β-Actin Messenger RNA Localization and Protein Synthesis Augment Cell Motility

In chicken embryo fibroblasts (CEFs), β-actin mRNA localizes near an actin-rich region of cytoplasm specialized for motility, the lamellipodia. This localization is mediated by isoform-specific 3′-untranslated sequences (zipcodes) and can be inhibited by antizipcode oligodeoxynucleotides (ODNs) (Kislauskis, E.H., X.-C. Zhu, and R.H. Singer. 1994. J. Cell Biol. 127: 441–451). This inhibition of β-actin mRNA localization resulted in the disruption of fibroblast polarity and, presumably, cell motility. To investigate the role of β-actin mRNA in motility, we correlated time-lapse images of moving CEFs with the distribution of β-actin mRNA in these cells. CEFs with localized β-actin mRNA moved significantly further over the same time period than did CEFs with nonlocalized mRNA. Antizipcode ODN treatment reduced this cell translocation while control ODN treatments showed no effect. The temporal relationship of β-actin mRNA localization to cell translocation was investigated using serum addition to serum-deprived cultures. β-actin mRNA was not localized in serum-deprived cells but became localized within minutes after serum addition (Latham, V.M., E.H. Kislauskis, R.H. Singer, and A.F. Ross. 1994. J. Cell Biol. 126:1211–1219). Cell translocation increased over the next 90 min, and actin synthesis likewise increased. Puromycin reduced this cell translocation and blocked this induction in cytosolic actin content. The serum induction of cell movement was also inhibited by antizipcode ODNs. These observations support the hypothesis that β-actin mRNA localization and consequent protein synthesis augment cell motility.     

Two ZBP1 KH domains facilitate beta-actin mRNA localization,granule formation,and cytoskeletal attachment

Chicken embryo fibroblasts (CEFs) localize beta-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of beta-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.     

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.     

Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype

We have characterized the structure and function of RNA sequences that direct beta-cytoplasmic actin mRNA to the cell periphery were mapped to two segments of 3'-untranslated region by expression of LacZ/beta-actin chimeric mRNAs in chicken embryo fibroblasts (CEFs). A 54-nt segment, the "RNA zipcode," and a homologous but less active 43-nt segment each localized beta-galactosidase activity to the leading lamellae. This zipcode contains the full activity, and mutations or deletions within it reduce, but do not eliminate, its activity, indicating that several motifs contribute to the activity. Two of these motifs, when multimerized, can regenerate almost full activity. These sequences are highly conserved in evolution, since the human beta-actin zipcode, positioned identically in the 3'UTR localizes equally well in chicken cells. Complementary phosphorothioate oligonucleotides against the zipcode delocalized endogenous beta-actin mRNA, whereas those complementary to the region just outside the zipcode, or sense oligonucleotides, did not. Actin mRNA or protein levels were unaffected by the antisense treatments, but a dramatic change in lamellipodia structure, and actin stress fiber organization was observed using the same antizipcode oligonucleotides which delocalized the mRNA. Hence, discrete 3'UTR sequences direct beta-actin isoform synthesis to the leading lamellae and affect cell morphology, presumably through the actin cytoskeleton.     

How and why does β‐actin mRNA target?

beta-Actin mRNA is localized near the leading edge in several cell types where actin polymerization is actively promoting forward protrusion. The localization of the beta-actin mRNA near the leading edge is facilitated by a short sequence in the 3'UTR (untranslated region), the 'zipcode'. Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zipcode) in the 3'UTR leads to delocalization of beta-actin mRNA, alteration of cell phenotype and a decrease in cell motility. The dynamic image analysis system (DIAS) used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zipcode showed that net pathlength and average speed of antisense-treated cells were significantly lower than in sense-treated cells. This suggests that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zipcode-directed antisense oligonucleotides. We postulate that delocalization of beta-actin mRNA results in delocalization of nucleation sites and beta-actin protein from the leading edge followed by loss of cell polarity and directional movement. Hence the physiological consequences of beta-actin mRNA delocalization affect the stability of the cell phenotype.     

