes (Fig. 6). The vesicular location of
macrophage ankyrin-3 (Fig. 7 B) differs dramatically from
ankyrin-1, which is found in its characteristic position on
the plasma membrane (Fig. 7 A). The presence of ankyrin1 in macrophages was confirmed by immunoblotting (data
not shown).
Ankyrin-3-positive Vesicles Are Lysosomes
Because the dense appearance of the ankyrin-3-positive
vesicles suggested they might be lysosomes (56, 69), we
first tested their accessibility to endocytic tracers by incubating macrophages with soluble fluorescein-conjugated
dextran (average Mr = 10,000 daltons). After a brief 15min labeling pulse, we washed the cells and incubated
them for various chase times up to 24 h. We then fixed the
macrophages, localized ankyrin-3 with the Ank3-R1 antibody, and visualized it with rhodamine-conjugated antiIgG. None of the ankyrin-3-positive vesicles were filled
with fluorescent dextran at very early time points (Fig. 8,
A and B, arrowheads), and only a small fraction of the vesicles contained both fluorophores after a 6-h chase (Fig. 8,
C and D, arrows). In contrast, after 24 h, all the fluorescent dextran was located in ankyrin-3-positive vesicles
(Fig. 8, E and F, arrows). This indicates the vesicles are in
the endocytic pathway (5, 23, 24, 30, 38, 57, 72, 73). However, endocytic tracers can be chased out of endosomes and into lysosomes within a few hours (25, 68, 71), while it
takes 24 h for dextran-containing endosomes to reach the
ankyrin-3-positive compartment. This suggests ankyrin-3-
positive vesicles are lysosomes instead of endosomes.
To examine this further, we stained macrophages for
rab9, a marker of late endosomes (47). Rab9 associates
with large, phase-light vesicles that were completely different in appearance from ankyrin-3-positive or dextran-positive vesicles (data not shown). We also stained macrophages
with acridine orange, a marker for acidic compartments (53). All phase-dense vesicles were acidic (data not shown), which supports the argument that phase-dense, ankyrin-3-
positive vesicles are lysosomes.
Lastly, we stained macrophages with antibodies to
LAMP-1, a well-characterized lysosomal membrane glycoprotein that has been used frequently as a marker for lysosomal compartments (9, 10, 20). After the 24 h chase, a
time point at which all ankyrin-3-positive vesicles are
filled with dextran, saponin-permeabilized cells reveal that
LAMP-1-positive vesicles are also filled with fluorescent dextran (data not shown). Additionally, by simultaneously
colocalizing LAMP-1 and ankyrin-3 in macrophages that
were not exposed to dextran, we show that both of these
proteins associate with the same intracellular vesicles (Fig.
9, A and B, arrows). The LAMP-1 staining shown here is
not optimal in its intensity because we had to use higher concentrations of saponin than those normally used for
LAMP-1 immunolocalization use to allow ankyrin-3 antibodies to localize simultaneously (see Materials and Methods for more details). Also, under more optimal conditions, LAMP-1 stains many phase-light lysosomes in the
perinuclear region of the cytoplasm.
Together these experiments demonstrate that ankyrin-3
is expressed in bone marrow-derived macrophages as two
isoforms that lack the NH2-terminal repeats and, within
these cells, resides on the intracellular membranes of
acidic, phase-dense, LAMP-1-positive lysosomes. The
presence of an ankyrin, which is normally associated with
the plasma membrane and membrane skeleton, on the surface of lysosomes raises the possibility that structures like
the membrane skeleton may also form on intracellular
membranes and influence their function.
Ankyrin-3 Expression
Ankyrins have been localized to polarized plasma membrane surfaces in a wide range of tissue types, including
cardiac and skeletal muscle, intestine, retinal, renal, gastric, and airway epithelium, MDCK cells, initial segments
and the node of Ranvier in axons, dendrites, and the Torpedo electrocyte (4, 19, 22, 26, 29, 31, 34, 44, 54, 60, 67).
