1 W. M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences,
The University of Iowa, Iowa City, IA 52242, USA
2 Department of Genetics, Cell Biology and Development, The University of
Minnesota, Minneapolis, MN 55455, USA
* Author for correspondence (e-mail: david-soll{at}uiowa.edu)
Accepted 3 June 2003
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Summary |
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Key words: Basic cell motility, Chemotaxis, Myosin I, Myosin A, Myosin B, Myosin F, Dictyostelium discoideum, Functional redundancy
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Introduction |
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Actin plays a fundamental role in these complex behaviors, and an ever
increasing number of actin-associated molecules and regulatory pathways have
been implicated both in motor function, and in regulating the temporal and
spatial dynamics of actin polymerization
(Pollard et al., 2000;
Small et al., 2002
). One group
of actin-based motor proteins implicated in the regulation of pseudopod
formation during basic motile behavior is the family of myosin I proteins
(Albanesi, 1985; Pollard and Korn,
1973
; Zot et al.,
1992
; Uyeda and Titus,
1997
; Mermall et al.,
1998
; Jung et al.,
1993
; Morita et al.,
1996
; Fukui et al.,
1989
). The class I myosins possess an N-terminal motor domain and
a light chain binding neck region. The motor activity of the lower eukaryotic
class I myosins is regulated by phosphorylation of a single serine or
threonine in the TEDS rule site by a PAK family member kinase
(Novak and Titus, 1997
). There
are two distinct types of myosin I, each distinguished by differences in their
C-terminal tail domain. The tail of the `amoeboid' form possesses a polybasic
region that interacts with anionic phospholipids, then a domain typically rich
in the amino acids glycine, proline alanine or glutamate (GPA) that binds
actin, and an SH3 (src homology 3) domain. The `short' forms of
myosin I only possess a polybasic domain in their tail. Dictyostelium
expresses seven class I myosins (Uyeda and
Titus, 1997
; de la Roche and
Cote, 2001
), three amoeboid (MyoB, MyoC and MyoD), two short
(MyoA, MyoE and MyoF), and an additional tail-less form (MyoK).
The actin-based motor function of these myosins suggested that they played
roles in cellular locomotion. Because of their multiplicity, the possibility
of functional redundancy has been entertained
(Jung et al., 1993;
Jung et al., 1996
;
Ostap and Pollard, 1996
;
Dai et al., 1999
). If two forms
of myosin I could substitute functionally for each other (i.e., if they were
functionally redundant), then a null mutant of a single myosin I gene would
exhibit no behavioral phenotype, while a double null mutant of the two myosin
I genes would result in an aberrant phenotype, assuming similar levels of
expression. Alternatively, if the two myosins could not substitute for each
other (i.e., if they were not functionally redundant), then each individual
null mutant would exhibit aberrant behavior, and the double null mutant would
exhibit a combination of the aberrant behaviors, again assuming similar levels
of expression.
The first computer-assisted analysis of cellular locomotion of a myosin I
mutant (Wessels et al., 1991)
was performed on a MyoB null mutant (Jung et al., 1990), lacking one of the
three amoeboid myosins. It was demonstrated that when translocating in buffer,
myoB mutant cells turned twice as frequently as control cells
(Wessels et al., 1991
). Since
the extension of lateral pseudopods is responsible for the majority of sharp
turns made by a cell crawling in buffer
(Wessels et al., 1994
), these
results suggested that myoB mutant cells were defective in
suppressing lateral pseudopod formation during basic motile behavior. MyoB has
been shown to associate with the plasma membrane during locomotion
(Senda et al., 2001
), and has
been localized to actin-rich regions, most notably the anterior pseudopod
(Jung et al., 1996
) and
eupodia (Fukui and Inoue,
1997
). It has also been shown to recruit components of the Arp 2/3
actin polymerization machinery to the plasma membrane through an interaction
with an adapter protein, CARMIL (Jung et
al., 2001
), suggesting that it plays a role in regulating or
focusing actin polymerization during pseudopod extension.
The second computer-assisted analysis
(Titus et al., 1992) was
performed on a null mutant lacking the short myosin I MyoA
(Titus et al., 1989
).
Surprisingly, the behavioral defects of myoA mutant cells
translocating in buffer were similar to those of myoB mutant cells
(Titus et al., 1992
), in spite
of the differences between their overall structure. These results suggested
that both MyoA and MyoB cooperated in a common process that suppresses lateral
pseudopod formation and turning during cellular locomotion. In the case of
myoA mutant cells, it was further demonstrated that the increase in
lateral pseudopod formation was restricted to those pseudopods formed on the
substratum, and that this defect was also manifested during chemotaxis
(Titus et al., 1992
).
