From the Department of Cell Biology, Baylor College of Medicine,
Houston, Texas 77030
Class I myosins function in cell motility,
intracellular vesicle trafficking and endocytosis. Recently, it was
shown that class I myosins are phosphorylated by a member of the
p21-activated kinase (PAK) family. PAK phosphorylates a conserved
serine or threonine residue in the myosin heavy chain. Phosphorylation
at this site is required for maximal activation of the actin-activated Mg2+-ATPase activity in vitro. This
serine or threonine residue is conserved in all known class I myosins
of microbial origin and in the human and mouse class VI myosins. We
have investigated the in vivo significance of this
phosphorylation by mutating serine 371 of the class I myosin heavy
chain gene myoA of Aspergillus nidulans.
Mutation to glutamic acid, which mimics phosphorylation and therefore
activation of the myosin, results in an accumulation of membranes in
growing hyphae. This accumulation of membranes results from an
activation of endocytosis. In contrast, mutation of serine 371 to
alanine had no discernible effect on endocytosis. These studies are the
first to demonstrate the in vivo significance of a
regulatory phosphorylation on a class I myosin. Furthermore, our
results suggest that MYOA has two functions, one dependent and one
independent of phosphorylation.
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INTRODUCTION |
Class I myosins have been implicated in a variety of cellular
processes, including cell locomotion, contractile vacuole function, receptor-mediated endocytosis, protein secretion, and intracellular vesicle transport (1). We have been studying the function of the class
I myosin gene, myoA, of the filamentous fungus
Aspergillus nidulans (2). We constructed a conditional null
mutant myoA strain using the promoter of the inducible
alcohol dehydrogenase gene alcA (2). Using this strain, we
showed that myoA is an essential gene in A. nidulans and that it was required for the establishment of
polarized hyphal growth. We also showed that strains lacking
myoA were deficient for the secretion of acid phosphatase
into the medium. In contrast, work on class I myosin function in the
budding yeast Saccharomyces cerevisiae has suggested that
they function in endocytosis and possibly protein secretion (3, 4). In
S. cerevisiae there are two genes, MYO3 and
MYO5, that code for class I myosin polypeptides. Deletion of
either of these genes is not lethal, suggesting that they have
overlapping or redundant functions. Deletion of both genes produces
strains that either grow poorly (4) or are inviable (3). A strain that
has a temperature-sensitive myo5 mutation is defective for endocytosis but not for protein secretion (3).
Studies of class I myosin function and their actin-activated ATPase
activity in Dictyostelium discoideum and Acanthamoeba castellanii have shown that these class I myosins are regulated by
phosphorylation at a specific and highly conserved serine or threonine
residue on the heavy chain (5, 6). This phosphorylation site is also
found in other unconventional myosins like the class VI myosins from
humans and mice (Fig. 1). Recent studies
have shown that the kinase responsible for phosphorylation of the
amoeboid class I myosins is a member of the
PAK/STE201 family of
serine-threonine protein kinases (5, 6). Binding of the GTP-bound form
of the small GTPases, Rac or Cdc42 (7, 8), stimulates the activity of
PAKs. Activated forms of these small GTPases when transfected into
tissue culture cells have dramatic effects on the organization of the
actin cytoskeleton (7, 8).

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Fig. 1.
Alignments of class I and class VI myosin
sequences around the conserved PAK phosphorylation site. The
conserved serine or threonine residue that can be phophorylated is
bold and is underlined. The single letter amino
acid code has been used. Conservative amino acid substitutions are
indicated by a +, and identities are indicated by the letter. All
similarities are derived from comparisons to the A. nidulans
MYOA. Abbreviations for the sequences used to make the alignment are:
MYOA, A. nidulans; MYO3, S. cerevisiae; MYO5,
S. cerevisiae; DDIC, D. discoideum IC; ACIB,
A. castellanii IB; MusVI, mouse myosin VI; and HumVI, human
myosin VI.
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The demonstration that a PAK regulates the activity of class I myosin
provides a link to how members of the small GTP-binding proteins, like
Rac, mediate some of their effects on the actin cytoskeleton and
membrane ruffling of vertebrate cells in culture (7-9). Class I or
other unconventional myosins, like the class VI myosins, may in turn be
the functional connection between the regulatory activity of PAKs and
the motor activity necessary for changes in the actin cytoskeleton and
membrane ruffling. The morphological changes previously attributed to
PAKs may in fact be due to phosphorylation of myosins by PAK-like
kinases.
