BACKGROUND
dynamic rearrangements of the actin cytoskeleton are required for cell migration, cell polarization, phagocytosis, adhesion, and cytokinesis  <cit> .
this reorganization involves f-actin assembly from soluble monomers in the cytoplasm and their subsequent turnover through depolymerization to replenish the precursor pool  <cit> .
cells use the force generated from new actin assembly to deform the cell membrane, changing the cell shape to extend the leading edge of the cell.
polymerization of new actin filaments requires actin nucleating factors – arp2/ <dig> complex and formins – that catalyze new actin assembly, and thus play a key role in inducing morphological changes  <cit> .
however, maintenance of the appropriate shape of the cell likely depends on actin cross-linkers to provide mechanical resistance so that focused force production occurs in the right direction.
dynacortin, an actin filament cross-linking protein, was discovered in dictyostelium discoideum in a genetic screen for suppressors of the cytokinesis defect of cortexillin-i mutants  <cit> .
dynacortin localizes to the cortex and is especially enriched in dynamic protrusions built by the actin cytoskeleton, such as pseudopodia, lamellipodia, and phagocytic cups  <cit> .
from a variety of genetic, in vivo and in vitro analyses, dynacortin has been found to be an actin cross-linking protein that generates mechanical resistance in the cortex that controls cytokinesis contractility dynamics  <cit> .
because of dynacortin's localization to cell surface protrusions in vegetative cells, we speculated that it might play a role in chemotaxis.
here, we use epifluorescence and total internal reflection fluorescence imaging to demonstrate that dynacortin is localized to the actin network, including the leading edges of chemotaxing dictyostelium.
cells depleted of dynacortin can sense chemoattractant but have trouble polarizing normally.
using purified proteins, we demonstrate that dynacortin directly stabilizes actin in vitro.
overall, dynacortin is an actin cross-linking protein that facilitates cell polarization during chemotaxis.
RESULTS
dynacortin localization in chemotaxing dictyostelium
in vegetative cells, dynacortin localizes to the cell cortex and is especially enriched in protrusions such as pseudopodia, filopodia, lamellipodia, macropinocytic crowns, and cell-substrate structures called feet or eupodia.
when cells change direction, green fluorescent protein fused with dynacortin  redistributes to the leading edge of the cell  <cit> .
this dynamic redistribution of gfp-dynacortin led us to speculate that dynacortin may have a role in chemotaxis.
to determine the subcellular localization of dynacortin during chemotaxis, we expressed gfp-dynacortin in wild-type cells.
we then imaged gfp-dynacortin distribution during the movement of aggregation-competent cells toward camp released from a micropipette.
epifluorescence microscopy revealed that gfp-dynacortin was diffusely localized throughout the cortex with occasional increases in concentration at the leading edge .
the linescan in figure 1b demonstrates dynacortin enrichment at the front of the migrating cell.
to obtain higher resolution images, we examined the distribution of dynacortin in the cell surface using total internal reflection fluorescence  microscopy .
tirf imaging allows the cortical layer to be imaged, greatly reducing the signal contribution from soluble gfp-dynacortin in the cytosol .
dynacortin is organized into fibrous, punctuate structures, which are the actin-rich network near the cell surface that make up the actin feet  <cit> .
thus, the actin cross-linker dynacortin is recruited to highly dynamic regions of the cytoskeleton during chemotaxis.
dynacortin is required for cell polarization in response to camp
given the localization of dynacortin during chemotaxis, we tested the chemotaxis ability of cells depleted of dynacortin.
wild-type control cells were able to chemotax with high efficiency and assumed a highly polarized shape in response to camp .
here, we define cell polarization based on cell morphology in which the cells become highly elongated , and we use quantitative metrics  to assess cell shape .
cells that expressed a dynacortin hairpin plasmid had no detectable dynacortin and had a quantitative defect in cell polarization in response to camp   <cit> .
though we frequently observed that dynacortin-deficient cells had highly impaired motility, failing to become polarized , the degree of immobility was highly variable and wild-type cells also had some highly rounded cells on occasion.
thus, we focused our quantitative analysis on motile cells from wild-type and dynacortin-deficient cells and measured the velocity, chemotactic index, directional persistence of the movement and the roundness .
of the motile cells, dynacortin-depleted cells had statistically indistinguishable velocity, chemotactic index and directional persistence as compared to wild-type cells .
however, dynacortin-deficient cells  were significantly more round than wild-type cells .
thus, dynacortin is required for the cell to fully polarize in response to camp.
we observed similar trends at  <dig>  and  <dig> h of starvation, indicating that the defect in polarization is independent of developmental stage; significant increase in polarization is observed during this time period in wild-type cells  <cit> .
further, even though the dynacortin-deficient cells did not polarize as fully as wild-type control cells, gfp-myosin-ii partially enriched in the rear half of the polarized, but not non-polarized, dynacortin-deficient cells .