A Rho-dependent signaling pathway operating through myosin localizes beta-actin mRNA in fibroblasts

BACKGROUND: The sorting of mRNA is a determinant of cell asymmetry. The cellular signals that direct specific RNA sequences to a particular cellular compartment are unknown. In fibroblasts, beta-actin mRNA has been shown to be localized toward the leading edge, where it plays a role in cell motility and asymmetry. RESULTS: We demonstrate that a signaling pathway initiated by extracellular receptors acting through Rho GTPase and Rho-kinase regulates this spatial aspect of gene expression in fibroblasts by localizing beta-actin mRNA via actomyosin interactions. Consistent with the role of Rho as an activator of myosin, we found that inhibition of myosin ATPase, myosin light chain kinase (MLCK), and the knockout of myosin II-B in mouse embryonic fibroblasts all inhibited beta-actin mRNA from localizing in response to growth factors. CONCLUSIONS: We therefore conclude that the sorting of beta-actin mRNA in fibroblasts requires a Rho mediated pathway operating through a myosin II-B-dependent step and postulate that polarized actin bundles direct the mRNA to the leading edge of the cell.     

Rapid WAVE dynamics in dendritic spines of cultured hippocampal neurons is mediated by actin polymerization

The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE) complex has been proposed to link Rho GTPase activity with actin polymerization but its role in neuronal plasticity has never been documented. We now examined the presence, distribution and dynamics of WAVE3 in cultured hippocampal neurons. WAVE3 was localized to dendritic spines via its N-terminal domain. Green fluorescent protein (GFP)-tagged WAVE3 clusters demonstrate an F-actin-dependent high rate of local motility. Constitutive Rac activation translocates WAVE3 (via the N-terminus), to the leading edge of lamellipodia. Also, spinogenesis is associated with actin-based motility of the WAVE3 protein. Brain specific WAVE1 showed similar localization and effects on spine density. Cytoplasmic fragile X mental retardation protein interacting protein (CYFIP) and non-catalytic region of tyrosine kinase adaptor protein 1 (NCK-1), proteins that are assumed to complex with WAVE, have a somewhat similar cellular distribution and motility. We propose that the WAVE complex is a downstream effector of the Rac signaling cascade, localized to sites of novel synaptic contacts by means of its N-terminal domain, to guide local actin polymerization needed for morphological plasticity of neurons.     

Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes

We have developed in situ hybridization methodology for nonisotopically labeled oligonucleotide probes to detect cellular mRNA with improved speed, convenience, and resolution over previous techniques. Previous work using isotopically labeled oligonucleotide probes characterized important parameters for in situ hybridization (Anal Biochem 166:389, 1987). Eleven oligonucleotide probes were made to coding and noncoding regions of chick beta-actin mRNA and one oligonucleotide probe to chick alpha-cardiac actin mRNA. All the probes were 3' end-labeled with bio-11-dUTP using terminal transferase, and the labeled probes were hybridized to chicken myoblast and myotube cultures. The hybridized probe was detected using a streptavidin-alkaline phosphatase conjugate. Our assay for the success of probe hybridization and detection was the demonstration of beta-actin mRNA highly localized in the lamellipodia of single cells (Lawrence and Singer, Cell 45:407, 1986) as well as the expression of alpha-cardiac actin mRNA and the repression of beta-actin mRNA in differentiating myoblasts and in myotubes. With the alpha-cardiac probe, we found that this mRNA was distributed all over the cytoplasm of myotubes and differentiated (bipolar) single cells and negative in undifferentiated single cells and at the ends of myotubes. When beta-actin probes were used, two of 11 probes were highly sensitive, and, in pooling them together, the localization of beta-actin mRNA in fibroblastic single cells was evident at the leading edge of the motile cells, the lamellipodium. beta-Actin mRNA was not detected in myotubes except at the ends where contact was made with substrate. This indicates that both beta and cardiac actin mRNA can coexist in the same myotube cytoplasm but at different locations.     

Neurotrophin regulation of beta-actin mRNA and protein localization within growth cones.

Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of beta-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that beta-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of beta-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA. Localization of beta-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.     

