Cell migration plays an important role in tumor progression [1
]. During invasion and metastasis, migration is driven by soluble extracellular cues like epidermal growth factor (EGF). EGF is a well-known chemoattractant [2
]; however, uniform doses also stimulate chemokinetic responses. EGF’s control of cell motility originates from its regulation of adhesion and protrusion [5
]. This occurs through altering adhesive attachments called focal adhesions (FAs) [8
] as well as actin cytoskeleton organization.[8
] The response to EGF at the level of cell migration is dose dependent, but there exists a range of maximal stimulation concentrations. Often migration saturates at 2–10 nM EGF [2
], but some of the studies showed an inhibition of migration at EGF concentrations >2-10 nM [7
]. This is in agreement with other work demonstrating that in certain contexts, EGF can inhibit migration [16
]. Within each study there is wide diversity in migration behavior, even among cells observed during the same experiment [14
]. Interestingly, the distribution in migration speed and persistence time appears to be dependent on EGF stimulation [18
], suggesting that EGF controls not only the mean response, but also the amount of cell-to-cell variability. Cell-to-cell variability has been widely observed, and has drawn much attention due to its influence on physiology [19
] and pharmacology [21
]. Consequently, mathematical models [19
] have been used to show that even small changes in the distribution of protein concentrations yield enhanced wound healing or metastasis due to the selection of an optimal subpopulation. When the subpopulation is defined based on migration speed, it will not only be beneficial to examine the distribution of protein concentrations, but also higher level characteristics like focal adhesion (FA) properties and cytoskeletal dynamics.
FAs are dynamic, macromolecular structures that serve as both mechanical linkages and centers of intracellular signal transduction [22
]. They assemble as nascent adhesions, mature into focal complexes, focal adhesions and fibrillar adhesions and disassemble [23
]. Consequently, FAs exhibit different morphological maturation states throughout their lifetime and this is thought to regulate their behavior. For example, small, nascent FAs, transmit strong forces and serve as traction points for propulsive forces to move the cell body forward [25
]. They also generate signals for protrusion by activating actin accessory proteins [27
]. Under tension, these small FAs can mature into larger focal complexes, focal adhesions and fibrillar complexes with different force transmission characteristics and propensities for protrusion signaling [32
]. Several morphological characteristics have been used to predict traction force and cell migration speed including FA protein density, number per cell, sliding speed, lifetime, size and elongation [22
]. These morphological characteristics have begun to be quantitatively measured [36
] and the distributions properly quantified [38
]. However, their direct correlation to migratory states as well as their response to extracellular cues like EGF is unknown.
Protrusion is mediated by actin polymerization, whereas retraction is driven through myosin II activity and actin depolymerization [39
]. Protrusion and retraction can either occur continuously in spatially confined regions as in keratocyte migration or it can occur in cycles or waves of protrusion that move laterally along the edge [41
]. This has been characterized in several cell types when cells are either spreading [44
] or migrating [41
]. In fact a recent paper has shown that slower migrating keratocytes employ lateral protrusion waves [43
]. While the timing of the cycles and the propagation of the waves is dependent on intracellular pathways, very little work has been done to examine how protrusion is quantitatively altered in response to extracellular stimuli like EGF.
To understand the relationship between EGF-stimulated cell migration, FA properties and protrusion dynamics, we imaged metastatic (MTLn3) and non-metastatic (MTC) cell lines. We analyzed the cell migration speed and persistence under various EGF stimulation conditions and found that EGF moderately increased the median migration rate and persistence of MTLn3 cells, whereas it had no significant effect on the speed and persistence of MTC cells. Interestingly, higher concentrations of EGF broadened the distributions and increased the coefficient of variation of both the migration rate and persistence of MTLn3 cells, but not MTC cells. When data was binned based on EGF stimulation conditions, FA intensity and FA number per cell showed the largest difference among stimulation groups. FA intensity decreased with increasing EGF concentration and FA number per cell was highest under intermediate stimulation conditions. No difference in protrusion behavior was observed. However, when data was binned based on cell migration speed, FA intensity and not FA number per cell showed the largest difference among groups. FA intensity was lower for fast migrating cells. Additionally, waves of protrusion tended to correlate with fast migrating cells. Consequently, low FA intensity and waves of protrusion are markers for fast migrating cells, but these characteristics are only partially predictive of EGF stimulation conditions because of the large cell-to-cell variability in response to EGF.