We have shown that the morphological oscillations of rounded cells result from compression and dilation of the coupled membrane-cortex layer. The PM has a limited capacity for areal extension (<4%) before rupture (Sheetz et al., 2006
) but a high degree of plasticity with a low bending energy (Lipowsky, 1991
; Farsad and De Camilli, 2003
), which makes it a good candidate for folding. The actin cortex is capable of supporting a variety of shapes (Michelot and Drubin, 2011
), it being a dynamic, signaling-regulated structure where myosin and other actin binding proteins determine the network architecture. We demonstrated for the first time that the periodic protrusive phenotype results from membrane–cortical actin traveling waves. The observation of traveling cortical waves is novel and may represent, for example, how rounded cells choose directionally in response to chemoattractants. The cortical wave may also be analogous to actin wave propagation in the lamellae of leukocytes or fibroblasts (Dunn and Zicha, 1995
; Servant et al., 1999
; Driscoll et al., 2012
We envision that the cortical actin traveling wave plays at least two important roles in regulating net migration of a periodically protruding cell. First, the traveling wave established the position of the future protrusion by releasing the membrane-cortex layer from the heavily folded region (). Second, the traveling wave directs the cytosol flow used to inflate the protrusion by transiently positioning the contractile region of the cortex that drives the flow; this flow, in turn, determines the speed and, to a certain extent, the size of the protrusion.
Figure 10. Schematic of hypothetical mechanisms for the traveling wave that leads to periodic protrusions. (A) Postulated locations on the periphery of a cell exhibiting a cortical actin traveling wave where feedbacks and folding dynamics occur. (B) Schematic representations (more ...)
presents a schematic view for how the cortical traveling wave may be related to net cell migration. If there is sufficient adhesion in the central region of the cell proximal to the contractile region to promote traction, the rounded protrusion, inflated by cytosolic flow, will move the center of mass of the cell in the direction of the protrusion (, left). Subsequent movement of the contractile wave may result in rupture of the initial adhesive region but for migration to be sustained another region of transient adhesion must be established forward of the initial adhesion (, right). Note that the cartoon in represents the case when the traveling wave propagates in the direction parallel to the substrate. In reality, cortical waves can travel in any fixed direction along the cell periphery, which may necessitate a modification of our simple cartoon view. For example, if the wave is traveling in the plane normal to the substrate, it would create visual similarity with the “walking” cell ( and Video 8).
For periodic protrusions to result in net migration, cell adhesions to the substrate need to be dynamic and coordinated with the movement of the protrusion. Without sufficient adhesion, the cortical traveling wave will propagate around the periphery without producing net migration and if cells are too adherent, they will oscillate without net displacement. Additionally, to create a directionality, some form of cell polarity must be established either externally (chemotactic/ haptotactic gradient) or internally (for example, by asymmetrically distributed adhesions or cortical flexibility; Lorentzen et al., 2011
How might the cortical traveling wave be propagated in terms of the signaling pathways? Microtubule status clearly plays a role in the oscillatory phenotype as deliberately depolymerizing them increases the amplitude of the oscillations (Costigliola et al., 2010
). Whether this is caused solely by the release of guanine nucleotide exchange factor H1 to activate RhoA or caused also by a structural effect remains to be determined. In general, traveling waves require positive feedback and some form of negative feedback. We assume that a positive feedback loop operates globally to increase cortical density and folding until a local negative feedback mechanism rapidly dissipates cortical density and folding. These loops are hypothesized to be initiated through phosphoinositide metabolism in the regions depicted schematically in . Indeed, our preliminary studies with inhibitors of PI3K and PLC (Wortmannin, PI3K-IV, and U73122) indicate an important role for inositol lipids processed by these enzymes. In general, phosphoinositides provide regulation of the subcellular localization of many scaffolding and actin severing proteins that are involved in the interplay between the actin cytoskeleton and PM. In turn, these proteins can provide feedback from the actin cortex to the phosphoinositides and regulate their spatial distribution through restricted diffusion or sequestration (Sheetz et al., 2006
; Hilgemann, 2007
; Golebiewska et al., 2008
). Thus, spatiotemporal regulation of the membrane concentration of phosphoinositides might produce the control required to sustain the periodic protrusive phenotype. This regulation would be coupled to the RhoA–MLCK–MLCP–myosin II pathway, known regulators of the oscillatory phenotype, via guanine nucleotide exchange factors and GTPase activating proteins.
The notion of compression and dilation of the membrane-cortex couple has several antecedants. It was previously suggested that cells use surface, reserved in membrane “protuberances,” for spreading and initiating cell locomotion (Erickson and Trinkaus, 1976
; Gauthier et al., 2011
). The migration of fibroblasts was shown to require “retraction-induced spreading” (Chen, 1981
), whereby excess cell surface accumulated during tail retraction was stored in dorsal folds, which relaxed when the cell underwent the next round of lamellipodial spreading.
Periodically protruding cells show several characteristics that are remarkably similar to cells undergoing the blebby-type amoeboid migration in which rounded protrusions prevail. 3D computer reconstructions of Lifeact-GFP fluorescence ( and Videos 7 and 8) also showed morphological similarity to the blebby type of amoeboid migration (Mierke et al., 2008
; Friedl and Wolf, 2009
). In addition, both phenotypes lack strong focal adhesions and stress fibers that pull on the substrate to generate movement (Friedl and Wolf, 2010
). Both have been shown to be regulated by the phosphoinositides (Vemuri et al., 1996
; Saarikangas et al., 2010
) and the Rho family of small GTPases, in particular RhoA, plays a major role, whereas Rac appears to be less important (Sahai and Marshall, 2003
; Costigliola et al., 2010
; Paňková et al., 2010
). The fact that periodically protruding cells can migrate adds credence to this phenomenon being a model for the blebby type of amoeboid locomotion that is tractable from both experimental and theoretical points of view (Wang et al., 2012
We presume that compression–dilation and classical blebbing are two distinct mechanisms that may be related in the following way. Membrane-cortex relaxation and unfolding is a global cell process to be contrasted to more local blebbing (Charras and Paluch, 2008
; Bergert et al., 2012
). The process of compression–dilation requires a highly flexible and elastic cortex, capable of rapid and global transformation and it is likely that microtubule depolymerization is one of the possible ways to achieve this flexibility. In contrast, if the cortical cytoskeleton is more rigid, global shape transformation through the compression–dilation mechanism would be less likely but blebbing could still occur.
The compression–dilation mechanism provides a simple but efficient way to transform cell shape and offers the advantage that the position of the transformation or protrusion can be spatiotemporally regulated in a precise fashion. In addition, in both developmental and metastatic cancer contexts, amoeboid-like cells must negotiate the interstices of the filamentous structures composed of extracellular matrix proteins. Cell movement through these structures in all likelihood requires greater structural strength than would be furnished by an initially unsupported PM bleb but could be provided by the protrusive PM being supported by a subjacent cortical layer. We anticipate that the cortical compression–dilation hypothesis will find applicability not only for cell migration but for various types of cell shape transformations, including those involved in cell division, differentiation, and spreading