*differences in fraction of motile cells between control and dynhp cells for each genetic background were significant .
†differences in roundness between control and dynhp cells for each genetic background were significant .
all values are mean ± sem.
to quantify the motility defect further, we used a skeleton representation  to characterize the dynamic changes in cellular morphology during chemotaxis .
the skeletons of ax <dig> control cells consisted primarily of a single segment indicating a lack of lateral protrusions .
with the skeleton representation, we could separate the extension  and retraction  of cell surface protrusions.
the extending tips were relatively long-lived  and the angles  of the extending tips co-aligned with the chemoattractant gradient .
in contrast, the skeletons of ax2:dynhp cells showed multiple branches characteristic of cells with multiple protrusions .
these projections occurred at a broader range of angles relative to the gradient and had a shorter duration .
moreover, though these projections were also in alignment with the chemoattractant gradient on average, a broader distribution of extending protrusion angles was observed  .
similarly, ax <dig> control  and ax3:dynhp cells  also produced protrusions with a similar angle relative to the gradient but ax3:dynhp cells extended the protrusions with a larger variation in angles .
thus, without dynacortin, cells generate more protrusions over a much broader distribution of angles than wild-type cells do.
the defect in polarization of dynacortin-deficient cells is similar to that observed for ly <dig>  treatment  <cit> .
therefore, we investigated the effect of ly <dig> treatment of gfp-dynacortin localization .
using  <dig> -h cells, wild-type cells expressing gfp-dynacortin were elongated with gfp-dynacortin enriched at the leading edge of the cell .
within about  <dig> s of treating the cells with 50-μm ly <dig>  the cells nearly completely rounded up as described previously   <cit> .
however, by bringing the needle containing  <dig> μm camp close to the rounded cell, the cell accumulated gfp-dynacortin on the side facing the needle and extended a pseudopod within  <dig> s .
within 2– <dig> min, the cells adapted to the ly <dig> and began to chemotax towards the needle but never fully repolarized .
significantly, gfp-dynacortin accumulated at the front end of the chemotacting ly29004-treated cells, suggesting that dynacortin accumulation at the cell front may be independent of pi <dig> kinase activity.
we also treated dynacortin-deficient cells with ly29004; however, while the cells again showed lower overall polarization relative to wild-type cells, they were still able to chemotax towards the needle .
for a population assessment, we examined the development on db-agar.
the dynacortin-depleted cells appeared largely indistinguishable from the wild-type control cells, forming normal looking fruiting bodies.
however, at decreasing densities, the wild-type control cells repeatedly formed large, extensive streams whereas the dynacortin-depleted cells seldom formed streams, and when they did they were smaller than the wild-type control.
thus, in this population assay, dynacortin is required for development at low cell densities, consistent with the overall polarization and motility defects observed in the needle assays.
dynacortin does not affect bulk actin polymerization in response to camp stimulation
as dynacortin localizes to actin-rich dynamic cortical domains, we determined whether dynacortin contributes to the bulk actin polymerization dynamics in the cell.
we compared the kinetics of in vivo bulk actin polymerization of dynacortin-silenced cells  to control wild-type cells in response to a chemotactic stimulus .
within  <dig> s of stimulation with 1-μm camp, wild-type and dynacortin-depleted cells showed a transient assembly of actin , and then a decrease to basal levels by  <dig> s, followed by a smaller second  peak at  <dig> s. thus, dynacortin does not have a detectable effect on the bulk actin assembly dynamics in response to camp.
further, basal actin levels were similar between wild-type and dynacortin-depleted cells .
dynacortin drives polymerization under low-ionic strength conditions
because dynacortin enriches at the leading edge of chemotaxing cells and these motile cells are known to assemble new filamentous actin when stimulated with camp, we wondered whether dynacortin has an effect on actin assembly.
to do this, we investigated the effect of dynacortin on actin assembly in vitro using pyrene-actin assays.
in these experiments, the fluorescence of pyrene-actin increases as it assembles into filaments.
