1 Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242,
USA
2 Department of Cell and Molecular Biology, Northwestern University Medical
School, Chicago, IL 60611, USA
* Author for correspondence (e-mail: david-soll{at}uiowa.edu )
Accepted 29 January 2002
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Summary |
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Key words: Myosin light chain, Myosin phosphorylation, Cell motility, Chemotaxis, Dictyostelium discoideum
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Introduction |
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During chemotaxis in natural aggregation territories of
Dictyostelium, the regulation of lateral pseudopod formation and
polarity play key roles in the behavioral responses of cells to the different
phases of each natural cAMP wave (Wessels
et al., 1992; Wessels et al.,
2000a
; Wessels et al.,
2000b
). Since myosin II is involved in both pseudopod formation
and cell polarity, it must play an underlying role in chemotaxis. The rapid
addition of the chemoattractant cAMP to cells in buffer results in
phosphorylation of the myosin heavy chain
(Berlot et al., 1985
;
Berlot et al., 1987
), which in
turn results in the depolymerization of myosin II thick filaments
(Kuczmarski and Spudich, 1980
;
Cote and McCrea, 1987
;
Ravid and Spudich, 1989
).
Conversion of the three mapped threonine phosphorylation sites in the MHC tail
to nonphosphorylatable alanines in the mutant 3XALA results in an increase in
myosin II localization to the cell cortex and increased cortical tension
(Egelhoff et al., 1996
). It
also results in behavioral defects in buffer and in spatial gradients of cAMP
consistent with an increase in cortical tension, and a significant decrease in
chemotactic efficiency (Stites et al.,
1998
). Together, these results suggest that the
phosphorylation/dephosphorylation of MHC plays a critical role in the
maintenance of cell shape and motility in buffer, and in chemotaxis in a
spatial gradient of cAMP.
Cyclic AMP also stimulates myosin regulatory light chain (RLC)
phosphorylation (Kuczmarski and Spudich,
1980; Berlot et al.,
1985
; Berlot et al.,
1987
), increasing myosin's actin-activated Mg2+ ATPase
activity (Griffith et al.,
1987
; Trybus,
1989
). To investigate the role of RLC phosphorylation in motility
and chemotaxis, we expressed either wild-type RLC or RLC in which serine 13
was substituted with alanine (S13A) in an RLC null mutant
(Ostrow et al., 1994
). Myosin
II from mutant S13A cells exhibited only 30% of the actin-activated
Mg2+ATPase activity of wild-type myosin II
(Ostrow et al., 1994
).
Nevertheless, S13A cells underwent cell division, localized myosin II to the
cortex of locomoting cells and formed fruiting bodies
(Ostrow et al., 1994
),
suggesting that RLC phosphporylation was not essential for growth or
cytoskeletal organization during locomotion and development.
Under the assumption that a modification of myosin II mediated through occupancy of the cAMP receptor must play a role in chemotaxis, we subjected S13A cells to high resolution computer-assisted motion analysis, employing a set of experimental protocols that tested, first, the basic motile behavior of mutant cells in the absence of an extracellular cAMP signal and, second, the responses of mutant cells to the different spatial, temporal and concentration components of the natural cAMP wave (Fig. 1). The results of these experiments demonstrate that RLC phosphorylation plays a role in the basic motile behavior of cells in the absence of an extracellular chemotactic signal, and in the normal response of cells to the peak and back of a natural chemotactic wave. The incapacity of mutant S13A cells to phosphorylate RLC at the peak and in the back of the wave results in less efficient chemotaxis in natural waves early in mutant cell aggregation, and to the fragmentation of streams late in aggregation. These results support a model of chemotactic regulation in which independent regulatory pathways emanating from the distinct phases of the natural chemotactic wave elicit a sequence of specific cellular behaviors that together represent the natural chemotactic response.
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Materials and Methods |
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Maintenance and development of control, mutant and rescued
strains
Spores of JH10, S13A and WT-res strains were frozen in 10% glycerol and
stored at -80°C. For experimental purposes, cultures were generated from
spores every three weeks (Sussman,
1987). Cells were initially grown in HL-5 medium alone for two
days, then in HL-5 medium containing 10 µg per ml of G418, to a final cell
concentrations of 2x106 per ml. To initiate development,
cells were washed in buffered salt solution (BSS) containing 20 mM KCl, 2.5 mM
MgCl2 and 20 mM KH2PO4 (pH 6.4) and dispersed
on a black filter pad saturated with BSS at a density of
5x106 cells per cm2
(Soll, 1987
). For all analyses
of single cell behavior, except those in which the developmental regulation of
motility was monitored, cells were harvested at the ripple stage, which in
dense cultures represents the onset of aggregation
(Soll, 1979
), the time at
which Dictyostelium amoebae attain their highest average velocity
(Varnum et al., 1986
).
Analysis of the basic motile behavior of mutant cells (protocol 1,
Fig. 1B)
The behavior of cells in the absence of an extracellular cAMP signal, which
we will refer to henceforth as the `basic motile behavior' of a cell, was
analyzed according to methods previously described
(Varnum et al., 1985;
Varnum-Finney et al., 1987a
;
Wessels et al., 2000a
;
Wessels et al., 2000b
). In
brief, 1.1 ml of dilute cell suspension were inoculated into a Sykes-Moore
chamber (Bellco Glass, Vineland, NJ). The chamber was then inverted and
positioned on the stage of an upright microscope fitted with long-range
objectives and condenser. For motion analysis, cell behavior was either
video-recorded or digitized directly through a 10x objective or
25x objective. The chamber was perfused with BSS at a rate that replaced
the liquid volume every 15 seconds to ensure that cells did not condition the
medium. This flow rate was demonstrated not to interfere with normal cellular
translocation.