Roles of Rho-associated kinase and myosin light chain kinase in morphological and migratory defects of focal adhesion kinase-null cells

Fibroblasts derived from focal adhesion kinase (FAK)-null mouse embryos have a reduced migration rate and an increase in the number and size of peripherally localized adhesions (Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., and Yamamoto, T. (1995) Nature 377, 539-544). In this study, we have found that Y27632, a specific inhibitor for Rho-associated kinase (Rho-kinase), dramatically reversed the round cell morphology of FAK(-/-) cells to a spread fibroblast-like shape in 30 min and significantly enhanced their motility. The effects of Y27632 on the FAK(-/-) cell morphology and motility were concomitant with reorganization of the actin cytoskeleton and redistribution of focal adhesions. Conversely, the expression of the constitutively active Rho-kinase in FAK(+/+) cells led to round cell shape and inhibition of cell motility. Furthermore, coincident with the formation of cortical actin filaments, myosin light chain (MLC), Ser-19-phosphorylated MLC, and MLC kinase mainly accumulated at the FAK(-/-) cell periphery. We found that the disruption of actin filaments by cytochalasin D prevented the peripheral accumulation of MLC kinase and that inhibition of myosin-mediated contractility by 2,3-butanedione monoxime induced FAK(-/-) cells to spread. Taken together, our results suggest that Rho-kinase may mediate the formation of cortical actomyosin filaments at the FAK(-/-) cell periphery, which further recruits MLC kinase to the cell periphery and generates a non-polar contractile force surrounding the cell, leading to cell rounding and decreased motility.     

Actin mRNA localizes in the absence of protein synthesis

Actin mRNA is localized in chicken embryo fibroblasts to the distal regions of leading lamellae, but not within the ruffling edges. In this investigation we have addressed the role of actin translation in this process. The translocation of actin mRNA to the cell periphery was studied by monitoring the distribution of actin mRNA in cells during spreading. Within 90 min, actin mRNA moved from a perinuclear to a peripheral distribution. Formation of lamellipodia preceded actin mRNA localization, indicating that localization is not a prerequisite for this event. Neither puromycin (which dissociates ribosomes from mRNA) nor cycloheximide (which stabilizes ribosomes on mRNA) had any effect on this movement of actin mRNA. Anchoring of actin mRNA was studied using cells with peripherally localized actin mRNA. No change in actin mRNA localization was observed for 30 min in the same inhibitors. These data indicate that the presence of the nascent polypeptide is not necessary for translocation of actin mRNA to the cell periphery, or anchoring at that site. This suggests that the mRNA contains information concerning its spatial distribution within the cytoplasm.     

Protocadherin FAT1 binds Ena/VASP proteins and is necessary for actin dynamics and cell polarization

Cell migration requires integration of cellular processes resulting in cell polarization and actin dynamics. Previous work using tools of Drosophila genetics suggested that protocadherin fat serves in a pathway necessary for determining cell polarity in the plane of a tissue. Here we identify mammalian FAT1 as a proximal element of a signaling pathway that determines both cellular polarity in the plane of the monolayer and directed actin-dependent cell motility. FAT1 is localized to the leading edge of lamellipodia, filopodia, and microspike tips where FAT1 directly interacts with Ena/VASP proteins that regulate the actin polymerization complex. When targeted to mitochondrial outer leaflets, FAT1 cytoplasmic domain recruits components of the actin polymerization machinery sufficient to induce ectopic actin polymerization. In an epithelial cell wound model, FAT1 knockdown decreased recruitment of endogenous VASP to the leading edge and resulted in impairment of lamellipodial dynamics, failure of polarization, and an attenuation of cell migration. FAT1 may play an integrative role regulating cell migration by participating in Ena/VASP-dependent regulation of cytoskeletal dynamics at the leading edge and by transducing an Ena/VASP-independent polarity cue.     

Trypanosoma cruzi infection affects actin mRNA regulation in heart muscle cells

We have previously described alterations in the cytoskeletal organization of heart muscle cells (HMC) infected with Trypanosoma cruzi in vitro. Our aim was to investigate whether these changes also affect the regulation of the actin mRNAs during HMC differentiation. Northern blot analysis revealed that alpha-cardiac actin mRNA levels increased during cell differentiation while beta-actin mRNA levels declined. Nonmuscle cells displayed beta-actin mRNA signal localized at the cell periphery, while alpha-cardiac actin mRNA had a perinuclear distribution in myocytes. Trypanosoma cruzi-infected cells showed 50% reduction in alpha-cardiac actin mRNA expression after 72 h of infection. In contrast, beta-actin mRNA levels increased approximately 79% after 48 h of infection. In addition, in situ beta-actin mRNA was delocalized from the periphery into the perinuclear region. These observations support the hypothesis that Trypanosoma cruzi affects actin mRNA regulation and localization through its effect on the cytoskeleton of heart muscle cells.     

LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility

Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro‐inflammatory mediators. We examined mechanisms of LPS‐stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage‐like J774 cells. A wound‐healing assay revealed that LPS also activated directed cell movement that was followed by TNF‐α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5‐bisphosphate. Fractionation of Triton X‐100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin‐regulatory proteins, paxillin on tyrosine 118 by 80% and N‐WASP on serine 484/485 by 20%, and these events preceded activation of NF‐κB. LPS‐induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N‐WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS‐stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF‐α production suggesting that LPS‐induced cell motility is not required for TNF‐α release. J. Cell. Biochem. 113: 80–92, 2012. © 2011 Wiley Periodicals, Inc.     

Autoregulation of actin synthesis requires the 3'-UTR of actin mRNA and protects cells from actin overproduction

Monomeric (G) actin was shown to be involved in inhibiting its own synthesis by an autoregulatory mechanism that includes enhanced degradation of the actin mRNA [Bershadsky et al., 1995; Lyubimova et al., 1997]. We show that the 3'-untranslated region (3'-UTR) of beta-actin mRNA, but not its 5'-untranslated region, is important for this regulation. The level of full-length beta-actin mRNA in cells was reduced when actin filaments were depolymerized by treatment with latrunculin A and elevated when actin polymerization was induced by jasplakinolide. By contrast, the level of actin mRNA lacking the 3'-UTR remained unchanged when these drugs modulated the dynamics of actin assembly in the cell. Moreover, the transfection of cells with a construct encoding the autoregulation-deficient form of beta-actin mRNA led to very high levels of actin expression compared with transfection with the control actin construct and was accompanied by characteristic changes in cell morphology and the structure of the actin cytoskeleton. These results suggest that the autoregulatory mechanism working via the 3'-UTR of actin mRNA is involved in controlling the maintenance of a defined pool of actin monomers that could be necessary for the proper organization of the microfilament system and the cytoskeleton-mediated signaling.     

Cloning and characterization of betaCAP73, a novel regulator of beta-actin assembly

In non-muscle cells, the isoactins are differentially localized, with beta-actin specifically enriched at the cell cortex within motile structures, such as lamellae, while gamma-actin shows no specific localization. To understand the sorting and regulation of beta-actin within moving cells, we previously isolated betaCAP73, a novel beta-actin-specific binding protein (Cell Motil. Cytoskel. 35 (1996) 175). Here, we have cloned and characterized the 4718 nucleotide betaCAP73 cDNA from an endothelial cell library. betaCAP73 cDNA contains six predicted ankyrin-like repeats at the amino terminus and is partially homologous to three previously reported sequences of unknown function. Northern analysis reveals betaCAP73 expression in all tissues tested, with highest levels in skeletal muscle. Consistent with previously demonstrated interactions between native betaCAP73 and beta-actin filament barbed-ends, recombinant betaCAP73 inhibits pyrene-actin assembly in an isoactin-specific manner. Compared to stationary cells betaCAP73 mRNA is down regulated in crawling cells. Similarly, motility-defective cells have increased betaCAP73 protein. Overexpression of full-length betaCAP73 induces the formation of novel membrane protrusions that are enriched in betaCAP73, while overexpression of betaCAP73 domains alters cell morphology. Combined, these results indicate that betaCAP73 modulates isoactin dynamics to regulate the morphological alterations required for cell growth and motility.     

Cancer cell motility--on the road from c-erbB-2 receptor steered signaling to actin reorganization.

Cell migration depends mainly on actin polymerization and intracellular organization, which are influenced by a vast variety of actin binding proteins (ABPs). Regulation of ABP activity is mediated by second messengers such as phosphoinositides and calcium. Signaling via these second messengers is initiated and regulated by membrane receptors, e.g., receptor tyrosine kinases (RTKs), and by adhesion molecule interactions (e.g., integrins and selectins) and focal adhesion kinases. A major role in steering second-messenger signaling and thus in actin cytoskeleton reorganization and motility of cancer cells is played by the RTK c-erbB-2. This occurs through a number of signaling pathways which involve mainly enzymes, e.g., phospholipase Cgamma1 and GTPases, which modify signaling molecules. Furthermore large multiprotein complexes including actin-related protein 2/3, Wiskott-Aldrich syndrome protein, profilin, and capping protein among others play an important role in regulating actin reorganization. The complex picture of the mode of actin reorganization, which is involved in tumor cell migration, is slowly emerging from the mists of cellular signaling pathways, but this is still by no means a clear view.