Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes

We have examined the role of feedback-regulation in the expression of the nonmuscle actin genes. C2 mouse myoblasts were transfected with the human beta- and gamma-actin genes. In gamma-actin transfectants we found that the total actin mRNA and protein pools remained unchanged. Increasing levels of human gamma-actin expression resulted in a progressive down-regulation of mouse beta- and gamma-actin mRNAs. Transfection of the beta-actin gene resulted in an increase in the total actin mRNA and protein pools and induced an increase in the levels of mouse beta-actin mRNA. In contrast, transfection of a beta-actin gene carrying a single-point mutation (beta sm) produced a feedback-regulatory response similar to that of the gamma-actin gene. Expression of a beta-actin gene encoding an unstable actin protein had no impact on the endogenous mouse actin genes. This suggests that the nature of the encoded actin protein determines the feedback-regulatory response of the mouse genes. The role of the actin cytoskeleton in mediating this feedback-regulation was evaluated by disruption of the actin network with Cytochalasin D. We found that treatment with Cytochalasin D abolished the down-regulation of mouse gamma-actin in both the gamma- and beta sm-actin transfectants. In contrast, a similar level of increase was observed for the mouse beta-actin mRNA in both control and transfected cells. These experiments suggest that the down-regulation of mouse gamma-actin mRNA is dependent on the organization of the actin cytoskeleton. In addition, the mechanism responsible for the down-regulation of beta-actin may be distinct from that governing gamma-actin. We conclude that actin feedback-regulation provides a biochemical assay for differences between the two nonmuscle actin genes.     

Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.     

Quantitative changes in cytoskeletal beta- and gamma-actin mRNAs and apparent absence of sarcomeric actin gene transcripts in early mouse embryos

Actin is known to be synthesized both during oogenesis and in cleavage-stage embryos in mice. Cytoskeletal beta-actin appears to be the major component, followed by gamma-actin, but the synthesis of alpha-actin has also been inferred from protein electrophoretic patterns. We have studied the expression of cytoskeletal (beta- and gamma-) and sarcomeric (alpha-cardiac and alpha-skeletal) actin genes at the level of the individual mRNAs in blot hybridization experiments using isoform-specific RNA probes. The results show that there are about 2 x 10(4) beta-actin mRNA molecules in the fully grown oocyte; this number drops to about one-half in the egg and less than one-tenth in the late two-cell embryo but increases rapidly during cleavage to about 3 x 10(5) molecules in the late blastocyst. The amount of gamma-actin mRNA is similar to that of beta-actin in oocytes and eggs but only about 40% as much in late blastocysts, indicating a differential accumulation of these mRNAs during cleavage. The developmental pattern of beta- and gamma-actin mRNA provides a striking example of the transition from maternal to embryonic control that occurs at the two-cell stage and involves the elimination of most or all of the maternal actin mRNA. There was no detectable alpha-cardiac or alpha-skeletal mRNA (i.e., less than 1,000 molecules per embryo) at any stage from oocyte to late blastocyst, suggesting that the sarcomeric actin genes are silent during preimplantation development.     

Colony-stimulating factor-1 (CSF-1) rescues osteoblast attachment, survival and sorting of beta-actin mRNA in the toothless (tl-osteopetrotic) mutation in the rat