Analysis of mutant cell chemotaxis in a spatial gradient of cAMP
(protocol 2, Fig. 1B)
The motile behavior of cells in a spatial gradient of cAMP generated in a
single cell spatial gradient chamber
(Zigmond, 1977) was analyzed
according to methods previously described
(Varnum and Soll, 1984
;
Varnum-Finney et al., 1987b
;
Wessels et al., 2000a
;
Wessels et al., 2000b
). In
brief, cells were dispersed on the bridge of a Plexiglas gradient chamber, in
which one of the two troughs bordering the bridge contained BSS (sink) and the
other trough contained BSS plus 10-6 M cAMP (source). Cells were
video-recorded through a 25x objective with bright field optics for a 10
minute period following an initial 5 minute incubation period necessary for
establishing a steep gradient (Shutt et
al., 1998
).
Analysis of mutant cell behavior in temporal waves of cAMP (protocol
3, Fig. 1B)
The motile behavior of cells in a series of temporal waves of cAMP, which
simulate the temporal dynamics of natural waves in the absence of spatial
gradients, was analyzed according to methods previously described
(Varnum et al., 1985;
Varnum-Finney et al., 1987a
;
Wessels et al., 2000b
). In
brief, cells were inoculated into a Sykes-Moore chamber as described for the
analysis of cell behavior in buffer. To generate temporal waves of cAMP, cells
were perfused with increasing, then decreasing, temporal gradients of cAMP,
and the process repeated three times. Cells were first perfused with 5 ml of
BSS, then with 2 ml of BSS containing 7.8x10-9 M cAMP over a
30 second period. At 30 second intervals thereafter, cells were perfused with
2 ml of a new solution containing twice the cAMP concentration of the
preceding solution, terminating at 10-6 M cAMP, the last step in
the increasing phase. Cells were then treated with 2 ml increments of BSS
containing half the previous concentration of cAMP at 30 second intervals,
terminating at 10-8 M cAMP. The second, third and fourth waves were
generated in a similar fashion. The periodicity of simulated temporal waves
was, therefore, 7 minutes. Fields of cells were video-recorded or directly
digitized through a 10x or 25x objective. The concentration of
cAMP in the chamber through the four simulated waves was assessed by
fluorescent dye experiments as previously described
(Wessels et al., 2000b
).
Analysis of mutant cell behavior after the rapid addition of
10-6 M cAMP (protocol 4, Fig.
1B)
The motile behavior of cells before and after the rapid addition of
10-6 M cAMP was analyzed according to methods described above for
analysis of the basic motile behavior in buffer with one modification. That
is, following perfusion for 10 minutes with BSS, the perfusion solution was
rapidly switched to BSS containing 10-6 M cAMP. The concentration
of cAMP in the Sykes-Moore chamber was assessed by fluorescent dye experiments
as previously described (Wessels et al.,
2000b). Cell behavior prior to and after addition of cAMP was
continuously video recorded or digitized directly through a 10x or
25x objective.
Analysis of mutant cell behavior in self-generated waves of cAMP
(protocol 5, Fig. 1B)
The motile behavior of cells in selfgenerated waves of cAMP was analyzed
according to methods previously described
(Escalante et al., 1997), with
the exception that the plastic surface of the tissue culture dish was not
coated with agar (Wessels et al.,
2000b
). In brief, 2 ml of a cell suspension
(2.4x106 per ml BSS) were dispersed on a 35 mm tissue culture
dish. After 30 minutes of incubation, 1.0 ml of fluid was withdrawn and the
dish placed on the stage of an inverted microscope. Cell behavior was
continuously video-recorded or directly digitized through a 10x
objective. Individual cells positioned in the same area of the field that
exhibited no cell-cell contacts were selected for analysis. For streaming
experiments late in aggregation, a 2.5x objective was employed.
Analysis of mutant cell behavior in wild-type waves of cAMP (protocol
6, Fig. 1B).
The motile behavior of mutant cells in natural waves of cAMP generated by
wild-type cells was analyzed according to methods previously described
(Wessels et al., 2000a;
Wessels et al., 2000b
). In
brief, S13A cells were stained with the vital dye DiI (Molecular Probes,
Eugene, OR), mixed with a majority of unstained JH10 cells, at a ratio of 1:9,
and 2 ml of the cell mixture (2.4x106 per ml BSS) dispersed
on a 35 mm tissue culture dish. After 30 minutes, 1 ml of fluid was withdrawn
and the dish positioned on the stage of an Axiovert 100STV Zeiss microscope
equipped for epifluorescent analysis. Cell behavior was analyzed with
brightfield and fluorescence microscopy according to methods previously
described (Wessels et al.,
2000b
). In a control experiment, unstained mutant cells were mixed
with stained wild-type cells.
Computer-assisted analysis of cell motility
Video images were digitized at a rate of 15 frames per minute (i.e. at 4
second intervals) onto the hard disc of a Macintosh G4 computer (Apple
Computers, Cupertino, CA) equipped with a Data Translation framegrabber board
(Data Translation Inc., Marlboro, MA) and 2D-DIAS software
(Soll, 1995;
Soll and Voss, 1998
).
