Our observations support a working model for adhesion assembly during cell migration (). Nascent adhesions assemble in the lamellipodium in a single concerted step as the protrusion advances. The assembly rate is linked to the protrusion rate and probably to actin polymerization. The nascent adhesions are stable only within dendritic actin and disassemble as the wave of depolymerizing actin at the rear of the lamellipodium passes by them with the advancing protrusion. Both the assembly and stability of the nascent adhesions in the lamellipodium are myosin II-independent. When protrusion pauses, a subset of nascent adhesions grow and elongate centripetally from the base of the lamellipodium. The elongation is directed by actin filaments. α-Actinin associates with the emerging actin filaments and organizes and orients them centripetally. These α-actinin–actin filaments function as a template for the hierarchical addition of other adhesion components. The actin crosslinking properties of myosin II also have a major role in the formation of the template and initial stages of adhesion maturation.
Figure 8 Working model for adhesion assembly, turnover and maturation (a) During protrusion, adhesions initially assemble as punta (blue circle) in the lamellipodium (gray band); their formation is driven by or linked to actin polymerization. Following assembly, (more ...)
Our data show that nascent adhesions assemble in the lamellipodium as diffraction-limited puncta and then undergo myosin II-dependent maturation near the lamellipodium–lamellum interface. The prominent, readily visualized adhesions (for example, focal complexes reported to be present at the lamellipodium–lamellum interface11,12
) differ from nascent adhesions as they are larger, more stable and myosin-II-dependent. The CHO cell is particularly useful for studies of adhesion assembly as the nascent adhesions have long lifespans and only a fraction of them mature. Presumably, this is due in part to lowered or localized myosin II activity. In contrast, nascent adhesions in MEFs or U2OS cells reside in the dendritic actin for only a few seconds (Supplementary Information, Fig. S4
, Movie 12
and data not shown); most stabilize and mature at the lamellipodium–lamellum interface. Myosin II is a determining factor as blebbistatin inhibits the probability of adhesion maturation and promotes the assembly and turnover of the nascent adhesions in both cell types (Supplementary Information, Fig. S4
and data not shown). Conversely, MIIA overexpression in CHO.K1 cells inhibits protrusion and increases the probability that nascent adhesions grow and elongate, which occurs almost immediately after they form14
(Supplementary Information, Movie 13
). In all of these cells, however, the elongating adhesions in protrusions arise from nascent adhesions. Previous observations also implicate myosin II in adhesion assembly12,20
and the periodic interruption and retraction of protrusions12,14,30
The formation and stability of nascent adhesions within the lamellipodium, the correlation between the assembly and protrusion rates, and the inhibition of both protrusion and nascent adhesion assembly by cytochalasin-D suggest that adhesion assembly is mechanistically and kinetically linked to actin polymerization in the lamellipodium; this is also observed when net protrusion is driven without myosin II mediated retrograde flow. Two components of nascent adhesions, vinculin and FAK, interact with the Arp2/3 complex, which nucleates actin polymerization within dendritic actin and thereby provide a potential mechanism for the coupling31–33
. A recent study showed actin-polymerization-based, protrusion-independent lateral movement of integrins to filopodia-like ripples in the lamellipodium34
; however, they did not report a similar mechanism for adhesions outside of ripples.
The lamellipodium–lamellum interface emerges as a critical region where adhesion fate is determined. Nascent adhesions either disassemble or mature as the dendritic actin passes by them. It is also the region where the dendritic actin turns over35
, suggesting that nascent adhesions are physically linked to dendritic actin and disassemble in response to its turnover.
We present a mathematical model, which assumes that an adhesion precursor binding to the dendritic actin is a limiting step for adhesion assembly, and that the adhesion disassembly is mechanically coupled to dendritic actin disassembly (Supplementary Information, Text
). We also assumed that the Arp2/3 mediated branching takes place near the leading edge, either on adhesions, where the branching points are firmly anchored to the substratum, or in the immediate vicinity of adhesions (so that ‘daughter’ filaments branch off ‘mother’ filaments anchored to the substratum). The solutions of mathematical equations derived from these assumptions reproduced our quantitative observations. That is, as the branching rate is almost constant near the leading edge, total dendritic actin filament length builds up almost linearly from the leading edge towards the rear. Adhesion assembly lags by the characteristic time of the precursor binding to actin behind the front of the actin band. Actin–ATP hydrolysis, cofilin action and decrease of the branching activity behind the leading edge determine the rear of the dendritic actin band, where both actin and adhesions disassemble in synchrony (). Consistent with our data, the model further predicts that the adhesion assembly rate is proportional to the leading edge extension rate, independent of the disassembly rate from the speed of protrusion, and correlates with the inverse duration of the adhesion stability phase with the speed of protrusion.
Nascent adhesions at the lamellipodium–lamellum interface can also grow and elongate, presumably in response to a changing or different organization of actin. Elongated, centripetal adhesions at the periphery are a hallmark of maturing adhesions22,36
. Short, linear actin filaments emanate from this region and provide a template for the maturation of nascent adhesions. These filaments could arise from either the reorganization of existing filaments in the dendritic actin or from local polymerization.
α-Actinin and myosin II are essential for the formation and organization of the actin template. In the absence of α-actinin, actin filaments are abnormally short, discontinuous and misoriented. α-Actinin also positions adhesions along actin filaments as adhesion components in α-actinin
knockdown cells are no longer restricted to the ends of actin filaments and appear as puncta spread along the entire filament. α-Actinin has been reported previously to participate in the later stages of adhesion maturation by forming large stress fibres and adhesions at their ends29,37,38
. An earlier observation that α-actinin is a late entry into the larger adhesions was made with wide-field optics, which would not have seen the templates and early events described here38
Myosin II is also required for the growth and elongation of nascent adhesions. Neither actin templates nor the elongation of nascent adhesions are observed in MIIA-deficient or inhibited cells14
. Several reports suggest that myosin II-mediated contraction has a major role in the maturation of adhesions by tension-induced alterations in the conformation of adhesion-related proteins14,15,24,39,40
. Our study shows that the actin crosslinking activity of myosin II is important in the initial stages of adhesion maturation, presumably by organizing and clustering actin and actin-associated adhesion components. Thus, although myosin II-mediated contractility seems to promote the formation of thick actin filament bundles and large adhesions at later stages of maturation, the contractile activity could function synergistically with myosin II-mediated actin crosslinking at early stages by organizing actin filaments. Myosin II-mediated actin crosslinking can also transmit distally generated actomyosin contractility to adhesions. Others have also ascribed roles for myosin II crosslinking: an ATPase-deficient myosin II restores cortical integrity in Dictyostelium discoideum41
; a motor-impaired MIIB mutant rescues hydrocephalus in MIIB
; the bundling function of myosin in adhesion assembly was proposed previously43
In summary, the data presented here provide new insights into the mechanism of adhesion assembly. They identify and characterize a new class of adhesions, ‘nascent adhesions’, which reside in the lamellipodium and serve as precursors for other adhesions in the protrusion. Moreover, they clarify the role of myosin II in adhesion maturation, lead to a ‘template’ model for centripetal adhesion elongation along actin–α-actinin filaments, and demonstrate the importance of the actin-bundling activity of myosin II.