To investigate the in vivo significance of phosphorylation
of class I myosin heavy chain in myosin function, we have mutated the
class I myosin gene myoA of A. nidulans and
examined the consequences of these changes on cell growth, cell
morphology, protein secretion, and endocytosis. We made these mutants
because in vitro the myosin I kinase from A. castellanii phosphorylates a synthetic peptide with the sequence
of this region of MYOA.2 We
generated mutant forms of MYOA containing either alanine or glutamic
acid substitutions at the conserved, PAK-phosphorylated serine 371 of
MYOA (2). These mutations were introduced back into the chromosomal
myoA locus by homologous recombination. Strains were
identified that express only the mutant forms of the class I myosin
(S371E or S371A), and the phenotypes of these strains were
characterized. Our results indicate that mutation of serine 371 to
glutamic acid leads to constitutive activation of endocytosis.
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EXPERIMENTAL PROCEDURES |
Mutagenesis--
S371A and S371E myoA mutations were
made using a polymerase chain reaction (PCR)-based mutagenesis method
(10). The names and sequences of the primers used in these studies are
given in Table I. The primers used to
make the mutations in myoA were Ser-Ala,
Ser-Ala2, Ser-Glu, and Ser-Glu2. Ser-Ala and
Ser-Glu were phosphorylated with T4 polynucleotide kinase prior to
being used in the PCR. The flanking primers used were myoA3, and myoA7.
The conditions for the PCR reaction were 95 °C for 5 min to denature
the plasmid. This was followed by 30 cycles of 95, 55, and 72 °C for
1, 1, and 2 min, respectively. The reactions consisted of 10 ng of
plasmid DNA template in 50 mM KCl, 10 mM
Tris-HCl, pH 9.0, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 0.1 mg/ml gelatin, and
100 ng of the appropriate primer pair in a final volume of 0.05 ml. The
final PCR product was digested with NheI and MluI
and cloned into plasmid pmyo4. The presence of the specific mutations
and the absence of other changes were confirmed by DNA sequencing. A
KpnI-SmaI fragment containing the mutation was
used to replace the equivalent wild type sequence in the plasmid pmyo3
to generate pmyo3-Ala and pmyo3-Glu. These plasmids are based on the
vector pRG3 and used for transformation into A. nidulans
(13). The PCR conditions to determine which myoA allele was
being expressed used the same conditions described above but used 100 ng of genomic DNA as template in the first PCR reaction. The primers
used in the first PCR reaction were myoA5 and myoA7. The product of
this reaction was 1274 bp (base pairs) in length. The primer myoA5 is
not contained in the sequences of the integrative vector and would not
produce a PCR product with the expected restriction polymorphism unless
the crossover during homologous recombination was upstream of the site
of the mutation. Thus, non-homologous integration events and those in which the crossover occurred downstream of the site of the mutation would yield the wild type sequence lacking the restriction polymorphism expected for the mutant allele. The product from the first PCR reaction
was purified using a Qiaquick PCR purification kit (Qiagen, Inc.). A
portion of the purified PCR product was used to prime a second reaction
with the primers myoA24 and myoA7 to produce a product of 190 bp. The
time of incubation at each temperature were reduced by half for this
second PCR reaction.
Microscopy--
Differential interference contrast micrographs
were collected from hyphal tips grown in 0.5% yeast extract, 1%
dextrose, and 1.5% agar and placed as thin layers on sterile
microscope slides. The slides were incubated overnight in a humid
chamber to allow germination of the conidia. Hyphae were observed using
a Zeiss Axiophot, and images were collected as described below. To look at endocytosis, conidia were germinated overnight at room temperature (14-16 h). They were incubated on ice in medium containing 32 µM FM 4-64 (11). Germlings were subsequently observed
using a Zeiss Axiophot. Digital images were collected using a Hamamatsu Model C2400 camera, and images were obtained using a SCSI adapter and
twain driver with AdobeTM Photoshop 3.0. Samples for electron microscopy were grown in 0.5% yeast extract, 20 mM glucose
for 20 h at room temperature. Germlings were fixed in 6%
glutaraldehyde, PBS for 2 h. They were washed three times in PBS,
post fixed in 1% osmium tetroxide in PBS for 1 h, and rinsed in
three changes of distilled water. The fixed sample was dehydrated
through a series of increasing concentrations of ethanol and then into
absolute ethanol and propylene oxide. They were infiltrated with a 1:1 mixture of propylene oxide and Epon resin overnight at room temperature and then embedded in Epon resin. Sections were cut using a diamond knife on an RMC ultramicrotome at 80-nm thickness and viewed on a
Phillips 410 electron microscope after staining with uranyl acetate and
lead citrate.