Perimeters were automatically outlined and converted to beta-spline
replacement images (Soll,
1995
; Soll and Voss,
1998
; Soll et al.,
2000
). Motility parameters were computed from centroid positions
and morphology parameters from perimeter contours
(Soll, 1995
). Instantaneous
velocity of a cell in frame n was computed by drawing a line from the
centroid in frame n-1 to the centroid in frame n+1 and
dividing the length of the line by twice the interval time (15 seconds)
between frames. For simplicity, instantaneous velocity will be referred to
simply as `velocity' in the text. 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°.
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 summed area in the expansion zones of a
difference picture divided by the total cell area in frame n and
multiplied by 100 represents positive flow. The period between overlapping
images in difference pictures was 1 minute. This parameter provides a measure
of cellular translocation that is independent of cell centroid movement
(Soll, 1995;
Soll and Voss, 1998
).
Maximum length was the longest chord between any two points along the
perimeter of a cell. Roundness was computed by the formula
10x4xarea/perimeter2. Chemotactic index (CI) in a
spatial gradient of chemoattractant was the net distance moved towards the
source of 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. In measuring the
frequency of lateral pseudopod formation, 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
(Wessels et al., 1996
;
Wessels et al., 2000a
;
Wessels et al., 2000b
).
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 3 µm per minute. For all strains in all tested situations, this represented over 70% of each population.
Myosin II localization
Cells were stained for myosin II according to methods previously described
(Wessels et al., 2000b). In
brief, cells were subjected to three simulated temporal waves of cAMP. Midway
through the increasing phase, at the peak and midway through the decreasing
phase of the last of these waves in independent cultures, the chambers were
perfused with 4% paraformaldehyde in phosphate buffer solution supplemented
with 0.01% saponin. After an antigen retrieval procedure
(Wessels et al., 2000b
), cells
were incubated with rabbit anti-myosin II antibody, a generous gift of Arturo
DeLozanne (University of Texas, Austin, TX), and stained with FITC-labeled
antirabbit antibody (Jackson ImmunoResearch, West Grove, PA). DIC and confocal
images were captured at 1 µm intervals beginning at the substratum with a
Zeiss 510 laser-scanning confocal microscope in the Central Microscopy
Facility at the University of Iowa. To measure the distribution of myosin II
across a cell, intensity plots were derived along a line that did not cross
the cell nucleus, using Zeiss 510 software.
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Results |
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The basic motile behavior of mutant cells is aberrant
During Dictyostelium development, the velocity of individual
wild-type amoebae increases to a maximum at the onset of aggregation
(Varnum et al., 1986;
Wessels et al., 2000b
;
Escalante et al., 1997
). To
test whether mutant cells behaved similarly, JH10, S13A and WT-res cells were
removed from developing cultures at various times and analyzed for mean
instantaneous velocity in buffer. All three strains attained maximum
instantaneous velocity at the onset of aggregation, between 8 and 9 hours of
development (Fig. 2),
demonstrating that the developmental regulation of velocity was intact in the
absence of RLC phosphorylation. However, the maximum instantaneous velocity
achieved by S13A cells at the onset of aggregation was at least 30% higher
than that of either JH10 or WT-res cells
(Fig. 2). The difference in
both cases was significant (P<0.01, Student t-test). To
address the possibility that the increase simply reflected the proportion of
motile cells, the mean instantaneous velocity of only those cells moving
faster than 3 µm per minute was computed for all three cell lines. This
velocity threshold has been used previously to eliminate cells not
persistently translocating (Wessels et
al., 1996
; Wessels et al.,
2000a
; Wessels et al.,
2000b
). When applied, the peak velocity (±s.d.) of JH10,
S13A and WT-res cells was 8.3±5.6 (n=46), 10.3±5.3
(n=38) and 7.4±4.6 (n=52) µm per minute,
respectively. Again, the peak of S13A cells was 24% higher than that of JH10
cells and 39% higher than that of WT-res cells. These differences were
significant (P<0.02, Student t-test).
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Cell velocity can be affected by the rate of pseudopod expansion
(Cox et al., 1992;
Cox et al., 1996
) and the
frequency of lateral pseudopod formation, the latter correlating with the
frequency of turning (Varnum-Finney et
al., 1987b
). In buffer, the directional change parameter, an
indicator of turning frequency (Soll,
1995
; Soll and Voss,
1998
), was consistently 10-20% lower in S13A cells than JH10 cells
translocating in buffer. Increased turning can depress instantaneous velocity,
while decreased turning can elevate it. We tested whether the increase in
velocity of S13A cells in buffer was accompanied by a decrease in lateral
pseudopod formation by counting the number of lateral pseudopods formed over a
10 minute period. JH10 and S13A cells retracted old anterior pseudopods and
extended new lateral pseudopods in a qualitatively similar manner
(Fig. 3A and B, respectively).
However, S13A cells formed lateral pseudopods at only onethird the rate of
JH10 cells (Table 1). These
results suggest that the increase in velocity of S13A cells in buffer may be
due, at least in part, to the decreased rate of lateral pseudopod
formation.
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Mutant cells chemotax efficiently in a spatial gradient of cAMP
To test whether S13A cells chemotax efficiently in a spatial gradient of
cAMP, the mechanism presumed to be basic to the directional decision in phase
A of the natural wave (Fig.