We have extended our analysis of the roles of the class I myosins in the
basic motile behavior of cells and chemotaxis. The two major questions to be
addressed were (1) whether or not two distinct class I myosins (MyoA, MyoB)
played functionally redundant roles during motility and (2) whether two
similar class I myosins (MyoA, MyoF) played functionally redundant roles
during motility. These questions have been addressed through a detailed
analysis and comparison of single and double mutants. We have compared the
three single mutants myoA, myoB and myoF and the two double
mutants myoA/myoB and myoA/myoF, using
computer-assisted methods to reconstruct and analyze the motion of cells
(Soll, 1995;
Soll and Voss, 1998
;
Heid et al., 2002
) and
experimental protocols to measure the behavior of individual cells in buffer,
in response to natural waves of cAMP and in response to the spatial, temporal
and concentration components of the natural wave
(Wessels et al., 2000b
;
Soll et al., 2003
;
Zhang et al., 2002
;
Zhang et al., 2003
). The
results reveal an unexpected variety of shared, unique and redundant functions
of the three class I myosins. Similarities between the defects exhibited by
the five myosin I mutants analyzed here and a mutant expressing constitutive
PKA activity suggest that PKA may play a role in the regulation of all three
class I myosins.
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Materials and Methods |
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Strain maintenance and development
Spores of both control and mutant strains were stored in 10% glycerol at
-80°C. For experimental purposes, cells were reconstituted every 3 weeks
(Sussman, 1987). Cells were
grown in 150 mm Petri dishes in HL-5 medium
(Cocucci and Sussman, 1970
) to
confluency. To initiate development, cells were washed free of nutrients in
basic salts solution (BSS: 20 mM KCl, 2.5 mM MgCl2, 20 mM
KH2PO4, pH 6.4), then dispersed at a density of
5x106 cells per cm2 on filter pads
(Soll, 1987
). Filters were
incubated in a humidity chamber and harvested for experimental purposes at the
ripple state, which represents the onset of aggregation
(Soll, 1979
).
Analysis of basic motile behavior
The methods were similar to those previously described
(Wessels et al., 2000b;
Zhang et al., 2002
;
Zhang et al., 2003
). Briefly,
cells were washed from filter pads, diluted into BSS, and inoculated into a
Sykes-Moore chamber (Bellco Glass, Vineland, NJ). The chamber was then
perfused with BSS at a rate that replaced a chamber volume-equivalent of
buffer every 15 seconds. Cell images were digitized into a Macintosh computer
equipped with a frame-grabber at a rate of 4 frames per minute for 10 minutes
for subsequent analysis.
Analysis of chemotaxis in a spatial gradient of cAMP
The methods were similar to those previously described
(Varnum and Soll, 1984;
Wessels et al., 2000b
;
Zhang et al., 2002
;
Zhang et al., 2003
). Briefly,
cells were dispersed on the bridge of a plexiglass chemotaxis chamber
(Varnum and Soll, 1984
;
Zigmond, 1977
). The bridge was
bordered by two troughs, one containing BSS (sink) and the other containing
10-6 M cAMP in BSS (source). After 5 minutes, cell images were
digitized into a Macintosh computer equipped with a frame-grabber at a rate of
4 frames per minute for 10 minutes for subsequent analysis. This period
represents the time during which the spatial gradient of cAMP was steep enough
to elicit a maximal response (Shutt et
al., 1998
).
Analysis of behavioral responses to the different phases of simulated
temporal waves of cAMP
The methods were similar to those previously described
(Varnum et al., 1985;
Wessels et al., 1992
;
Wessels et al., 2000a
;
Zhang et al., 2002
;
Zhang et al., 2003
). Briefly,
cells were dispersed on the glass wall of a Sykes-Moore chamber. One port of
the chamber was attached to a gradient maker and an opposing port to a
peristaltic pump. To simulate the temporal dynamics of a wave, amoebae were
perfused with 2 ml of BSS containing 8x10-9 M cAMP, then at
30- second intervals with 2 ml of BSS containing twice the concentration of
cAMP to that of the previous solution. When the peak concentration of a
natural wave (10-6 M) was reached, amoebae were perfused at
30-second intervals with 2 ml of BSS containing half the concentration of cAMP
of the previous solution. This procedure was repeated four times, generating a
sequence of four simulated temporal waves with a periodicity of 7 minutes.
Cell behavior was recorded as described in previous sections for DIAS
analysis.
Analysis of behavior in self-generated and wild-type-generated waves
of cAMP
The methods for the analysis of cell behavior in response to self-generated
waves were similar to those previously described
(Escalante et al., 1997;
Wessels et al., 2000b
;
Zhang et al., 2003
). Briefly,
2 ml of a cell suspension at a density of 2.4x106 per ml were
dispersed on the surface of a 35 mm plastic Petri dish. After 6 hours, cell
images were continuously digitized at low magnification for a subsequent 6- to
9-hour period into a Macintosh computer at a rate of 6 frames per minute. Wave
propagation was analyzed using the vector flow plot program of DIAS
(Escalante et al., 1997
;
Soll, 1999
;
Zhang et al., 2003
). The
methods for the analysis of responses to wild-type-generated waves were
similar to those previously described
(Zhang et al., 2003
). In
brief, DiI-labeled mutant cells were mixed with unlabeled wild-type cells,
plated on a 35 mm Petri dish and imaged through a NORAN laser scanning
confocal microscope. Transmitted and fluorescent images were simultaneously
collected every 20 seconds, averaged using Intervision software, and saved on
the hard drive in Silicon Graphics format (SGI Inc., Mountain View, CA). SGI
movies were then converted to QuickTime format, and the behavior of labeled
and unlabeled cells analyzed.