traditionally, these assays are performed in high-salt f-buffer, which contains  <dig> mm kcl and  <dig> mm mgcl <dig>  however, while performing some control experiments, we discovered that dynacortin drove actin assembly in g-buffer, which contains only  <dig> mm tris-hcl and  <dig>  mm cacl <dig>  because g-actin normally does not assemble under these low-salt g-buffer conditions, this dynacortin-mediated assembly represents a nearly infinite rate enhancement.
therefore, we tested dynacortin's role in actin assembly under both g- and f-buffer conditions and report these findings here.
we first measured the critical concentration of g-actin required for polymerization in the presence and absence of dynacortin.
the critical concentration of g-actin assembled by  <dig>  μm dynacortin under g-buffer conditions was only  <dig>  μm, which is significantly lower than the  <dig>  μm critical concentration for actin alone assembled by the addition of f-buffer .
because the addition of dynacortin to f-actin caused a partial quenching of pyrene fluorescence, the critical concentration of actin under f-salt conditions could not be determined accurately .
then, we investigated the impact of dynacortin on the kinetics of actin polymerization under g-buffer conditions.
we mixed increasing amounts of dynacortin with a constant amount of pyrene-labeled ca2+-actin  and the rate of polymerization was monitored over time by fluorescence intensity measurements.
at dynacortin concentrations above  <dig> μm , the g-actin assembled into polymers, and dynacortin increased the steady state amount of assembled actin in a dynacortin-concentration-dependent manner .
because of the absence of an apparent lag phase , the observed rate of polymerization could be obtained by fitting the curves to an exponential rise equation .
the rate of polymerization increased linearly with increasing dynacortin concentration until a maximal value  was reached at 5-μm dynacortin.
further increase in dynacortin concentration resulted in no increase in rate beyond the maximal value.
considering the k+ and the slope of the concentration-dependent phase, the km was calculated to be  <dig> μm, which is similar to the measured  <dig>  μm critical concentration for dynacortin to assemble polymers in g-buffer .
these results suggest that under low ionic strength conditions, dynacortin scaffolds actin monomers into filaments.
further, the absence of an apparent lag phase suggests that the mechanism of dynacortin-mediated actin assembly is different than the conventional nucleation-elongation mechanism of actin assembly.
next, we verified that the increase in fluorescence was not due to an effect on the fluorescent probe but that dynacortin indeed bound actin under these low-salt conditions, using a co-sedimentation assay .
the solutions from the pyrene assay were centrifuged at high-speed centrifugation to pellet all the assembled actin and bound dynacortin.
in the case of g-actin alone, no actin precipitated; most of the actin assembled using f-buffer precipitated at this speed.
in the presence of g-actin and dynacortin, dynacortin co-precipitated with actin and had a saturation stoichiometry of one dyn <dig> dimer per actin monomer.
this saturation stoichiometry is identical to the saturation stoichiometry for dynacortin-mediated actin bundling  <cit> .
more importantly, the amount of actin that assembled and pelleted increased as the dynacortin concentration increased.
to independently confirm the presence of actin filaments, we examined the samples under the electron microscope and observed bundles of actin filaments in the presence of dynacortin under low salt conditions .
staining actin filaments with rhodamine-phalloidin and visualizing them under the fluorescence microscope also revealed the presence of bundles .
taken together, these data demonstrate that dynacortin drives assembly of g-actin into polymers under conditions that disfavor assembly and that the amount of polymer formed is proportional to the amount of dynacortin available.
thus, dynacortin acts as a scaffold or molecular staple that holds the actin monomers together under these low salt g-buffer conditions.
we also tested whether dynacortin can nucleate actin under standard f-buffer conditions.
under these conditions, the conventional lag phase associated with the nucleation-elongation reaction is observable.
as the dynacortin concentration was increased, the lag phase for actin assembly decreased slightly .
the lag phase is derived from the time required for actin nuclei to form.
to analyze this more quantitatively, we determined the time for the actin fluorescence signal to increase to 10% of the maximum signal, indicating that the actin is 10% assembled .
the reciprocal of the time to 10% has been demonstrated to be a close approximation of the nucleation rate  <cit> .
dynacortin weakly increased the apparent nucleation rate in a dynacortin-concentration-dependent manner .
overall, we conclude that dynacortin's primary role is in actin filament cross-linking with a possible effect on filament stability.
discussion
chemotaxis is an important cellular process for organisms, ranging from bacteria and protists to higher multicellular animals  <cit> .
in mammals, it is critical for normal immune function, while in the simple protozoan dictyostelium discoideum, it is important for coalescence of individual cells into multicellular structures that allow the organism to respond to environmental stresses such as nutrient starvation.