Genetic, Molecular Genetic, and Other Methods--
Methods for
the growth and genetic manipulation of A. nidulans have been
described in detail elsewhere (12, 13). Similarly, those methods used
in the manipulation of plasmids and molecular cloning have been
described elsewhere (13). The determination of the number of conidia
produced in culture was determined using the method previously
described (14).
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RESULTS |
Detection of the S371A and S371E Mutations in the
Genome--
Primary transformants were purified three times to single
spores on selective medium. A PCR-based assay was designed to determine whether the wild type or mutant allele of myoA was being
expressed (Fig. 2A). Our assay
uses two rounds of PCR to detect the allele being expressed from the
promoter of myoA. Both the S371A and S371E mutations
generate a restriction polymorphism that was readily identified
following PCR amplification from the expressed gene. The S371A mutation
introduces a HaeII site, which results in two fragments of
105 and 85 bp following digestion of the PCR product. The S371E
mutation introduces a BslI site, which produces three fragments of 105, 59, and 26 bp while the wild type produces two fragments of 131 and 59 bp (Fig. 2B). The nested PCR
procedure made detection of the restriction polymorphism reproducible.
Genomic Southern analysis was performed on strains expressing the
mutant allele to confirm that a single integration event had occurred at the homologous myoA locus (data not shown). The phenotype
of the S371A and S371E mutations was confirmed for at least three independent isolates.

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Fig. 2.
Map for expected integration of pmyo3 and
myoA and detection of the restriction polymorphism of the
mutant gene from the expressed locus. A, maps of the
plasmid pmyo3 and the myoA locus before and after
integration of pmyo3 by homologous recombination. The approximate
location and direction for DNA synthesis primed by each of the primers,
myoA5, 7, and 24 is indicated below the restriction maps.
B, agarose gel showing the restriction polymorphism from the
expressed mutant myoA genes by a nested PCR strategy. The
primers myoA5 and myoA7 were used in the first PCR reaction and will
only amplify genomic sequences from the expressed gene. The primers
myoA24 and 7 were then used in a second round of PCR to amplify the
region containing the restriction polymorphism. M, DNA
molecular weight markers, pUC19 HinfI digest, Wild
type, S371A, and S371E lanes are DNA
products from the PCR reaction. The DNA fragments were then digested
(lanes labeled Cut) with HaeII,
left half of the figure, or BslI, right
half of the figure. Lanes labeled Uncut were
not treated with the restriction endonuclease.
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The S371E Mutant Accumulates Intracellular Membranes--
The
S371A and S371E mutants and a control strain were examined by light
microscopy. We found that the S371E mutation had a dramatic effect on
the appearance of the cytoplasm by differential interference contrast
(DIC) microscopy. Hyphal tips of the S371E mutant strain displayed a
grainy appearance, suggestive of a major change in the composition of
the cytoplasm, whereas the hyphal tips of the S371A and control strains
appeared similar to one another (Fig. 3).
Many things could account for the differences in the appearance of the
cytoplasm between the control strains and the S371E mutant strain.
Among these would be changes in organization of cytoskeletal elements,
the accumulation of additional membranous organelles, or a change in
the amount of solute in the cytoplasm.

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Fig. 3.
Differential interference contrast
micrographs of hyphal tips of a control strain, an S371A mutant strain,
and an S371E mutant strain. We always observe the difference in
the appearance of the cytoplasm between the control and the S371E
mutant strains. The bar in the lower left panel
is 5 µm long.
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To determine the reason for the changes in the cytoplasm, we examined
hyphae by electron microscopy. Thin section electron micrographs of
control and S371E mutant hyphae confirmed our light microscopic
observations, suggesting a change in the organization of the cytoplasm
(Fig. 4). Vesicles were visible in the
tips of the control and S371A strains, but they were present in greater numbers along with tracks of membranes, presumably formed by
invagination, in the S371E mutant. In addition to the membrane tracks
in the S371E mutant, other intracellular membranes take on a rougher appearance (Fig. 4). To show that this difference is not restricted to
those images we present, we quantified the difference in the numbers of
vesicles and membrane tracks in the control and mutant strains. The
number of vesicles and membrane tracks was determined for a fixed area
in eight hyphal tips from the control and the two mutant strains. The
S371A mutant strain had 70% of the vesicles and 75% of the membrane
tracks observed in the control strain. In contrast, the S371E mutant
strain had 2.6 times the number of vesicles and 5.8 times the number of
membrane tracks seen in the control strain.

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Fig. 4.
Thin section electron micrographs (11) of
hyphal tips of a control strain (A), an S371A mutant strain
(B), and a S371E mutant strain (C and
D). The bar in panel D is 1 µm
long.