1A), the behavior of JH10, S13A, and WT-res cells were compared in
spatial gradients of cAMP generated in a chamber consisting of a bridge that
supports the cells, and two bordering troughs, one filled with attractant (the
source) and the other with buffer (the sink)
(Zigmond, 1977;
Varnum and Soll, 1984
;
Shutt et al., 1998
). Cell
behavior was analyzed in a 10 minute time window (the 5-15 minute period
following filling of the chamber troughs), when the evolving gradient of cAMP
across the bridge elicits the maximum chemotactic response
(Shutt et al., 1998
). S13A
cells translocated in spatial gradients of cAMP at a velocity significantly
higher than that of JH10 or WT-res cells
(Table 2). S13A cells also
exhibited a mean positive flow value, approximately twice that of either JH10
or WT-res cells (Table 2).
Positive flow is a measure of area displacement in a 4 second period that
provides a measure of translocation that is independent of the cell centroid
(Soll, 1995
). Furthermore,
S13A cells exhibited a directional change parameter 60% that of JH10 cells and
70% that of WT-res cells (Table
2), indicating that S13A cells turned less frequently than the
other two cell types during chemotaxis. Finally, both the mean chemotactic
index and the proportion of the population exhibiting a positive chemotactic
index (percent positive chemotaxis) were higher in S13A cells
(Table 2). The higher mean
chemotactic index (Table 2) was
due to the very high proportion of S13A cells with chemotactic indices
>0.8, as demonstrated in the histogram in
Fig. 4.
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The differences in both velocity and chemotactic efficiency were reflected in perimeter tracks. The perimeter tracks of the three S13A cells with the highest chemotactic indices in a spatial gradient of cAMP were more persistent in the direction of the source of chemoattractant and included fewer sharp turns (Fig. 5B) than the perimeter tracks of the three JH10 cells (Fig. 5A) and the three WT-res cells (Fig. 5C) with the highest chemotactic indices. In addition, the perimeters of S13A cells (Fig. 5B) were less tightly stacked than those of JH10 cells (Fig. 5A) or WT-res cells (Fig. 5C), indicating higher average velocities. The reduction in sharp turns in the S13A perimeter tracks suggested that, as in buffer, S13A cells formed fewer lateral pseudopods per unit time in a spatial gradient than either JH10 or Wt-res cells. To test this prediction, direct counts were made of the number of lateral pseudopods formed in a 10 minute period. The results demonstrated that the frequency of lateral pseudopod formation by S13A cells was half that of JH10 cells during chemotaxis in a spatial gradient of cAMP (Table 1).
|
Mutant cells respond abnormally to the peak and back of temporal
waves
The results obtained in spatial gradient chambers suggest that mutant
cells, which cannot phosphorylate the myosin regulatory light chain in
response to a cAMP signal, can still orient and chemotax efficiently up a
spatial gradient of cAMP, the presumed mechanism for orientation and
polarization in phase A of the natural wave
(Fig. 1A). The behavior of
cells in phases B, C and D of the natural wave, however, are in response to
the temporal and concentration characteristics of the wave
(Wessels et al., 1992)
(Fig. 1A). In response to the
increasing temporal gradient in the front of each wave (phase B), cells
suppress lateral pseudopods (Varnum-Finney
et al., 1987a
; Wessels et al.,
1992
; Wessels et al.,
2000b
) and move in a highly persistent and directional manner
towards the aggregation center. When cells encounter the high concentration of
cAMP at the peak of the wave (phase C), they round up, lose polarity and stop
translocating. Finally, in response to the decreasing temporal gradient in the
back of the wave (phase D), cells again extend pseudopods, but remain
relatively apolar, resulting in little net movement in any direction
(Varnum-Finney et al., 1987a
;
Wessels et al., 1992
,
2000b
). These responses
restrict the movement of cells towards the aggregation center during natural
aggregation to phase B of the natural wave. The responses to the temporal and
concentration components of phases B, C and D of the natural wave are readily
assessed by subjecting cells to sequential increasing and decreasing gradients
of cAMP generated in a purfusion chamber
(Fig. 1B, protocol 3)
(Varnum et al., 1985
;
Varnum-Finney et al., 1987a
;
Wessels et al., 1992
;
Wessels et al., 2000b
).
Because of the round shape of the chamber and perfusion rate, the temporal
waves are generated in the absence of spatial gradients. In
Fig. 6A and B, the
instantaneous velocity of a representative JH10 and S13A cell, respectively,
and the estimated concentration of cAMP, are co-plotted as functions of time
through four successive simulated temporal waves. The average velocity of the
representative JH10 cell (Fig.
6A) and the S13A cell (Fig.
6B) remained depressed through the first simulated temporal wave,
increased at the onset of the second wave, peaked at the midpoint of the
increasing phase of the second wave, decreased at the peak of the second wave
and remained depressed through the remaining decreasing phase of the second
wave. Behaviors in the different phases of the third and fourth waves were
similar to those in the second wave. Similar results were obtained with WT-res
cells (data not shown). The velocity data suggest, therefore, that S13A cells
respond normally to the temporal dynamics of the chemotactic wave. However,
scrutiny of cell shape during the different phases of the temporal wave
revealed that the instantaneous velocity plots did not provide the full story.