In addition to diverse tissue expression, ankyrin-3 is characterized by its unique isoforms, some of which are missing the 89-kD repeat domain (60) responsible for interactions with integral membrane ion channels or adhesive
proteins (7, 12, 44, 50, 55). Since the isolated 62-kD
spectrin-binding domain of ankyrin-1 retains its stability
and capacity to bind spectrin (2, 62, 74), it is easy to believe
that the 120- and 105-kD isoforms of ankyrin-3, which contain the spectrin-binding and regulatory domains, would be
stable and functional as well. It follows that if the domain
responsible for binding to plasma membrane-associated proteins is missing, these spliced isoforms might localize to membranes other than the plasma membrane.
Ankyrin-3 is expressed in epithelial cells as multiple isoforms ranging in size from 215 to 100 kD. The major difference between these isoforms is the presence or absence of
the NH2-terminal repeat domain. In cells that only express
the smaller two isoforms of ankyrin-3 that lack the repeat
domain, ankyrin-3 appears to be concentrated in the cytoplasm, not the plasma membrane (60). One of these cells is
the macrophage and, as shown here, macrophage ankyrin3 localizes exclusively to the intracellular membranes of
lysosomal vesicles. We are confident this is ankyrin-3, and not another ankyrin, since antibodies raised to two different regions of ankyrin-3 localize to the same membrane
surface and antibodies to ankyrin-1 show a different (and
more typical) plasma membrane staining pattern.
It is interesting that the 5 Ankyrin-3-positive Lysosomes
It is well established that soluble tracer molecules tagged
with fluorescent markers are useful in following the fluid
phase through the endocytic pathway over time (23, 30,
38, 57, 68, 71). Since we can chase one of these endocytic tracers, fluorescent dextran, into ankyrin-3-positive vesicles, the vesicles must be part of the endocytic
pathway.
The 24-h chase time required to obtain complete colocalization of endocytically incorporated dextrans and
ankyrin-3 (Fig. 8, E and F) suggests the ankyrin-3-positive
vesicles are lysosomes (25, 68, 71). This was confirmed by
the fact that dextran-containing vesicles are also positive
for LAMP-1 at the 24-h chase time and by the fact that
LAMP-1 and ankyrin-3 colocalize when the staining conditions for each antigen are carefully adjusted so that both can be stained at the same time.
The colocalization experiment is difficult because immunofluorescence of ankyrin-3 and LAMP-1 staining varies depending on the lysis conditions used. The number of
LAMP-1- and/or dextran-positive vesicles in macrophages
decreases when high concentrations of saponin or Triton
X-100 are used for lysis (conditions that are optimal for
ankyrin-3 labeling), while ankyrin-3 cannot be detected when low concentrations of saponin are used (optimal conditions for LAMP-1). In addition, the dark, phase-dense
appearance of macrophage lysosomes correlates with the
vesicles' ability to retain ankyrin-3 staining (Hoock, T.C.,
unpublished observations). It is important to note that
macrophages grown from bone marrow do not all contain
phase-dense vesicles. Anti-ankyrin-3 antibodies do not react with cells that lack phase-dense vesicles. We have tried
many cytokines and stimulatory agents to induce their expression: tumor necrosis factor We do not understand what controls the expression of
phase-dense vesicles in macrophages; however, they seem
to increase in number when cells become fully differentiated and stop dividing, and when replated from primary
culture before cells reach confluent density (Hoock, T.C.,
unpublished observations). Furthermore, resident macrophages isolated from the peritoneal cavity of mice also
show that ankyrin-3 associated with intracellular vesicles, while an immortal macrophage cell line, J774, does not
stain for intracellular ankyrin-3 (data not shown). Preliminary data from experiments in which we fed increasing
amounts of unlabeled dextran to macrophages show a correlation with increasing numbers of ankyrin-3-positive vesicles (Hoock, T.C., unpublished data). This experiment
suggests that cells respond to an increase in endocytic material by increasing the number of endocytic vesicles to
house the material. It remains to be seen whether an increase in the expression of ankyrin-3 is observed before or
after an increase in endocytic material being ingested.
Intracellular Membrane Skeletons
Using the red cell plasma membrane skeleton as a model,
one might expect to find other skeletal proteins on vesicle
membranes. Preliminary experiments suggest the vesicles
contain Band 3 (32), In macrophages, anti-ankyrin-3 antibodies did not stain
plasma membranes and were never found in a perinuclear
position, suggesting that macrophage ankyrin-3 isoforms,
which differ from those expressed in MDCK cells, do not
reside in the Golgi apparatus or the ER. Based on these
observations, we hypothesize that ankyrin-3 is translated
in the cytoplasm on demand and quickly diffuses or is
shuttled to lysosomal membranes.
