Transcript of Myosin regulatory light chain and motility
Myosin regulatory light chain and motilityIntroduction In a
translocating Dictyosteliumamoeba, myosin II forms thick filaments
that localize to the cortex of the cell body (Yumura and Fukui,
1985; Fukui, 1990). Myosin II does not localize to an expanding
pseudopod, although it does appear transiently in a retracting
pseudopod (Moores et al., 1996). Through the motion analysis of
myosin heavy-chain-deficient mutants (DeLozanne and Spudich, 1987;
Knecht and Loomis, 1987), it has been demonstrated that mysoin II
is necessary for normal cell polarity, cell motility and
chemotaxis, and plays a role in the spatial distribution and
dynamics of pseudopod extension and retraction (Wessels et al.,
1988; Wessels and Soll, 1990; Spudich, 1989; Sheldon and Knecht,
1996). It has been proposed that myosin II plays a role in the
suppression of lateral pseudopod formation (Wessels et al., 1988,
Wessels et al., 2000b; Stites et al., 1998; Chung and Firtel,
1999), presumably through the generation of cortical tension
(Clarke and Spudich, 1974; Fukui and Yumura, 1986; Pasternak et
al., 1989; Egelhoff et al., 1996).
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;
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The myosin regulatory light chain (RLC) of Dictyostelium discoideum
is phosphorylated at a single serine site in response to
chemoattractant. To investigate the role of the phosphorylation of
RLC in both motility and chemotaxis, mutants were generated in
which the single phosphorylatable serine was replaced with a
nonphosphorylatable alanine. Several independent clones expressing
the mutant RLC in the RLC null mutant, mlcR–, were obtained. These
S13A mutants were subjected to high resolution computer-assisted
motion analysis to assess the basic motile behavior of cells in the
absence of a chemotatic signal, and the chemotactic responsiveness
of cells to the spatial, temporal and concentration components of
natural cAMP waves. In the absence of a cAMP signal, mutant cells
formed lateral pseudopods less frequently and crawled faster than
wild-type cells. In a spatial gradient of cAMP, mutant cells
chemotaxed more efficiently than wild-type cells. In the front of
simulated temporal and natural waves
of cAMP, mutant cells responded normally by suppressing lateral
pseudopod formation. However, unlike wild-type cells, mutant cells
did not lose cellular polarity at the peak and in the back of
either wave. Since depolarization at the peak and in the descending
phase of the natural wave is necessary for efficient chemotaxis,
this deficiency resulted in a decrease in the capacity of S13A
mutant cells to track natural cAMP waves relayed by wild-type
cells, and in the fragmentation of streams late in mutant cell
aggregation. These results reveal a regulatory pathway induced by
the peak and back of the chemotactic wave that alters RLC
phosphorylation and leads to cellular depolarization. We suggest
that depolarization requires myosin II rearrangement in the cortex
facilitated by RLC phosphorylation, which increases myosin motor
function.
Key words: Myosin light chain, Myosin phosphorylation, Cell
motility, Chemotaxis, Dictyostelium discoideum
Summary
Phosphorylation of the myosin regulatory light chain plays a role
in motility and polarity during Dictyostelium chemotaxis Hui Zhang
1, Deborah Wessels 1, Petra Fey 2, Karla Daniels 1, Rex L. Chisholm
2 and David R. Soll 1,* 1Department of Biological Sciences,
University of Iowa, Iowa City, Iowa 52242, USA 2Department of Cell
and Molecular Biology, Northwestern University Medical School,
Chicago, IL 60611, USA *Author for correspondence (e-mail:
david-soll@uiowa.edu)
Accepted 29 January 2002 Journal of Cell Science 115, 1733-1747
(2002) © The Company of Biologists Ltd
Research Article
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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.
Materials and Methods Origin of strains The RLC null mutant mlcR–
was generated from parental strain JH10 by targeting the RLC gene
(mlcR) as previously described (Chen et al., 1994). The S13A
mutants, which contained unphosphorylatable RLC, were generated by
transforming mlcR– with the Dictyostelium integrating vector
pBVN5115, which contained a mutated version of RLC [in which Ser13
(TCA) was changed to Ala (GCC), under the regulation of the actin
15 promoter] and a neomycin resistance gene for G418 selection of
transformed cells (Ostrow et al., 1994). Three S13A mutants were
generated in independent transformations, S13A- 1, S13A-2 and
S13A-3. A control strain, WT-res, representing the RLC deletion
strain rescued with wild-type RLC was generated by transforming
mlcR– with the Dictyostelium integrating vector pBVN5133, which
contained a wild-type version of RLC under the regulation of the
actin 15 promoter, and a neomycin resistance gene for the selection
of transformed cells (Ostrow et al., 1994). It was demonstrated
that the WT-res RLC, but not the S13A RLC, could be phosphorylated
with the myosin regulatory light chain kinase MLCK in vitro (Ostrow
et al., 1994). In the computer-assisted analysis of mutant
behavior, the first characterizations were performed in detail on
mutant strain S13A-1, and aberrant behaviors then verified less
rigorously in mutant strains S13A-2 and S13A-3.
