From the Department of Cell Biology, Duke University
Medical Center, Durham, North Carolina 27710, the ¶ Department of
Biochemistry, Queens University, Kingston, Ontario, K7L 3N6 Canada, and
the ** Department of Genetics, Cell Biology and Development, University
of Minnesota, Minneapolis, Minnesota 55455
Received for publication, September 4, 2000
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ABSTRACT |
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The Dictyostelium class I myosins,
MyoA, -B, -C, and -D, participate in plasma membrane-based
cellular processes such as pseudopod extension and
macropinocytosis. Given the existence of a high affinity
membrane-binding site in the C-terminal tail domain of these motor
proteins and their localized site of action at the cortical
membrane-cytoskeleton, it was of interest to determine whether each
myosin I was directly associated with the plasma membrane. The membrane
association of a myosin I heavy chain kinase that regulates the
activity of one of the class I myosins, MyoD was also examined.
Cellular fractionation experiments revealed that the majority of the
Dicyostelium MyoA, -B, -C and -D heavy chains and the
kinase are cytosolic. However, a small, but significant, fraction
(appr. 7. -15%) of each myosin I and the kinase was associated with
the plasma membrane. The level of plasma membrane-associated MyoB, but
neither that of MyoC nor MyoD, increases up to 2-fold in highly motile,
streaming cells. These results indicate that Dictyostelium
specifically recruits myoB to the plasma membrane during directed
cell migration, consistent with its known role in pseudopod formation.
The class I myosins are expressed in a wide range of organisms and
cell types where they have roles in moving membranes along actin
filaments (1). They possess a conserved N-terminal motor domain, 1-6
light chain-binding sites, and a C-terminal tail that has a region rich
in basic residues (the polybasic domain). The polybasic domain binds
directly to either pure anionic phospholipid vesicles or stripped
native plasma membranes with high affinity, in the 100 nM
range, in vitro (2-4). Phylogenetic analyses reveals that
there are at least four myosin I subclasses. The amoeboid subclass is
the most widely expressed and its members are distinguished by the
presence of two additional C-terminal tail domains (5, 6). The first is
a region rich in glycine, proline, and alanine (or serine or
glutamate), referred to as the GPA domain, that binds to actin in an
ATP-insensitive manner, and the second is a Src homology 3 (SH3)1 domain either at the
extreme C terminus or within the GPA domain (4, 7-9). The SH3 domain
is essential for myosin I function (10-12), however, in the case of a
Dictyostelium and a mammalian myosin I, it does not play a
role in localization (12, 13) but does in the case of a yeast myosin I
(11).
Molecular genetic analysis of myosin I function in yeast,
Aspergillus, and Dictyostelium reveals that the
individual members of this family have distinct, yet overlapping roles
in mediating the functions of the cortical actin cytoskeleton (14-18).
These include nonreceptor mediated fluid-phase uptake (i.e.
macropinocytosis (15-18)), exocytosis (14, 16, 19), the orderly
extension of pseudopodia during cell migration (20-22),
and regulation of the distribution of cortical F-actin (15, 17). The
nature of the roles played by myosin Is suggests that they may
specifically interact with the plasma membrane and intracellular
transport vesicles such as early endosomes or lysosomes.
The mechanism by which myosin I interacts with membranes in
vivo and how that interaction is regulated remains unclear. The ability of the polybasic domain to mediate the binding of myosin I to
membranes via electrostatic interactions (2-4) indicates that this
motor protein could interact nonspecifically with any membrane that
contains negative phospholipids such as phosphatidylserine. This
suggests that in Dictyostelium, for example, myosin I could be associated with the contractile vacuole, the plasma membrane, and
lysosomes, all compartments that contain least 15-20% acidic phospholipids (23). However, the amoeboid myosin Is are discretely localized to particular membrane compartments as well as in regions enriched for actin (3, 17, 24-28). Therefore, there must be a
mechanism to direct the specific membrane association of myosin I
in vivo, such as a receptor.
A complete understanding of the mechanism(s) by which myosin I may be
localized within the cell requires the isolation of a myosin
I-containing membrane fraction and identification of factors that may
interact with myosin I either in the cytosol or on specific membranes
to determine its localization in vivo. Dictyostelium has emerged as an excellent system for such
studies as several of the amoeboid myosin Is have been purified and
analyzed (29) and information regarding their in vivo roles
has been obtained by molecular genetic methods (30).
Therefore, an analysis of the membrane association of several
Dictyostelium myosin Is, MyoA, -B, -C, and -D, and a myosin
I heavy chain kinase (MIHCK) that regulates the activity of MyoD (31)
has been performed as the first step in elucidating how their
subcellular distribution and, by extension, their function, may be controlled.
