InteractiveFly: GeneBrief

Myocardin-related transcription factor: Biological Overview | References

Gene name - Myocardin-related transcription factor

Synonyms - MAL-D

Cytological map position 62F2-62F3

Function - Transcription factor

Keywords - cofactor for serum response factor, oogenesis, tracheal branching, wing

Symbol - Mrtf

FlyBase ID: FBgn0052296

Genetic map position - 3L:2,719,685..2,766,480 [-]

Classification - SAP domain, RPEL repeat

Cellular location - cytoplasmic and nuclear

NCBI link: EntrezGene
Mrtf orthologs: Biolitmine
Recent literature
Faria, L., Canato, S., Jesus, T. T., Goncalves, M., Guerreiro, P. S., Lopes, C. S., Meireles, I., Morais-de-Sa, E., Paredes, J. and Janody, F. (2023). Activation of an actin signaling pathway in pre-malignant mammary epithelial cells by P-cadherin is essential for transformation. Dis Model Mech 16(2). PubMed ID: 36808468
Alterations in the expression or function of cell adhesion molecules have been implicated in all steps of tumor progression. Among those, P-cadherin is highly enriched in basal-like breast carcinomas, playing a central role in cancer cell self-renewal, collective cell migration and invasion. To establish a clinically relevant platform for functional exploration of P-cadherin effectors in vivo, a humanized P-cadherin Drosophila model was generated. This study reports that actin nucleators, Mrtf and Srf, are main P-cadherin effectors in the fly. These findings were validated in a human mammary epithelial cell line with conditional activation of the SRC oncogene. Prior to promoting malignant phenotypes, SRC induces a transient increase in P-cadherin expression, which correlates with MRTF-A accumulation, its nuclear translocation and the upregulation of SRF target genes. Moreover, knocking down P-cadherin, or preventing F-actin polymerization, impairs SRF transcriptional activity. Furthermore, blocking MRTF-A nuclear translocation hampers proliferation, self-renewal and invasion. Thus, in addition to sustaining malignant phenotypes, P-cadherin can also play a major role in the early stages of breast carcinogenesis by promoting a transient boost of MRTF-A-SRF signaling through actin regulation.

Cells migrating through a tissue exert force via their cytoskeleton and are themselves subject to tension, but the effects of physical forces on cell behavior in vivo are poorly understood. Border cell migration during Drosophila oogenesis is a useful model for invasive cell movement. This migration requires the activity of the transcriptional factor serum response factor (SRF) and its cofactor MAL-D and evidence is presented that nuclear accumulation of MAL-D is induced by cell stretching. Border cells that cannot migrate lack nuclear MAL-D but can accumulate it if they are pulled by other migrating cells. Like mammalian MAL, MAL-D also responds to activated Diaphanous, which affects actin dynamics. MAL-D/SRF activity is required to build a robust actin cytoskeleton in the migrating cells; mutant cells break apart when initiating migration. Thus, tension-induced MAL-D activity may provide a feedback mechanism for enhancing cytoskeletal strength during invasive migration (Somogyi, 2004).

Cell migration is a process that is critically dependent on mechanical force production by the cytoskeleton. It also requires signaling to and controlled assembly of the cytoskeletal components. These two aspects of the cytoskeleton must be regulated and integrated for proper migration. Regulation of cellular processes by mechanical force, mechanosensitivity, has been established in several contexts. Specialized mechanosensory cells employ mechanically gated ion channels as sensors. Muscle cells also respond to tension both acutely and over the long term. In muscle, proteins of the Z disc may be responsible for tension sensing. Studies in tissue culture cells have revealed tension-sensitive signaling events. Focal adhesion complexes (cell-matrix interaction) and adherence junctions (cell-cell interaction) have been argued to function as loci of mechanosensing. Application of external mechanical force can also change gene expression in developing embryos. In the context of physiological cell migration events, there is limited understanding of how mechanical forces influence cell behavior and what regulatory mechanisms are involved. When cells migrate through a tissue, the forces produced by the cell must be sufficient to allow invasion but also appropriate for the resistance of the substratum. The migrating cells as well as substrate should remain intact while active movement occurs. Migrating cells may aid this process by selecting substrata with appropriate molecular and physical properties (Somogyi, 2004).

The migration of border cells during Drosophila oogenesis is a simple but very useful model system for studying invasive cell migration in vivo. The genetic tractability of this system has allowed many steps of the migration to be dissociated and analyzed. Border cells are a group of about eight follicle cells that delaminate from the follicular epithelium as a cluster, invade the underlying germline tissue, and migrate directionally to the oocyte. The two central cells of the cluster (the polar cells) induce migratory behavior in the approximately six surrounding (outer) cells but are themselves not migratory. The activities of several transcription factors are required for border cells to become specified as actively migrating cells. Border cells migrate upon other cells (germline cells) and use DE-cadherin for specific adhesion to the migration substratum. The migration is guided by signaling through two receptor tyrosine kinases, PDGF/VEGF receptor (PVR) and EGFR. Border cells display a very prominent actin cytoskeleton as they migrate and like other migratory cells require nonmuscle myosin for cell translocation. This study showed that, during the migration process, cell integrity and the actin cytoskeleton are regulated by a transcription factor complex consisting of MAL-D and serum response factor (SRF) in response to perceived tension (Somogyi, 2004).

Mutants that cause changes in bristle morphology in Drosophila have been found to encode actin regulatory proteins such as profilin (chickadee) and cofilin phosphatase (slingshot) as well as myosins (crinkled). EP37532, a P element insertion, was identified based on its recessive bristle defects. EP37532 was inserted in the predicted gene CG32296, now renamed mal-d. A stronger allele, mal-dΔ7, was generated by removing the first exon of the gene. mal-dΔ7 mutant flies show a more penetrant bristle phenotype and are in addition female sterile. An antibody directed against MAL-D protein was generated and affinity purified. It showed a single band on a Western blot, which was not detectable in mal-dΔ7 mutant ovaries and was enhanced upon mal-d overexpression. To look at the phenotype on a cellular level, clones of mal-dΔ7 mutant cells were generated within the follicular epithelium, a simple monolayer epithelium in the ovary. The mutant cells proliferate and differentiate properly. However, the basal network of actin filaments prominent in the differentiated epithelial cells is reduced. Other F-actin-rich structures, such as cortical F-actin and ring canals in germline cells, were slightly reduced in mutant ovaries (Somogyi, 2004).