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 2×106 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 5×106 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
10× objective or 25× 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 25× 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 7.8×10–9 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
Journal of Cell Science 115 (8)
1735Myosin regulatory light chain and motility
simulated temporal waves was, therefore, 7 minutes. Fields of cells
were video-recorded or directly digitized through a 10× or 25×
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 10× or 25× objective.
Analysis of mutant cell behavior in self-generated waves of cAMP
(protocol 5, Fig. 1B) The motile behavior of cells in self-
generated 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 (2.4×106 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 10× 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 2.5× 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 (2.4×106 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.
Experimental ProtocolTested Behavior
PHASE A
PHASE B
PHASE C
PHASE D
Front of Wave: Increasing temporal and increasing spa- tial
gradients of cAMP
Back of Wave: Decreasing temporal and decreasing spa- tial
gradients of cAMP
Peak
Re-extension of pseudopods in random directions; maintenance of
depolarized state; no net movement in any direction (response to
decreasing temporal gradi- ent of cAMP)
Cell depolarization, cessation of translocation (response to peak
concentration of cAMP).
Rapid, directed translocation towards source of wave; suppression
of lateral pseudopods (response to increasing temporal gradient of
cAMP.)
Direction towards aggregation center estab l ished, ce l lu lar
polarization (response to posi- tive spatial gradient of
cAMP).
A. Behavioral responses to the different phases of the natural
wave.
B. Experimental protocols for determining the behavioral defects of
S13A mutants in basic motile behavior and chemotaxis.
1. Perfusion of amoebae with buffer in perfusion chamber.
2. Spatial gradient of cAMP generated on the bridge of a single
cell spatial gradient chamber.
3. Exposure to a sequence of interspersed increasing and decreasing
temporal gradients of cAMP that mimic the temporal dynamics of
natural waves in the absence of established spatial
gradients.
4. Rapid addition of 10-6M cAMP to cells crawling in buffer.
5. Aggregation in a dilute monolayer on a plastic surface.
6. Aggregation in which fluorescently stained mutant cells are
mixed with unstained wild type cells in a 1 to 9 ratio.
1. Basic motile behavior in the absence of an extracellular cAMP
signal.
2. Capacity to assess the direction of a spatial gradient of cAMP
(Phase A).
3. Behavior in the middle of the front of the wave in response to
increasing temporal gradient of cAMP (Phase B), the response to the
concentration of cAMP at the peak of the wave (Phase C) and the
behavior in the back of the wave (Phase D).
x
4. Response to the concentration of cAMP at the peak of the wave
(Phase C).
5. Behavior in all phases (A, B, C and D) of self-generated natural
waves of cAMP.
6. Behavior in response to all phases (A, B, C and D) of natural
waves generated by wild type cells.
Direction of relayed wave
center
Fig. 1. (A) The behavioral responses of wild-type cells to the
spatial, temporal and concentration components of the different
phases of the natural cAMP wave, derived from results obtained in
prior studies (Varnum-Finney et al., 1987a; Varnum-Finney et al.,
1987b; Wessels et al., 1992; Wessels et al., 2000b). (B) Protocols
used in this study to determine the behavioral defects of S13A
mutant cells.
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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
10×4π×area/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
Journal of Cell Science 115 (8)
0 2 4 6 8 10 12 0
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Fig. 2.Motility is developmentally regulated in the myosin
regulatory light chain phosphorylation mutant S13A. Cells were
removed from developing JH10, S13A-1 and WT-res cultures at noted
times, dispersed on the wall of a perfusion chamber and analyzed
for cell velocity over a 10 minute period while perfused with
buffer. The mean instantaneous velocity (Inst. Vel.) was computed
at each time point from the average instantaneous velocity of 20 to
30 amoebae selected at random without a velocity threshold. Results
similar to those for S13A-1 cells were obtained for cells of the
independent mutant strains S13A-2 and S13A-3.