Cell Growth and Maintenance--
The Dictyostelium
Ax3 strain was used for these studies and standard methods for the
maintenance of Dictyostelium cells were employed (32). Cells
were grown in suspension in HL5 medium at 220 rpm. Transformants
expressing green fluorescent protein (GFP)-MyoA or -MyoB tail
fragments were maintained in HL5 supplemented with 10 µg/ml G418
(Geneticin; Life Technologies, Inc., Gaithersburg, MD).
Preparation of Membrane Fractions--
A slight modification of
the procedures described by Cardelli et al. (33) and Zhu and
Clark (34) was employed for the preparation of total cellular
membranes. Briefly, cells were collected by centrifugation, washed once
with 20 mM MES (pH 6.8), 2 mM MgSO4 and 0.2 mM CaCl2 and resuspended with 20 mM TES (pH 7.5), 25 mM KCl, 5 mM
MgCl2, 0.1 mM CaCl2, and 10 mM MgATP (TKMC-ATP buffer) containing 0.25 M
sucrose and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.1 mM
L-1-tosylamido-2-phenylethyl chloromethyl ketone, 0.1 mM
N Extraction of Myosin I and MIHCK from Membranes--
Either the
total membrane fraction or the pooled myosin I-containing membrane
fractions were diluted with TKMC-ATP buffer and the suspension spun at
14,000 × g for 20 min. The pelleted membranes were
resuspended with TKMC-ATP buffer, and then incubated with either 150 mM NaCl, 1 M NaCl or 1% Triton X-100 in 150 mM NaCl for 5 min at room temperature. Following
centrifugation of the membrane suspension at 14,000 × g for 20 min, the supernatants were carefully collected and
proteins precipitated by the addition of trichloroacetic acid to a
final concentration of 5%. The trichloroacetic acid pellets and
membrane pellets were solubilized by the addition of an equal volume of
SDS sample buffer containing 6 M urea (USB). Equal volumes
from both the supernatant and pellet samples were analyzed by
immunoblotting (see below).
Marker Enzyme Assay and Immunoblotting--
An equal volume of
each sucrose gradient fraction was assayed for marker enzymes. The
presence of contractile vacuole membranes was determined by assaying
for alkaline phosphatase, and lysosomal membranes were detected by acid
phosphatase activity as described (36). The microsomal
fractions were identified based on Generation of Dictyostelium Cells Expressing MyoB Tail Domains or
GFP-MyoA--
Expression constructs encompassing either the entire
MyoB tail domain, the combined GPQ/SH3 domains, or GFP -MyoA were
generated using standard molecular biological techniques (39) using
enzymes obtained from New England Biolabs (Beverly, MA). All polymerase chain reaction products were sequenced to confirm that no mistakes were
present. Polymerase chain reaction was used to incorporate an
EcoRI restriction site and an in-frame initiator ATG at the 5' end of the region of the myoB gene encoding either the
entire tail (beginning at nt. 2620) or GPA/SH3 domain (beginning at
nucleotides 3131) and including the endogenous MyoB terminator. The
actin 15 promoter from pDH (40) was added to the 5' end of each
fragment, and the final MyoB tail or GPA/SH3 expression vectors (pDPb14 and pDPb15, respectively) generated by cloning the promoter/tail regions into pLittle (41), a Dictyostelium low copy number
extrachromosomal expression plasmid.
The GFP-MyoA expression vector was generated by using polymerase chain
reaction to introduce a unique restriction site and in-frame initiator
ATG into the 5' end of second exon of the myoA gene (42).
The altered myoA gene was then cloned into p40B.2 (43), a
vector carrying the gene for GFP driven by the actin 15 promoter,
fusing the 5' end of myoA to gfp. The gene
encoding the full-length GFP-MyoA fusion protein was then ligated to
pLittle (pDTa49).
The pDPb14, pDPb15, and pDTa49 expression plasmids were transformed
into Ax3 cells by electroporation (44, 45). Following growth in 10 µg/ml G418 for 10-14 days, individual colonies were picked into
24-well plates and analyzed for the expression of the MyoB tail
fragments or GFP by immunoblotting. Several individual transformants
were analyzed using standard phenotypic assays (15).
Quantification of MyoB--
The entire MyoB tail domain fused
in-frame to glutathione S-transferase was used as a
calibration standard for quantification of MyoB levels. Bacteria
expressing the fusion protein were lysed using an N2
nebulizer (Glas-Col, Terre Haute, IN) and the high-speed supernatant of
the lysate incubated with glutathione-agarose beads (Sigma). After
washing the beads with phosphate-buffered saline and phosphate-buffered
saline, 1 M NaCl, the fusion protein was extracted
from the beads by incubation with 3 M NaSCN. The fusion protein was then dissolved with USB, separated on 10%
SDS-polyacrylamide gel, and purified to homogeneity by electroelution
(Electro-Eluter, Bio-Rad). The purified fusion protein was stored at
Association of the Myosin I with Cellular Membranes--
The
fraction of Dictyostelium myosin Is and an MIHCK associated
with membranes was first determined using quantitative immunoblotting with antibodies specific for the Dictyostelium amoeboid
myosin Is, MyoB, -C, and -D (15, 29) and MIHCK (31). The membrane association of a Dictyostelium myosin I that lacks the
C-terminal GPA and SH3 domains, MyoA (42), was also analyzed using a
strain of cells that express GFP-MyoA. The GFP-MyoA strain was
generated as all attempts to produce MyoA-specific antibodies proved unsuccessful.