While most follicle cells appeared to function normally without mal-d, border cell migration was severely perturbed in mutant females. In mal-dΔ7 mutant animals, border cell clusters either did not initiate migration at all or migrated very poorly. Clonal analysis showed that this defect is cell autonomous. When migrating, border cells normally display a particularly robust actin cytoskeleton with higher F-actin levels than nonmigrating follicle cells. This enhanced F-actin accumulation is completely absent in mal-dΔ7 mutant border cells. Ubiquitous expression of a mal-d cDNA rescued the mal-dΔ7 mutant phenotypes completely (normal bristles, normal border cell migration), confirming the gene identification. Thus, the mal-d gene product affects F-actin accumulation in multiple cell types and is required for border cell migration (Somogyi, 2004).

MAL-D is related to mammalian MAL/MRTF-A/MKL1/BSAC, MAL16/MRTF-B/MKL2, and Myocardin proteins (Cen, 2003; Ma, 2001; Mercher, 2001; Miralles, 2003; Sasazuki, 2002; Selvaraj, 2003; Wang, 2001; Wang, 2002), with an N-terminal MAL homology domain (MHD) containing three RPEL motifs and a SAP domain, as well as a less well-defined basic region. Mammalian MAL family proteins have been found to interact with SRF and serve as transcriptional cofactors for SRF. Ternary complex factors (TCF), which are ETS domain proteins, represent another type of SRF cofactor in mammalian cells. Cofactors had not been identified for Drosophila SRF; specifically, there was no evidence for a TCF gene in the sequenced Drosophila genome. MAL-D appears to be the only MAL family protein in Drosophila. In transfected Schneider cells, it was found that Drosophila SRF and MAL-D could be coimmunoprecipitated and cooperate to activate transcription from a serum response element (SRE)-containing reporter plasmid. This indicates that MAL-D is a bona fide SRF cofactor in Drosophila. To investigate this in vivo, phenotypes of mal-d and SRF mutants were compared (Somogyi, 2004).

SRF is essential for viability in flies and for proper tracheal and wing development. The mal-dΔ7 mutation removes a 5' noncoding exon of mal-d that is required for expression in the ovary, and homozygous flies are viable but female sterile. However, the coding region is not altered in mal-dΔ7 mutant flies, and mal-d is still expressed at other times during development. To determine whether mal-d is an essential gene, additional mal-d alleles were generated by ems mutagenesis. Three alleles were used for further analysis: mal-dS9 and mal-dS2 both have a stop codon in the middle of the open reading frame (L659 and Q675 to stop, respectively), and mal-dF2 has a frameshift at position A1364. The mal-d ems alleles were homozygous and transheterozygous early larval lethal, with mal-dF2 larvae surviving longer, suggesting that this might be a hypomorphic allele. The mal-d mutants could be rescued by a mal-d cDNA, expressed ubiquitously under control of an alpha-tubulin promoter. Thus, like Drosophila SRF, mal-d is required for development. Clones of cells mutant for SRF or the new alleles of mal-d showed essentially the same phenotypes as mal-dΔ7 in border cell migration, F-actin accumulation, and bristle morphology. However, none of the mal-d alleles showed blistering when clones were induced in the wing primordium, as found for SRF mutant clones. In patterning the intervein region of wings, SRF may therefore act alone or with a different cofactor. Overall, these results indicate that SRF and MAL-D act together during development to control specific processes that are highly dependent on the actin cytoskeleton. In particular, SRF and MAL-D are required for accumulation of a robust actin cytoskeleton during border cell migration and for this invasive migration event to be effective (Somogyi, 2004).

SRF immunoreactivity is detected in most or all nuclei of both germline and somatic cells. Although a potential transcriptional cofactor, MAL-D protein was detected mainly in the cytoplasm even when highly overexpressed. Mammalian MAL was also found to be cytoplasmic in serum-starved NIH/3T3 cells. Removal of the N terminus of mammalian MAL with the conserved RPEL motifs renders it nuclear and active. The corresponding change in MAL-D (MAL-D-ΔN) had the same effect: MAL-D-ΔN was largely nuclear and highly transcriptionally active in a transfection assay. Expression of MAL-D-ΔN in follicle cells induces excessive F-actin accumulation, an effect opposite that from the mal-d loss-of-function phenotype. Overexpressing high levels of wild-type MAL-D has a similar but milder effect on F-actin. These results indicate that, as for the mammalian MAL, MAL-D protein can accumulate in the cytoplasm, but the nuclear form is the active one. Taken together with the loss-of-function analyses, this indicates that a transcription factor complex consisting of SRF and MAL-D positively regulates genes important for establishing a robust F-actin cytoskeleton (Somogyi, 2004).

To understand why cells in a tissue might need a robust F-actin cytoskeleton, border cell migration, which shows a strong dependence on MAL-D and SRF, was examined. At the initiation of migration, border cells normally produce an actin-rich long cellular extension. Formation of this extension requires proper cell specification, directional signals via the guidance receptors EGFR and PVR, and substrate adhesion via DE-cadherin, but it does not require force generation by myosin, functionally separating these steps. mal-d mutant border cells did produce long cellular extensions, indicating that guidance and adhesion were occurring properly. Subsequently, mal-d mutant border cells showed a unique defect. Large, round cytoplasmic fragments (without nuclei) appeared to 'break off' from the extension. At later stages, the cell fragments were detected progressively further along the normal migratory path. Inspection of intervening confocal sections showed no evidence of any connection between the cytoplasmic fragments and the rest of the cell. The spherical shape also suggested that these fragments were unattached. Thus, failure to augment the cytoskeleton in mal-d mutants led to fragmentation of the long cellular extensions. The border cell fragments continued to move directionally, leaving the cell body and nucleus behind (Somogyi, 2004).