Computer-assisted reconstruction and motion analysis of individual
cells
The methods for digitizing video images, reconstructing cells as
beta-spline representations, and motion analyzing the images with 2D-DIAS
software have been described in detail in previous publications
(Soll, 1995;
Soll and Voss, 1998
). For 2D
analyses, cell perimeters were automatically outlined and converted to
beta-spline replacement images. The centroid (center of area) of a cell at
each time point was computed for each replacement image. Motility and dynamic
shape parameters were computed from the cell centroid and contour,
respectively (Soll, 1995
;
Soll and Voss, 1998
).
Instantaneous velocity of a cell in frame n was computed by
drawing a line from the centroid in frame n-1 to that in n+1
and dividing the length of the line by twice the interval time (15 seconds)
between frames. For the analysis of instantaneous velocity as a function of
developmental time, all cells in the population were motion analyzed. For all
other experiments, motion analysis parameters were computed at 4-second
intervals only for those cells crawling at instantaneous velocities above 2
µm per minute. For all strains in all tested situations, this represented
over 70% of each population. Directional change was computed as the direction
in the interval (n-1, n) minus the direction in the interval
(n, n+1). Directional change values >180° were subtracted from
360°, providing a positive value between 0° and 180°. Maximum
length was considered the longest chord between any two points along the
perimeter of a cell. Maximum width was considered the longest chord at a
90° angle to the maximum length chord. Roundness was computed by the
formula 100x4xarea/perimeter2. Chemotactic index
(CI) in a spatial gradient of chemoattractant was the net distance moved
directly towards the source of the chemoattractant divided by the total
distance moved in the same time period. Percent positive chemotaxis was the
proportion of the cell population exhibiting a positive CI over the period of
analysis. A lateral pseudopod was considered to be a projection formed from
the main axis of translocation at an angle
30° that attained a minimum
of 5% total cell area and initially contained nonparticulate cytoplasm. The
main axis of translocation was determined by drawing a line between the
centroid of the cell in the frame 15 seconds earlier and the centroid of the
cell in the present frame. Difference pictures were generated by superimposing
the image in frame n on the image in frame n-1. The regions
of the cell image in frame n not overlapping the cell image in frame
n-1 were considered the `expansion zones'. The period between
overlapping images in difference pictures was 1 minute. 3D direct image
reconstructions of living cells were performed by methods previously described
(Wessels et al., 1998
;
Soll et al., 2000
;
Heid et al., 2002
). In brief,
thirty optical sections were collected in a 2 second period and repeated every
5 second. Images were obtained using differential interference contrast (DIC)
microscopy. The edge of the cell in each optical section was automatically
determined and only the image within the outline retained. These processed
images were then stacked for each reconstruction and viewed.
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Results |
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To identify motility and chemotaxis defects in each myosin I mutant, a set
of experimental protocols has been developed
(Fig. 1B)
(Wessels et al., 2000b;
Zhang et al., 2002
;
Soll et al., 2003
;
Zhang et al., 2003
). To assess
basic motile behavior in the absence of a chemotactic signal, mutant cells
were analyzed in a chamber in which they were perfused with buffer at a flow
rate that excluded conditioning of the soluble microenvironment. To assess the
capacity to read a positive spatial gradient, the presumed mechanism for
determining polarity and the subsequent direction of translocation at the
onset of the wave (phase A), cells were motion analyzed in a spatial gradient
generated on the plexiglass bridge of a spatial gradient chamber
(Fig. 1B)
(Zigmond, 1977
;
Varnum and Soll, 1984
). To
assess the capacity of a cell to respond to the increasing temporal gradient
of cAMP in the front of the wave (phase B), the high concentration of cAMP at
the peak of the wave (phase C) and the decreasing temporal gradient of cAMP in
the back of the wave (phase D), cells were treated with four temporal waves of
cAMP that mimicked the approximate temporal dynamics and concentration range
of a series of natural waves of cAMP (Fig.
1B) (Varnum et al.,
1985
; Wessels et al.,
1992
). These simulated temporal waves were generated in the
absence of spatial gradients in a round perfusion chamber using gradient
makers. To assess behavior during natural cell aggregation, homogeneous
monolayers of cells were motion analyzed at low magnification
(Escalante et al., 1997
;
Wessels et al., 2000b
;
Zhang et al., 2003
). Finally,
to assess responsiveness to the different phases of a natural wave generated
by wild-type cells, mixed monolayers of vitally stained mutant cells and
unstained wild-type cells at a ratio of 1:9, respectively, were imaged
simultaneously through DIC microscopy and laser scanning confocal microscopy
(Wessels et al., 2000b
;
Zhang et al., 2003
). In each
of the above protocols, the behavior of mutant cells was quantitatively
compared to that of relevant control cells under identical conditions using
DIAS software.