Overall, the presence of these membrane skeleton proteins, previously thought to only associate with the plasma
membrane, on intracellular organelles entices speculation
about their possible functions. If part of a vesicular membrane skeleton, ankyrin could: (a) strengthen the membrane of the vesicle and prevent "accidental" leakage of its
contents into the cytoplasm; (b) prevent spontaneous or
promiscuous fusion or exocytosis of vesicles; (c) influence trafficking of endocytic/trafficking vesicles by interacting
with motor and/or motor docking proteins; (d) localize a
specific membrane channel to establish or maintain the intravesicular milieu; or perhaps (e) contribute to the regulation of vesicle/organelle biogenesis. Continued work on
these questions will no doubt yield interesting roles for
this class of proteins in the biology of intracellular membranes.
-spectrin and the plasma membrane skeleton (3, 4, 13, 33). This linkage is critical for the
stability of the lipid bilayer (61). Defects in ankyrin-1 or its
attached proteins result in a spherocytic hemolytic anemia
(11, 21, 49, 75) due to excessive vesiculation of RBC membrane lipids. Ankyrin-1 is also expressed in muscle (6), endothelial cells (Lux, S.E., unpublished data), cerebellar
Purkinje cells and granule cells, and a subset of spinal cord
and hippocampal neurons (34, 59).
Materials and Methods
Ank3, respectively (60). Anti-human ankyrin-1
antibodies were prepared against a mixture of native and SDS-denatured
ankyrin as previously described (66). Anti-hemagglutinin (HA) mAb
(12CA5) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Anti-LAMP-1 sera were obtained from the Developmental
Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences (Johns Hopkins University School of Medicine, Baltimore, MD), and from the Department of Biological Sciences
(University of Iowa, Iowa City, IA) (under contract N01-HD-6-2915 from
the National Institute of Child Health and Human Development). The
anti-rab9 antiserum was provided by Dr. Angela Wandinger-Ness (Northwestern University, Evanston, IL). Anti-
-spectrin, anti-adducin, and anti-
protein 4.1 antibodies were provided by Dr. Shih-Chun Liu (St. Elizabeth's Hospital, Brighton, MA) (17,46). The adducin antiserum was raised against both
- and
-adducin. Anti-canine erythrocyte
-spectrin and
ankyrin antibodies, provided by Dr. Kenneth Beck (Stanford University,
Stanford, CA) (1), detect Golgi spectrin and ankyrin, respectively, in
MDCK cells (1).
-actin were provided
by Dr. Ira Herman (Tufts University School of Medicine, Boston, MA)
(28). Anti-GP-260 antibodies, which were raised against an Acanthamoeba protein immunologically related to
-spectrin, were provided by
Dr. Tom Pollard (Johns Hopkins University School of Medicine, Baltimore, MD) (63). Anti-human brain fodrin antibodies were provided by
Dr. Jon Morrow (Yale University School of Medicine, New Haven, CT)
(27). Anti-bovine brain fodrin antibodies were provided by Dr. Shin Lin
(Johns Hopkins University) (45). Affinity-purified anti-guinea pig brain
fodrin antibodies were provided by Dr. Mark Willard (Washington University School of Medicine, St. Louis, MO) (43). All of the fodrin antisera
were raised against both
- and
-fodrin.
Fig. 1.
Schematic diagram of ankyrin-3 protein isoforms and
location of antibody epitopes and PCR primers. Ank3-B is a peptide antibody against the B insert, 5Ank3 is a peptide antibody
directed against the NH2-terminal region of the spectrin-binding
domain and the short 5
exon from the 120- and 105-kD isoforms
present in kidney, and Ank3-R1 is a polyclonal antibody against
the regulatory domain. The positions of the antigens used for
those antibodies are displayed as well as the positions of the forward and reverse primers used in RT-PCR experiments.