This behavior of mal-d mutant border cells indicates that fragments of invasive, migratory cells have sufficient autonomy to respond to guidance cues and move through a tissue. The fragments appear to move less efficiently than normal border cells (reach the oocyte at a later stage). This could either be due to the cell fragments being fragments and not whole cells or be due to their mutant origin. It has previously been shown that anucleate leukocyte fragments (cytoplasts) can perform chemotaxis in vitro, demonstrating that cytoplasmic fragments can have considerable autonomy from the nucleus with respect to migration in vitro. Specific transcription factors are required for cells to differentiate and acquire migratory/invasive behavior during development. In addition, the MAL-D/SRF complex is required for cells to acquire a robust cytoskeleton and remain intact when performing an invasive migration. However, nuclei and therefore transcriptional changes are apparently not essential for guided movement in vitro or in vivo (Somogyi, 2004).

MAL-D activity might simply be required to stimulate F-actin accumulation and thus contribute to trigger border cell migration. However, expression of constitutively active MAL-D-ΔN in border cells effectively blocks migration, indicating that MAL-D activity needs to be regulated. As a first step in understanding this regulation, when endogenous MAL-D could be detected in the nucleus was investigated as an indication of when MAL-D/SRF might be active. Endogenous MAL-D was detectable in nuclei of some migrating border cells. Nuclear MAL-D can be detected in cells initiating migration or during migration but not when migration is complete (stage 10). About half of the migrating border cell clusters contained one or more nuclei clearly positive for MAL-D, but no specific stage of the migration was always positive. Thus, nuclear MAL-D apparently dies not reflect the cluster position in the egg chamber or developmental stage. During migration, outer border cells could be positive, but the central polar cells were always negative. The polar cells are part of the border cell cluster but are not actively migrating. Both front and rear border cells could be positive. This suggested that MAL-D accumulation is regulated in some dynamic way related to migration. It was noticed that clusters that were elongated or stretched had a high probability of positive nuclei, whereas rounded clusters were less likely to show staining. To quantify this, the length of midmigration clusters was measured as an indicator of stretching. The correlation to MAL-D-positive nuclei was statistically highly significant. Thus, nuclear accumulation of MAL-D correlates with the stretched shape of the migrating cell cluster. Stretching of the cell cluster would be expected to reflect external force application and tension within the cell (Somogyi, 2004).

To further investigate conditions for MAL-D nuclear accumulation, border cells genetically unable to initiate migration were analyzed. slbo is a transcription factor that is required for border cell migration. None of the clusters in which all cells were mutant for slbo (n = 20 clusters) had nuclear MAL-D, regardless of developmental stage. Thus, border cells that were genetically unable to initiate migration were unable to accumulate nuclear MAL-D (Somogyi, 2004).

To determine whether the lack of nuclear MAL-D in slbo mutant cells was due to cell genotype or due to the physical state of the cell, an in vivo 'pulling experiment' was performed. This experiment takes advantage of the fact that border cells migrate as a cluster of strongly adherent cells and not as individual cells. If nonmigratory slbo mutant cells are found in a border cell cluster with wild-type cells, the mutant cells can be pulled along by the wild-type cells. This 'piggy-back' behavior is observed for a variety of different mutants affecting border cell migration -- in fact, it occurs in all genotypes that have been tested. The slbo mutant cells are always in the rear and delay migration of the border cell cluster in proportion to their abundance. Thus, the mutant cells do not become migratory as such but are pulled along by the actively migrating cells. Remarkably, slbo mutant cells that were pulled into migration by wild-type cells did accumulate nuclear MAL-D. They did so at a frequency similar to that of wild-type migrating cells. Migration of mixed clusters is often delayed and may occur during stage 9 or stage 10. In both situations, nuclear MAL-D accumulation was observed. Finally, even mutant cells that had not (yet) invaded the germline could be positive if attached to migrating wild-type cells. This, together with the observations in wild-type cells, shows that border cell position does not control MAL-D accumulation. Thus, nuclear MAL-D accumulation is not directly dependent on cell genotype, on cell position, or on developmental stage. However, nuclear MAL-D accumulation is only observed in nonmotile mutant border cells if they are being pulled by other cells. These results support the idea that cell deformation or perceived tension regulates MAL-D accumulation (Somogyi, 2004).

The conserved protein structure, in particular the conserved RPEL motifs (MHD), as well as the functional interactions with SRF suggested that mammalian and fly MAL proteins might be regulated in similar ways. In a series of interesting experiments, activation of mammalian SRF and nuclear accumulation of MAL have shown to respond to changes in actin dynamics in NIH-3T3 cells. The N-terminal RPEL motifs of MAL were required for this regulation, which has also been called the Rho-actin pathway. One of the strongest activators of MAL/SRF was an activated form of Diaphanous, which acts downstream of Rho. To determine whether MAL-D could be subject to the same regulation, a corresponding activated form of Drosophila Diaphanous (HA-diaCA) was made and overexpressed in border cells. Border cell migration was blocked by HA-diaCA; however, nuclear accumulation of MAL-D was nevertheless stimulated. This effect was observed on endogenous MAL-D but was most obvious when looking at border cell clusters cooverexpressing MAL-D and HA-diaCA. In border cells, as in follicle cells, overexpressed MAL-D was predominantly cytoplasmic. In contrast, when HA-diaCA was present, MAL-D was predominantly nuclear. When both proteins were expressed at high levels, the nuclear pool of MAL-D was still detectable, but MAL-D was mainly cytoplasmic, suggesting that nuclear translocation was saturable. Thus, the ability of the Rho pathway to activate MAL proteins appears to be conserved in Drosophila (Somogyi, 2004).

