Isotropic cell spreading is a process during which a cell exhibits a small number of motility modules in coordination with two sharp transitions in global behavior (summarized in ). Detailed characterization of the motility modules during spreading (summarized in ) reveals that they are similar to those observed during more general motile phenomena. For instance, cells can exhibit membrane blebbing during mitosis 
, development 
, and cancer cell movement 
. Lamellipodial dominated motility very similar to P1 continuous spreading has been observed in post-mitotic cell spreading 
and keratocyte migration 
, as well as in tumor-derived epithelial cell lines exposed to epidermal growth factor, which undergo a two minute long period of rapid actin polymerization 
. Furthermore, we showed previously that periodic contractions similar to P2 were present in migrating fibroblasts and endothelial cells, and is one of the most fully understood motility modules in migrating cells 
. Thus, the quantitative characterization of spreading motility modules can provide an important aid in comparing mathematical models of motility 
to experiment. In addition, we have begun the development of a set of kinematic (–&), mechanical () and molecular (,) fingerprints for different motility modules that, combined with an understanding of the molecular machines that contribute to those motility modules 
, will allow us to probe the molecular-level function of a given perturbation based on our relatively low resolution, high-throughput, quantitative spreading assay. Such an approach could provide fast, highly interpretable functional screens for chemical libraries, siRNA libraries 
, or tumor cells.
Correspondence between spreading phases and motility modules.
Properties of Motility Modules.
Spreading assays owe their interpretability to the homogenous nature of motility during isotropic cell spreading. Sharp temporal transitions occur between phases, and we have shown that this transition is the result of a rapid shift in the combination of motility modules exhibited. In P0, we observed that cells exhibit a filopodial motility module or a blebbing motility module, but not both, and that the phenotype was dependent on Rho-kinase activity. A sharp transition from blebbing in P0 to the continuous protrusion motility in P1 was observed, with very little overlap between blebbing and continuous protrusion. However, in P1, while many untreated cells contained small regions that did not exhibit continuous protrusion, these regions had sharp boundaries and did not alter the behavior of the continuous protrusion proceeding on either side. Upon cytochalasin D treatment, continuous protrusion in P1 decreased in edge protrusion speed and regions of quiescence were formed. However, the boundary between the regions of continuous protrusion and quiescence regions were generally very sharp, with certain regions exhibiting continuous protrusion while other regions remained quiescent. In P2, we found that discrete regions of the cell exhibit protrusive motility modules, and that these regions can maintain coherence over long times and distances (). Protrusive motility in P2 can consist of periodic contractions, continuous protrusion, and large membrane ruffles, but each maintains the distinct molecular identity of a motility module (). Together, these observations support our hypothesis that each motility module represents a dynamic state of the cytoskeleton that is stable over a range of biochemical and biophysical conditions, but that when this range is exceeded, a transition to a different motility module occurs. In future work, the velocity map could be replaced by a module map, in which velocity information in the velocity map would be replaced simply by an indication of the motility module exhibited at that particular location and time on the cell edge. This high level of data reduction and abstraction would be highly advantageous for statistical analyses of cytoskeletal behavior.
The hypothesis of discrete motility modules is consistent with our proposed model of hierarchical motility regulation 
. At the lowest level of the hierarchy are actin and proteins that directly modify actin dynamics (e.g., actin polymerization factors such as VASP, myosin motors). Particular complexes of these molecules give rise to stable states of actin dynamics, resulting in a stereotypic kinematic signature of the cell edge. At the higher levels of the hierarchy are molecules that lead to global organizational changes such as those observed in transitions between spreading phases and those factors that control the generation of traction forces. Candidate molecules are the Rho family GTPases 
or calcium signals induced by integration of chemical or mechanical signals 
, both of which exhibit abrupt changes in activity or concentration in response to cell spreading and may be involved in the global regulation of spreading phases. However, motility modules can also be organized locally, as occurs during polarization and migration when lamellipodial contractions and ruffling are switched on only in the protruding regions of the cell. Such modular regulation has been observed in the switching between migrational modes in neurons 
, amoeba 
, immune synapses 
, tumor cells 
, and Dictyostelium 
. That these many different cell types share many motility modules in common suggests that these stable states of cytoskeleton dynamics are relatively conserved, and that differences between cells is primarily achieved at the higher levels of the regulatory hierarchy. In this paper, we have provided the biophysical and biochemical characterization of motility modules required to test this hypothesis.
Generation of traction forces on the substrate was shown previously to be primarily dependent upon myosin II A and B isoforms 
. We find that relatively little force is generated during phase 1, which is consistent with the slow rate of actin retrograde flow 
and the ability of cells to spread in this mode with no requirement for further integrin binding 
. The rapid rise in force exposes a major change in cell state from phase 1 to phase 2. We postulate that this transition reflects a global activation of myosin through a RhoA kinase pathway, although further experimentation is required to differentiate such a biochemical hypothesis from a more biophysical mechanism such as a sudden increase in membrane tension as the cell spreads. Additionally, based on our pharmacological perturbations, we found intriguing difference between the Rho-kinase and MLCK myosin II pathways in P0. However, none of these cells exhibited an impaired ability to initiate P1; indeed, cells treated with impaired or ablated myosin II activity were able to initiate P1 
. We propose that while myosin force generation in P0 plays a role in promoting early adhesion formation () and could be of functional importance under conditions where fluid flow shears the cell, integrin signaling alone is sufficient to initiate P1.
The property of continuous protrusion during P1 represents an experimental system that can be used to test biophysical models for how actin generates edge protrusion (see Appendix S1
). In general, testing the predictions of models against in vivo
experiments is difficult because cells rarely undergo large scale, steady-state protrusion. Therefore, most of the experimental constraints on these models are based on in vitro
data. However, during P1 continuous spreading, the actin cytoskeleton is in a spatially homogenous protrusive steady-state. These protrusions are independent of substrate adhesion 
, exhibit much less traction force on the substrate, and are independent of myosin II activity 
. Thus, P1 continuous spreading represents a motility module in which actin polymerization against the membrane is the dominant motile event, making P1 spreading an ideal cell state on which to test the predictions of mathematical models of cytoskeletal protrusion.
While the study of the continuous protrusion motility module can experimentally isolate actin mechanochemistry, the lateral propagation of activity over a long-distance in both P0 blebbing and P2 periodic contractions make these systems ideal for studying the biophysical nature of these propagations. Theoretical models have suggested that lateral propagation can be generally explained by a combination of myosin motors and polymerization-stimulating membrane proteins aggregating in regions of convex membrane curvature 
. Our results suggest that this model may not apply in the case of lateral propagation of blebbing motility, since high concentrations of CD would likely disrupt polymerization from a membrane bound protein but do not disrupt the lateral propagation of blebbing in P0. Interestingly, myosin II is required for normal function of both of these motility modules, though blebbing motility depends on Rho kinase and not the myosin light chain kinase that was previously shown to disrupt periodic lamellipodial contractions 
. At present however, we do not understand the cause of lateral propagation in P0 blebbing.
In a recent editorial on the state of systems biology, George Church asks how the rest of biology can “reach the enviable status of bioinformatics and crystallography?” and suggests that sharing data is a crucial step towards achieving this goal 
. All data for the cells analyzed in this paper, along with their corresponding two-dimensional velocity maps and the open source software CellMAP, are available at http://cellmap.sourceforge.net
. In the spirit of projects such as the Open Microscopy Project (http://www.openmicroscopy.org
), we hope that making our data and software freely available will provide a model for a collaborative future in the field of cell motility and guide the way to a systematic approach for storing and distributing cell image data, such as already exists in the fields of protein biophysics.