Basic motile behavior in buffer
2D-DIAS analysis of basic motile behavior in buffer revealed decreases in
the mean instantaneous velocity of all three single myosin I mutants
(myoA, myoB, myoF) and both double myosin I mutants
(myoA/myoB, myoA/myoF)
(Table 1). The percentage
decrease in instantaneous velocity when compared to the relevant control
strains ranged from 16% for the myoF mutant to 42% for the
myoA/myoF mutant, and in all cases were statistically
significant (Table 1). The
extent of the observed decreases for the myoA, myoB and
myoA/myoB mutants moving in buffer is similar to what was
reported previously (Wessels et al.,
1991; Titus et al.,
1992
; Novak et al.,
1995
). The decreases in instantaneous velocity were accompanied by
increases in the frequency of lateral pseudopod formation for all five mutant
strains (Table 1). The
percentage increase ranged from 37% for the myoF mutant to 72% for
the myoA/myoB mutant
(Table 1). The decreases in
instantaneous velocity were apparent in mutant cell perimeter tracks that
were, on average, more contracted than those of relevant control cells
(Fig. 2).
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2D-DIAS analysis also revealed a major defect in cell shape in the
myoF and myoA/myoF mutants not manifested in
myoA, myoB or myoA/myoB mutant cells. The mean
maximum length was reduced by 29% in myoF mutant cells and 33% in
myoA/myoF mutant cells
(Table 1). In addition, the
mean roundness parameter was 36% higher in the myoF mutant cells and
51% higher in myoA/myoF mutant cells than in control cells
(Table 1). A statistical
analysis using the Student's t-test revealed that the defects in
shape of myoF and myoA/myoF cells were significant
(P values <0.05). A comparison of 3D-DIAS direct image
reconstructions (Wessels et al.,
1998; Soll, 1999
;
Soll et al., 2000
;
Heid et al., 2002
) of control
and mutant cells supported this conclusion. While representative JH10 cells
translocating in buffer were long and flat, myoF and
myoA/myoF mutant cells were rounder and hemispherical
(Fig. 3). In contrast, 3D
direct image reconstructions of myoA, myoB and
myoA/myoB mutant cells were generally elongate and flat,
like their relevant control cells (data not shown) and cells of control strain
JH10 (Fig. 3A). In addition,
myoA, myoB and myoA/myoB mutant cells also had mean
length and width parameters similar to those of control cells and
myoA and myoB cells had mean roundness parameters similar to
control cells (Table 1). While
the mean roundness parameter of myoA/myoB cells was
significantly larger than that of control cells, the 3D morphology was similar
to that of control cells and the roundness parameter was significantly lower
than that of myoF and myoA/myoF cells.
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These results demonstrate that MyoA, MyoB and MyoF each play a role in facilitating rapid cellular translocation and in suppressing lateral pseudopod formation in the absence of chemoattractant. There was no indication of functional redundancy, first because each single mutant exhibited a similar defect, and second because the cell defects of the double mutant were not significantly greater than those of the single mutant in the combination with the most severe defect (e.g., while the percentage decrease in instantaneous velocity was 38% for the myoA/myoB mutant, it was 34% for the single myoA mutant; and while the percentage decrease was 42% for the myoA/myoF mutant, it was 32% for the myoA mutant) (Table 1). In other words, the single mutant defects were not additive in the double mutants. The analysis of behavior in buffer also revealed that MyoF plays an additional, unique role in maintaining the elongate shape of cells translocating in buffer.
Chemotaxis in a spatial gradient of cAMP
Prior analysis of myoA mutant cells in a spatial gradient of cAMP
revealed that they formed more lateral pseudopodia that contacted the
substratum than control cells did (Wessels
et al., 1996). While the myoB and
myoA/myoB mutants were also found to translocate at lower
velocities than control cells in buffer
(Wessels et al., 1991
;
Novak et al., 1995
), their
behavior in a spatial gradient of cAMP was unknown. Thus, the motility of each
mutant plus that of the myoF and myoA/myoF mutants
in a spatial gradient of cAMP was analyzed. When wild-type cells are placed on
the bridge of a spatial gradient chamber, the majority move in the direction
of increasing cAMP concentration between 5 and 20 minutes of incubation, the
time window during which the gradient is steep enough to elicit a positive
chemotactic response (Shutt et al.,
1998
). There were strong average chemotactic indices (CI) for the
three control cell lines, ranging from +0.47 to +0.76, and percent positive
chemotaxis measurements ranging from 89 to 93%
(Table 2). The mean CIs for all
mutant cell lines were positive, ranging between +0.18 and +0.31
(Table 2), demonstrating that
all were able to assess and move in a directed fashion up the cAMP gradient.
However, the mean CI for every mutant was lower than that for each relevant
control strain (Table 2). The
reduction in CI varied from 52% for myoA cells to 76% for
myoA/myoB cells (Table
2). A comparison of the histograms of chemotactic indicies
revealed that cells of each of the five mutant lines were less efficient in
attaining high-end chemotactic indices than relevant control cells
(Fig. 4). In addition, all
mutant cell lines had a lower proportion of cells with positive CIs than the
relevant control cell lines (Table
2). Together these results demonstrate that cells of all five
mutant lines were capable of responding positively up a spatial gradient of
cAMP, but all were less efficient.