It is therefore proposed that the transcription factor complex of MAL-D and SRF is responsible for a regulatory mechanism by which physical pulling force upon and tension within an invasively migrating cell induces a compensatory strengthening of its cytoskeleton. Mutant analysis has shown that MAL-D and SRF are required for migrating border cells to build up a robust cytoskeleton and remain intact during invasive migration. Regulation via MAL-D may be particularly critical for cells that perform force-demanding processes such as invasive cell migration. At least, this is the case for border cells. It will be of interest to determine whether this regulation also plays a role in pathologically invasive migrations such as in metastasis. While they migrate, border cells normally display a very robust F-actin cytoskeleton. It is suggested that this F-actin accumulation results from multiple rounds of MAL-D activation during migration. Failure to augment the cytoskeleton leads to fragmentation of the long cellular extensions leading the invasion and production of migrating 'cytoplasts'. Although these fragments are not produced by normal cells, their behavior can be useful in determining what cells can do in vivo without a nucleus. Production of platelets by megakaryocytes is an example of physiological production of non-migratory cell fragments (Somogyi, 2004).

How does MAL-D/SRF regulate the actin cytoskeleton and cell integrity? Studies in mammalian cells give some indications of what the critical target genes might be in Drosophila. Cytoskeletal actin and vinculin genes can be regulated by a feedback mechanism, and these genes are regulated by SRF (and MAL). In mouse ES cells, SRF is important for the production of actin-directed cytoskeletal structures and cell motility. No changes in total actin levels were detected in mal-d mutant tissues. However, actin polymerization and actin filament organization is highly regulated in cells. MAL-D- and SRF-dependent changes in F-actin accumulation could therefore be due to changes in levels of any of the many actin-regulating and actin-interacting proteins, including myosins. Dictyostelium amoebae mutant for myosin II heavy chain display loss of cortical integrity and cell fragmentation when cells migrate under restrictive environments, apparently due to loss of the actin-crosslinking activity of myosin II. Further analysis of transcription profiles is required to pinpoint the exact target genes of MAL-D/SRF. Interestingly, several MAL family proteins as well as SRF are important for muscle-specific gene expression. Also, stretching of mammalian myogenic cells in culture leads to a complex set of trophic and differentiation responses, including increased production of SRF. It is tempting to speculate that the prominent role of MAL/SRF in muscle differentiation is related to its regulatory role in tension-dependent gene expression in nonmuscle cells, muscle being a dedicated actin/myosin-dependent contractile tissue (Somogyi, 2004).

What is the molecular mechanism for MAL-D regulation by tension? Given that the actin cytoskeleton and tension or cell shape changes are interdependent, it is likely that this regulation is related to the regulation of MAL/SRF by actin dynamics (the Rho pathway). Two models were proposed to explain the effect of actin on MAL and SRF. The simplest model is that free G-actin sequesters MAL in the cytoplasm, and depletion of this G-actin pool by actin polymerization results in MAL translocation/activation. Observations in border cells do not fit very well with this simple model. In normal cells, even very highly overexpressed MAL-D is almost exclusively cytoplasmic, indicating practically unlimited capacity in the cytoplasm. Expression of constitutively active Diaphanous, which should 'release' MAL-D by causing actin polymerization, did cause accumulation of MAL-D in the nucleus. But further overexpression of MAL-D led to more protein in the cytoplasm, not in the nucleus as would be expected if G-actin depletion in the cytoplasm (induced by active Diaphanous) were the trigger for nuclear translocation. Finally, even though endogenous MAL-D is expressed at low levels, overexpression of a nonpolymerizable form of actin in border cells did not appear to sequester MAL-D in the cytoplasm. These data seem more consistent with the alternative 'active' model of MAL activation, wherein a subpopulation of actin or an actin protein complex accumulates when actin polymerization is favored, leading to MAL nuclear translocation and activity (Somogyi, 2004).

There are two general ways in which regulation of MAL by actin and by tension might be related. Changes in actin dynamics, as induced by activated Diaphanous, may induce changes in tension, which could then affect MAL. For example, RhoA activation can induce formation of stress fibers, which are contractile structures. Conversely, changes in cell tension could affect RhoA, Diaphanous, and thereby actin dynamics, which then in turn directly regulate MAL. In fact, RhoA and Diaphanous, two of the most potent activators of SRF/MAL, have been shown to be important mediators of mechanosensitive changes at focal adhesions. The physical interaction observed between the conserved N-terminal domain of MAL and unpolymerized forms of actin suggests that regulation of MAL by actin is quite direct and thus supports this type of relationship. Tension applied to cell-matrix attachments or cell-cell interactions may also locally increase actin polymerization by other means and thereby activate MAL. A more speculative link to MAL regulation is offered by actin itself. A specific conformation of actin, or a specific protein complex containing actin, may be induced by tension and serve as the signal that is perceived by MAL. This would be consistent with the idea that a particular subpopulation of actin is responsible for the active regulation of MAL. It would be an elegant way for hard-working migratory cells to regulate strength as needed by the actin cytoskeleton. It is usually thought that actin-myosin supplies force and tension; the MAL/SRF system suggests a role for the complex actin cytoskeleton in force perception as well (Somogyi, 2004).

A myocardin-related transcription factor regulates activity of serum response factor in Drosophila

Serum response factor (SRF) regulates genes involved in cell proliferation, migration, cytoskeletal organization, and myogenesis. Myocardin and myocardin-related transcription factors (MRTFs) act as powerful transcriptional coactivators of SRF in mammalian cells. An MRTF from Drosophila, called DMRTF, is described that shares high homology with the functional domains of mammalian myocardin and MRTFs. DMRTF forms a ternary complex with and stimulates the activity of Drosophila SRF, which has been implicated in branching of the tracheal (respiratory) system and formation of wing interveins. A loss-of-function mutation introduced into the DMRTF locus by homologous recombination results in abnormalities in tracheal branching similar to those in embryos lacking SRF. Misexpression in wing imaginal discs of a dominant negative DMRTF mutant also causes a diminution of wing interveins, whereas overexpression of DMRTF results in excess intervein tissue, abnormalities reminiscent of SRF loss- and gain-of-function phenotypes, respectively. Overexpression of these DMRTF mutants in mesoderm and in the tracheal system also perturbs mesoderm cell migration and tracheal branching, respectively. It is concluded that the interaction of MRTFs with SRF represents an ancient protein partnership involved in cytoplasmic outgrowth and cell migration during development (Han, 2004).