Total cell lysates were prepared from either wild type or GFP-MyoA
cells and experiments were performed using buffers containing 10 mM MgATP to prevent the precipitation of myosin I with
actin via its motor domain. Lysates were applied to a sucrose step
gradient to separate the cytosol from total cellular membranes.
Quantitative immunoblotting revealed that, surprisingly, only a
relatively small proportion (6.8-8.7%) of each of the three amoeboid
myosin Is (MyoB, -C, and -D) was present in the total membrane fraction (Fig. 1). In contrast, a significant
proportion of GFP-MyoA (32%) was present in the total membrane
fraction. However, since it is not known if the GFP-MyoA levels are
significantly higher than endogenous MyoA levels, this value might be
an overestimate. Indeed, cells expressing excess MyoB using the same
extrachromosomal vector (10) have a 2-fold greater amount of
membrane-associated MyoB than wild type cells (14 versus
6.8%). This would suggest that the amount of membrane-associated MyoA
in wild type cells is approximately half that observed in the GFP-MyoA
cells, or 16%.
The existence of an apparently nonspecific electrostatic
membrane-binding site in all myosin I tail regions (5) suggested that
myosin Is could potentially be associated with any membrane compartment
that contains sufficient amounts of anionic phospholipids. The
intracellular compartment that contains bound myosin Is was identified
by subfractionation of the total cellular membranes on a linear
30-70% sucrose gradient (33, 34). The resulting fractions were
analyzed for the presence of endoplasmic reticulum, lysosomal,
contractile vacuole, and mitochondrial enzymes by a series of enzymatic
assays (36-38). Several distinct peaks of enzyme activity were
reproducibly found for endoplasmic reticulum, lysosomes, contractile
vacuole, and mitochondria (Fig.
2A). A broad peak of biotin
(that marks the plasma membrane) was routinely found between the peaks
of lysosome and contractile vacuole activity (Fig. 2, A and
C). Analysis of the protein composition of the fractions
(Fig. 2B) revealed the presence of a 45-kDa band in fractions 3-10 that was identified as actin by immunoblotting (data
not shown). Immunoblotting with myosin I-specific antibodies was used
to detect the position of MyoB, -C, and -D in the gradient. A peak of
MyoB immunoreactivity consistently coincided with that of the plasma
membrane marker (Fig. 2C). The MyoB peak was somewhat broader than that of the plasma membrane peak, suggesting that a
fraction of the MyoB was associated with actin present in the heavier
fractions (numbers 8-10; Fig. 2B). The MyoC and MyoD heavy chains were in the same position as MyoB (Fig. 2D)
indicating that these myosin Is were also associated with the plasma
membrane. Analysis of fractionated membranes from GFP-MyoA expressing
cells revealed that MyoA was also associated with the plasma membrane, based on its co-fractionation with MyoB (Fig.
3A). Therefore, despite
differences in their C-terminal tail regions, the amoeboid myosin Is
and MyoA are all associated specifically with the plasma membrane.
Given the ability of the amoeboid myosin Is to bind to F-actin via
their GPA domains (4, 7-9), it was important to establish whether
myosin I was truly associated with membranes. The myosin I-containing
membrane fractions were pooled, incubated with 1% Triton X-100 to
solubilize all membranes, and then subjected to centrifugation. The
majority of the MyoB and -C heavy chains were released into the
supernatant (Fig. 4), confirming that
these myosins were indeed membrane-associated and not simply present in
the membrane fraction via association with F-actin. While a substantial
amount of the MyoD heavy chain was also solubilized (Fig. 4), a larger
proportion than observed for MyoB and -C remained in the pellet. This
could be due to association of MyoD with actin via its GPA domain.
GFP-MyoA behaved in a similar manner to MyoB and -C, with the majority
of this myosin I being released into the supernatant following Triton
treatment (Fig. 4).
The binding of Acanthamoeba myosin I to membranes in
vitro is salt-sensitive (2, 3). The total membrane fraction was incubated with 150 mM or 1 M NaCl to test if
each myosin I associated with membranes via an electrostatic
interaction. Only a small amount of the MyoB heavy chain was
solubilized following salt treatment (Fig. 4) and, surprisingly,
neither the MyoC nor the MyoD heavy chains were released into the
supernatant (Fig. 4). Similarly, GFP-MyoA was not released from the
membrane following salt treatment (Fig. 4). These results suggest that
the interaction of the Dictyostelium myosin I with isolated
cellular membranes does not occur through electrostatic interactions.