Fig. 3.Mutant S13A-1 cells retract anterior pseudopods and extend
lateral pseudopods in a manner similar to that of wild-type JH10
cells. Retraction of the original anterior pseudopod and extension
of a new lateral pseudopod over a 24 second period for a
representative JH10 (A) and S13A (B) cell imaged through
differential interference contrast optics. a, original anterior
pseudopod, at time zero, and new anterior pseudopod at 24 seconds;
u, uropod. Arrows indicate direction of retraction of original
anterior pseudopod and direction of expansion of new lateral
pseudopod for both cell types. Time is indicated in seconds in
upper left hand corners of panels. Similar results were obtained
for cells of strains WT-res, S13A-2 and S13A-3.
1737Myosin regulatory light chain and motility
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
anti- rabbit 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.
Results Strains analyzed To obtain mutant cells containing a myosin
regulatory light chain (RLC) that cannot be phosphorylated, the RLC
deletion mutant mlcR–, derived from the parental wild-type strain
JH10, was rescued with a mutated form of RLC, S13A, in which serine
13 (TCA) was substituted with alanine (GCC), under the control of
the constitutive actin 15 promoter (Ostrow et al., 1994). Three
independent S13A mutants were generated, S13A-1, S13A-2 and S13A-3.
In addition, the mlcRmutant was rescued with wild-type RLC to
generate the control strain WT- res. The parent strain JH10, the
mutant strains S13A-1, S13A- 2 and S13A-3, and the rescued strain
WT-res were then analyzed for basic motile behavior and chemotaxis.
In every condition in which aberrant behavior was demonstrated in
strain S13A-1, it was also demonstrated in the additional mutants
S13A-2 and S13A-3. For simplicity, mutant S13A-1 will be referred
to as strain S13A in the Results, and as strain S13A-1 in the
figure legends and tables, where corroborative results with strain
S13A-2 and S13A-3 are reported.
The basic motile behavior of mutant cells is aberrant During
Dictyosteliumdevelopment, 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).
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
Table 1. Lateral pseudopod formation by cells crawling in buffer or
in a spatial gradient of cAMP* Average frequency of
Number 0 Lateral pseudopods 1 Lateral pseudopod 2 Lateral
pseudopods >2 lateral pseudopods lateral pseudopod Condition
Cell type of cells per 10 min (%)† per 10 min (%)† per 10 min (%)†
per 10 min (%)† per cell per 10 min
Buffer JH10 24 4 8 8 79 3.4 S13A 24 29 29 38 4 1.2
Spatial gradient JH10 20 40 45 15 0 0.75 S13A 28 64 36 0 0
0.36
*Cells were imaged at 25× magnification. For the definition of a
lateral pseudopod, see Materials and Methods. Cells were analyzed
in all cases for 10 minutes. †A Chi square test was performed
between JH10 and S13A cells on the combined data of the four
categories of lateral pseudopod formation. The difference
between JH10 and S13A cells both in buffer and spatial gradients of
cAMP was found to be highly significant (10–12 and 4×10–3,
respectively).
Table 2. Motility, dynamic morphology and chemotaxis parameters in
a spatial gradient of cAMP Instantaneous Directional Percent
velocity Positive flow change Area Maximum length positive
Chemotactic Cell type Cell number (µm/min) (%/min) (deg./min) (µm2)
(µm) Roundness (%) chemotaxis† index
JH10 20 7.7±4.6 8.0±4.6 25±13 100±18 17±3 68±11 90 0.53±0.32 S13A
28 13.6±5.1 17.6±19.4 15±9 88±17 18±3 57±8 100 0.73±0.24 WT-res 29
9.9±6.8 9.8±6.8 22±15 95±27 18±5 63±14 83 0.49±0.47 P values* JH10
vs S13A 0.0001 0.006 0.003 0.03 0.04 0.00001 0.001 S13 vs WT-res
NS(0.01) NS(0.03) 0.001 NS NS 0.04 NS(0.02)
*Significance was determined by the Student t-test for all measured
parameters except ‘percent positive chemotaxis’. A P value greater
than 0.05 was considered non-significant (NS), but values close to
0.05 are shown in parenthesis.
†A Chi-square test found the difference between JH10 and S13A close
to significant and the difference between WT-res and S13A
significant.
1738
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 one- third 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.
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.
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
Journal of Cell Science 115 (8)
Fig. 4.Mutant S13A-1 cells chemotax more efficiently than JH10
cells in a spatial gradient of cAMP. A histogram of chemotactic
indices indicates that S13A cells attain high CIs (>0.8-1.0)
more frequently than JH10 cells or WT-res cells. The number of
JH10, S13A-1 and WT-res cells analyzed was 20, 28 and 29,
respectively. Results similar to those for S13A-1 cells were
obtained for S13A-2 and S13A-3 cells.