The Drosophila tracheal system is comprised of a network of interconnected epithelial tubes that undergo sequential sprouting. Terminal branches of the tracheal network are formed from individual cells that express high levels of DSRF and extend long cytoplasmic processes toward target tissues. Signaling from fibroblast growth factor (FGF) to SRF has been shown to regulate cytoplasmic outgrowth of terminal cells during tracheal branching. The similarities between the tracheal branching phenotypes of DMRTF and DSRF mutant embryos suggest that DMRTF and DSRF may function together during terminal tracheal branching. The small GTPase Rac, which regulates cell adhesion and actin-based cytoskeletal motility, has also been shown to act in a signaling pathway that interconnects FGF signaling with SRF during tracheal branching. The DMRTF RNAi tracheal phenotype is similar to that of embryos lacking Rac1 and -2, suggesting DMRTF may act in the Rac signaling pathway during tracheal development. Given the role of the mammalian DMRTF ortholog, MRTF-A/MAL, in transduction of growth signals and changes in actin treadmilling to SRF, these findings point to a similar role for DMRTF during cytoplasmic branching of the tracheal system in Drosophila (Han, 2004).

The wings of Drosophila are derived from sheets of epithelial cells that become subdivided into vein and intervein cells, giving rise to a stereotypical pattern of veins that provide structural support to the wings. Intervein cells are required for adherence of the two surfaces of the wing. As in the developing tracheal system, signaling by FGF to SRF has been shown to be required for vein/intervein formation; the absence of either of these factors results in a deficiency of intervein cells (Han, 2004).

Consistent with the proposed role of DMRTF as a coactivator of SRF, DMRTF mutant embryos or embryos expressing dominant negative DMRTF displayed a loss of intervein cells. Conversely, misexpression of hypermorphic DMRTF alleles results in excess intervein cells and the concomitant loss of vein cells. The latter phenotype was also observed in response to misexpression of DSRF and DMRTF together (Han, 2004).

Mesodermal cells form in the ventral region of the Drosophila embryo and migrate in a dorsolateral direction to give rise to a uniform monolayer. This process is essential for the regional specification of different mesodermal derivatives, such as cardiac, somatic, and visceral muscles. Like the dependence of tracheal branching on FGF signaling, the mesoderm-specific FGF receptor Heartless has been shown to be required for mesoderm migration. Recently, a guanyl nucleotide exchange factor, Pebble, was found to be required for mesoderm migration, likely through modification of a small GTPase such as Rho and Rac. Interestingly, the activity of DMRTF, like that of its mammalian orthologue MRTF-A/MAL, is also stimulated by Rho-actin signaling in Drosophila cultured cell assays. Because misexpression of dominant negative and hyperactive forms of DMRTF results in opposite migratory phenotypes, DMRTF may act downstream of heartless and pebble in mesoderm migration (Han, 2004).

The target genes of DMRTF responsible for the diverse DMRTF gain- and loss-of-function phenotypes remain to be determined. It is imagined that many of these target genes are directly regulated by SRF and, indeed, several such genes with multiple SRF-binding sites in their control regions have been identified. However, it is also possible that transcription factors in addition to SRF serve as cofactors for DMRTF (Han, 2004).

SRF integrates diverse signals for cell growth, migration, cytoskeletal organization, and myogenesis via its association with transcriptional cofactors. The mammalian MRTF MAL has been shown to associate with G-actin in the cytoplasm and to translocate to the nucleus in response to growth factor signaling and actin treadmilling. It is intriguing that embryonic stem cells lacking SRF display defects in spreading, adhesion, and migration, all of which correlate with abnormalities in actin stress fibers. Thus, the striking parallels between the roles of SRF and myocardin family members in mammalian cells and Drosophila suggest that these factors comprise an ancient and evolutionarily conserved system for coupling changes in cell shape and extracellular signaling with cell migration during development (Han, 2004).

Myocardin is a bifunctional switch for smooth versus skeletal muscle differentiation

Skeletal and smooth muscle can mutually transdifferentiate, but little molecular insight exists as to how each muscle program may be subverted to the other. The myogenic basic helix-loop-helix transcription factors MyoD and myogenin (Myog) direct the development of skeletal muscle and are thought to be dominant over the program of smooth muscle cell (SMC) differentiation. Myocardin (Myocd) is a serum response factor (SRF) coactivator that promotes SMC differentiation through transcriptional stimulation of SRF-dependent smooth muscle genes. This study shows by lineage-tracing studies that Myocd is expressed transiently in skeletal muscle progenitor cells of the somite, and a majority of skeletal muscle is derived from Myocd-expressing cell lineages. However, rather than activating skeletal muscle-specific gene expression, Myocd functions as a transcriptional repressor of Myog, inhibiting skeletal muscle differentiation while activating SMC-specific genes. This repressor function of Myocd is complex, involving histone deacetylase 5 silencing of the Myog promoter and Myocd's physical contact with MyoD, which undermines MyoD DNA binding and transcriptional synergy with MEF2. These results reveal a previously unrecognized role for Myocd in repressing the skeletal muscle differentiation program and suggest that this transcriptional coregulator acts as a bifunctional molecular switch for the smooth versus skeletal muscle phenotypes (Long, 2007).

Skeletal muscle identity is controlled primarily by four skeletal muscle-specific myogenic regulatory factors (MRFs), MyoD, myogenin (Myog), Myf5, and MRF4, which cooperate with the myocyte enhancer factor-2 (MEF2) transcription factor to activate skeletal muscle gene expression. Although the MRFs act in a dominant manner and can convert a variety of cell types, including smooth muscle, into skeletal muscle, there are settings in which skeletal muscle can be induced to transdifferentiate into other cell types, suggesting that the MRFs may be subordinate to other cell-specific transcription factors. Much of the work related to transcriptional regulation of smooth muscle cell (SMC) differentiation has focused on serum response factor (SRF), a widely expressed transcription factor that binds the CArG [CC (A/T-rich)6GG] box found in the regulatory regions of many SMC-specific genes. Genetic inactivation of SRF and CArG mutagenesis studies in transgenic mice have confirmed the necessity of CArG-SRF in controlling SMC differentiation. However, SRF is only a weak transcriptional activator and requires interacting cofactors that recruit proteins to promote a permissive state for gene transcription. One such cofactor is myocardin (Myocd), which is expressed primarily in cardiac and SMCs and displays high transcriptional activity. Myocd can activate SMC-specific genes (Pipes 2006), and genetic deletion of Myocd in mice leads to defective vascular SMC differentiation (Li, 2003). Thus, Myocd displays features of a master regulator of the SMC phenotype (Long, 2007).

