Lymphocyte migration in vivo
relies on coordinated regulation of actin polymerization and branching, as suggested by work on T cells deficient in upstream regulators of Arp2/3 such as WASP27
, and Coronin-1A29,30
. On the other hand, the role of class-II myosin during interstitial T cell migration was previously unknown. Our work is the first indication of a requirement for MyoIIA function in lymphocyte migration and trafficking in vivo
. Importantly, it provides novel insight into the extent and dynamics of surface contacts as important factors regulating 3D lymphocyte migration.
Microfabricated channels enabled us to recapitulate some aspects of environmental confinement that lymphocytes may encounter in vivo
and permitted us to demonstrate the existence of a confinement optimum for amoeboid motility. The presence of integrin ligands had only a limited effect on this optimization, in contrast to what is predicted from the haptokinetic model and in agreement with recent studies of leukocyte migration in vivo7,9
. Indeed, faced with low adhesive substrates, amoeboid cells can adapt by increasing actin polymerization rates during protrusion to compensate for reduced force-coupling31
. This is also consistent with the idea of ‘chimneying’ described in cells crawling between adjacent coverslips32
Under ideal confinement, we observed migration speeds greater than 20µm/min; these are higher than average speeds but equivalent to the fastest instantaneous speeds reported for motility in lymph nodes. This could be due to the fact that individual microchannels provide a consistent and in some cases optimized (i.e. ~8µm channels) environment for T cell migration. In contrast, within lymph nodes, lymphocytes likely encounter varying microenvironments with different characteristics which may modulate motility speeds and limit peak velocities.
Furthermore, in the absence of MyoIIA function T cells can’t achieve optimal motility within microchannels, likely due to hyper-adhesion and spreading which cause increased friction and drag. In this light, MyoIIA regulation of cortical tension and adhesion can modulate the ability of lymphocytes to adapt their morphology to optimize their interaction with the environment during motility.
We propose that the motility optimum is reached at confinement levels where three things coincide. First, the ability to contact and exert force on multiple adjacent surfaces is maximized, optimizing frictional coupling without the need for highly specific protein-protein interactions. Second, inefficiencies from actin-based protrusion along incorrect axes are minimized. Third, the cell can restrict the extent of cortical contact with the substrate, minimizing excessive drag. Under extreme confinement, the size and rigidity of the nucleus may limit how thin the cell can become, forcing the cortex to drag along even when not in use for force-coupling. It has previously been proposed that MyoIIA might overcome such nuclear-induced drag when dendritic cells migrate through dense collagen matrices7
. That report, however, speculated that MyoIIA functioned as a ‘squeezing’ force which may be a separate function for this motor.
Morphologically, the ‘walking’ mode facilitated by MyoIIA utilizes multiple contacts that may be somewhat similar to the ‘eupodia’ described in Dictyostelium33
. We previously showed by molecular imaging that clusters of MyoIIA moved inward towards the center of these contacts and that this movement was coincident with the extinguishing of the contact and release from the substrate14
. Our data is in agreement with work showing that 2D motility in the amoeba Dictyostelium
relies on myosin-II contractility to provide cortical tension and de-adhesion from the substrate34
. Further evidence for MyoIIA in disassembling actin and thereby potentially functioning to extinguish cortical adhesive contacts with the substrate has recently been reported35
. A ‘millipede’ motility mode during migration along the inner face of HEVs36
may also be a variant of this ‘walking’ mode.
A general theme emerging from our work is a role for MyoIIA in controlling adhesion independently of substrate composition. Thus, T cells lacking MyoIIA over-adhere to HEVs, other T cells, and artificial substrates, whether coated with integrin ligands or blocked from integrin binding. This is partly consistent with reports suggesting that MyoIIA plays a role in uropodal detachment from highly adhesive integrin ligand-coated 2D surfaces15,16
. This has been proposed to involve MyoIIA linkage to LFA-1 to mediate de-attachment, without affecting LFA-1 affinity regulation15
. While not excluding such a possibility, we prefer the interpretation that MyoIIA’s role in adhesion may be indirect, and not strictly linked to integrin adhesion. The specific effects that were confined to the uropod in the previous studies could be due to the specific adhesive properties of the surfaces, cells, or drugs used in these different experimental systems.
In sum our data suggests that the increased turning and reduced velocity of MyoIIA cKO T cells in vivo may be determined by a diminished capacity to limit contacts with their environment. We did not find evidence that the FRC network was a substrate for this increased adhesion in vivo. On the other hand, our results indicated that the over-adhesive properties of MyoIIA-deficient T cells can cause an abnormal level of attachment to other T lymphocytes. In the lymph node, even brief spreading of the cell cortex of one T cell onto the cortex of another, particularly when moving with opposite trajectories, would oppose efficient forward movement.
While it has been reported that loss of MyoIIA can affect neutrophil polarity37
, and alter T cell morphology13
, our data suggests that loss of polarity is not the primary defect of MyoIIA-depleted T cells. In particular, within microchannels we did not observe excessive directional changes of MyoIIA-inhibited cells, and the reduced response to a chemotactic gradient is likely secondary to their over-adhesion.
Overall, our findings suggest that by limiting surface adhesion and providing contractile force, MyoIIA optimizes lymphocyte crawling efficiency within the multiple environments that T cells must pass through during their trafficking. In contrast to an integrin-only mediated switch in motility rate as predicted by haptokinetic models, we propose a switch in motility rate affected by the density (degree of confinement) of the tissue and by modulation of MyoIIA function. Such a motility switch for T cells might also occur during early activation which leads to phosphorylation of the myosin heavy chain in response to calcium signaling13
. This can result in the reorganization of myosin filaments38
, leading to the regulation of the ability of MyoIIA to crosslink and contract the actin network. Modulation of MyoIIA activity can therefore be considered as an important regulator of T cell trafficking and potentially surveillance by modulating cell stopping.