[View Larger Version of this Image (31K GIF file)]
-ACTGGCAGTATGACCAAGTGGTCCTGGACTGAC-3
(Tm = 70.7°C); REGF1; 5
-CACATAAAAAGGCTGAGAAGGCAGACAGACGCC-3
(Tm = 70.7°C); SPR1;
5
-GGACTCATGCTGGGTTCAGTCAAGTAGCTGTAG-3
(Tm = 70.0°C); SPINTF1; 5
-GAAGATGCCATCACAGGGGACACTGACAAG-3
(Tm = 69.5°C); SP7F1; 5
-ACAAAATGAATGTCCCAGAAACGATGAATGAAG-3
(Tm = 63.3°C); SPEC5F1; 5
-GGACGGCTTACTCTAAACCCCTGCTTAAGGAAT-3
(Tm = 69.4°C); MEMF1;
5
-GATCTCCTGCCTCGTCTCAACTCCCCTGATCTC-3
(Tm = 73.2°C).
-RACE PCR
end of macrophage ankyrin-3 was carried out as
described by the manufacturer using the 5
/3
RACE kit (Boehringer
Mannheim Biochemicals). Briefly, RACE products were generated by
MEMR3 priming the reverse transcription, and MEMR1 and MEMR2
priming the primary and nested PCR reactions. Additional reactions used
the MEMR2 primer for the reverse transcription, and 5PRR1 and 5PRR2
(which are located at the extreme 3
end of the repeat domain) for priming the primary and nested PCR reactions.
-ACCTTCTGCTGGCAGGGAGTCATCACCTAGCTC-3
(Tm = 72.9°C); MEMR2; 5
-AAGGTCCTGTGGCCCGAGATACTTGTCAGTGTC-3
(Tm = 71.9°C); MEMR3;
5
-GTAGGACCTATCCGAACTGAAGGAGCGGAGGC-3
(Tm = 71.3°C); 5PRR1; 5
-AGGCGTTGTTCTGAAGCAAGACATTGATGATAT-3
(Tm = 65.7°C); 5PRR2; 5
-CCTGCTGAGCAGCCTGGTGCAGTGCTGTGTATC-3
(Tm = 74.4°C).
Results
Fig. 2.
Western blot analysis.
(A) Mouse kidney membranes
(50 µg) stained with a 1:500 dilution of the Ank3-R1 antibody.
(B) Mouse kidney membranes
stained with ANK3-R1 plus a
200-fold molar excess of GST.
(C) Mouse kidney membranes
stained with ANK3-R1 plus a 200-fold molar excess of the GST-
ankyrin-3 regulatory domain fusion peptide used to generate
Ank3-R1. Note that there are
five major isoforms of ankyrin-3
expressed in the kidney, all of
which are detected by and specific to the Ank3-R1 antibody.
(D) Mouse bone marrow macrophage extract (50 µg) stained
with Ank3-R1. (E) Mouse bone
marrow macrophage extract
stained with ANK3-R1 plus a 200fold molar excess of the Ank3R1 epitope. Note that only the 100- and 120-kD isoforms are
expressed in bone marrow macrophages.
[View Larger Version of this Image (28K GIF file)]
Fig. 3.
RT-PCR analysis of bone marrow macrophage mRNA.
(A) Regulatory domain RT-PCR using REGF1 and REGR1
primers (See Materials and Methods and Fig. 1). Note two bands
(arrows) of ~1,025 and 1,600 bp. These were subcloned (lanes H
and G, respectively) and sequenced. (C) Spectrin-binding domain
RT-PCR using SPINTF1 and SPR1 primers. Note one band of
~1,800 bp (arrow), which was subcloned (lane F) and sequenced.
Other bands shown in lane C were nonspecific products. (Lanes
B, D, and E) DNA size standards.
[View Larger Version of this Image (57K GIF file)]
untranslated region of the short kidney
ankyrin-3 transcripts that lack the repeat domain (Fig. 1),
fails to yield any PCR product from macrophage cDNA. This
experiment suggests that the 5
end of the macrophage
cDNA is distinctive from the kidney clones. Analysis of
this region of macrophage ankyrin-3 via 5
-rapid amplification of cDNA ends (RACE) PCR confirms the presence
of upstream coding sequence (Fig. 4 B). Four separate RACE reactions were performed using two different RNA
samples and two sets of primers (see Materials and Methods). Products generated from these experiments (Fig. 4 A)
end at base 2,280 (60) located at the 3
end of the 22nd
repeat. These data suggest the start site for macrophage
ankyrin-3 is 29 amino acids upstream of the spectrin-binding domain (Fig. 4 B). There are three other possible start
sites downstream to the first methionine encoded by the
sequence revealed in our 5
-RACE experiment. All of
these methionine residues are in adequate Kozak contexts
(for review see 39) for efficient translation initialization.