In an effort to define the cells of the cardiovascular system derived from Myocd-dependent lineages, lineage tracing was performed in mouse embryos by introducing Cre recombinase into the Myocd locus and monitoring the expression of a Cre-dependent lacZ from the ROSA26 reporter (R26R) mouse line. Consistent with previous expression data, cardiac and vascular SMCs are derived from Myocd-dependent lineages. Surprisingly, skeletal muscle in these mice also expressed lacZ, indicating its derivation from a Myocd-dependent lineage. However, rather than functioning as an activator of skeletal muscle gene expression, Myocd represses MyoD-mediated stimulation of the Myog promoter and blocks skeletal muscle differentiation in vitro. At the same time, Myocd transactivates SMC contractile protein genes, thereby converting skeletal myoblasts to an SMC phenotype. These results suggest that Myocd acts as a bifunctional switch for muscle differentiation by concurrently opposing the gene program for skeletal muscle differentiation and specifying a SMC fate (Long, 2007).

The results of this study reveal Myocd to be an early marker of skeletal muscle lineages and a negative regulator of the transcriptional program for skeletal muscle differentiation. Skeletal myoblasts overexpressing Myocd either transiently or stably are refractory to terminal differentiation and acquire an SMC-like phenotype. The ability of Myocd to block skeletal muscle differentiation can be attributed, at least in part, to its transcriptional repression of Myog, an essential activator of skeletal muscle gene expression. This transcriptional repression appears to involve multiple mechanisms involving the recruitment of HDAC5, as well as an obstruction to both MEF2-MyoD functional association and MyoD DNA binding to the Myog promoter. The transient expression of Myocd within the somitic compartment of the mouse embryo provides a biological context where Myocd may function in the transdifferentiation of skeletal myoblasts to a SMC-like lineage. These results support the concept of Myocd acting as a bifunctional switch for smooth versus skeletal muscle differentiation (Long, 2007).

Myocd was first reported to be a strong transactivator through physical association with SRF bound to CArG elements in the regulatory regions of cardiac- and SMC-restricted genes. This study reports that Myocd displays repressor activity over the Myog promoter even when the conserved CArG element is mutated or deleted, indicating that transrepression is independent of CArG-SRF. This finding is further supported by Myocd-mapping studies demonstrating that the repressive activity of Myocd did not require the basic domain of Myocd, which mediates SRF binding. Thus, Myocd displays a broader role in regulating gene expression than previously thought. Consistent with this concept, a microarray screen in human skeletal myoblasts transduced with Myocd revealed repression of many genes, including Myog. Moreover, Myocd inhibits cell growth and malignant transformation, although it is unclear whether these effects are due to repression of growth-regulatory genes (Long, 2007).

The mechanisms for Myocd's transrepression of the Myog promoter are complex. Myocd interacts with positive (p300) and negative (HDAC5) coregulators of chromatin remodeling to effect changes in SMC gene expression (Cao, 2005). This study shows that the HDAC5 interacting domain of Myocd (poly Q) is partly required for transrepression. Further, antisense-HDAC5 rescues the suppression of Myog in cells stably transfected with Myocd. Interestingly, repression cannot be rescued with p300, which physically binds to the TAD of Myocd and is a crucial coactivator of MyoD-dependent gene expression (Sartorelli, 1997). This outcome implies that repression is not the result of Myocd binding limiting amounts of p300. Because the TAD of Myocd is essential for its transrepression activity, this domain may interact with another protein to mediate transcriptional repression. One potential candidate is MyoD, which has been shown by GST pulldown and coimmunoprecipitation assays to physically associate with Myocd. MyoD-Myocd complexes likely account for attenuated MEF2-MyoD functional association and reduced MyoD binding to E-boxes in the Myog promoter (Long, 2007).

Skeletal muscle differentiation and the MRFs are considered to be dominant over other cell types, including cardiac and smooth muscle. Importantly, the apparent dominance of the skeletal muscle program over SMCs only has been shown in vitro, where levels of Myocd are low. It is suggested that the stoichiometry of Myocd is critical in maintaining SMC differentiation. When Myocd levels are low, SMCs lose their differentiated phenotype and may take on other cell fates. However, when Myocd levels are elevated, cells are more likely to adopt an SMC fate. Interestingly, MyoD has no effect on Myocd-dependent transactivation of SMC-restricted promoters, suggesting that Myocd can override the actions of MRFs and the skeletal muscle program of differentiation (Long, 2007).

Retrospective clonal analysis in mice has shown some embryonic aortic SMCs to arise from the dermamyotome of somites, thereby providing evidence for the existence of a common progenitor for smooth and skeletal muscle. Recently, quail-chick transplantation studies also found the sclerotome compartment of the somite to be a source of aortic SMCs. LacZ expression directed by Myocd-Cre was detected in both the dermamyotome and the sclerotome. Regardless of the somitic origins of aortic SMCs, the expression of Myocd-Cre suggests that Myocd is expressed in somitic progenitor cells that may give rise to aortic SMCs. Therefore, it is possible that a subpopulation of Myocd-expressing cells within the somites is prevented from differentiating into myotubes or other cell types (e.g., bone), thus allowing these cells to migrate to the dorsal aorta and differentiate into vascular smooth muscle. Because Myocd expression is not detected in the skeletal muscle lineage beyond E9.0, it is proposed that Myocd is required only transiently in a common progenitor of skeletal and smooth muscle lineages, and that its subsequent repression is required for skeletal muscle development (Long, 2007).