We have shown that in the osteopetrotic rat mutation toothless (tl) osteoblasts are absent from older bone surfaces in mutants and that mutant osteoblasts in vivo lack the prominent stress fiber bundles polarized along bone surfaces in osteoblasts from normal littermates. Our recent data demonstrate that in normal osteoblasts in vitro beta- and gamma-actin mRNAs have different, characteristic intracellular distributions and that tl (mutant) osteoblasts fail to differentially sort these mRNAs. Because bone resorption and formation are highly interdependent and injections of CSF-1, a growth factor, increase bone resorption and growth in tl rats, we examined the effects of CSF-1 treatment on osteoblast survival and ultrastructure in vivo and ability to sort actin mRNAs in vitro. Neonatal CSF-1 treatment of mutants restores osteoblasts on older bone surfaces, normalizes the intracellular distribution of stress fibers in osteoblasts in vivo and promotes normal sorting of beta-actin mRNA in mutant osteoblasts in vitro without normalizing gamma-actin distribution. These data suggest the beta- and gamma-actin mRNAs in osteoblasts are sorted by different mechanisms and that the differential sorting of beta-actin mRNA is related to the characteristic polarization of stress fibers in osteoblasts and their survival on bone surfaces. This experimental system can be used to explore the relationships and regulation of these aspects of cell and tissue biology.     

Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes.

The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.     

Serum-induced signal transduction determines the peripheral location of beta-actin mRNA within the cell

Cell motility is dependent upon the reorganization of the cellular cytoskeleton. Actin filaments form the major component of the cytoskeleton and respond rapidly to serum growth factors. We have previously shown that myoblasts sort the two cytoskeletal beta- and gamma-actin isoform mRNAs to different intracellular regions and that only beta-actin mRNA was associated with peripheral regions of cell motility (Hill, M.A. and P. Gunning. 1993. J. Cell Biol. 122: 825-832). We now show by in situ hybridization that 3T3 fibroblasts similarly sort actin isoform mRNAs and that peripheral beta-actin mRNA is regulated by serum. In the absence of serum, we could not detect beta- actin mRNA at the periphery. Addition of serum rapidly redistributed beta-actin mRNA to the periphery. gamma-actin mRNA distribution was not altered by serum addition at any time. Both proteins, as identified by immunochemistry with isoform-specific antibodies, were found in similar cellular structures. Serum-stimulated cell motility is mediated through the GTPase signal transduction pathway. We find that an RNA-binding protein, p62, that is part of this pathway, displays a localization pattern similar to beta-actin mRNA. Our results suggest a new biological mechanism which integrates signal transduction with the supply of an architectural component required for membrane remodeling. We propose that active transport of beta-actin mRNA to regions of cell motility is one possible objective of these signal transduction pathways.     

High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture

The impact of the human beta- and gamma-actin genes on myoblast cytoarchitecture was examined by their stable transfection into mouse C2 myoblasts. Transfectant C2 clones expressing high levels of human beta-actin displayed increases in cell surface area. In contrast, C2 clones with high levels of human gamma-actin expression showed decreases in cell surface area. The changes in cell morphology were accompanied by changes in actin stress-fiber organization. The beta-actin transfectants displayed well-defined filamentous organization of actin; whereas the gamma-actin transfectants displayed a more diffuse organization of the actin cables. The role of the beta-actin protein in generating the enlarged cell phenotype was examined by transfecting a mutant form of the human beta-actin gene. Transfectant cells were shown to incorporate the aberrant actin protein into stress-fiber-like structures. High level expression of the mutant beta-actin produced decreases in cell surface area and disruption of the actin microfilament network similar to that seen with transfection of the gamma-actin gene. In contrast, transfection of another mutant form of the beta-actin gene which encodes an unstable protein had no impact on cell morphology or cytoarchitecture. These results strongly suggest that it is the nature of the encoded protein that determines the morphological response of the cell. We conclude that the relative gene expression of beta- and gamma-actin is of relevance to the control of myoblast cytoarchitecture. In particular, we conclude that the beta- and gamma-actin genes encode functionally distinct cytoarchitectural information.     

Identification and developmental expression of a smooth-muscle gamma-actin in postmeiotic male germ cells of mice.

Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.     

Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation.

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.     