In simulated temporal wave two to four, JH10 and WT-res cells exhibited the
sequence of shape changes previously reported
(Wessels et al., 2000b). In
the increasing temporal gradient in the front of waves two to four (phase B),
JH10 cells were highly elongate with a dominant anterior pseudopod
(Fig. 7A-C). At the peak of the
wave (phase C), JH10 rounded up, exhibiting a loss of polarity
(Fig. 7D-F). In the decreasing
temporal gradient in the back of the wave (phase D), JH10 cells again extended
pseudopods, but in random directions, reflecting a lack of polarity
(Fig. 7G-I). WT-res cells
progressed through shape changes identical to those of JH10 cells in phases B,
C and D (data not shown). In the increasing temporal gradients in the front of
waves two to four (phase B), S13A cells were elongate on average
(Fig. 7A'-C'),
similar to JH10 (Fig. 7A-C) and
WT-res cells (data not shown). However, the majority of S13A cells were still
elongate and polar at the peak of the wave (phase C), each with a dominant
anterior pseudopod (Fig.
7D'-F'), and remained elongate and polar during the
decreasing phase of the wave (phase D)
(Fig. 7G'-I').
|
|
Therefore, although S13A cells exhibited a decrease in instantaneous velocity at the peak and in the decreasing phase of the second to fourth simulated temporal wave in a series, they did not undergo the normally associated loss of cellular polarity. The abnormal maintenance of polarity resulted in a defect in the motile behavior of S13A cells at the peak and in the decreasing phase of temporal waves. The perimeters of JH10 cells (Fig. 8A) and WT-res cells (data not shown) at the peak and in the back of simulated temporal waves became on average relatively round and polar, and tended to stack one on top of the other in a time series, indicating little net translocation in any one direction. However, the perimeters of S13A cells (Fig. 8B) remained on average elongate and polar, and generated tracks with a directional component, indicating that S13A cells continued to crawl abnormally at the peak and in the back of simulated temporal waves, albeit at reduced velocity.
|
Myosin distribution in the peak and back of temporal waves
Myosin II localizes to the cortex of normal elongate cells translocating in
the front of a wave and is more generally distributed in less polar cells not
actively suppressing lateral pseudopod formation
(Wessels et al., 2000b). In
the front of simulated temporal waves of cAMP, myosin II localized to the
cortex of the posterior two-thirds of rapidly translocating, elongate JH10
cells (Fig. 7J) and S13A cells
(Fig. 7J'). In the back
of simulated temporal waves of cAMP, although myosin II was more evenly
distributed throughout the cytoplasm of apolar, nontranslocating JH10 cells
(Fig. 7K), it was still
localized in the cortex of the posterior two-thirds of the abnormally elongate
S13A cells (Fig. 7K').
Similar results were attained when the same analysis was performed on nine
additional JH10 cells and nine additional S13A cells in each phase of the
wave.
Mutant cells respond abnormally to the rapid addition of
10-6 M cAMP
In the increasing phase of a natural wave, a cell experiences an increase
in the concentration of cAMP from less than 10-8 M at the trough to
10-6 M at the peak over a period of several minutes (Tomchik and
Deverotes, 1981). At the peak of a wave, a normal cell loses polarity and
stops translocating (Wessels et al.,
1992). One approach that has been commonly used to assess the
cellular response to the peak of the wave is to add cAMP (10-6 M)
rapidly to cells in buffer (e.g. Ross and
Newell, 1981
; Hall et al.,
1988
; Wessels et al.,
1989
). When cAMP is added rapidly to cells crawling in a perfusion
chamber, so that the concentration increases from 0 to 10-6 M in
less then 8 seconds, the cells stop translocating, round-up and lose cellular
polarity within 20 seconds from the time cAMP first enters the chamber
(Wessels et al., 1989
). These
behavioral changes are similar to those of cells responding to the peaks of
simulated temporal waves of cAMP and to the peaks of natural waves
(Wessels et al., 1992
). JH10
cells responded to the rapid increase in cAMP in a manner similar to that
described for other wild-type strains of Dictyostelium
(Wessels et al., 1989
;
Wessels and Soll, 1990
;
Cox et al., 1992
;
Escalante et al., 1997
).
Prior to the addition of 10-6 M cAMP, the centroid tracks of JH10 cells reflected relatively persistent and rapid translocation (Fig. 9A). Within 10 seconds after the addition of cAMP to the chamber, centroids clustered, reflecting the cessation of cellular translocation (Fig. 9A). After the addition of cAMP, perimeters stacked one on top of the other, again reflecting the cessation of cellular translocation (Fig. 8C). Perimeters also became rounder, reflecting the loss of cellular polarity (Fig. 8C).
|
Prior to the addition of 10-6 M cAMP, the centroid tracks of the S13A cells also reflected persistent translocation (Fig. 9B). However, after the addition of 10-6 M cAMP the centroids did not cluster tightly like those of JH10 cells. Rather, they reflected continued translocation, albeit at reduced velocity. This interpretation was reinforced in perimeter tracks. After the rapid addition of cAMP, S13A cells retained their elongate morphologies and translocated in a persistent manner (Fig. 8D), similar to S13A cells responding to the peak and back of simulated temporal waves (Fig. 8B). Therefore, S13A cells abnormally retained an elongate, polar morphology and continued to translocate (albeit at reduced velocity), after the rapid addition of 10-6 M cAMP, the same abnormalities exhibited at the peak of simulated temporal waves of cAMP.