There are several instances during development where SMCs transdifferentiate into skeletal muscle. It is hypothesized that a critical prerequisite for SMC-skeletal muscle transdifferentiation is the silencing of Myocd expression. In support of this premise, cultured SMCs with a propensity to transdifferentiate into skeletal muscle display low-level expression of Myocd. It will be interesting to investigate whether the types of repressive mechanisms observed in this study also are operative in settings of SMC phenotypic modulation, as occurs during pathological vascular remodeling in vivo. Finally, VEGF can promote the transdifferentiation of skeletal myoblasts or muscle-derived stem cells into functional SMCs. Because VEGF is under study in a variety of angiogenesis clinical trials, it may be prudent to evaluate skeletal muscle function in patients undergoing this type of therapy (Long, 2007).

In summary, a new function is described for Myocd related to the transcriptional repression of Myog and the respecification of skeletal myoblasts to a SMC-like lineage. It is proposed that Myocd functions as a bifunctional molecular switch for muscle differentiation, advancing SMC differentiation while repressing the skeletal muscle differentiation program. These studies have important implications for understanding the molecular underpinnings associated with transdifferentiation of skeletal muscle and smooth muscle during development and the derivation of these cell types from stem cells (Long, 2007).

Myocardin-related transcription factors regulate the Cdk5/Pctaire1 kinase cascade to control neurite outgrowth, neuronal migration and brain development

Numerous motile cell functions depend on signaling from the cytoskeleton to the nucleus. Myocardin-related transcription factors (MRTFs) translocate to the nucleus in response to actin polymerization and cooperate with serum response factor (Srf) to regulate the expression of genes encoding actin and other components of the cytoskeleton. This study shows that MRTF-A (Mkl1) and MRTF-B (Mkl2) redundantly control neuronal migration and neurite outgrowth during mouse brain development. Conditional deletion of the genes encoding these Srf coactivators disrupts the formation of multiple brain structures, reflecting a failure in neuronal actin polymerization and cytoskeletal assembly. These abnormalities were accompanied by dysregulation of the actin-severing protein gelsolin and Pctaire1 (Cdk16) kinase, which cooperates with Cdk5 to initiate a kinase cascade that governs cytoskeletal rearrangements essential for neuron migration and neurite outgrowth. Thus, the MRTF/Srf partnership interlinks two key signaling pathways that control actin treadmilling and neuronal maturation, thereby fulfilling a regulatory loop that couples cytoskeletal dynamics to nuclear gene transcription during brain development (Mokalled, 2010).

The core and conserved role of MAL is homeostatic regulation of actin levels

The transcription cofactor MAL (Myocardin-related transcription factor or Mrtf) is regulated by free actin levels and thus by actin dynamics. MAL, together with its DNA-binding partner, SRF, is required for invasive cell migration and in experimental metastasis. Although MAL/SRF has many targets, this study provides genetic evidence in both Drosophila and human cellular models that actin is the key target that must be regulated by MAL/SRF for invasive cell migration. By regulating MAL/SRF activity, actin protein feeds back on production of actin mRNA to ensure sufficient supply of actin. This constitutes a dedicated homeostatic feedback system that provides a foundation for cellular actin dynamics (Salvany, 2014).

The transcription cofactor MAL is regulated by cellular actin dynamics and confers this regulation on the activity of its DNA-binding partner, SRF. Free G-actin directly binds to MAL via RPEL motifs at the N terminus of MAL and negatively regulates its activity. This regulation is conserved from mammals to insects (Somogyi 2004). In the physiological context of the animal, the function of MAL and related proteins (MRTF-A and MRTF-B in mammals and mal-d/mrtf in Drosophila) appears conserved as well, related to active changes in the cytoskeleton. For example, initiation of invasive cell migration is essentially abolished in the absence of MAL or SRF in border cell migration in the Drosophila ovary (Somogyi 2004) or in mouse bipolar neurons exiting the subventricular zone of the brain and for cancer cells in three-dimensional (3D) invasion assays and experimental metastasis (Medjkane, 2009; Salvany, 2014).

Actin is a very abundant and exquisitely conserved protein in eukaryotic cells. Cycling of actin between G-actin and F-actin pools is controlled by a vast array of regulators, which have been the focus of considerable attention. Actin protein synthesis is also a regulated process. Actin mRNA localization and localized protein synthesis are important for cell migration and axonal growth and guidance. This study presents evidence that regulation of actin gene transcription is itself a key regulatory step in the control of invasive cell migration. Using a genome-wide approach, Actin5C was identified as a major target of the MAL/SRF transcription factor complex. Loss or reduction of MAL activity impairs invasive migration in Drosophila and human cancer cell models. It was found that restoring actin expression can be sufficient to replace the requirement for MAL to support invasive migration in these models. Thus, actin and MAL form a conserved homeostatic feedback system to ensure that actin levels are appropriate to support the actin dynamics required for complex cell behavior (Salvany, 2014).

To understand why MAL is essential for invasive migration and whether the apparent similarity of its role in different organisms reflects a conserved molecular mechanism, attempts were made to identify Drosophila MAL target genes at the genome level. A combination was used of chromatin immunoprecipitation (ChIP) and gene expression analysis and focus was placed on MAL, as SRF has functions independent of MAL/mrtf in mammals and flies. To perform analysis in the relevant tissue context, a mutant in the single Drosophila mal-d gene was used that abolishes expression in the ovary (mal-dΔ7) (Somogyi, 2004). This specifically blocked invasive migration by border cells and caused overall ovary growth defects. Ubiquitous expression of a GFP-tagged version of Mal-d completely rescued the mal-d mutant phenotypes, showing that the fusion protein provides normal Mal-d function. The MAL-GFP transgene also allowed efficient identification of MAL-GFP-bound regions in the Drosophila genome by immunoprecipitation with GFP. Key MAL-GFP-bound regions were confirmed in independent samples, and their recovery was dependent on the presence of the transgene. In parallel, genome-wide expression analysis of wild-type versus mal-d mutant ovaries identified genes whose expression was dependent on MAL. These two complementary data sets allowed genome-wide identification of MAL target genes (Salvany, 2014).