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Since all mutant cell lines exhibited depressed instantaneous velocities and increases in the frequency of lateral pseudopod formation when translocating in buffer, the possibility was entertained that both of these defects were also manifested by mutant cells in a spatial gradient of cAMP, and were the cause in each case of the decrease in chemotactic efficiency. This proved to be the case. All mutant cell lines exhibited decreases in instantaneous velocity and increases in the frequency of lateral pseudopod formation in a spatial gradient of cAMP (Table 2). The increases in the frequency of lateral pseudopod formation varied from 54% for myoF to 240% for myoA/myoB mutant cells (Table 2). These defects in cell behavior were apparent in the perimeter tracks of mutant cells moving up spatial gradients of cAMP (Fig. 5). The perimeter tracks of mutant cells were more compressed and included more sharp turns than those of control cells.
|
These results demonstrate that although none of the class I myosins tested is essential for chemotaxis in a spatial gradient of cAMP, they all facilitate efficient chemotaxis. Since all of the single and double mutants exhibited similar behavioral defects either in a spatial gradient of cAMP or in buffer, it is likely that the decreases in chemotactic efficiency in both cases were due to the general incapacity to suppress lateral pseudopod formation. As in the analysis of behavior in buffer, there was no indication of functional redundancy, since each single mutant exhibited a relatively similar defect, and these defects were not additive in double mutants (Table 2).
Responses to the temporal and concentration components of the cAMP
wave
When wild-type cells were challenged with four successive temporal waves of
cAMP that roughly imitated the temporal dynamics of natural waves, but in the
absence of spatial gradients, they rarely surged in the front of the first
wave, but then exhibited velocity surges in the front of each of the last
three waves (Varnum et al.,
1985; Wessels et al.,
1992
; Wessels et al.,
2000b
; Zhang et al.,
2002
; Zhang et al.,
2003
). Velocity surges began at roughly the onset of the front of
a wave and ended just prior to the peak of the wave. Cells of wild-type
strains will exhibit clearly identifiable surges in the front of two or all of
the last three in a series of four waves. All three control cell lines
exhibited velocity surges on average in the front of the last three waves in a
series of four temporal waves, as is evident in the time plot of velocity data
averaged for a number of cells of each control strain
(Fig. 6A-C;
Table 3). The proportions of
control cells that exhibited velocity surges in the front of each wave are
presented in Table 3. The
average proportions of control cells exhibiting surges in waves 2, 3 and 4
were 82%, 81% and 56%. For all three control cell lines (nh6, KAX3, JH10),
cells in the front of the wave (phase B) were elongate, suppressed lateral
pseudopod formation and translocated in a persistent fashion through the
expansion of a dominant anterior pseudopod along the substratum, as is evident
in pictures in which expansion zones are color-coded green
(Fig. 6A-C -phase B). Cells at
the peak of the wave (phase C) retracted their dominant pseudopod, rounded up
and stopped translocating (Fig.
6A-C - 204 to 220 seconds, phase C). Cells in the back of the wave
(phase D) reinitiated pseudopod formation, but remained apolar, extending
pseudopods of diminished volume in all directions
(Fig. 6A-C - 232 to 352
seconds, phase C). Control cells made no net progress in any direction in the
back of the wave.
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|
The myoA mutant
Time plots of velocity data averaged for a number of myoA mutant
cells revealed velocity surges in the front of the last three in a series of
four temporal waves of cAMP (Fig.
7A), a response pattern similar to control cells
(Fig. 6A;
Table 3). However,
myoA mutant cells formed more lateral pseudopods than control cells
in the front of the wave (phase B), resulting, on average, in a less elongate
shape than hb6b control cells (compare phase B in
Fig. 7A with phase B in
Fig. 6A). myoA mutant
cells also turned approximately three times as frequently as control cells
(nh6b) in the front of the wave (Table
3), presumably as a result of the increased frequency of lateral
pseudopod formation. The shape changes of myoA mutant cells at the
peak and in the back of waves were similar to those of control cells (compare
phases C and D in Fig. 7A with
phases C and D in Fig. 6A).
|
The myoB and myoA/myoBmutants
Time plots of velocity data revealed that both myoB and
myoA/myoB mutant cells did not exhibit a velocity surge in
either the first or second in a series of four temporal waves of cAMP
(Fig. 7B,C). They did respond
to the third and fourth wave (Fig.
7B). The delay in the response of myoB and
myoA/myoB mutant cells is evident in the proportions of
cells exhibiting velocity surges in each wave
(Table 3). Like the
myoA mutant cells, myoB and myoA/myoB
mutant cells formed more lateral pseudopods than control cells when responding
to the front of a wave (phase B in Fig.
7B,C), resulting on average in a less elongate shape than control
cells (compare with phase B in Fig.
6B). myoB and myoA/myoB mutant cells
also turned more frequently than control cells (KAX3) in the front of a wave
(Table 3), presumably as a
result of the increased frequency of lateral pseudopod formation. The shape
changes of myoB and myoA/myoB mutant cells at the
peak of the wave (204-216 seconds in Fig.