Mutant cells exhibit defects at the peak and in the back of
self-generated natural waves of cAMP
Based on the behavioral phenotypes of S13A cells in buffer, in a spatial
gradient of cAMP and in simulated temporal waves of cAMP, one would expect
S13A cells to orient correctly at the onset of each natural wave (phase A,
Fig. 1A) and translocate in a
persistent fashion towards the aggregation center in the front of the wave
(phase B, Fig. 1A), but
abnormally remain elongate (i.e. not undergo cellular depolarization) and
abnormally continue to translocate at the peak and in the back of the wave
(phases C,D, Fig. 1A). To
assess the behavior of mutant cells in natural waves, we employed a submerged
culture protocol (Escalante et al.,
1997) that allowed comparison of the behavior of individual S13A,
JH10 and WT-res cells in self-generated natural waves of cAMP with similar
average periodicity (5 minutes for S13A cells, 6 minutes for JH10 cells and 5
minutes for WT-res cells). Time plots of velocity for JH10 cells
(Fig. 10A), WT-res cells (data
not shown) and S13A cells (Fig.
10B) contained peaks and troughs at relatively constant intervals
reflecting increased velocity in the front of the waves (phase B) and
decreased velocity at the peak (phase C) and in the back (phase D) of
waves.
|
Centroid tracks of both JH10 and S13A cells pointed in the general direction of their respective aggregation centers during each rapid translocation segment (phase B) (Fig. 10C and 10D, respectively), demonstrating that S13A cells assessed the correct direction of the spatial gradient of cAMP at the onset of each self-generated natural wave (phase A). However, neighboring S13A centroid tracks did not appear to exhibit on average the overall accuracy of JH10 cells (i.e. maintain the same level of directionality towards the deduced aggregation center; data not shown). In addition, S13A cells abnormally retained polarity and continued to translocate, albeit at reduced velocity, in the deduced peak and back of each self-generated wave, just as they did in the back of simulated temporal waves. The tracks of JH10 cells (Fig. 10C) included segments in which centroids were separated and aligned in the direction of the aggregation center (arrow), representing behavior in the front of each wave (phase B), interspersed with segments in which the centroids were highly clustered, reflecting little net translocation in any one direction at the peak (phase C) and in the back (phase D) of each natural wave (Fig. 10B). Outlined images of a representative JH10 cell through a wave revealed an elongate morphology during the translocation segment in the deduced front of the wave (phase B), and the loss of polarity during centroid clustering at the deduced peak and in the deduced back of the wave (phase C and D; Fig. 10E). The centroid tracks of S13A cells (Fig. 10E) also included segments in which the centroids were separated and aligned in the general direction of the aggregation center, representing behavior in front of each wave (phase B), interspersed with contracted segments in which the distances between centroids were reduced. The contracted segments still exhibited alignment, reflecting slower but still persistent translocation at the peak and in the back of the wave, the same abnormal behavior observed at the peak and in the back of simulated temporal waves of cAMP. Outlined images of a representative S13A cell through a wave revealed the abnormal maintenance of an elongate, polar morphology at the peak and in the back of the wave (Fig. 10F), the same abnormality exhibited at the peak and in the back of a simulated temporal wave (Fig. 8B).
S13A cells respond abnormally to wild-type waves
If S13A cells respond abnormally to the peaks and backs of self generated
cAMP waves in aggregation territories, they should also respond abnormally to
the peaks and backs of cAMP waves generated by wild-type cells. To test this
prediction, S13A cells were stained with the vital dye DiI, mixed at a 1:9
ratio with unlabeled JH10 cells, and analyzed by transmitted light and
fluorescence microscopy. The results (Fig.
11) were similar to those collected for the two cell types in self
generated waves. In the centroid tracks of the dominant cell type JH10,
expanded, persistent segments (phase B) were interspersed with highly
clustered segments (phase C and D). The net direction of the representative
JH10 track in Fig. 11, was
towards the aggregation center. The tracks of nine additional JH10 cells
analyzed in the same manner exhibited the same general characteristics. In the
centroid track of a neighboring S13A cell, expanded persistent segments (phase
B) were interspersed with less extensive, but still persistent segments
(phases C and D). As in simulated temporal waves and self-generated natural
waves, S13A cells continued to translocate at the peak and in the back of
natural waves generated by JH10 cells. In addition, although the track of the
representative S13A cell was in the general direction of the aggregation
center, its accuracy was not as great as that of the neighboring JH10 cells
(Fig. 11). The tracks of nine
additional S13A cells analyzed in the same manner exhibited the same general
characteristics as the representative S13A cell in
Fig. 11, and were, on average,
also less on track in phase B than neighboring JH10 cells (data not
shown).
|
Streaming is defective during S13A aggregation
In the previous sections, we demonstrated behavioral defects associated
with single cell chemotaxis, which occurs early in the aggregation process.
However, late in the aggregation process, cells coalesce into multicellular
streams, in which they move, still in a pulsatile fashion, into the final
aggregate (Reitdorf et al.,
1997). To test whether streaming was normal in late aggregating
S13A cell populations, fields of cells were video-recorded at low
magnification. Whereas JH10 cells formed normal contiguous streams late in
aggregation that grew thicker as aggregation progressed, S13A cells formed
streams that fragmented along their lengths
(Fig. 12).
|
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Discussion |
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Dissecting the complex behavior of Dictyostelium amoebae in
natural chemotactic waves
The chemotactic responsiveness of Dictyostelium amoebae has been
assessed by a variety of in vitro protocols, including the rapid addition of
10-6 M cAMP, slow release of cAMP from a micropipette and the
genesis of a spatial gradient of cAMP in a gradient chamber. However, the
actual chemotactic signal a cell experiences in nature is quite different. In
a natural aggregation territory, individual cells respond to nondissipating,
symmetrical waves of cAMP relayed from the aggregation center outwardly
through the cell population (Tomchik and
Devreotes, 1981). A cell responds to each phase of a natural wave
in a relatively different fashion (Fig.