We have assigned methionine806 (60) as the start site because it is the first in-frame methionine encoded in our 5
RACE product. The cDNA sequence of full-length macrophage ankyrin-3 has been deposited in (GenBank No.
U89275). The calculated molecular mass of this protein is 128,323 daltons, which is close to its estimated size of 120 kD based on mobility in SDS polyacrylamide gels. This
NH2 sequence corresponds to the extreme COOH-terminal end of the membrane ("repeat") domain (60), beginning at the end of the last repeat. The 100-kD isoform corresponds to the full-length protein minus the acidic regulatory domain insert (GenBank No. U89274; calculated molecular mass 106,827 daltons).
Fig. 4.
RACE analysis of
the 5 end of macrophage
ankyrin-3. (A) 378-bp 5
RACE product using
A3MEMR3 for the reverse
transcription, and A3MEMR1
and A3MEMR2 for primary
and nested PCR reactions, respectively. (B) Schematic
diagram of sequence encoded by the 5
end of macrophage ankyrin-3.
[View Larger Version of this Image (17K GIF file)]
Ank3, which also recognizes both isoforms of macrophage ankyrin-3 (data not shown). 5
Ank3
was raised against a synthetic peptide representing the six
unique NH2-terminal residues of the small (105 and 120 kD) isoforms of kidney ankyrin-3, plus the first nine residues of the spectrin-binding domain (60) (Fig. 1). In contrast, an antibody directed against an alternatively spliced
exon of ankyrin-3 that is only present in the full-length ankyrin-3 isoforms (Ank3-B; Fig. 1) (60) does not stain
macrophage membranes at all (data not shown). This observation supports the RT-PCR and immunoblotting experiments, identifying vesicular ankyrin-3 as truncated isoforms.
Fig. 5.
Localization of ankyrin-3 in mouse bone marrow macrophages. (A and C) Phase-contrast photomicrographs. (B) Macrophage
stained with Ank3-R1 antibody (inset, high power view). (D) Preimmune serum. Note the intense staining surrounding phase-dense vesicles throughout the macrophage cytoplasm (A and B, arrowheads). Bar, 10 µm.
[View Larger Version of this Image (83K GIF file)]
Fig. 6.
Localization of epitope-tagged macrophage ankyrin-3
expressed in COS cells. (A) Phase-contrast photomicrographs.
(B) COS cell stained with 12CA5 mAb against the HA epitope
that has been fused to macrophage ankyrin-3. Note the staining
pattern surrounding vesicles shown in phase contrast (arrows).
Bar, 10 µm.
[View Larger Version of this Image (71K GIF file)]
Fig. 7.
Comparison of ankyrin-3 and ankyrin-1 (erythrocyte
ankyrin) localization in mouse bone marrow macrophages. (A)
Ankyrin-1 antibody. (B) Ank3-R1 antibody. Note that the
ankyrin-1 antiserum does not localize to intracellular membranes
and stains most of the surface plasma membrane, while the
ankyrin-3 antiserum only labels intracellular vesicles. Bar, 10 µm.
[View Larger Version of this Image (69K GIF file)]
Fig. 8.
Simultaneous localization of ankyrin-3 and endocytically incorporated, FITC-labeled dextran. (A, C, and E) Ank3-R1 antibody. (B, D, and F) FITC-dextran. Cells were pulsed for 15 min with 1 mg/ml FITC-dextran, and then washed thoroughly and chased in BMM media for 0 h (A and B), 6 h (C and D), and 24 h (E and F). Arrows and arrowheads indicate ankyrin-3-positive vesicles that do
or do not colocalize with FITC-dextran, respectively. Note that at 6 h a small subset of ankyrin-3-positive vesicles contain FITC-dextran, but complete coincidence between ankyrin-3 and vesicular FITC-dextran does not occur until the 24-h chase period. Bar, 10 µm.