chemotaxis can be separated into three components: directional sensing, polarization and motility  <cit> .
although a high level of understanding has been achieved for directional sensing and motility, little is known about how cells polarize and how these three processes are integrated is not at all understood.
certainly, evidence exists that the actin network reinforces directional sensing  <cit> .
because of the complexity of actin assembly, motility, and signaling, it is possible that cooperativity and redundancy have obscured critical players in genetic screens geared to uncover the connections between sensing and motility.
also, the usual suspects for motility  play a role in the chemotactic response  <cit> .
our results demonstrate that the actin cross-linking protein, dynacortin, plays a direct role in cell polarization during chemotaxis.
dynacortin is a novel actin cross-linking protein that enriches in cell surface protrusions as well as punctate surface structures that are the dictyostelium equivalents of focal adhesions  <cit> .
dynacortin contributes to cell cortex mechanics and interacts antagonistically with myosin-ii driven dynamic rearrangements of the cell cortex  <cit> .
by enriching at sites of dynamic actin cytoskeleton, dynacortin-mediated cross-linking and polymer stabilization may create the appropriate level of viscoelastic resistance for the cell to polarize and/or maintain a polarized shape.
cortical viscoelasticity may also contribute to chemotaxis by setting a timescale over which a cell can change shape as it sends out a pseudopod.
a cell with greater viscoelasticity would be expected to be slower at assembling and disassembling a pseudopod whereas a cell with lower viscoelasticity should be able to form pseudopods much more rapidly.
indeed, the softer dynacortin cells form many more pseudopods per unit of time and extend them in a greater number of directions.
different levels of mechanical resistance between the leading and trailing edges of chemotactic cells have been documented previously, and the membrane-actin tether, talin, contributes significantly to the cortical resistance at the rear of the cell  <cit> .
different regulatory systems modulate the actin responses during chemotaxis.
in mammalian cells, the initial actin response is thought to be mediated by cofilin  <cit> .
however, in dictyostelium, actin assembly appears to be regulated at least in part by pten phosphatase and the pi <dig> kinases.
inhibition of these phosphoinositol-regulating enzymes may alter the general cytoskeletal dynamics, leading to defects in chemotaxis  <cit> .
dynacortin, in contrast, may act downstream of the signaling networks to directly modulate the viscoelasticity of the actin network thereby promoting polarization in response to chemotactic stimulation.
in addition to cross-linking, our in vitro data suggest that dynacortin may serve as a scaffolder of actin assembly.
under low-salt conditions, dynacortin drove monomeric actin into polymers in a concentration-dependent manner.
further, the amount of assembled polymer was dependent on the dynacortin amounts and assembly occurred with a 1: <dig> stoichiometry, indicating that dynacortin scaffolded the actin.
other examples of actin scaffolding proteins include nebulin, which controls the length of the actin filaments in the muscle sarcomere  <cit> , and the salmonella sipa, which promotes cytoskeletal reorganization for entry into the cell  <cit> .
thus, while this scaffolding activity is unusual, it is not unprecedented.
CONCLUSIONS
regulated actin assembly is the essential component for directed cell motility.
because of its importance for normal cell physiology, numerous players contribute to the assembly and organization of the leading edge actin network.
while arp2/ <dig> and its regulators have critical roles, other accessory proteins such as actin cross-linkers also contribute, ensuring robustness.
our results demonstrate that the actin cross-linker dynacortin should be considered in chemotaxis for its role in cross-linking and stabilizing the dynamic actin network, promoting cell polarization.
indeed, the cytoskeleton is the prototypical example of biocomplexity where many proteins that are physically connected by cytoskeletal filaments interact to promote complicated cell behavior.