7B,C) and in the back of the wave (232-352 seconds in
Fig. 7B,C) were similar to
those of control cells (Fig.
6B). Hence, myoA/myoB mutant cells exhibited the
defects of myoB cells.
The myoF mutant
Time plots of velocity data revealed that myoF mutant cells, like
control and myoA mutant cells, responded normally to a sequence of
four temporal waves of cAMP. The myoF mutant cells surged in the
front of the last three in a series of four successive waves
(Fig. 7D;
Table 3). However,
myoF mutant cells, like myoA, myoB and
myoA/myoB cells, were less elongate than control cells when
responding to the front of a wave and formed lateral pseudopods more
frequently (phase B in Fig.
7D), resulting in a higher frequency of turns than control cells
(Table 3). The myoF
mutant cells responded normally to the peak and back of waves (phases C and D
in Fig. 7D).
The myoA/myoF mutant
myoA/myoF mutant cells, in sharp contrast to
myoA mutant, myoF mutant and control cells, did not surge in
any of the four successive temporal waves of cAMP
(Fig. 7E,
Table 3). In the front of the
last three in a series of four temporal waves, cells were less elongate and
formed lateral pseudopods at frequencies higher than control cells (phase B in
Fig. 6E), resulting in a higher
frequency of turns (Table 3).
The myoA/myoF mutant cells responded normally to the peak
and back of waves (phase C and D in Fig.
7E).
Together, these results demonstrate, first, that the major behavioral defect manifested in buffer and in a spatial gradient of cAMP by all mutant cells, namely, an increase in the frequency of lateral pseudopod formation and turning, is also manifested in the front (phase B) of temporal waves of cAMP. Second, they suggest that MyoB, although not essential, facilitates priming of cells for the velocity response (surge) to phase B of a temporal wave. Third, they suggest that either MyoA or MyoF, both short myosins, is essential for the velocity response to phase B, and that the two can substitute for one another in this role. The fact that cells with either single deletion responded normally, but the double mutant did not respond at all, represents a clear case of functional redundancy.
Natural aggregation in homogeneous territories
The defects displayed by the single and double mutants in response to the
various spatial and temporal components of the natural wave should impact
natural aggregation. To test this prediction, cells were dispersed as a
submerged monolayer on the surface of a plastic Petri dish
(Escalante et al., 1997;
Zhang et al., 2003
). Cells of
control strains nh6b and KAX3 began to aggregate after 6 hours, underwent
streaming, rarely exhibited disruptions in the aggregation process within a
territory and formed normal-sized, large aggregates
(Table 4). Cells of the control
strain JH10 began to aggregate after 7 hours, formed slightly smaller streams
than the other two control strains, rarely exhibited disruptions in the
aggregation process, and formed aggregates slightly smaller than those of the
other two control strains (Table
4). The myoA, myoB, myoA/myoB and myoF
mutant cell lines all exhibited the same characteristics in the natural
aggregation process. Cells of the four mutant cell lines underwent
aggregation, but all formed streams smaller than those of relevant control
cells. The streams of all four mutants frequently fragmented
(Table 4). Finally, all formed
aggregates smaller than those of their relevant control strains
(Table 4). In marked contrast
to the other four mutant cell lines, myoA/myoF mutant cells
neither streamed nor aggregated (Table
4).
|
In the process of natural aggregation, cells respond to outwardly moving
nondissipating waves of cAMP by moving in surges towards the aggregation
center. The surges are in response to the increasing temporal gradients in the
fronts (phase B) of each successive wave relayed through the cell population
(Thomchik and Devreotes, 1982; Soll et
al., 2003). To test this characteristic in mutant cell lines,
aggregating cell monolayers were videorecorded at low magnification after 6
hours of incubation. Selected regions of the monolayers were analyzed using
vector flow technology (Escalante et al.,
1997
; Soll et al.,
2000
; Zhang et al.,
2003
). In vector flow plots the behavior of hundreds of cells in a
small area are simultaneously vectored. Relative net movement of all cells in
the selected area towards (+) or away from (-) the aggregation center are
plotted. A cell responding correctly to a series of waves will exhibit a
succession of peaks in the positive range with relatively constant
periodicity. A cell moving randomly will exhibit a series of peaks randomly
distributed in the positive and negative zone with erratic time intervals
between peaks. All three control cell lines established surges (a series of
positive peaks) towards the aggregation center with relatively constant
periodicity after 6 to 7 hours of incubation
(Table 4). An example of the
vector flow plot of control cell line nhb6 is shown in
Fig. 8A. The myoA, myoB,
myoA/myoB and myoF mutant cell lines all established
cyclic behavior (positive peaks in vector flow plot) with relatively constant
periodicity after 6 to 9 hours of incubation
(Table 4). In all of these
mutant lines, there were frequent examples of transient disruption of cyclic
behavior, sometimes followed by a change in vector direction. Examples of the
vector flow plots of myoA and myoF mutant cells are shown in
Fig. 8B and 8C, respectively.