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Intracellular Membranes Accumulate in the S371E Mutant by
Endocytosis--
In yeast, class I myosin has been shown to be
important for endocytosis (3). Therefore, we hypothesized that the
S371E mutant internalizes more plasma membrane by endocytosis. To test this, we examined endocytosis in germlings of the control and the S371A
and S371E mutants by measuring the uptake of the fluorescent molecule
FM 4-64. FM 4-64 is a lipophilic styryl dye that has been used to
follow bulk membrane internalization and transport in yeast (11).
Germlings were incubated in FM 4-64 at 0 °C to label the plasma
membrane and then warmed to room temperature. Internalization of the
dye was assessed at various times by fluorescence microscopy. We
observed that FM 4-64 was rapidly internalized in S371E germlings,
whereas in the control its internalization was much slower
(Fig. 5). After 10 min, the internalized
membrane in the S371E mutant appeared as large fluorescent patches that continued to increase in number out to 20 min. In contrast, the number
and size of fluorescent patches in the control were reduced at all time
points. Thus, the S371E mutant strain showed a dramatic increase in
plasma membrane internalization. To demonstrate that this
internalization of plasma membranes was F-actin dependent, we treated
germlings with cytochalasin D, a compound that depolymerizes F-actin.
Cytochalasin D treatment of control and S371E mutant germlings blocked
FM 4-64 internalization (Fig. 5). Together these experiments
demonstrate that FM 4-64 uptake is a MYOA-dependent process that also requires F-actin.

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Fig. 5.
Fluorescence micrographs of a control strain
and an S371E strain stained with the lipophilic dye FM 4-64 on ice and
imaged at 0, 10, and 20 min after transfer to room temperature.
The two panels at the far right are the control
and S371E treated in the same manner but in the presence of
cytochalasin D after 15 min. We always see this difference in FM 4-64
uptake between the control and the S371E mutant strains. The
bar in the lower left panel is 5 µm long.
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The S371E Mutant Endocytosis Phenotype Is Dominant--
We
predicted that the S371E mutation resulted in a constitutively active
myosin, and therefore the phenotype would be dominant. We tested this
hypothesis by constructing a diploid between an S371E mutant and a
control strain, FGSC 122 (Fungal Genetics Stock Center, University of
Kansas Medical Center, Kansas City, KS). Diploids heterozygous for the
wild type and S371E MYOA proteins displayed the same grainy appearance
of the cytoplasm characteristic of the S371E myoA mutant
strains when examined by DIC microscopy (data not shown). Thus,
constitutive activation of MYOA function by mutation of serine 371 to
glutamic acid leads to a dominant activation of endocytosis.
The Effects of the S371A and S371E Mutations on Growth,
Development, and Protein Secretion--
The growth characteristics of
strains identified as expressing only the S371A or S371E mutant forms
of MYOA were examined under a variety of conditions. The S371A and
S371E mutations had no discernible effects on the growth of the fungus
on normal solid media. In contrast to the apparently normal growth
observed for these strains on standard solid media, we noticed that the
mutant strains grew less well in submerged liquid cultures than did the control strains. Both the S371E and S371A mutant strains took longer to
germinate, as measured by the timing of germtube emergence. At 5 h
of growth, approximately 20% of the control spores and less than 2%
of the mutant spores had germinated. Similarly, both mutants showed
delays in sending out a second germtube from the conidium and in hyphal
branching when compared with control strains. Though the mutants grew
more slowly than control strains they were morphologically normal and
were able to reach the same hyphal mass in culture given a sufficient
growth period.
We also noted that both the S371A and S371E mutant strains appeared to
conidiate less well on solid medium containing high concentrations of
potassium chloride. We quantified the effect of higher potassium
chloride on conidiation in the S371A and S371E strains by determining
the number of conidia produced by each of the mutant strains and a
control wild type transformant on medium with increasing concentrations
of potassium chloride (Fig. 6). The S371E
mutant displayed the most dramatic response to the presence of
increasing concentrations of potassium chloride in the medium.
Conidiation in the S371E mutant strain was 70% of that observed for
the control on 0.3 M potassium chloride. Conidiation in
this mutant strain remained at 50-70% of the control strain at
potassium chloride concentrations up to 1.2 M. Similarly,
the S371A mutant strain showed reduced levels of conidiation but not until the concentration of potassium chloride reached 0.9 M
or higher. The effect of potassium chloride on conidiation was not simply the result of increased osmolarity of the medium because a
similar effect on conidiation was not seen when sucrose was substituted
for potassium. We conclude from this that efficient conidial
development in high medium requires that the activity of MYOA be
precisely regulated.

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Fig. 6.