 dynamic rearrangements of the actin cytoskeleton are required for cell migration, cell polarization, phagocytosis, adhesion, and cytokinesis. this reorganization involves f-actin assembly from soluble monomers in the cytoplasm and their subsequent turnover through depolymerization to replenish the precursor pool. cells use the force generated from new actin assembly to deform the cell membrane, changing the cell shape to extend the leading edge of the cell. polymerization of new actin filaments requires actin nucleating factors – arp2/ complex and formins – that catalyze new actin assembly, and thus play a key role in inducing morphological changes. however, maintenance of the appropriate shape of the cell likely depends on actin cross-linkers to provide mechanical resistance so that focused force production occurs in the right direction. dynacortin, an actin filament cross-linking protein, was discovered in dictyostelium discoideum in a genetic screen for suppressors of the cytokinesis defect of cortexillin-i mutants. dynacortin localizes to the cortex and is especially enriched in dynamic protrusions built by the actin cytoskeleton, such as pseudopodia, lamellipodia, and phagocytic cups. from a variety of genetic, in vivo and in vitro analyses, dynacortin has been found to be an actin cross-linking protein that generates mechanical resistance in the cortex that controls cytokinesis contractility dynamics. because of dynacortin's localization to cell surface protrusions in vegetative cells, we speculated that it might play a role in chemotaxis. here, we use epifluorescence and total internal reflection fluorescence imaging to demonstrate that dynacortin is localized to the actin network, including the leading edges of chemotaxing dictyostelium. cells depleted of dynacortin can sense chemoattractant but have trouble polarizing normally. using purified proteins, we demonstrate that dynacortin directly stabilizes actin in vitro. overall, dynacortin is an actin cross-linking protein that facilitates cell polarization during chemotaxis.  dynacortin localization in chemotaxing dictyostelium in vegetative cells, dynacortin localizes to the cell cortex and is especially enriched in protrusions such as pseudopodia, filopodia, lamellipodia, macropinocytic crowns, and cell-substrate structures called feet or eupodia. when cells change direction, green fluorescent protein fused with dynacortin redistributes to the leading edge of the cell. this dynamic redistribution of gfp-dynacortin led us to speculate that dynacortin may have a role in chemotaxis. to determine the subcellular localization of dynacortin during chemotaxis, we expressed gfp-dynacortin in wild-type cells. we then imaged gfp-dynacortin distribution during the movement of aggregation-competent cells toward camp released from a micropipette. epifluorescence microscopy revealed that gfp-dynacortin was diffusely localized throughout the cortex with occasional increases in concentration at the leading edge. the linescan in figure 1b demonstrates dynacortin enrichment at the front of the migrating cell. to obtain higher resolution images, we examined the distribution of dynacortin in the cell surface using total internal reflection fluorescence microscopy. tirf imaging allows the cortical layer to be imaged, greatly reducing the signal contribution from soluble gfp-dynacortin in the cytosol. dynacortin is organized into fibrous, punctuate structures, which are the actin-rich network near the cell surface that make up the actin feet. thus, the actin cross-linker dynacortin is recruited to highly dynamic regions of the cytoskeleton during chemotaxis. dynacortin is required for cell polarization in response to camp given the localization of dynacortin during chemotaxis, we tested the chemotaxis ability of cells depleted of dynacortin. wild-type control cells were able to chemotax with high efficiency and assumed a highly polarized shape in response to camp. here, we define cell polarization based on cell morphology in which the cells become highly elongated, and we use quantitative metrics to assess cell shape. cells that expressed a dynacortin hairpin plasmid had no detectable dynacortin and had a quantitative defect in cell polarization in response to camp. though we frequently observed that dynacortin-deficient cells had highly impaired motility, failing to become polarized, the degree of immobility was highly variable and wild-type cells also had some highly rounded cells on occasion. thus, we focused our quantitative analysis on motile cells from wild-type and dynacortin-deficient cells and measured the velocity, chemotactic index, directional persistence of the movement and the roundness. of the motile cells, dynacortin-depleted cells had statistically indistinguishable velocity, chemotactic index and directional persistence as compared to wild-type cells. however, dynacortin-deficient cells were significantly more round than wild-type cells. thus, dynacortin is required for the cell to fully polarize in response to camp. we observed similar trends at and h of starvation, indicating that the defect in polarization is independent of developmental stage; significant increase in polarization is observed during this time period in wild-type cells. further, even though the dynacortin-deficient cells did not polarize as fully as wild-type control cells, gfp-myosin-ii partially enriched in the rear half of the polarized, but not non-polarized, dynacortin-deficient cells. *differences in fraction of motile cells between control and dynhp cells for each genetic background were significant. †differences in roundness between control and dynhp cells for each genetic background were significant. all values are mean ± sem. to quantify the motility defect further, we used a skeleton representation to characterize the dynamic changes in cellular morphology during chemotaxis. the skeletons of ax control cells consisted primarily of a single segment indicating a lack of lateral protrusions. with the skeleton representation, we could separate the extension and retraction of cell surface protrusions. the extending tips were relatively long-lived and the angles of the extending tips co-aligned with the chemoattractant gradient. in contrast, the skeletons of ax2:dynhp cells showed multiple branches characteristic of cells with multiple protrusions. these projections occurred at a broader range of angles relative to the gradient and had a shorter duration. moreover, though these projections were also in alignment with the chemoattractant gradient on average, a broader distribution of extending protrusion angles was observed. similarly, ax control and ax3:dynhp cells also produced protrusions with a similar angle relative to the gradient but ax3:dynhp cells extended the protrusions with a larger variation in angles. thus, without dynacortin, cells generate more protrusions over a much broader distribution of angles than wild-type cells do. the defect in polarization of dynacortin-deficient cells is similar to that observed for ly treatment. therefore, we investigated the effect of ly treatment of gfp-dynacortin localization. using -h cells, wild-type cells expressing gfp-dynacortin were elongated with gfp-dynacortin enriched at the leading edge of the cell. within about s of treating the cells with 50-μm ly the cells nearly completely rounded up as described previously. however, by bringing the needle containing μm camp close to the rounded cell, the cell accumulated gfp-dynacortin on the side facing the needle and extended a pseudopod within s. within 2– min, the cells adapted to the ly and began to chemotax towards the needle but never fully repolarized. significantly, gfp-dynacortin accumulated at the front end of the chemotacting ly29004-treated cells, suggesting that dynacortin accumulation at the cell front may be independent of pi kinase activity. we also treated dynacortin-deficient cells with ly29004; however, while the cells again showed lower overall polarization relative to wild-type cells, they were still able to chemotax towards the needle. for a population assessment, we examined the development on db-agar. the dynacortin-depleted cells appeared largely indistinguishable from the wild-type control cells, forming normal looking fruiting bodies. however, at decreasing densities, the wild-type control cells repeatedly formed large, extensive streams whereas the dynacortin-depleted cells seldom formed streams, and when they did they were smaller than the wild-type control. thus, in this population assay, dynacortin is required for development at low cell densities, consistent with the overall polarization and motility defects observed in the needle assays. dynacortin does not affect bulk actin polymerization in response to camp stimulation as dynacortin localizes to actin-rich dynamic cortical domains, we determined whether dynacortin contributes to the bulk actin polymerization dynamics in the cell. we compared the kinetics of in vivo bulk actin polymerization of dynacortin-silenced cells to control wild-type cells in response to a chemotactic stimulus. within s of stimulation with 1-μm camp, wild-type and dynacortin-depleted cells showed a transient assembly of actin, and then a decrease to basal levels by s, followed by a smaller second peak at s. thus, dynacortin does not have a detectable effect on the bulk actin assembly dynamics in response to camp. further, basal actin levels were similar between wild-type and dynacortin-depleted cells. dynacortin drives polymerization under low-ionic strength conditions because dynacortin enriches at the leading edge of chemotaxing cells and these motile cells are known to assemble new filamentous actin when stimulated with camp, we wondered whether dynacortin has an effect on actin assembly. to do this, we investigated the effect of dynacortin on actin assembly in vitro using pyrene-actin assays. in these experiments, the fluorescence of pyrene-actin increases as it assembles into filaments. traditionally, these assays are performed in high-salt f-buffer, which contains mm kcl and mm mgcl however, while performing some control experiments, we discovered that dynacortin drove actin assembly in g-buffer, which contains only mm tris-hcl and mm cacl because g-actin normally does not assemble under these low-salt g-buffer conditions, this dynacortin-mediated assembly represents a nearly infinite rate enhancement. therefore, we tested dynacortin's role in actin assembly under both g- and f-buffer conditions and report these findings here. we first measured the critical concentration of g-actin required for polymerization in the presence and absence of dynacortin. the critical concentration of g-actin assembled by μm dynacortin under g-buffer conditions was only μm, which is significantly lower than the μm critical concentration for actin alone assembled by the addition of f-buffer. because the addition of dynacortin to f-actin caused a partial quenching of pyrene fluorescence, the critical concentration of actin under f-salt conditions could not be determined accurately. then, we investigated the impact of dynacortin on the kinetics of actin polymerization under g-buffer conditions. we mixed increasing amounts of dynacortin with a constant amount of pyrene-labeled ca2+-actin and the rate of polymerization was monitored over time by fluorescence intensity measurements. at dynacortin concentrations above μm, the g-actin assembled into