Myosin II plays important roles in regulating a multitude of cellular activities, which contribute to a diverse array of biological processes. It is fascinating that for all the myosin II-dependent cellular activities that have been described, the basic molecular functions of myosin II are only two, namely, the assembly of monomers into filaments and ATPase-driven motor activity (Conti and Adelstein, 2008
). How cells spatially and temporally regulate these two functions appropriately to orchestrate biological processes as different as cytokinesis and migration has long been a subject of scientific inquiry. In this study we sought to characterize how the isoform-distinct subcellular distribution of IIA and IIB seen in migrating cells is regulated, as this difference between the isoforms has been proposed to contribute to proper cell motility.
The results presented here demonstrate that the C-terminal region of the myosin heavy chain plays a primary role in directing isoform-specific distribution. This was a surprising finding because previous results suggested that the different motor activities of IIA and IIB led to their distinct localizations. For example, IIA is frequently observed to move faster and further into protrusions than IIB, and the former is known to exhibit higher motor activity than latter (Kelley et al., 1996
). Moreover, IIA and IIB no longer exhibit distinct distribution in cells treated with small molecules that inhibit myosin II motor activity (Kolega, 2003
). However, our observation that the chimera IIA-Bacd, a molecule that is ~91% IIA and 9% IIB by amino acid sequence, distributes in a manner highly similar to IIB, with the same being true for the inverse chimera, IIB-Aacd, strongly suggests that functions of the extreme C-terminal tail region supersede differences in motor activity in controlling isoform-specific distribution.
That the C-terminal region contributes to myosin II distribution in an isoform-specific manner should not be surprising, because within this region of the heavy chain the amino acid sequences of IIA and IIB diverge most significantly and protein binding and phosphorylation events occur that have been suggested previously to be important factors regulating myosin II distribution (Li and Bresnick, 2006
; Rosenberg and Ravid, 2006
). However, our chimeric data are the first to suggest that this region not only contributes to but also plays the primary role in determining how IIA and IIB distribute throughout a moving cell. It is important to note, however, that although IIA-Bacd is most similar to IIB in TX-100 solubility, turnover properties and subcellular distribution, the two are not identical. For example, although the TX-100 solubility of IIA-Bacd is closer to that of IIB than IIA, the solubility of the chimera was still significantly greater than that of IIB. One explanation for this is that the C-terminal region used here only includes one of two described ACDs (Nakasawa et al., 2005
). Perhaps if the swapped regions were expanded slightly to include both of the ACDs, the similarities would be even greater. This supposition is supported by our observation that the chimera in which the larger region was swapped, IIA-Btail, functioned nearly identically to IIB.
Further evidence that a larger C-terminal region would induce the IIA-Bacd chimera to behave more like full-length IIB comes from studies examining the expression of IIB truncation fragments. For example, when expressed in cells, a fragment of the C-terminal portion of the IIB heavy chain that is slightly larger than, and fully encompasses, the C-terminal region used here was shown to disrupt the function of IIB, leading to cytoskeletal phenotypes similar to those that occur upon depletion of IIB (Sato et al., 2007
). In contrast, when we expressed the C-terminal regions of IIA or IIB from this study in cells neither produced any obvious effects on cell morphology (data not shown), consistent with a previous report in which a similar, slightly smaller fragment of IIB was expressed in cells (Ben-Ya'acov and Ravid, 2003
). Therefore, the C-terminal region from our study seems to impart isoform-specific function when in the context of a full-length chimera, but this region is not enough to disrupt the function of the full-length isoform when expressed as a fragment. Interestingly, in contrast to the discrepancy between IIA-Bacd and IIB, the chimera IIB-Aacd behaved very similarly to IIA. This may simply reflect that changes to the structure of the C-terminal region within the myosin heavy chain (i.e., swapping the domains between the isoforms) by default more easily lead to increases rather than decreases in solubility. Alternatively, IIB may be dually regulated by the C-terminal region and changes in RLC phosphorylation (see more on this topic below).