Fig. 5.Mutant S13A-1 cells migrate faster and with fewer turns in a
spatial gradient of cAMP. Computer-generated tracks are presented
of the three JH10 (A), S13A-1 (B) and WT-res (C) cells with the
highest chemotactic indices. Cells were selected from 20, 28 and 29
analyzed cells, respectively. Cell perimeters are drawn every 4
seconds. Results similar to those for S13A-1 cells were obtained
for strains S13A-2 and S13A-3 cells.
1739Myosin regulatory light chain and motility
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
A. JH10
/L )
10-8
10-7
10-6
10-9
2
12
22
Fig. 6.Mutant S13A cells respond to a sequence of simulated
temporal waves of cAMP generated in the absence of spatial
gradients by increasing and decreasing velocity in a manner similar
to wild-type JH10 cells. The instantaneous velocity is plotted as a
function of time for a representative JH10 cell (A) and a
representative S13A-1 cell (B) during four simulated waves. The
estimated cAMP concentration, measured in dye experiments (Wessels
et al., 2000b), is presented as a function of time through the four
waves. Note that the velocity of neither JH10 nor S13A-1 cells
increases in the front of the first simulated wave, a result
previously reported for wild-type cells (Varnum et al., 1985).
Instantaneous velocity was measured at 5 second intervals.
Instantaneous velocity plots were smoothed 10 times with Tukey
windows of 10, 20, 40, 20 and 10. Results similar to those for the
representative JH10 and S13A-1 cells were obtained for nine
additional cells of each respective cell line. Results similar to
those for S13A-1 cells were obtained with S13A-2 cells.
1740
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
Journal of Cell Science 115 (8)
Fig. 7.Cell morphology and myosin II localization during the
different phases of a simulated temporal wave of cAMP generated in
the absence of a spatial gradient. A to C, D to F, and G to I
represent differential interference contrast (DIC) microscopy
images of representative JH10 cells fixed in the front, peak and
back, respectively, of a simulated temporal wave of cAMP. A′ to C′,
D′ to F′ and G′ to I′ are DIC images of representative S13A-1 cells
fixed in the front, peak and back, respectively, of a simulated
temporal wave of cAMP. J and K are representative JH10 cells in the
front and the back, respectively, of a simulated temporal wave of
cAMP stained with anti-myosin II antibody (first panel in each set)
and scanned along the white line shown in the first panel for
staining (pixel) intensity (second panel in each set). J′ and K′
are representative S13A-1 cells in the front and the back,
respectively, of a simulated temporal wave of cAMP stained and
analyzed in a fashion similar to the JH10 cells in panels J and K.
Over 100 JH10 and 100 S13A-1 cells were analyzed for morphology in
the front, peak and back of simulated waves, and found to exhibit
the morphologies of the representative cells in this figure. Nine
additional JH10 and S13A-1 cells in the front and back of simulated
temporal waves were found to exhibit the distribution of myosin
demonstrated for the representative cells in the figure.
1741Myosin regulatory light chain and motility
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 (10–6M) 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.,
Fig. 8.S13A cells abnormally fail to lose polarity and abnormally
continue to translocate, albeit at diminished velocity, at the peak
and in the back of a simulated temporal wave of cAMP and after the
rapid addition of 10–6 M cAMP. (A,B) Perimeter tracks of
representative JH10 and S13A cells, respectively, at the peak and
in the back of the second and third waves in a series of four
simulated temporal waves generated in the absence of a spatial
gradient. (C,D) Perimeter tracks of representative JH10 and S13A
cells, respectively, after the rapid addition of 10–6
M cAMP.
1742
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
Journal of Cell Science 115 (8)
A. JH10 B. S13A
0 M cAMP 0 M cAMP 10-6 M cAMP 10-6 M cAMP
50 µm 50 µm
Fig. 9.Centroid tracks of representative JH10 cells (A) and S13A
cells (B) prior to (–10 to 0 minutes) and after (0 to +10 minutes)
the rapid addition of 10–6 M cAMP. Time interval between centroids
is 10 seconds. Similar results were obtained for 17 additional JH10
and S13A cells analyzed in the same fashion. Results similar to
those of JH10 were obtained for WT-res cells analyzed in the same
fashion and results similar to those for S13A-1 cells were obtained
for S13A-2 and S13A-3 cells analyzed in the same fashion.
1743Myosin regulatory light chain and motility
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).
Discussion Disruption of the gene mlcR, which encodes the myosin
regulatory light chain (RLC) in Dictyostelium discoideum, resulted
in defects in cytokinesis, morphogenesis and motility (Chen et al.,
1994). These defects were similar to those obtained with the
original disruption of the myosin heavy chain (DeLozanne and
Spudich, 1987; Knecht and Loomis, 1987; Wessels et al., 1988).