polymers, and dynacortin increased the steady state amount of assembled actin in a dynacortin-concentration-dependent manner. because of the absence of an apparent lag phase, the observed rate of polymerization could be obtained by fitting the curves to an exponential rise equation. the rate of polymerization increased linearly with increasing dynacortin concentration until a maximal value was reached at 5-μm dynacortin. further increase in dynacortin concentration resulted in no increase in rate beyond the maximal value. considering the k+ and the slope of the concentration-dependent phase, the km was calculated to be μm, which is similar to the measured μm critical concentration for dynacortin to assemble polymers in g-buffer. these results suggest that under low ionic strength conditions, dynacortin scaffolds actin monomers into filaments. further, the absence of an apparent lag phase suggests that the mechanism of dynacortin-mediated actin assembly is different than the conventional nucleation-elongation mechanism of actin assembly. next, we verified that the increase in fluorescence was not due to an effect on the fluorescent probe but that dynacortin indeed bound actin under these low-salt conditions, using a co-sedimentation assay. the solutions from the pyrene assay were centrifuged at high-speed centrifugation to pellet all the assembled actin and bound dynacortin. in the case of g-actin alone, no actin precipitated; most of the actin assembled using f-buffer precipitated at this speed. in the presence of g-actin and dynacortin, dynacortin co-precipitated with actin and had a saturation stoichiometry of one dyn dimer per actin monomer. this saturation stoichiometry is identical to the saturation stoichiometry for dynacortin-mediated actin bundling. more importantly, the amount of actin that assembled and pelleted increased as the dynacortin concentration increased. to independently confirm the presence of actin filaments, we examined the samples under the electron microscope and observed bundles of actin filaments in the presence of dynacortin under low salt conditions. staining actin filaments with rhodamine-phalloidin and visualizing them under the fluorescence microscope also revealed the presence of bundles. taken together, these data demonstrate that dynacortin drives assembly of g-actin into polymers under conditions that disfavor assembly and that the amount of polymer formed is proportional to the amount of dynacortin available. thus, dynacortin acts as a scaffold or molecular staple that holds the actin monomers together under these low salt g-buffer conditions. we also tested whether dynacortin can nucleate actin under standard f-buffer conditions. under these conditions, the conventional lag phase associated with the nucleation-elongation reaction is observable. as the dynacortin concentration was increased, the lag phase for actin assembly decreased slightly. the lag phase is derived from the time required for actin nuclei to form. to analyze this more quantitatively, we determined the time for the actin fluorescence signal to increase to 10% of the maximum signal, indicating that the actin is 10% assembled. the reciprocal of the time to 10% has been demonstrated to be a close approximation of the nucleation rate. dynacortin weakly increased the apparent nucleation rate in a dynacortin-concentration-dependent manner. overall, we conclude that dynacortin's primary role is in actin filament cross-linking with a possible effect on filament stability. discussion chemotaxis is an important cellular process for organisms, ranging from bacteria and protists to higher multicellular animals. in mammals, it is critical for normal immune function, while in the simple protozoan dictyostelium discoideum, it is important for coalescence of individual cells into multicellular structures that allow the organism to respond to environmental stresses such as nutrient starvation. chemotaxis can be separated into three components: directional sensing, polarization and motility. although a high level of understanding has been achieved for directional sensing and motility, little is known about how cells polarize and how these three processes are integrated is not at all understood. certainly, evidence exists that the actin network reinforces directional sensing. because of the complexity of actin assembly, motility, and signaling, it is possible that cooperativity and redundancy have obscured critical players in genetic screens geared to uncover the connections between sensing and motility. also, the usual suspects for motility play a role in the chemotactic response. our results demonstrate that the actin cross-linking protein, dynacortin, plays a direct role in cell polarization during chemotaxis. dynacortin is a novel actin cross-linking protein that enriches in cell surface protrusions as well as punctate surface structures that are the dictyostelium equivalents of focal adhesions. dynacortin contributes to cell cortex mechanics and interacts antagonistically with myosin-ii driven dynamic rearrangements of the cell cortex. by enriching at sites of dynamic actin cytoskeleton, dynacortin-mediated cross-linking and polymer stabilization may create the appropriate level of viscoelastic resistance for the cell to polarize and/or maintain a polarized shape. cortical viscoelasticity may also contribute to chemotaxis by setting a timescale over which a cell can change shape as it sends out a pseudopod. a cell with greater viscoelasticity would be expected to be slower at assembling and disassembling a pseudopod whereas a cell with lower viscoelasticity should be able to form pseudopods much more rapidly. indeed, the softer dynacortin cells form many more pseudopods