The results presented herein are consistent with the hypothesis that β-actin mRNA localization has a physiologically significant role in fibroblast motility. Presumably, the mRNA augments cell motility by providing synthesis of new actin monomers for polymerization at the leading edge. Several results support this interpretation. First, cells with β-actin mRNA localized in the leading lamellae moved farther than cells that did not, previous to fixation and in situ hybridization. Second, inhibition of this localization with specific antizipcode ODNs retarded translocation distances relative to controls, independent of the growth conditions of the cells. Third, serum addition to starved cells rapidly induced both mRNA localization and motility. Fourth, the amount of actin per cell increased significantly after serum stimulation. Fifth, this increase was inhibited by puromycin, as was the increase in cell motility.
Because F-actin and actin mRNA appear in the lamellipodia and leading lamellae, respectively, within 2 min of serum addition, both the actin protein and mRNA appear to sort simultaneously. That β-actin mRNA localization does not require protein synthesis (Sundell and Singer, 1990
) and puromycin did not inhibit formation of the lamellipod shows that these events are independent, initially. Later (>30 min), disruption of actin mRNA localization and inhibition of protein synthesis did have an effect on cell translocation. Therefore, it is reasonable to propose that translation of localized β-actin mRNA is important to achieve maximal translocation.
Contemporary models of cell motility have not seriously considered a role for translation (Lauffenburger and Horwitz, 1996
; Mitchison and Cramer, 1996
). This is in part because protein synthesis inhibitors were not observed to completely prevent motility or protrusion of the lamellipodia (Spooner et al., 1971
; Albrecht-Buhler, 1980
). Over time, quantitation of translocation distance demonstrated that inhibition of protein synthesis resulted in a partial effect (~50%) on motility in primary fibroblasts.
How could protein synthesis influence the dynamics of the cellular actin pool? In response to serum stimulation, protein synthesis rates have been estimated to increase fourfold. At the peak rate of synthesis, 4–6 h after serum addition, actin synthesis accounts for 15% of the total cell constituents (Riddle et al., 1979
). If ribosomes are spaced 15 nucleotides apart, synthesis of one actin/s/mRNA is well within the established translation rate of a polysome/ mRNA complex, estimated to be about five amino acid residues per second (Darnell et al., 1995
). This would result in the synthesis of about 150,000 actin molecules/min/cell, assuming ~2,500 mRNAs/cell (Latham et al., 1994
). We have measured the average actin content per cell to be 10.5 pg, which represents ~1.5 × 108
actin molecules/cell. This is also the number of actin molecules calculated per cell by another independent method, based on ~1.8 pl volume/cell and an actin concentration of 135 μM. At a rate of synthesis of 150,000 actin/min, a cell could increase its actin content by 6% per hour (0.9 × 107
actin molecules/ h). Growing CEFs divide approximately every 20 h, allowing sufficient time for the actin content to double. These calculations are consistent with the amount of actin synthesis we actually observed per cell. We observe that actin content after serum stimulation increased at the rate of 1 pg/h/cell, or ~1.4 × 107
molecules/h, nearly a 10% increase per hour. Based on an estimated 2,500 mRNAs, this is the equivalent of 3,900 actin molecules synthesized per second, or ~1.5 actins/s/mRNA. Most important, however, is the obvious conclusion that all the synthesis is restricted to where the mRNA is localized (i.e., the lamella). The generation of over 105
actin molecules/min in a cytoplasmic compartment that represents only a few percent of the total cell volume may have significant consequences for this region of the cell, the region most involved in cell motility (see below).
The serum induction of mRNA localization, and subsequent actin synthesis, provides the basis for a model of how translation may promote cell motility. Serum induces a burst of rapid actin polymerization in the leading lamella that precedes protrusion of the lamellipod. In mammary adenocarcinoma cells stimulated with an upshift of EGF concentration, polymerization is nucleated from severed actin filaments, and actin monomers are estimated to add to the leading edge at the rate of between 60,000 and 600,000 per second (Chan, A.Y., S. Raft, M. Bailly, J.B. Wyckoff, J.E. Segall, and J.S. Condeelis, manuscript submitted for publication), in an area within 1.5 μm of the leading edge membrane (Condeelis, 1993
; Segall et al., 1996
). This polymerization persists for 1 min, possibly using as much as 36 million monomers. Because the leading lamella is a minor portion of the total cytoplasm, this rapid reduction of actin monomers may deplete the local concentration, requiring either sorting of recycled actin or new synthesis. In cells moving in a gradient of EGF or spontaneously in culture, repetitive cycles of actin polymerization at the leading edge are expected. Localized actin synthesis could influence this cyclical reaction over time by augmenting the supply of free actin monomers necessary for preferential polymerization at the leading edge. This continuing, highly localized synthesis may become significant in maintaining the persistence of movement. In this model, it would follow that inhibition of mRNA localization, or of actin translation, would reduce the constant asymmetric supply of monomers, eventually affecting the translocation of the cell. The data presented here are consistent with this model since the initial protrusion events (polymerization of actin in lamellipodia) upon serum induction were unaffected by either the antizipcode ODNs or puromycin treatment (data not shown). However, after 60 min, inhibition of protein synthesis began to negatively affect motility, suggesting that the continued supply of new actin monomer replenishes the leading edge.
β-Actin mRNA localization could affect other structures that are important in motility (Farmer et al., 1983
; Bershadsky et al., 1995
). Focal adhesions influence locomotion by providing traction for the contractile force. Synthesis of actin (and other proteins) in association with this structure likewise may provide an anchoring point for filament elongation during protrusion. Fibroblasts, although slow in their migration, can generate very high traction forces because of their strong substrate adherence (Lauffenburger and Horwitz, 1996
). The localized supply of actin monomers at the leading edge could also facilitate the synthesis and assembly of proteins involved in forming the focal adhesions.
This work has focused on the β-isoform of actin, implicated in cell motility (Hoock et al., 1991
; Herman, 1993
). Other actin isoforms such as α- and γ- are also components of the cytoskeleton in particular cell types (Otey et al., 1988
). The ratio of the various isoforms within the cell has a profound effect on cell structure and function (Schevzov et al., 1992
; Lloyd and Gunning, 1993
). While each actin isoform is highly conserved in amino acid sequence, the 3′ UTRs of their respective mRNAs differ. Possibly, each isoform is synthesized in its respective cytoplasmic compartment, and protein sorting results (von Arx et al., 1995), in part, from the distribution of their respective mRNAs. mRNA localization therefore represents a spatial component of gene expression in the cytoplasm dictating where a protein will be synthesized. This level of control of gene expression could be as significant to cell structure and function as which protein is expressed.