It is tempting to speculate as to how the C-terminal region regulates isoform-distinct distribution. One explanation is that in regulating each isoform's assembly into filaments, the different C-terminal regions of IIA and IIB control the pool of each isoform that is available to move into a new protrusion. Thus, when a new protrusion is formed in a migrating cell due to the extension of actin filaments, the nascent protrusion is temporarily devoid of myosin II until soluble myosin II molecules, filamentous myosin II being relatively stationary, redistribute there, although not to the very leading edge () because the compact nature of the actin meshwork in this region is thought to prohibit the movement of myosin II (DeBiasio et al., 1988
). Because nearly 90% of the cellular pool of IIA, compared with ~30% of IIB, exists in the soluble fraction, the concentration gradient for the movement of IIA into the protrusion is larger than that of IIB. Furthermore, it is relevant to note that in each of the cells types used in this study there is greater than 500 times more IIA than IIB at the mass level, whereas smaller ratios were observed in other cell types (Supplemental Figure S3). However, whether the dramatically different levels of IIA and IIB observed in the cell types used here contribute to their unique distributions is not clear at this time.
The above-mentioned idea raises the question, How do soluble IIA and IIB redistribute into the protrusion? FRAP data shown here, as well as in previous studies (Kolega and Taylor, 1993
), indicate that diffusion of soluble myosin II is quite rapid and could account for the movement of these molecules within the cell. For example, in Y27632-treated cells the fluorescence signals of both GFP-IIA and GFP-IIB recovered very rapidly and nearly completely after photobleaching. Moreover, the recovery curves of GFP-IIAΔacd and GFP-IIBΔacd, deletion mutants that lack the C-terminal region and thus the ability to assemble into filaments (Supplemental Figure S2), are highly similar to those of their full-length counterparts in the presence of Y27632. Thus, under conditions of decreased motor activity and/or decreased filament assembly IIA and IIB exhibit similarly rapid movement kinetics, consistent with the idea that diffusion of soluble, nonfilamentous molecules can account for rapid movements of these isoforms within the cell. However, because the diffusion rates of soluble IIA and IIB seem to be similar, diffusion alone cannot explain the enrichment of IIA over IIB in more anterior regions of protruding cells. So, because the diffusion rates of IIA and IIB are similar, if simple diffusion was the only factor then the ratio of IIA to IIB in the protrusion would be the same as the region from where they came. Thus, it is necessary to consider active movement as a mechanism of myosin II redistribution into protrusions, especially because diffusion of myosin II-sized molecules is limited in protrusions (Luby-Phelps et al., 1987
). However, the differences in the motor properties of IIA and IIB do not seem to be what controls their distinct redistribution because IIB-Aacd and IIB harbor the same motor domain and the former distributes more anteriorly than does the latter.
With regard to this issue, it is our interpretation that the main factor directing the differential distribution of IIA and IIB is not how these isoforms move into the protrusion but rather how much of each is available to move. Accordingly, the FRAP experiments demonstrate that in untreated cells the turnover of IIA within a given region occurs more rapidly and to a greater extent compared with IIB. Therefore, under normal conditions when soluble IIA and IIB move into a newly formed protrusion, effectively decreasing the amount of soluble IIA and IIB in the more posterior region, the re-equilibration of the ratio of filamentous to soluble IIA will be more rapid and occur to a greater extent than that of IIB in a given period. Thus, as IIA and IIB move out of the region directly posterior to the protrusion and into the protrusion, the pool of IIA in the more posterior region that is soluble and able to redistribute into the new protrusion will increase relative to IIB, facilitating the enrichment of IIA over IIB in the protrusion. A prediction from this model is that if protrusion ceases, IIB will eventually re-equilibrate with IIA in the protrusion. This prediction is supported by (also see Supplemental Video 1), in which mChe-IIA and GFP-IIB are observed to briefly overlap in a small region at the front of the migrating cell where protrusion is temporarily stalled, but then the two isoforms quickly separate again once protrusion is resumed.