[View Larger Version of this Image (78K GIF file)]
Fig. 9.
Colocalization of LAMP-1 and ankyrin-3 in mouse
macrophages. (A) Ankyrin-3. (B) LAMP-1. In this representative
cell, note the almost identical staining pattern in this region of the
cell (A and B, arrows). Bar, 10 µm.
[View Larger Version of this Image (44K GIF file)]
Discussion
end of macrophage ankyrin-3
differs from the NH2-terminal truncated kidney 105- and
120-kD isoforms. Our 5
-RACE analysis reveals that macrophage ankyrin-3 encodes a short 29-amino acid sequence at its NH2-terminal end that corresponds to the final 29 amino acids of the repeat domain. These amino acids do not encode a repeat and are not conserved between ankyrins, suggesting a gene-specific function, but
could contain the lysosomal targeting sequence. This hypothesis is supported by the vesicular localization of epitopetagged ankyrin-3 transiently expressed in COS cells. This
experiment demonstrates that macrophage ankyrin-3 contains sequence information that can localize it to an intracellular surface in cells other than macrophages. Since all
of the sequence of macrophage ankyrin-3 is contained
within the full-length (~210 kD) ankyrin-3, which is found
on plasma membranes in various tissues (37, 60), the intracellular targeting sequence(s) must be blocked or inactivated in the larger protein.
, interleukin (IL)-1
, IL-4,
IL-10, IL-13, gamma interferon, and PMA. While many of
these had effects on cell morphology and growth rate,
none increased the number of ankyrin-3-positive, phasedense lysosomes.
-spectrin (two of 10 antisera tested) (data not
shown). One of these antibodies, raised against
-spectrin
from canine red cells, also associates with the intracellular
membranes of the Golgi apparatus in MDCK cells (1).
Macrophage lysosomes were not stained by antisera raised
against
-spectrin (0 of nine antisera), fodrin (0 of three antisera), protein 4.1 (0 of two antisera), adducin (0 of one antiserum), or
-actin (0 of one antiserum). Numerous attempts to identify interactions between ankyrin-3 and
spectrin by immunoprecipitation combined with Western
blotting were unsuccessful, in part because we were unable
to completely inhibit macrophage protease activities with
multiple combinations of protease inhibitors, including diisopropylfluorophosphate.
-spectrin (1), and, more recently, ankyrin-3
(18) have been localized to Golgi membranes in MDCK
cells. The isoform of ankyrin associated with Golgi in kidney and muscle has recently been identified and is a previously unknown isoform of ankyrin-3 (AnkG119). It contains
the last 13 of the 24 repeats in the NH2-terminal domain,
the complete spectrin-binding domain, and a unique, truncated (5 kD) regulatory domain. AnkG119 binds avidly to
1 (erythrocyte/muscle) spectrins (Kd = 4.2 nM) but not
to
-spectrin (18). Since the spectrin-binding domains of AnkG119 and lysosomal ankyrin-3 are highly conserved, it
is likely that lysosomal ankyrin-3 also binds
-spectrin. In
fact, as noted in the previous paragraph, our preliminary
experiments show that mouse macrophage lysosomes stain
with some
-spectrin antisera, including an antiserum that
sees Golgi spectrin (1).
Received for publication 16 November 1996 and in revised form 23 December 1996.
Address all correspondence to Samuel E. Lux, Division of Hematology/ Oncology, Children's Hospital, Enders 7, 300 Longwood Avenue, Boston, MA 02115. Tel.: (617) 355-7904. Fax: (617) 355-7262.We thank Joel Swanson for all his help and suggestions over the course of this study; John Hartwig for facilitating much of the photography shown here and for his encouragement; and Kathryn John, whose assistance in the lab greatly contributed to the success of these experiments.
This work was supported by National Institutes of Health grants DK34083 (to S.E. Lux), HL32262 (to S.E. Lux), and HL55321 (to L.L. Peters), and by a March of Dimes grant 5-FY94-0921 (to L.L. Peters).
aa, amino acid; BMM, bone marrow macrophage medium; GST, glutathione-S-transferase; HA, hemagglutinin; IL, interleukin; LAMP-1, lysosomal-associated membrane glycoprotein 1; RACE, rapid amplification of cDNA ends; RT, reverse transcription.