The Polybasic Domain Is Responsible for Myosin I Association with
Membranes in Vivo--
Total membranes from Dictyostelium
transformants expressing either the whole MyoB tail region or the
GPQ/SH3 domain (Fig. 5, A and
B) were fractionated using the same method described above,
to identify the domain responsible for the association of MyoB with
membranes. Immunoblotting revealed that ~25% of the MyoB tail was
present in the total membrane fraction. No significant diminution of
the endogenous MyoB heavy chain from the plasma membrane was observed,
suggesting that the excess tail does not successfully compete for
binding sites on the membrane with the native MyoB. The GPQ/SH3
fragment was not detected in the total membrane fraction (Fig.
5B), suggesting that the polybasic domain alone is required
for association of MyoB with membranes. Attempts to test this by
overexpression of the isolated polybasic domain were unsuccessful as
this domain appears to be
unstable.2 The MyoB tail
fragment was not released from the membrane fraction by treatment with
1 M NaCl, but was partially solubilized by detergent treatment (Fig. 4). Fractionation of the total membranes from MyoB tail
expressing cells revealed that the tail was associated with the plasma
membrane (Fig. 5C). Phenotypic analysis of cells expressing
either the full-length MyoB tail or the GPQ/SH3 domain revealed that
despite the expression of a 2-5-fold excess of either tail fragment,
no alteration in behavior was observed. Growth in suspension,
pinocytosis, development, and streaming were
normal.3
Membrane Association of MIHCK--
The physiological activity of
the lower eukaryotic amoeboid myosin I is tightly regulated by
phosphorylation of a single serine or threonine residue in the motor
domain (5). A Dictyostelium MyoD-specific MIHCK has been
identified (31) and found to be a member of the PAK family of
G-protein-regulated kinases (46). Recent experiments revealed that it
interacts with acidic phospholipids and a fraction of this kinase
cosediments with the membrane-cytoskeleton in a
salt-dependent manner (47). The presence of MIHCK in either the cytosolic or total membrane fractions was examined. Similar to what
was found for the myosin Is, the majority of MIHCK was found in the
cytosolic fraction with a small percentage of the total MIHCK (8.6%)
present in the total membrane fraction (Fig. 1). The identity of the
membranes containing the tightly bound MIHCK was determined by
subcellular fractionation and immunoblotting (Fig. 3B). The
MIHCK fractions largely overlapped with MyoB fractions (that mark the
plasma membrane), however, the peak of MIHCK immunoreactivity seems to
be shifted toward the denser fractions (Fig. 3B), suggesting that the kinase may be associated with more than one membrane compartment. Further analysis of the membrane-bound MIHCK revealed that
1 M salt treatment was not effective at releasing the
kinase from the membranes, but incubation with Triton X-100 was
effective (Fig. 4).
Increased Motility of Dictyostelium Coincides with an Increase in
the Membrane Association of Myosin I--
The onset of development by
starvation signals Dictyostelium cells to undergo directed
motility toward a source of chemoattractant, cAMP (i.e.
streaming) (48). The cells become polarized, highly motile, increase
their overall speed at least 2-fold, and migrate more persistently
(49). Loss of MyoA or MyoB from Dictyostelium results in
decreased rates of motility and a delay in streaming (20, 21, 50),
while loss of MyoC or -D does not appear to affect either streaming or
motility (51, 52). Consistent with these observations is the finding
that the overall levels of MyoB increase during the early, motile
stages of development whereas MyoC and -D levels increase only slightly
(19).4 The membrane
association of these three myosin I during streaming was examined to
determine whether one or more of the myosin I were preferentially
recruited to the plasma membrane during chemotaxis.
The percentage of MyoB, -C, and -D associated with membranes from
either growth phase or streaming cells was determined. The proportion
of the MyoB heavy chain associated with membrane at the onset of
streaming was increased, 1.9-fold, as determined by quantitative
immunoblotting (Table I). A smaller, but
still statistically significant, increase in the amount of
membrane-associated MyoD, 1.4-fold, was observed in streaming cells,
but no significant increase of MyoC was observed (Table I).
Interestingly, the levels of membrane-associated MIHCK also increased
by 1.5-fold (Table I).
The phenotype of Dictyostelium myosin I single null mutants
suggested that these motor proteins may have overlapping functions and
that the partial phenotypes observed in the
myoA Calculation of the Amount of Membrane-associated
MyoB--
Quantitative immunoblotting revealed that the growth phase
cells have 32 ± 4.6 ng of MyoB/106 cells (1.56 × 105 MyoB molecules/cell; n = 3). This
amount is 4-fold greater than the previously reported, 8 ng of
MyoB/106 cells (18), but the differences could be due to
slightly different conditions for the determination of MyoB levels. If
one assumes that the average diameter of Dictyostelium is
10.25 µm and the average volume of the cell is 565 µm3
(53), then the cellular concentration of MyoB is 460 nM.