Developmental change of protein constituents in chicken gizzards

Developmental change of protein constituents of chick gizzard smooth muscle was described by the fluorescent antibody technique and two-dimensional polyacrylamide gel electrophoresis. Myosin heavy chain, tropomyosin, and desmin were immunohistologically detected in 5-day-old gizzard primordia, but myoglobin was detected after 19 days of incubation. Two-dimensional polyacrylamide gel electrophoresis revealed that most structural proteins including beta- and gamma-actin are synthesized almost simultaneously in the primordium, and accumulate in three patterns by which the proteins examined are classified: (1) gradually increasing protein (gamma-actin, tropomyosin, desmin), (2) abruptly increasing protein at a certain stage (myosin, myoglobin), (3) decreasing or constantly kept protein (tubulin, beta-actin). Based on the quantitative analysis of protein constituents, the nature of regulatory system of protein synthesis in smooth muscle and the possible functional difference between beta- and gamma-actin are discussed.     

Amounts and modulation of actin mRNAs in mouse oocytes and embryos

In order to measure the content of beta- and gamma-actin mRNA in mouse oocytes and ovulated eggs, Northern and slot blots were hybridized to complementary RNA probes transcribed from mouse isotype-specific cDNA sequences. The blots included samples of isotype-specific sense strand RNA standards prepared from the same cDNA sequences. Total actin mRNA content was estimated to be 40 fg per preovulatory full-grown oocyte or egg, consisting of one-third beta-actin mRNA and two-thirds gamma-actin mRNA. Ninety per cent of the actin mRNA is on polysomes in full-grown oocytes. The per cent of actin mRNA in polysomal mRNA is similar to the per cent of actin in newly synthesized proteins. Measurements on other developmental stages showed that, in mid-growth-phase oocytes, each actin mRNA reaches a level twofold higher than in full-grown oocytes. Thereafter, all modulations of the two isotypic mRNAs occur in parallel; that is, they are maintained at constant levels during the late growth phase (oocytes from females 8-14 days old); gradually degraded in oocytes that have completed their rapid growth phase (oocytes from females 15-18 days old), in maturing oocytes, and in 1- and 2-cell embryos; and deadenylated after about 7 h of progression into meiotic maturation.     

Absence of gamma-actin expression in the mouse fibroblast cell line, L

Cytoplasmic isoactins of mouse fibroblast L-cells were examined by two-dimensional gel electrophoresis. In contrast to other cultured cell lines, which contain both beta- and gamma-actin, L-cells contained only beta-actin species. This unique phenomenon was due neither to the transformed status of this cell line nor to the characteristic nature of adipose tissue fibroblasts of C3H mice. When RNA of L-cells was translated in a nuclease-treated reticulocyte lysate system, actin synthesized in vitro also appeared as only one species, beta-actin. Therefore, the genetic information for gamma-actin is absent at the level of translatable mRNA in L-cells.     

Deficient polymerization in vitro of a point-mutated beta-actin expressed in a transformed human fibroblast cell line

HUT-14 cells, tumorigenic human fibroblasts, express a mutant beta-actin which has a single amino acid substitution at position 244 (glycine to aspartic acid), in addition to normal beta- and gamma-actin. In order to characterize the biochemical function of the mutant beta-actin, actins were extracted and purified from HUT-14 cells. The partially purified actin fraction contained beta-, gamma-, and mutant beta-actins in the ratio of 1:1:1, the same ratio as in the cells. When the actin of this fraction was purified through a polymerization step, mutant beta-actin was always less incorporated into actin filaments than beta- and gamma-actin. When the polymerization ability of purified HUT-14 actins was examined by sedimentation technique, it was lower than those of muscle and of cytoplasmic actins from another human cell line (HUT-11) which expresses only normal beta- and gamma-actin, in the ratio of 2:1. The deficient polymerization of mutant beta-actin was also observed by examining the ratio of beta-, gamma-, and mutant beta-actins incorporated into actin filaments. The ratio of mutant beta-actin in polymerized actins under all conditions examined was always less than that before polymerization. These results indicate that the single amino acid substitution at position 244 caused the reduction of incorporation of the mutant beta-actin into actin filaments in vitro.