which binds to G-actin with 5 nM kd, and phalloidin, which binds to actin (17). These stores of nonfilamentous actin are found at the leading edge and are located adjacent to sites of actin polymerization and in the region of the cell where the ß-actin mRNA is also present. Possibly these sites could result from islands of ß-actin synthesis.
ß-actin is found at the leading edge of crawling cells. ß-actin does not substitute for muscle actin in either the formation of stress fibers (17) or myofibrils in cardiomyocytes (18). In addition, it seems to interact more tightly with certain actin binding proteins that may function at the leading edge of crawling cells. Ezrin (19), profilin (20), thymosin ß 4 (21), and L-plastin (22) bind more strongly to ß-actin than a-actin. A capping protein, ß-cap 73, may cap the barbed end in an isoform-specific manner (23). There is growing evidence that the Arp2/3 complex is required for nucleation of actin filaments at the leading edge (12, 24–27). If the Arp2/3 complex is the dominant nucleation activity at the leading edge, a possible preference for the ß-actin isoform by the Arp2/3 complex would require local synthesis of ß-actin to supply the preferred monomer for polymerization. Therefore, the localization of ß-actin synthesis at the leading edge may be functionally important for polarity and motility.
Localization to the Leading Edge of Motility-Related mRNAs. The localization of ß-actin mRNA may be representative of the localization of a family of mRNAs with related 3' UTR zip codes, many of which function synergistically at the leading edge. Proteins coded for by these mRNAs therefore might have related functions. We have analyzed the 3' UTRs of mRNAs, which code for proteins believed to have actin binding functions at the leading edge, for the presence of the zip code consensus sequence. This sequence GACUX7–38ACACC is found in ß-actin mRNAs known to target to the leading edge from all vertebrates. Besides ß-actin mRNAs, mRNA for Arp3 and myosin IIB heavy chain contain the consensus sequence and are predicted to be recognized by the localization mechanism that targets ß-actin mRNA to the leading edge. It is known that the ACACCC consensus sequence, when mutated in ß-actin mRNA, results in a failure to localize the mRNA to the leading edge of cells (2, 7), even if the ß-actin coding sequence remains intact and is used as the reporter mRNA. Preliminary results indicate that Arp3 mRNA, like ß-actin mRNA, also localizes to the leading edge (G.Liu, W.Grant, D.Persky, V.L.Lathaur, R.H.S., and J.C., unpublished work). Serum-dependent localization of ß-actin mRNA suggests that signaling mechanisms are involved in the localization of motility-related mRNAs, thereby coordinating their temporal and spatial distribution and expression (28). Furthermore, it is possible that localized synthesis of, for instance, Arp3 could determine the localization of Arp2/3 complex in the leading edge of the cells even if mRNAs coding other components of Arp2/3 complex were more diffusely distributed. Arp2/3 complex and ß-actin, both localized in the leading edge, could determine the nucleation sites for actin polymerization. Newly formed actin filaments could interact with ß-actin isoform-specific binding proteins, thereby stabilizing the cell polarity and consequent directional motility (29).
The leading edge of the cell is a complex composite of asymmetrically distributed proteins many of which function in concert to produce the motility response. It is likely that other proteins like ß-actin also are synthesized asymmetrically and therefore would provide not only a differential concentration of these proteins but also an increased likelihood of interactions among relevant proteins in a cellular region where function depends on these interactions. We presume therefore that a panoply of mRNAs comprising a significant complexity of sequences is localized to the lamella to effect the complex events required by motility. It is our expectation that these sequences will contain a common motif and/or structure in the 3' UTR characterizing them as mRNAs for motility-related proteins. It is likely that further investigations will reveal the consensus sequences (see below).
The localization of ß-actin mRNA is not restricted to fibroblasts, but seems to be a feature of other localized cells. Neurons localize ß-actin mRNA to the growth cone of developing neurites (30, 31). The presence of the mRNA results in the specific translation of ß-actin protein in the growth cone. Like fibroblasts, the delocalization of the mRNA results in growth cone retraction and nondirectionality of growth cone guidance (37). In addition to the neuronal growth cones, embryonic neural crest cells might localize ß-actin mRNA to the front of the cell, in the direction of their migration. Disruption of the Xenopus homolog of ZBP1 appears to inhibit their migration and result in severe embryological defects in forebrain development (J.Yisraeli, personal communication). Furthermore, if the zip code for ß-actin mRNA is transferred to another protein, not normally at the leading edge, in this case vimentin, a distorted morphology results wherein the cell structure at the leading edge is branched and attenuated (32). These results argue that synthesis of the correct protein in the correct place (near the leading edge) is an important requirement for cell structure and polarity.
In addition to ß-actin mRNA localization in fibroblasts (1), the field of RNA localization has been advanced by the discovery of a number of systems where mislocalization of the RNA can lead to a significantly altered phenotype or lethality (33–36). In many of these cases mRNA localization is required for normal development and differentiation because the localized mRNA codes for nuclear factors and the resultant cell divisions segregate the mRNAs for these morphogenic determinants. However, the nature of the localization we describe here is important for a different reason: it determines the spatial orientation, morphology, and behavior of these somatic cells. In this second aspect of RNA localization, the complex of proteins involved in cell migration, cellular reaction to the environment and development of cell polarity are organized within the cytoplasm by virtue of the spatial segregation of their cognate mRNAs, and are not in the short term related to transcription of genes. In this way, components of the mechanism controlling cell behavior and structure can rapidly reassemble within the cell. In this model, the proteins involved in forming these multipolypeptide complexes (the nucleation complex, for instance) would be compartmentalized in response to environmental cues and subsequent signal transduction events and then synthesized in proximity to each other where they would interact preferentially because of their higher local concentrations. Possibly these higher concentrations of proteins could autoregulate their own synthesis. In this way, we propose that the localization of ß-actin mRNA represents one mechanism for the spatially compartmentalized assembly of cellular complexes.
We thank Michael Cammer in the Einstein Analytical Imaging Facility and Jeff Wyckoff and Shailesh M.Shenoy for technical help with light microscopy and the Dynamic Image Analysis System, Wayne Grant for technical help with experiments, and Steve Braut for synthesis of oligonucleotides. This work was supported by National Institutes of Health grants to R.H.S. and J.C.
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