The 6.8% of MyoB present on the plasma membrane (assuming an internal surface area of 331 µm2 at maximum) in growth phase cells
would then be equivalent to 32 molecules/µm2 of plasma membrane.
The membrane association of four Dictyostelium myosin
Is, MyoA, -B, -C, and -D, has been investigated using a fractionation approach. Despite the presence of a high affinity membrane-binding domain in their C termini, only a small fraction, 7-15%, of each is
membrane-associated (Fig. 1). The association membranes appears to be
mediated by the polybasic domain, as a MyoB tail fragment lacking this
domain is not membrane-associated (Fig. 5B). The finding
that the majority of each myosin I is found in the cytosolic fraction
is consistent with earlier observations that 80% of the total high
salt K+-ATPase activity (presumably contributed largely by
the myosin I) in a low salt Dictyostelium lysate is soluble
(29). Acanthamoeba myosin IA is also largely cytosolic, as
determined by quantitative immunoelectron microscopy (26). However, a
large proportion of the closely related Acanthamoeba myosin
Is, myosin IB and -IC, are membrane associated (26). Additionally, it
has been reported that two different forms of mammalian myosin I, Myr1
and Myr2 (nonamoeboid type myosin Is), are membrane associated in rat
liver and smooth muscle cells (54, 55). These myosins also appear to be
associated with more than one membrane compartment, although they are
predominantly found in Golgi (Myr2 in rat liver) or plasma membrane
(Myr1 in rat liver) fractions. This indicates that there must be
specific sequences in each myosin I that directs a particular myosin to
the appropriate membrane compartment and also regulates the proportion
of that myosin that is cytosolic or membrane-bound. These unidentified
distinctive tail sequences that most likely reside in the polybasic
domain could directly play a role in targeting these myosins to a
receptor on the appropriate membrane. Alternatively, they could be
responsible for associating with a cytosolic protein that prevents
binding to membranes or plays a role in mediating myosin I-myosin I
interactions in the lipid bilayer.
The MIHCK that regulates MyoD activity was also found to be largely
cytosolic under our fractionation conditions. Only a small proportion
was found in the total membrane fraction following lysis of the cells
(Fig. 1). In contrast to this result, a more significant amount of
MIHCK was found in association with a high speed pellet that contains
total membranes and the cytoskeleton when cells are directly lysed in
the presence of 20 mM salt (47). This binding was
significantly reduced, but not abolished, when the salt concentration
was raised to 100 mM, suggesting that MIHCK binding to the
membrane under low salt conditions can occur via electrostatic
interactions (47).
The Dictyostelium myosin Is cofractionate with plasma
membranes (Figs. 2, C and D, and 3A)
and do not appear to be bound to any other membrane compartment such as
lysosomes or contractile vacuoles, indicating that there is a mechanism
for specific association of the amoeboid myosin I with the plasma
membrane in Dictyostelium. These results are consistent with
immunolocalization experiments that found these myosins present at or
near the plasma membrane (25, 56). The localization of the four myosin
Is to the plasma membrane is consistent with mutant analyses that
implicate MyoA and MyoB in cell migration (20, 21) and all of these
myosin I in macropinocytosis (15, 18), functions that require the activity of the actin-rich membrane cortex of cells.
There is a specific recruitment of MyoB (2-fold, Table II) to the
plasma membrane during chemotaxis (Table I). The net increase in
membrane-associated MyoB is substantial if one takes into account the
observation that there is an overall 5-fold increase in the total
amount of this protein during streaming (18). Thus, streaming cells
have 10 times more MyoB on the plasma membrane than do normal growth
phase cells. This recruitment of MyoB may be required for controlling
appropriate pseudopod formation as myoB The finding that the deletion of a single amoeboid myosin I from either
yeast or Dictyostelium does not result in profound phenotypes, suggests that these motors share overlapping roles (15-18). In addition, compensation for the loss of one myosin I by
another could occur either up-regulation of the activity or amount of
one or more remaining myosin I. Examination of the remaining amoeboid
myosin Is (MyoC and MyoD) in the myoA The role of membrane association in myosin I function remains unclear.
A kinetic analysis of two Acanthamoeba myosin Is, IA and IB,
demonstrates that this class of motor protein is not proccessive, and
indicates that myosin I must tightly gather on membranes and/or the
actin cytoskeleton to power membrane movement or contraction of the
actin cortex (57). Ostap and Pollard (57) estimated that a cluster of
at least 20 molecules of myosin I would be necessary for membrane-based
myosin I function. The present results showing 32 molecules of MyoB
present per µm2 of plasma membrane would suggest that
this amount is insufficient to move the plasma membrane along actin if
it were uniformly dispersed across the inner plasma membrane surface.