Growth on medium with increasing
concentrations of potassium chloride reduces the number of conidia
produced by the S371A and S371E mutant strains relative to a control
strain.
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Previously, we showed that protein secretion was reduced in the absence
of MYOA function (2). For this reason we looked at the secretion of
invertase and acid and alkaline phosphatases. The secretion of all
three enzymes was not significantly different from that of control
strains when normalized for cell mass. Thus, the S371A and S371E
mutations do not have a measurable effect on protein secretion (data
not shown).
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DISCUSSION |
The experiments are the first to investigate fungal class I myosin
function in vivo. We have shown that the phosphorylation site for a PAK, serine 371 of the MYOA protein, plays a central role in
regulating class I myosin activity in vivo. Mutation of serine 371 to glutamic acid leads to constitutive activation of endocytosis in A. nidulans. Using both light and electron
microscopy, we showed that plasma membrane is internalized more rapidly
and to a greater degree in the S371E mutant strains. We also showed that membrane internalization in the S371E and control strains requires
the presence of filamentous actin, as would be predicted if MYOA was
functioning as the motor for membrane internalization.
We previously reported that, in A. nidulans, the class I
myosin, MYOA, functioned in polarized hyphal growth and protein
secretion (2). The studies we report here suggest a role for class I myosin in endocytosis. We attribute the differences observed in our
previous studies and those in budding yeast (3) to the kinds of
mutations used in the various studies. Our earlier studies which
suggested that MYOA functions in polarized cell growth and secretion
used a conditional null mutant strain (2), whereas our current studies
used a point mutation that would not be expected to grossly interfere
with MYOA function. Like our present studies, the studies in yeast were
conducted with a point mutation that confers heat-sensitive growth (3).
We would suggest that the diverse results obtained are the consequence
of the kind of mutation used to conduct the studies. Our previous
studies were conducted with a conditional null mutation in
myoA, whereas our current studies use point mutants. Thus,
the complete absence of a MYOA polypeptide has a more dramatic
phenotype than that observed for the point mutants. We propose that, in
the absence of MYOA polypeptide, cell polarity cannot be established
and that this is necessary for efficient growth and protein secretion.
In contrast, if a MYOA polypeptide is made, even if it is somewhat
defective as in this study, the establishment of cell polarity is not
impaired and protein secretion is normal. So why is the S371A mutant
viable if one requires activation of MYOA by phosphorylation by a PAK? One possibility is that the S371A mutation is not inactivating and that
basal activity is sufficient to support growth of the fungus.
Alternatively, MYOA motor function is not the essential activity and
there are other transport systems that overlap those of MYOA in
endocytosis. Thus MYOA has two functions, it is a motor protein
functioning in endocytosis, a nonessential activity of the myosin, and
it is an actin-binding protein that is required for polarized hyphal
growth and protein secretion. In yeast, many mutations that alter the
actin cytoskeleton have effects on bud site selection and the directed
transport of chitin to the growing cell wall, invertase secretion,
endocytosis, and mitochondrial distribution (4, 15-25). These
processes are similar to polarized hyphal growth and protein transport
for secretion.
The current studies are the first to establish that the PAK
phosphorylation site can play a significant role in regulating class I
myosin function in vivo. It further demonstrates that activation of class I myosin leads to increased endocytic activity. Since small GTP-binding proteins of the Rho family activate PAKs and
activated forms of these small GTP-binding proteins have dramatic effects on the organization of the actin cytoskeleton, it is
interesting to speculate that at least some of the changes are mediated
through activation of unconventional myosins. One inherent problem with this hypothesis is that class I myosins of vertebrates lack the conserved serine or threonine residue at the site of phosphorylation. Instead, they have a charged amino acid that would closely mimic the
S371E mutant we have used in this study (26). This makes it unlikely
that vertebrate class I myosins are regulated by a p21-activated
kinase. A more likely target for regulation by a PAK is the class VI
unconventional myosin family. The class VI myosins have the conserved
serine or threonine at the PAK phosphorylation site and thus could have
their activity regulated by phosphorylation (Fig. 1). We know of no
current studies that demonstrate that a class VI myosin family member
is the substrate for a PAK. It would be interesting to see what
consequences transfection of a class VI myosin that has its conserved
serine or threonine residue mutated to a charged amino acid is on the
actin cytoskeleton of a vertebrate cell in culture.
We thank Dr. Haisheng Lü for making the
initial DIC observations indicating that the S371E mutant had an
altered cytoplasm. We also thank the other members of the laboratory
for comments on the manuscript and helpful suggestions during the
course of these experiments. We thank Donna Turner for assistance with
the electron microscopy and Debbie Townley for assistance with figure reproduction.