In marked contrast to the relevant control and other mutant cell lines,
myoA/myoF mutant cells never established cyclic behavior
(Table 4,
Fig. 8D).
|
These results demonstrate that deletion of myoA alone, myoB alone, myoF alone or myoA plus myoB did not block the establishment of cyclic behavior, streaming or aggregation. However, in each mutant these aggregation-associated characteristics were less precise than in the relevant control cell lines, demonstrating that MyoA, MyoB and MyoF each plays a role in the precision of these processes. However, deletion of both myoA and myoF together resulted in a complete block in cyclic behavior, streaming and aggregation, a classic example of redundancy in which deletion of either gene alone does not block function, while simultaneous deletion of both genes completely blocks function. It should be noted that simultaneous deletion of both myoA and myoB resulted in the same minor defects observed in each of the single mutants, a classic example of the absence of redundancy.
Response of myoA/myoF mutant cells to natural wild-type
waves
Cyclic behavior in a monolayer can only be established if a cell line is
capable of emitting a pulsatile signal and responding to that signal in a
cyclic fashion. All of the single myosin I mutants and the
myoA/myoB double mutant could establish cyclic behavior in
homogeneous submerged monolayers, demonstrating that each was capable of both
pulsatile release of signal and cyclic surging in response to the signals.
However, the myoA/myoF double mutant did not establish
cyclic behavior. Hence, the myoA/myoB double mutant could be
defective in either the pulsatile release of signal, the surging response to
the signal, or both. To test whether myoA/myoF mutant cells
were defective in the surging response, they were labeled with DiI, a
fluorescent vital dye, and mixed with unlabeled control cells at a ratio of
1:9, respectively (Zhang et al.,
2003). The cell mixture was then dispersed as a monolayer on a
plastic tissue culture surface. Cell behavior was then monitored using
simultaneous light microscopy to visualize all cells, and laser scanning
confocal microscopy to distinguish mutant cells. The motions of labeled mutant
cells and neighboring unlabeled control cells were then analyzed. Under these
conditions, control cells generate outwardly moving, nondissipating waves of
cAMP that pass across the minority of labeled mutant cells as well as the
majority of neighboring control cells.
Control cells responded to the outwardly relayed waves with velocity surges readily identified in velocity plots (Fig. 9A). Neighboring myoA/myoF mutant cells did not respond to these waves with the same cyclic behavior (Fig. 9A). This defective behavior was evident in centroid tracks (Fig. 9B). While the tracks of control cells included persistent stretches of centroids in the direction of the aggregation center (phases A and B) separated by more densely packed clusters of centroids (phases C and D), the entire track of a neighboring myoA/myoF cell was clustered and exhibited no net directionality (Fig. 9B). These results demonstrate that the redundant function of MyoA and MyoF is essential for the surging response to the front of successive waves of cAMP during natural aggregation, virtually the same defect observed in simulated temporal waves of cAMP (Fig. 7E).
|
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Discussion |
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Shared, nonredundant function
The original observations that the myoA and myoB mutants
exhibited similar behavioral defects
(Wessels et al., 1991;
Titus et al., 1992
;
Wessels et al., 1996
) in
buffer were surprising. We have now added the myoF mutant to this
list. Since individual null mutations of all three genes cause the same
behavioral defects in buffer, namely a reduction in velocity and an increase
in the frequency of lateral pseudopod formation, and the
myoA/myoB and myoA/myoF double mutants
exhibit similar defects that were nonadditive (i.e., were not the sum of the
single mutant defects), we can tentatively conclude that all three myosins
play nonredundant roles in the same behavioral process.
It seems reasonable to suggest that the increase in the frequency of
lateral pseudopod formation exhibited by each of the five analyzed mutants in
buffer resulted, at least in part, in the associated decreases in velocity,
since an increase in the frequency of lateral pseudopod formation generally
causes an increase in the frequency of turning, which in turn causes a
decrease in centroid-based velocity measurements. This defect was manifested
not only in buffer, but also during chemotaxis by all five mutant cell lines
in spatial gradients of cAMP, the suggested mechanism for orientation in phase
A of the natural wave (Fig. 1B)
(Soll et al., 2003). In a
spatial gradient of cAMP, each mutant was fully capable of assessing the
direction of the gradient and moving in a directed fashion up it, but
presumably because of the increased frequency of lateral pseudopod formation
and turning, mutant cells did not stay on track, thus lowering the efficiency
of chemotaxis. These defects (i.e., increased lateral pseudopod
formation and decreased velocity) were also manifested by four of the mutant
cell lines (myoA, myoB, myoA/myoB, myoF) that responded to
increasing temporal gradients of cAMP with a velocity surge, the wave
information that stimulates rapid movement towards the aggregation center in
phase B of the natural wave (Fig.
1) (Soll et al.,
2003
). In an increasing temporal gradient of cAMP, each of these
four mutant cell lines was able to establish surging, but each mutant
continued to form pseudopods at increased frequency. This behavioral defect
has been demonstrated in other mutants to reduce the net progress a cell makes
towards an aggregation center in phase B of a natural wave, and hence the
efficiency of natural aggregation, resulting in small aggregates in monolayers
of developing cells (Wessels et al.,
2000b
; Zhang et al.,
2003
). This latter defect was also exhibited by the four myosin I
mutants.