Further support for the above-mentioned model can be found in previous studies demonstrating a correlation between myosin II assembly properties and distribution. In particular, a heavy meromyosin version of IIA, which dimerizes but does not form filaments, redistributes more quickly into protrusions than does full-length IIA (Kolega, 2006
). Moreover, a recent study examining the role of charge distribution within the C-terminal region in regulating IIB filament assembly demonstrated a correlation between filament assembly and IIB localization (Rosenberg et al., 2008
). Specifically, mutations of the charged regions in the C-terminal tail of IIB that decreased solubility in TX-100 relative to wild-type protein resulted in an increased tendency to accumulate in posterior regions of the cell, and vice versa for mutations that increased TX-100 solubility.
Although the primary role of the C-terminal region is thought to be regulation of assembly, we cannot rule out other ways in which this region could regulate isoform-specific distribution. For example, it is possible that as yet unidentified proteins selectively interact with the C-terminal region of IIA or IIB and shuttles or anchors the respective isoform to the appropriate cellular locale. Indeed, isoform-specific protein–protein interactions have been described to occur within this region. For example, the NHT of IIA exhibits a unique protein–protein interaction with the small calcium-binding protein S100A4 (Mts1), although the function of S100A4 binding also seems to be regulation of IIA assembly properties (Li and Bresnick, 2006
We also cannot rule out a role for RLC phosphorylation in isoform distribution. For example, the chimera GFP-IIA-Btail, which harbors the IQ domains and C-terminal region of IIB, exhibits solubility in TX-100 more similar to IIB than does GFP-IIA-Bacd. Because RLC phosphorylation is known to impact myosin II filament assembly, the above-mentioned result may indicate that the regulation of IIB solubility is multifactorial, involving regulatory events occurring both at the C-terminal tail and at the RLC. Interestingly, results from a previous study suggested that IIA and IIB may be differentially regulated by ROCK (Sandquist et al., 2006
). Moreover, it is shown here that Y27632 treatment increased the solubility of IIA and IIB to different extents, namely, IIA was nearly completely soluble after Y27632 treatment whereas ~25% of IIB remained insoluble under identical conditions. Thus, it is possible that tight spatiotemporal regulation of RLC phosphorylation within the cell may play a significant role in differential IIA and IIB distribution.
Conversely, it is perhaps not too surprising that the extent of the effect of Y27632 on the solubility of IIA and IIB is different considering the solubility of IIA in untreated cells is already much higher than that of IIB. However, the Triton-solubilities of IIA and IIB after blebbistatin treatment were much more similar, despite the basally higher level of IIA solubility. This discrepancy in the effects of Y27632 and blebbistatin is likely to reflect the fact that these drugs inhibit myosin II activity by different mechanisms. Y27632 decreases RLC phosphorylation via inhibiting ROCK, whereas blebbistatin directly binds ADP-bound myosin II, locking it in the low actin affinity state (Kovacs et al., 2004
). That blebbistatin produces a similar effect upon IIA and IIB solubility is consistent with previous studies demonstrating that the actin binding properties of IIA and IIB are similar (Kelley et al., 1996
; Kovacs et al., 2003
) and that blebbistatin inhibits the MgATPase activity of both isoforms equivalently (Straight et al., 2003
). The above-mentioned result thus serves as an important reminder that the solubility of myosin II in TX-100 may be modulated by more than just filament assembly but also by other factors such as specific binding to actin or even entrapment in actin networks. Clearly, much more work is needed to fully understand the mechanisms by which the C-terminal regions of IIA and IIB uniquely regulate the assembly of these isoforms, as well as how this region works in conjunction with phosphorylation, protein binding or other mechanisms yet to be discovered in regulating the differential distribution of IIA and IIB in migrating cells.