1A) (Varnum et al.,
1985
; Varnum-Finney et al.,
1987a
; Wessels et al.,
1992
; Wessels et al.,
2000b
). In the front of each natural wave, cells experience an
increasing spatial gradient of cAMP (increasing in the direction of the
aggregation center) and an increasing temporal gradient of cAMP (concentration
increasing with time). It has been proposed
(Wessels et al., 1992
) that
cells use the direction of the spatial gradient at the onset of the front of
the wave to polarize in the direction of the aggregation center. Once that
direction is set, cells repond to the associated increasing temporal gradient
of cAMP in the front of the wave by suppressing lateral pseudopod formation,
which facilitates rapid and directional movement in a blind fashion in the
direction of the aggregation center
(Wessels et al., 1992
). At the
peak of each natural wave, cells experience a cAMP concentration that has been
demonstrated to cause a loss of cellular polarity and a dramatic decrease in
instantaneous velocity (Varnum and Soll,
1984
; Wessels et al.,
1989
; Wessels et al.,
1992
). In the back of the wave, cells experience a decreasing
spatial gradient of cAMP (decreasing in the direction of the aggregation
center) and a decreasing temporal gradient of cAMP (concentration decreasing
with time). The decreasing temporal gradient suppresses cellular
repolarization, resulting in the formation of pseudopods in random directions
and no net translocation in any direction. This complex sequence of behavioral
responses to the different spatial, temporal and concentration components of
the four phases of the natural wave (Fig.
1A) confines directed cellular translocation towards the
aggregation center to the front of the wave. The variety of experimental
protocols employed in the present study
(Fig. 1B) have allowed us to
test which, if any, of the phase specific responses involve RLC
phosphorylation.
S13A cells are faster, even in the absence of a chemotactic
signal
We have found that, despite their inability to phosphorylate RLC, S13A
cells translocate faster than wild-type cells and form fewer lateral
pseudopods in the absence of a chemotactic signal. Although the decrease in
pseudopod formation must contribute to the observed increase in velocity, it
does not represent the entire explanation. The increased separation of
centroids and perimeters in plotted tracks of S13A cells in buffer, in spatial
gradients of cAMP and in the front of simulated temporal and natural waves of
cAMP suggests that the basic speed of individual mutant cells is greater than
that of wild-type cells, independent of lateral pseudopod formation and
turning. These results demonstrate that the serine phosphorylation site is
necessary for both the normal frequency of turning and the normal velocity of
a translocating cell in the absence of a cAMP signal, and that
phosphorylation/dephosphorylation of RLC plays a role in the basic motile
behavior of a cell in the absence of extracellular cAMP.
S13A cells migrate faster and with higher chemotactic efficiency in
spatial gradients of cAMP
S13A cells also exhibited a higher average chemotactic index than either of
the two control cell types. This observation was at first counter intuitive,
since one would have expected most mutations in cytoskeletal events downstream
of cAMP receptor occupancy to decrease the efficiency of chemotaxis. However,
it may not have been completely surprising given the inverse relationship
demonstrated between the efficiency of chemotaxis and the frequency of lateral
pseudopod formation. Varnum-Finney et al. demonstrated that as the chemotactic
index increases, the rate of lateral pseudopod formation decreases
(Varnum-Finney et al., 1987b).
S13A cells already exhibit depressed rates of lateral pseudopod formation in
their basic motile behavior, which appear to enhance chemotactic efficiency in
a spatial gradient. Therefore, if S13A cells can still assess the direction of
a spatial gradient and adjust direction by relying more heavily on biased
anterior pseudopod expansion than new lateral pseudopod formation, they may
chemotax more efficiently. Why, then, does Dictyostelium go to the
trouble of phosphorylating the RLC? One possible answer is that cells in vivo
must assess not only the spatial characteristics, but also the temporal
dynamics of a natural cAMP wave in order to chemotax properly, and that the
phosphorylation/dephosphorylation of RLC is intricately involved in this
complex process.
S13A cells respond abnormally to the peak and back of simulated
temporal and natural waves of cAMP
To test whether S13A cells were defective in responding to the temporal
characteristics of natural waves, they were subjected to a series of
increasing and decreasing temporal gradients that simulated the temporal
dynamics of sequential waves in the absence of spatial gradients
(Varnum-Finney et al., 1987a;
Wessels et al., 1992
). The
velocity responses of mutant cells were generally normal. Cells moved at peak
velocities in the front of waves, and at trough velocities at the peak and in
the back of waves. However, mutant cells failed to round up at the peak of the
wave and remained abnormally polarized (elongate) in the back of waves. Mutant
cells continued to move in a directed fashion at the peak and in the back of
simulated temporal waves, although at greatly reduced average velocity. These
results demonstrate that phosphorylation of RLC is necessary for the
morphological response to the high concentration of cAMP at the peak of the
natural wave, but is not essential for the general reduction in velocity.
Mutant cells also abnormally retained polarity in the back of the wave even
though velocity remained generally suppressed. To confirm that the absence of
depolarization in response to the peak of the wave represented a failure of
mutant cells to respond to the peak concentration of cAMP
(Varnum and Soll, 1984
), we
tested the response of mutant cells to the rapid addition of 10-6 M
cAMP (Wessels et al., 1989
).
Although the rapid increase in cAMP caused a dramatic reduction in velocity,
it did not elicit a loss of cellular polarity, as it did in wild-type cells.
This defect had an impact on the motile behavior of mutant cells at the peak
and in the back of both simulated temporal waves and natural waves of cAMP.