However, immunolocalization data indicate that all three of the
Dictyostelium amoeboid myosin Is are specifically
concentrated in one region of the cell (such as in an extending
pseudopod or macropinocytic ruffle) (25, 56). A highly motile,
chemotactic cell must have a high local concentration of MyoB on the
plasma membrane. This would result from a combination of a 5-fold
increase in levels of MyoB (when compared with growth phase cells) and
a 2-fold overall increase in MyoB on the plasma membrane during
streaming (18) (Table II). The average amount of plasma-membrane bound
MyoB in chemotactic cells, now increased to 320 molecules per
µm2, could occupy 0.1 µm2 (assuming that 20 molecules of myosin I occupy 25 × 25 nm on the plasma membrane;
Jontes and Milligan as cited in Ref. 57) of the membrane surface. In
other words, 10% of the unit membrane area would be covered by MyoB.
Given that the average length of F-actin in Dictyostelium is
0.2 µm (58), this appears to be sufficient to generate force for
motility along F-actin. Therefore, the membrane association of MyoB
could potentially be important in allowing this myosin I to participate
in the regulated formation of pseudopodia during directed migration,
consistent with the finding that deletion of MyoB results in
significant defects in pseudopod formation (20).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-p-tosyl-L-lysine
chloromethyl ketone, 10 µg/ml chymostatin, 10 µg/ml leupeptin, 10 µg/ml antipain, 10 µg/ml pepstatin, and 10 µM
3,4-dichloroisocoumarin) to a final density of 4 × 107 cells/ml. The cells were lysed by passage through
5-µm polycarbonate filters (Poretics Corp., Livermore, CA). In the
initial experiments, biotinylation of the cell surface proteins was
carried out by incubating cells with sulfo-NHS-biotin at 4 °C
(Pierce Chemical Co., Rockford, IL) prior to cell lysis (35). The
lysate was spun at 1,300 × g for 5 min to remove
unlysed cells and nuclei. The post-nuclear supernatant was loaded on a
step gradient of 15% sucrose over a 70% sucrose cushion, all in
TKMC-ATP buffer. Following centrifugation at 100,000 × g for 30 min, the cytosolic and membrane fractions were
carefully collected. The membrane fraction was resuspended with 1 ml of
TKMC-ATP buffer and hereafter referred to as total membranes.
Fractionation of total membranes was performed by loading this fraction
on a linear gradient of 30-70% sucrose in TKMC-ATP buffer and
centrifuging the samples at 100,000 × g for either 2.5 or 16 h. Fractions of equal volume were collected using a Auto
Densi-Flow II Fractionator (Labconco, Kansas City, MO). The sucrose
percentage was measured by a refractometer.
-glucosidase 2 activity (37), and
mitochondria by succinic dehydrogenase activity (38). SDS-PAGE
(polyacrylamide gel electrophoresis) samples for MyoB, -C, -D,
GFP-MyoA, and MIHCK were loaded on either a 6 or 12.5% gel and
transferred to either PVDF membrane (Immobilon P), or nitrocellulose
(Millipore, Bedford, MA). Polyclonal antibodies against the
Dictyostelium MyoB tail (15), purified MyoC, MyoD, and
purified MIHCK (29, 31) used for immunoblotting have been described
previously. Bands were detected using chemiluminescence (either ECL,
Amersham Pharmacia Biotech, or Super Signal, Pierce Chemical Co.)
followed by exposure to ECL film (Amersham Pharmacia Biotech) for
various time periods. Films were scanned using an Expression 636 scanner (Epson America Inc., Torrance, CA) and the bands quantified
using NIH Image 1.61 (Bethesda, MD). Exposures in the linear range of a
standard sample were used to determine the relative amount of myosin I
heavy chain present in a given gel lane. Biotinylated proteins
transferred to nitrocellulose were incubated with horseradish
peroxidase-labeled streptavidin (Pierce Chemical Co.) and visualized by chemiluminescence.
80 °C after adjusting the protein concentration, as determined
using the Bio-Rad DC protein assay, to 1 ng/µl with USB. A
Dictyostelium total cell lysate was prepared by suspending a
known number of cells with USB, and immunoblotting increasing amounts
of sample together with known amounts (0.1-10 ng range) of the fusion
protein on the same gel to generate a standard curve. After detection
and exposure, the intensities of each protein band were measured as described above. Only intensities in the linear range of the standard curve were used to determine the absolute amount of MyoB per number of
cells. Given that the same fusion protein used for generation of the
polyclonal antibody against MyoB was used for the quantification (15),
immunoreactivity to the fusion protein was assumed to be the same as
that to intact MyoB. Quantitative transfer of proteins in the range of
the full-length MyoB heavy chain and the fusion protein to
polyvinylidene difluoride membranes was confirmed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Membrane association of myosin Is and
MIHCK. Immunoblots of Dictyostelium cytosolic
(C) and membrane (M, concentrated 10-fold
relative to the cytosol) fractions with antisera specific for MyoB,
MyoC, MyoD, and MIHCK are shown. The proportion of each protein found
in the two fractions as a percentage of the total myosin I isoform is
shown below each panel. The position of the 116-kDa
molecular mass standard is indicated on the right.