MyoF and MyoB play unique as well as shared roles
Our results demonstrate that in addition to the shared roles played by
MyoA, MyoB and MyoF in the suppression of lateral pseudopod formation, MyoF
and MyoB play unique roles in other cell functions related to polarity and
motility. While myoA, myoB and myoA/myoB mutant
cells were elongate like control cells, myoF and
myoA/myoF mutant cells were constitutively hemispherical,
suggesting that MyoF plays a role in the maintenance of the elongate cell
shape. Our results also suggest that MyoB plays a unique role in the response
to the front of a wave. In the normal behavioral response to a series of four
temporal cAMP waves, a cell does not show a velocity surge in the front of the
first wave, but does so in subsequent waves, suggesting that a normal cell
must be `primed' with the first wave in order to respond to subsequent waves.
While myoA and myoF mutant cells exhibited a velocity surge
in the last three of a series of four temporal waves, as do control cells,
myoB and myoA/myoB mutant cells exhibited a
velocity surge only in the last two in a series of four waves. In addition,
the surge in the third wave was reduced, on average. These results suggest
that MyoB plays a role in priming cells to respond to the front of a wave.
The SH3 domain of MyoB interacts with CARMIL, an adaptor protein that binds
to both subunits of capping protein and the Arp2/3 complex
(Jung et al., 2001). CARMIL is
localized to the leading edge of chemotaxing cells and mutants lacking this
protein have reduced F-actin content and exhibit significant delays in
streaming. The loss of MyoB might result in the inefficient recruitment of
CARMIL to the leading edge which, in turn, could delay the response of the
cell to the first two waves of cAMP. Another mechanism, perhaps requiring one
of the two remaining amoeboid class I myosins, MyoC and MyoD, might then
account for the response to the later waves of cAMP.
The myoA/myoF mutant reveals redundant function
While myoA and myoF mutant cells exhibited normal surges
in response to the increasing temporal gradient in the front of the last three
in a series of four temporal waves of cAMP, the myoA/myoF
double mutant exhibited no surges. In addition, while myoA and
myoF mutant cells established normal cyclic behavior in a homogeneous
monolayer in response to self-generated cAMP waves,
myoA/myoF mutant cells did not, and while myoA and
myoF mutant cells aggregated, myoA/myoF mutant
cells did not. These results suggest that MyoA and MyoF, both short class I
myosins, perform the same function. Hence, when MyoA is deleted, MyoF fulfills
that function and vice versa. However, when MyoA and MyoF are simultaneously
deleted, there is complete loss of that function. Hence, they can substitute
for one another in facilitating the surges in velocity in the front of a wave
leading to cyclic behavior and aggregation in natural monolayers, and are,
therefore, redundant. Our results also demonstrate that MyoB, an amoeboid
myosin I, cannot substitute for either.
PKA and myosin I functions
pkaR mutant cells, which contain constitutively active PKA,
translocate in buffer and in a spatial gradient of cAMP at approximately half
the velocity of control cells, form lateral pseudopods at twice the frequency
of control cells both in buffer and in a spatial gradient of cAMP, exhibit an
average chemotactic index half that of control cells and do not suppress
lateral pseudopod formation sufficiently in response to the increasing
temporal gradient of cAMP in the front of a natural wave
(Zhang et al., 2003). These
defects are similar to the collective defects exhibited by the myoA, myoB,
myoA/myoB, myoF and myoA/myoF myosin I
mutants. PkaR- mutant cells also do not surge in the front
of any in a series of four temporal waves of cAMP, a defect shared with the
myoA/myoF double mutant. Finally, pkaR mutant cells
are constitutively ovoid, a defect shared with myoF and
myoA/myoF mutant cells. Normally, PKA activity is suppressed
when wild-type cells are crawling in buffer or in the front of a natural wave,
and activated at the peak of a natural wave, when the intracellular
concentration of cAMP peaks (Behrens et al., 1996;
Laub and Loomis, 1998
).
The behavioral phenotype of the pkaR mutant has led to the
proposal that PKA activity must be inhibited through its regulatory subunit in
order for cells to attain the normal elongate morphology and suppress lateral
pseudopod formation in a normal fashion in buffer, in a spatial gradient of
cAMP and in response to the increasing temporal gradient in the front of a
natural wave (Zhang et al.,
2003). The similarity between the defects exhibited by the
pkaR mutant and the combined defects of the five myosin I mutants
suggest that the suppression of PKA activity is necessary for normal function
of myoA, myoB and myoF mutants in buffer, in a spatial
gradient of cAMP and in the front of a natural wave. Conversely, the
activation of PKA at the peak of the wave would result in myosin I
dysfunction. Consistent with this proposed regulatory scenario, we have found
that all of the myosin I mutants, like the pkaR mutant, respond
normally to the peak and back of the wave. This indicates that PKA may be a
negative regulator of the class I myosins in Dictyostelium. This
suggested link between PKA regulation and myosin I function in motility and
chemotaxis warrants further exploration.
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Acknowledgments |
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