While there is very little net translocation by wild-type cells at the peak
and in the back of simulated temporal cAMP waves
(Varnum et al., 1985
;
Varnum-Finney et al., 1987a
)
and deduced natural waves (Wessels et al.,
1992
), abnormally elongate mutant cells continued to translocate
in a directed fashion, albeit at reduced velocity.
S13A cells respond less efficiently to natural cAMP waves
If normal depolarization at the peak of a natural wave and the normal
maintenance of the depolarized state in the back of the natural wave are
necessary components of chemotaxis, then S13A cells must be less efficient in
natural chemotaxis. Our results demonstrate that this is indeed the case. S13A
cells exhibited the same defects at the peak and in the back of self-generated
natural waves as those exhibited at the peak and in the back of simulated
temporal waves. In addition, the tracks of S13A cells, although generally
directed at a common aggregation center, were on average less on course than
tracks of JH10 cells responding to self-generated natural waves. When a
minority of labeled S13A cells were mixed with unlabeled JH10 cells, their
tracks were also generally directed towards the aggregation center, but again
were on average less on course than the tracks of neighboring JH10 cells. The
decrease in efficiency appeared to be in the decision on direction in deduced
phase A of each natural wave. This result suggests that cells may be more
efficient in making the correct directional decision at the onset of the front
of a natural wave if they have undergone depolarization at the peak and in the
back of the preceding wave, hence the importance of RLC phosphorylation
Late in aggregation the streams of S13A cells abnormally fragmented,
suggesting that depolarization in response to the peak and back of waves also
plays a role in maintaining the integrity of streams. This is not a surprising
result given the fact that changes in light defraction, which reflect cell
shape changes, move outwardly through streams in association with naturally
moving cAMP waves late in aggregation
(Reitdorf et al., 1997) (H.Z.,
D.W. and D.R.S., unpublished).
Mechanism
In buffer, S13A cells migrate with increased persistence as a result of a
decrease in the frequency of lateral pseudopod formation. Together,
localization of myosin II in crawling cells and the behavioral phenotype of
myosin heavy chain deletion mutants
(Wessels et al., 1988) suggest
that myosin is involved in the suppression of lateral pseudopod formation in
the posterior two thirds of a polarized cell. The results presented here
suggest that RLC phosphorylation is involved in overcoming this suppression.
In Dictyostelium, RLC phosphorylation increases myosin motor activity
by increasing the rate of actin-activated ATP hydrolysis. This increase in
motor function is manifested in in vitro motility assays as increased myosin
movement of actin. In vivo, localized RLC phosphorylation in a cortical region
may increase the relative mobility of myosin, producing a site where the
cortex is more conducive to the nucleation of actin assembly, resulting in
pseudopod extension. The S13A mutant, unable to increase myosin motor
function, would be deficient in the production of these sites, resulting in an
overall decrease in lateral pseudopod formation, as has been observed.
There is growing evidence that myosin activity facilitates pseudopod
extension. The movement of actin and myosin in a localized region in the
lateral cortex would produce a local decrease in rigidity, effectively
generating an opening for actin polymerization. Consistent with this model,
myosin heavy chain null mutants extend pseudopods in all directions (i.e. do
not suppress pseudopod formation in the posterior two thirds of the cell body)
(Wessels et al., 1988); myosin
heavy chain kinase, which promotes the disassembly of myosin filaments,
localizes in pseudopodial regions (Steimle
et al., 2001
); and PAKa, which promotes myosin assembly, localizes
to the posterior of the cell (Chung and
Firtel, 1999
).
As described here, wild-type cells undergo a loss in polarity at the peak
of simulated temporal and natural waves. The rapid addition of 10-6
M cAMP to wild-type cells also causes a rapid loss in polarity
(Wessels et al., 1988) and may
correlate temporally with RLC phosphorylation
(Berlot et al., 1985
). S13A
cells fail to exhibit this rapid loss of polarity at the peak of simulated
temporal and natural waves, and after the rapid addition of 10-6 M
cAMP, demonstrating that RLC phosphorylation is necessary for depolarization.
We suggest that, in response to the increasing temporal gradient of cAMP in
the front of a natural wave, there is an increased association of myosin with
the cortex. However, as the concentration of cAMP approaches 10-6
M, there is an increase in RLC phosphorylation that facilitates the general
relocalization of myosin and the loss of polarity. In the S13A mutant,
relocalization does not occur (i.e. myosin II remains localized in the cortex
of the posterior two-thirds of the elongate cell), providing an explanation
for the failure of S13A mutants to depolarize at the peak of a cAMP wave.
The identification of independent pathways emanating from different
phases of the natural wave
We recently demonstrated through the behavioral characterization of a
mutant of the internal phosphodiesterase RegA that a pathway emanating
specifically from the front of the wave is responsible for the suppression of
lateral pseudopods. The regA- mutant cannot suppress lateral
pseudopods in response to the increasing temporal wave in the front of the
wave and, therefore, cannot chemotax
(Wessels et al., 2000b). By
contrast, regA- cells respond normally to the peak and back of the
wave (Wessels et al., 2000b
).
Here, we have demonstrated that, whereas the RLC phosphorylation mutant S13A
responds normally to the front of the wave, it does not respond normally to
the peak and back of the wave. These results lead to a model in which
independent regulatory pathways emanating from different phases of the natural
wave effect very different behavioral responses in the complex sequence of
behavioral changes accompanying natural chemotaxis.
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Acknowledgments |
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