View larger version (32K):
[in a new window]
Fig. 2.
MyoB, -C, and -D are associated with the
plasma membrane. A, distribution of marker enzymes in
each fraction of Dictyostelium total membranes separated by
a 30-70% linear sucrose-density gradient. The following enzyme assays
were performed: alkaline phosphatase ( , contractile vacuole marker),
acid phosphatase (
, lysosome marker),
-glucosidase-2 (
,
endoplasmic reticulum marker), and succinic dehydrogenase (×,
mitochondria marker). The relative activities of marker enzymes were
plotted against the sucrose percentage of each fraction. The
asterisk indicates the position of the peak of MyoB
immunoreactivity. B, SDS-PAGE of fractionated membranes.
Equal volumes of fractions 1-10 from a 30-70% sucrose density
gradient were electrophoresed on 7.5% SDS-PAGE gel and stained by
Coomassie Blue. The position of known molecular weight standards, in
kDa, are indicated on the right. C,
co-fractionation of MyoB with the plasma membrane as determined by
quantitative immunoblotting. Biotinylated cell surface proteins (plasma
membrane marker) were detected by horseradish peroxidase-labeled
streptavidin. The relative intensities of MyoB (
) and biotinylated
proteins (+) were plotted against the sucrose percentage of each
fraction. The results shown are representative of four independent
experiments. D, MyoC and MyoD are also associated with the
plasma membrane. Equal amounts of each fraction of the sucrose density
gradient were analyzed for the presence of myosin I heavy chains by
immunoblotting. Shown is the cofractionation of the 125-kDa MyoB heavy
chain (top panel) with the 135-kDa MyoC and 125-kDa MyoD
heavy chains (bottom panel). The position of the 116-kDa
molecular mass standard is indicated on the right.
View larger version (40K):
[in a new window]
Fig. 3.
MyoA and MIHCK are also associated with the
plasma membrane. Equal volumes of fractions 1-12 from a linear
30-70% sucrose density gradient were analyzed for the presence of the
125-kDa MyoB, 136-kDa GFP-MyoA heavy chains, and the 110-kDa MIHCK by
immunoblotting. A, fractionation of MyoB and
GFP-MyoA. B, fractionation of MyoB and MIHCK. The total
membranes were separated by centrifugation for a shorter time period,
2.5 h as MIHCK was more susceptible to proteolysis than the myosin
I. The separation of individual membrane compartments was the same as
shown for the longer centrifugation as determined by enzymatic assays
and the position of MyoB in the fractions (data not shown;
B).
View larger version (45K):
[in a new window]
Fig. 4.
The myosin Is and MIHCK can be released from
the membrane fraction by detergent treatment. Membrane fractions
were incubated with 150 mM NaCl (150 mM), 1 M NaCl (1 M), or 1% Triton X-100
(TX) for 5 min and subjected to centrifugation. The
supernatant (S) and pellet (P) were collected and
equal volumes of the supernatant and pellet analyzed for the presence
of each myosin I (MyoB, -C, and -D, GFP-MyoA), MIHCK, and the MyoB tail
fragment (B tail) by immunoblotting.
View larger version (37K):
[in a new window]
Fig. 5.
The MyoB polybasic domain is required for
membrane association in vivo. A,
schematic diagram showing domains of intact MyoB, the whole MyoB tail
and the GPQ/SH3 domain. PB refers to the positively charged
membrane-binding polybasic domain, the striped box
represents the GPQ domain, and the stippled box the SH3
domain. B, the MyoB tail is associated with membranes. The
MyoB tail (tail) and the combined GPQ/SH3 (G/S) domains were
individually overexpressed in Ax-3 cells and their membrane association
analyzed by immunoblotting. The volume of membrane fraction was
adjusted to that of the cytosolic fraction, and equal amounts of the
cytosol (C) and the membrane (M) were analyzed.
This blot contains several nonspecific bands that are detected when the
nonaffinity purified antisera is employed for immunoblotting.
C, the MyoB tail is associated with plasma membranes.
Fractions from a linear 30-70% sucrose density gradient were
electrophoresed on a 12.5% gel and analyzed for the presence of MyoB
and the MyoB tail fragment by Western blotting. The position of known
molecular mass standards, in kDa, are indicated on the right.
Numbers above the lanes indicate the fraction number, M
corresponds to the total membrane sample, and T is the total
cell lysate. The arrows point to the major bands on the gel,
MyoB is the 125-kDa MyoB heavy chain, and MyoB tail is the 42-kDa MyoB
tail fragment. Note the correspondence between the position of the
intact MyoB heavy chain and the tail fragment.
