We describe a novel behavior of epithelial cells, specifically a patterned, oscillating, basal epithelial myosin assembly and contraction. Previous work suggested that the polarized, basal F-actin bundles and their integrin-mediated attachment to the surrounding basement membrane function as a corset, implying a static structure, that constrains growth of the egg chamber to the poles thus promoting tissue elongation10–12
. Here we show that, surprisingly, the “corset” is dynamic and is composed of periodic assembly and disassembly of myosin on the actin filaments, providing an explanation for the source of the necessary force. Contraction transiently diminishes the basal surface area of the affected follicle cells, however this is not permanent, and we propose that it is not the most significant consequence of the contraction. Instead, the force generated by the contraction propagates inward toward the germline and opposes the outward force caused by growth. Since the oscillations occur near the center of the egg chamber, expansion is directed preferentially to the poles. The oscillations are not synchronized, therefore different cells contract at different times and over the course of more than ten hours generate a sustained inward force.
These observations raise a number of interesting questions. For example, what is the biochemical mechanism of the oscillation? Myosin activity oscillates in many (but not all) biological contexts. For example cardiomyocytes beat in cell culture. However this oscillation does not display or require cycles of myosin assembly and disassembly, is driven by ion fluxes, and is much more rapid (150 beats per min) than the oscillations described here (average period of 6–7 minutes). Intriguingly, myosin has intrinsic biochemical properties that could in principle lead to oscillating assembly and disassembly on this time scale34
. Three properties, in combination, could contribute to oscillation: the intrinsic mechanochemical cycle of actin binding, power stroke, and dissociation from actin; thick filament assembly-disassembly dynamics; and actin filament anchoring. Myosin II assembly into thick filaments is tension-dependent35,36
. That is, as myosin begins to assemble on actin filaments, it exerts force upon them, generating tension if the filaments are anchored. If the resistance is great enough, myosin will stall in the isometric state rather than completing its power stroke and disassociating from the actin filament 34
. As a consequence, more and more myosin filaments assemble over time. In addition, myosin binding to actin becomes highly cooperative in response to tension. Thus more myosin molecules bind and they dissociate more slowly when there is tension. For myosin to sense and respond to tension, the actin filaments to which it is bound must be prevented from sliding. During Dictyostelium cytokinesis, the critical actin anchor is the actin crosslinker cortexillin37
. However in principle anchoring to the plasma membrane could also serve this purpose. In Drosophila follicle cells we found that myosin assembles on F-actin stress fibers that are attached via integrin, talin and paxillin to ECM fibers. This likely serves the critical function of anchoring actin filaments so that tension is generated when myosin binds. So what causes disassembly and leads to oscillations? When enough myosin molecules assemble such that the force per myosin head becomes small enough, then the myosins can complete their power strokes and disassociate from actin, resulting in myosin thick filament disassembly. Stochastically, new myosins bind, exert force, experience tension, recruit more myosin and the cycle repeats.
Increasing the load against which myosin works would be expected to increase the number of myosin molecules that assemble as well as the length of time until the force per unit molecule reduces to the point of disassembly. In other words increasing the mechanical resistance should increase both the amplitude and period of myosin oscillations. Our results suggest that the actin-integrin-ECM interaction provides the load, and explains why decreasing follicle cell-ECM adhesion reduces both the period and amplitude of the oscillation and why enhancing cell-ECM interaction increases both. This explanation is also consistent with the observation that the assembly-disassembly cycle that occurs during ventral furrow formation in the embryo has a shorter period (~1 minute). In this case the cycle occurs on the apical side of the cell where there is no basement membrane to provide mechanical resistance. Although there may be additional components to the oscillation mechanism, these elements would be sufficient in principle to cause oscillating myosin assembly and disassembly.
In contrast to most previously studied morphogenetic processes, in which cells change the shape of a tissue by altering their own geometry, follicle cells undergoing this basal contraction do not change their own shape permanently, but rather generate forces that constrain the shape of the underlying tissue (). Another morphogenetic process that involves two cell layers is branching morphogenesis of the developing mammary gland38
. In this case outer myoepithelial cells may help sculpt the underlying glandular epithelium constraining growth toward the terminal end buds. It will be of interest to determine whether basal actomyosin activity in the epithelial layer also contributes to the morphogenesis of this or other organs and tissues where expansion is constrained.
Figure 8 Model of tissue elongation controlled by basal actomyosin contraction Schematic representation of the distribution of molecules controlling oscillating basal contraction in an individual follicle cell and the organization of contractile forces into a (more ...)
The observation that this oscillation shares some characteristics with other actomyosin oscillations, such as that occurring during apical constriction, and yet differs in numerous respects, suggests that an intrinsic oscillator is subject to tissue specific regulation. This allows the oscillations to occur in some cells and at some stages of development but not others, near the apical cell surface in some cells or the basal side in others, and connected to a ratchet in some cells but not others. In addition, the period can be regulated in a cell-type specific manner by adjusting the resistance against which the motor pulls. In each cell type where it has been described, the observation of oscillations came as a surprise since intuitively a static contractile force might seem to suffice. Whether or not oscillation is essential remains to be clarified. In any case, a complete understanding of the temporal and spatial patterning, subcellular localization, and tuning of the oscillations will be necessary in order to realize the goal of reconstituting normal organ shapes and tissue architectures in vitro.