Although it is not clear whether the defects exhibited by the RLC
deletion mutant were due to the
Fig. 10.S13A cells remain abnormally elongate and continue to
translocate, albeit at reduced velocity, at the deduced peak and in
the deduced back of a self-generated natural wave of cAMP. (A,B)
Velocity plots of a representative JH10 cell and a representative
S13A-1 cell in respective homogeneous aggregation territories
responding to three natural sequential waves of cAMP. The phases of
the wave (A+B, C+D) are deduced from the velocity plots described
previously (Wessels et al., 1992). (C,D) Centroid tracks of the
representative JH10 cell and representative S13A cell through the
three successive natural waves (1,2,3) in which the deduced peak
plus back portions (phases C plus D) are boxed. Arrows point in the
direction of the interpreted aggregation centers. (E,F) Amplified
centroid tracks through one wave and associated cell morphologies.
Similar results were obtained for nine additional S13A-1 cells and
ten S13A-2 cells analyzed in a similar fashion.
1744
mislocalization of myosin II or to an alteration in motor function,
a recent analysis of mutants harboring RLCs with point mutations
suggested that RLC played a direct role in the motor properties of
myosin (Chaudoir et al., 1999). The DictyosteliumRLC is
phosphorylated in response to the rapid addition of chemoattractant
(Kuczmarski and Spudich, 1980; Berlot et al., 1985; Berlot et al.,
1987; Griffith et al., 1987), suggesting that phosphorylation plays
a role in chemotaxis. However, the gross defects of the mlcR–
mutant were reversed by the reintroduction of an RLC lacking the
single phosphorylation site at serine 13 (Ostrow et al., 1994),
suggesting that RLC phosphorylation was not required for myosin II
function. In vertebrates, bending of the smooth muscle and
nonmuscle myosin tail is regulated by RLC phosphorylation through a
Ca2+-calmodulin-dependent myosin light chain kinase, which
stimulates assembly and actin- activated ATPase (Suzuki et al.,
1978; Somlyo and Somlyo, 1981; Kamm and Stull, 1985; Ikebe et al.,
1987; Trybus and Lowery, 1987). In Dictyostelium, phosphorylation
of RLC regulates only enzymatic activity (Griffith et al., 1987).
The observations that RLC phosphorylation is tightly coupled to
cAMP receptor occupancy, and that it plays a role in enzymatic
activity led us to hypothesize that it must play a role in the
complex set of responses of Dictyosteliumamoebae to the natural
chemotactic wave (Wessels et al., 1992; Wessels et al., 2000a;
Wessels et al., 2000b).
Dissecting the complex behavior of Dictyostelium amoebae in natural
chemotactic waves The chemotactic responsiveness of
Dictyosteliumamoebae 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 respond 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/
Journal of Cell Science 115 (8)
Dire ct
io n
b
b
b
b
bS13A
JH10
Fig. 11.S13A cells respond abnormally to the deduced peak and back
of natural waves generated by JH10 cells in mixed cultures. Tracks
are presented of a representative labeled S13A-1 cell and a
representative unlabeled neighboring JH10 cell in an aggregation
territory that includes S13A-1 and JH10 cells in a ratio of 1:9.
JH10 cells exhibited tracks similar to those in homogeneous JH10
cell populations, suggesting that the waves relayed in the mixed
population conformed to that of the majority JH10 cell type. Note
that the labeled S13A-1 cell continued to translocate in a
persistent fashion, albeit at reduced velocity, at the peak and in
the back of deduced waves. Note also how the S13A cell veers off
track. The decrease in tracking efficiency was observed in a
majority of labeled S13A-1 cells in JH10 aggregation territories.
Reverse labeling experiments were performed that demonstrated that
labeling did not contribute to the observed effects.
1745Myosin regulatory light chain and motility
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 Dictyosteliumgo 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
Fig. 12.Streams of S13A cells late in aggregation fragment.
Homogeneous populations of JH10 and S13A-1 cells were
video-recorded at low magnification late in aggregation during
stream formation. Stream formation and fragmentation are obvious in
the S13A-1 cultures. In repeat experiments, S13A-1 and S13A-2
streams formed and fragmented, as in the representative panels in
B. Zero minutes represents the time at which video-recording was
initiated.
1746
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.
The authors are indebted to J. Swails for help in assembling the
manuscript. The research was supported by National Institutes of
Health grants HD-18577 (D.R.S.) and GM39264 (R.L.C.). The authors
acknowledge use of the W. M. Keck Dynamic Image Analysis Facility
at the University of Iowa, funded by the W.M. Keck
Foundation.
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