Membrane association of myosin and MIHCK during streaming
and myoB
cells
could be due to compensation for their loss by other members of the
myosin I family (30). Given that highly motile Dictyostelium have an increased level of membrane-associated MyoB (Table I), the
effect of deletion of MyoA or MyoB on the membrane association of MyoC
and -D, as well as MIHCK, was examined. Following the onset of
streaming in the myoA
or
myoB
null mutants, which is delayed and occurs
at 8 h after the initiation of starvation (15, 50, 52), the amount
of both the MyoC and MyoD heavy chains in the total membrane fraction
increases in both the myoA
and
myoB
cells by ~2-fold (Table
II). These increases in the amount of myosin I heavy chains on the plasma membrane are not due to a simple
increase of MyoC and MyoD expression in these cells during streaming
(Table II). Interestingly, there is also an almost 2-fold increase of
MIHCK in the myoA
and
myoB
total membrane fractions during the onset
of streaming (Table II). The membrane-associated MyoB is increased in
the myoA
cells by 2-fold (Table II), not
significantly different from what is observed for wild type cells
(Table I). Thus, loss of either MyoA or MyoB only results in an
increase in the amount of the MyoC and MyoD heavy chains with the
plasma membrane.
Relative levels and membrane association of myosin is and MIHCK in
myosin I null mutants
or
myoB
single mutants is shown ± S.D. The number of
trials for each sample is shown in parentheses. The fold increase in
the levels of membrane association during streaming is shown and the
asterisk (*) indicates a statistically significant increase
(p < 0.05).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mutants extend an increased number of pseudopodia (20). In contrast,
significantly less MyoC and MyoD is present on the plasma membrane in
streaming cells. The total amounts of these myosin I are lower than
that of MyoB and they do not increase during streaming (18), and a much
smaller increase in their association with the membrane is observed
(Table I). The lower levels of plasma membrane-associated MyoC and MyoD
in streaming cells are consistent with the finding that
myoC
and myoD
mutants
move at normal rates of speed and do not exhibit any delays in
streaming (51, 52).
and
myoB
cells and MyoB in the
myoA
cells reveals that the overall levels of
these myosin Is are not increased in the mutants (Table II). However,
there is a 2-fold increase in the amount of membrane-associated MyoC
and -D in the myoA
and
myoB
mutants (Table II). The availability of
free myosin I-binding sites on the plasma membrane may allow an
increased amount of MyoC and MyoD to bind to the plasma membrane.
Alternatively, the mutant cells may have a mechanism for increasing the
recruitment of the remaining myosin I to the plasma membrane during
streaming in an effort to compensate for the loss of either MyoA or
MyoB. The 2-fold increase in the levels of membrane-associated MyoC and
MyoD in the myoA
or
myoB
mutants (Table II) is clearly
insufficient to compensate for the loss of either of these myosin I. This is most likely due to the fact that the levels of MyoC and -D
expressed by the cell during streaming are significantly lower than
those of MyoB (18). However, there is evidence that the relatively
small increase in MyoC and -D on the plasma membrane could be playing a
role in maintaining some level of normal function related to motility in the mutant cells. The motility of a
myoB
/D
double mutant
does not appear to be worse than that of the
myoB
single mutant, however, the
myoB
/C
/D
triple mutant moves a bit more slowly and is more severely delayed in
streaming (18). Thus, MyoC may play a role in the efficient motility of
Dictyostelium myosin I mutants but, despite increased recruitment to the plasma membrane, it is not sufficient to fully compensate for the loss of MyoB.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Dan Kiehart, Pat Casey, Mike Sheetz, Vann Bennett, Arturo DeLozanne, and Gaku Ashiba for valuable discussions during the course of this study. We also thank Drs. Joe Kelleher and Richard Tuxworth for many helpful comments on the manuscript and Stephen Stephens for providing the GFP-MyoA strain used in this study.
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FOOTNOTES |
---|
* This work was supported by Canadian Institutes of Health Research Grant MOP-8603 (to G. P. C.) and National Institutes of Health Grant GM-46486 (to M. A. T.).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.
§ Present address: Merck Japan Ltd., LAB Division, Arco Tower 5F, 1-8-1 Shimomeguro, Meguro-ku, Tokyo 153-8927, Japan.
Present address: Dept. of Pharmacology, University of Texas
Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9041.
To whom all correspondence should be addressed: Depts. of
Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church St., SE., Minneapolis, MN 55455. Tel.:
612-625-8498; Fax: 612-624-8118; E-mail:
titus@lenti.med.umn.edu.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M008059200
2 M. D. Peterson and M. A. Titus, unpublished observations.
3 M. D. Peterson, S. Senda, and M. A. Titus, unpublished observations.
4 S. Senda and M. A. Titus, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SH3, Src homology domain 3; MIHCK, myosin I heavy chain kinase; MES, 4-morpholineethanesulfonic acid; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; GFP, green fluorescent protein.
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