per unit of time and extend them in a greater number of directions. different levels of mechanical resistance between the leading and trailing edges of chemotactic cells have been documented previously, and the membrane-actin tether, talin, contributes significantly to the cortical resistance at the rear of the cell. different regulatory systems modulate the actin responses during chemotaxis. in mammalian cells, the initial actin response is thought to be mediated by cofilin. however, in dictyostelium, actin assembly appears to be regulated at least in part by pten phosphatase and the pi kinases. inhibition of these phosphoinositol-regulating enzymes may alter the general cytoskeletal dynamics, leading to defects in chemotaxis. dynacortin, in contrast, may act downstream of the signaling networks to directly modulate the viscoelasticity of the actin network thereby promoting polarization in response to chemotactic stimulation. in addition to cross-linking, our in vitro data suggest that dynacortin may serve as a scaffolder of actin assembly. under low-salt conditions, dynacortin drove monomeric actin into polymers in a concentration-dependent manner. further, the amount of assembled polymer was dependent on the dynacortin amounts and assembly occurred with a 1: stoichiometry, indicating that dynacortin scaffolded the actin. other examples of actin scaffolding proteins include nebulin, which controls the length of the actin filaments in the muscle sarcomere, and the salmonella sipa, which promotes cytoskeletal reorganization for entry into the cell. thus, while this scaffolding activity is unusual, it is not unprecedented.  regulated actin assembly is the essential component for directed cell motility. because of its importance for normal cell physiology, numerous players contribute to the assembly and organization of the leading edge actin network. while arp2/ and its regulators have critical roles, other accessory proteins such as actin cross-linkers also contribute, ensuring robustness. our results demonstrate that the actin cross-linker dynacortin should be considered in chemotaxis for its role in cross-linking and stabilizing the dynamic actin network, promoting cell polarization. indeed, the cytoskeleton is the prototypical example of biocomplexity where many proteins that are physically connected by cytoskeletal filaments interact to promote complicated cell behavior. 
in particular, they send out multiple pseudopods that radiate at a broader distribution of angles relative to the chemoattractant gradient. dynacortin enriches with the actin, particularly at the leading edge of chemotaxing cells. we studied the effect of dynacortin on cell motility during chemotaxis and on actin dynamics in vivo and in vitro. dynacortin, an actin cross-linking protein, localizes to the cell cortex and contributes to cortical resistance, thereby helping to define the cell shape changes of cytokinesis. dynacortin contributes to cell polarization during chemotaxis. though dynacortin-deficient cells show normal bulk actin assembly upon chemoattractant stimulation, dynacortin can promote actin assembly in vitro. by cross-linking and possibly stabilizing actin polymers, dynacortin also contributes to cortical viscoelasticity, which may be critical for establishing cell polarity. by fluorescence spectroscopy, co-sedimentation and transmission electron microscopy, dynacortin acts as an actin scaffolder in which it assembles actin monomers into polymers with a stoichiometry of dyn2: actin under salt conditions that disfavor polymer assembly. dynacortin also becomes highly enriched in cortical protrusions, which are sites of new actin assembly. cells devoid of dynacortin do not become as polarized as wild-type control cells but move with similar velocities as wild-type cells. wild-type cells typically only send out one pseudopod at a time that does not diverge much from 0° on average relative to the gradient.
 cell shape changes during cytokinesis and chemotaxis require regulation of the actin cytoskeletal network. dynacortin, an actin cross-linking protein, localizes to the cell cortex and contributes to cortical resistance, thereby helping to define the cell shape changes of cytokinesis. dynacortin also becomes highly enriched in cortical protrusions, which are sites of new actin assembly.  we studied the effect of dynacortin on cell motility during chemotaxis and on actin dynamics in vivo and in vitro. dynacortin enriches with the actin, particularly at the leading edge of chemotaxing cells. cells devoid of dynacortin do not become as polarized as wild-type control cells but move with similar velocities as wild-type cells. in particular, they send out multiple pseudopods that radiate at a broader distribution of angles relative to the chemoattractant gradient. wild-type cells typically only send out one pseudopod at a time that does not diverge much from 0° on average relative to the gradient. though dynacortin-deficient cells show normal bulk actin assembly upon chemoattractant stimulation, dynacortin can promote actin assembly in vitro. by fluorescence spectroscopy, co-sedimentation and transmission electron microscopy, dynacortin acts as an actin scaffolder in which it assembles actin monomers into polymers with a stoichiometry of dyn2: actin under salt conditions that disfavor polymer assembly.  dynacortin contributes to cell polarization during chemotaxis. by cross-linking and possibly stabilizing actin polymers, dynacortin also contributes to cortical viscoelasticity, which may be critical for establishing cell polarity. though not essential for directional sensing or motility, dynacortin is required to establish cell polarity, the third core feature of chemotaxis. 