There were two key findings from this genome-wide analysis. First, only a small number of genes qualified as direct MAL targets, with MAL-GFP bound to the regulatory region and a significant decrease of mRNA expression in the mutant: the cytoplasmic Actin5C gene and five other genes, most encoding heat-shock proteins. A few additional genes encoding cytoskeletal proteins or regulators were identified as potential targets in the MAL-GFP-bound set. Second, three of the four most enriched MAL-bound regions in the whole genome were associated with Actin5C. The MAL-bound regions were conserved in other species, suggesting functional importance, and in each case, these sites bracketed gene-free upstream regions of ~10 kb. The latter is noteworthy because the Drosophila genome is dense, with most 'housekeeping genes' closely spaced, and large regulatory regions generally confined to developmental regulators. These findings focused the attention of this study on Actin5C (Salvany, 2014).

The gene expression arrays indicated a modest decrease of Actin5C mRNA levels in the mal-d mutant. Quantitative RT–PCR of carefully matched ovary samples showed a twofold decrease of mature Actin5C mRNA and a threefold to fourfold decrease of primary transcript in mal-d mutants, with no change in the closely related, but less highly expressed, Actin42A gene. In FACS-sorted migratory cells, including border cells, Actin5C levels were fourfold reduced. Analysis of the Actin5C promoter and upstream region in luciferase reporter assays showed robust promoter activity and 200-fold to 600-fold up-regulation by coexpression of SRF and activated MAL (mal-d ΔN -). Conversely, knockdown of MAL or SRF by RNAi gave 50-fold to 100-fold reduction in basal Actin5C expression. The Actin5C regulatory region also conferred responsiveness to drugs affecting actin dynamics, specifically induction by Cytochalasin D and inhibition by Latrunculin B, as observed for mammalian MAL/SRF-regulated genes. This type of reporter assay generally reveals regulatory potential at the transcriptional level. The large magnitude of regulation of the Actin5C promoter/enhancer region by MAL/SRF is consistent with the abundant binding of MAL to this region. In vivo, compensatory mechanisms may contribute to sustaining Actin5C mRNA levels upon loss of MAL activity. Thus, MAL and actin dynamics have the potential to regulate Actin5C transcription over a large dynamic range (Salvany, 2014).

The genomic data raised the possibility that Actin5C might not be just one of many cytoskeletal target genes for MAL regulation but the key target gene. If a transcription factor has one key target gene in vivo, re-expression of this gene should replace the need for the transcription factor. In genetic terms, expression of the target gene should rescue the phenotype of complete loss of function for the transcription factor in specific cells (Salvany, 2014).

To investigate this hypothesis functionally, the severe defect in invasive migration observed in mal-d mutant border cells was examined. Actin5C is the major cytoplasmic actin gene, and, as expected, mutating it perturbs border cell migration. To determine whether Actin5C was the sole required target gene of MAL, whether re-expression of Actin5C in cells that appear to be null for mal-d (mal-d S2) (Somogyi, 2004) could restore invasive migration was tested. Fly strains were used in which the Actin5C gene has a Gal4-responsive transposon, an 'EP element,' in the promoter region. Surprisingly, migration was indeed restored to normal when Actin5C was activated by Gal4 in mal-d mutant cells. Thus, as long as the Actin5C gene is induced at an adequate level, border cells do not need MAL to invade and migrate (Salvany, 2014).

In fully mutant ovaries (mal-d&Delta:7 homozygous females), restricted expression of Actin5C in terminally differentiated outer border cells using slbo-Gal4 provided significant but less efficient rescue of migration despite normal expression levels. This suggested that MAL also acts in other cells, consistent with the general oogenesis phenotype. The ORF of Actin5C in a Gal4-reponsive transgene (UAS-Actin-ORF) also provided some rescue of migration, whereas a construct with stop codons present did not. This confirmed that actin protein expression was responsible for the activity of the Actin5C locus in border cells (Salvany, 2014).

To test whether this finding extended to other tissues, another mal-d mutant phenotype, bent bristles, was examined. Bristles are 'hairs' organized by actin-rich structures and are characteristically defective in mal-d mutants (Somogyi, 2004). This effect of MAL deficiency was also restored to normal by ectopic expression of the Actin5C, accomplished by placing Actin5C cDNA under the control of a heat-shock promoter and rearing at 29°C. Thus, the requirement for MAL in these contexts reflects a specific need for MAL-driven induction of Actin5C expression. Regulation of other targets, direct or indirect, is not required. These findings indicate that the primary role of Drosophila MAL is to regulate actin levels in response to free actin fluctuations, a homeostatic feedback regulation (Salvany, 2014).

It was next asked whether this role of MAL as regulator of actin homeostasis is conserved in mammalian cells. The cytoplasmic β-actin and γ-actin genes are regulated by MAL/SRF in mammalian cells. As for Drosophila Actin5C, β-actin is essential for embryonic development and proper cell migration, whereas the related γ-actin can be compensated for. However, many other genes are more dramatically regulated by SRF and mrtfs, and some of these are important for cell migration and related functions. It is therefore assumed that MAL and SRF exert their function by regulating a battery of cytoskeletal genes. For SRF, the number of direct target genes is estimated at 200-300. For MAL and MRTFs, this is less clear, as systematic ChIP analysis is missing. Based on the findings in the fly model, the hypothesis was tested that a single target gene (β-actin) could be the key effector of mammalian MAL and that its expression could replace the need for MAL-driven gene regulation in an assay of invasive cell migration (Salvany, 2014).

The requirement for MAL is most pronounced in cases of active cell shape change and cytoskeletal challenge, such as tissue or matrix invasion (Somogyi, 2004; Medjkane, 2009; Pinheiro, 2011). Therefore, a simple cellular assay was sought that could test the ability of cells to invade into a confined environment. Migration under agarose provides mechanical resistance to movement and has been used to study migration of eukaryotic cells in a constrained space. The assay can easily be adapted to human tumor cells such as MDA-MB-231 breast cancer cells. MAL activity in these cells is provided by mrtf-a and mrtf-b, and reducing their expression severely reduces invasion in an organotypic assay and in experimental metastasis in mice (Medjkane, 2009). Simultaneous knockdown of mrtf-a and mrtf-b in MDA-MB-231 cells by siRNA reduced β-actin transcript levels. It also produced severe attenuation of migration under agarose, confirming that this assay interrogates MAL-dependent cell movement (Salvany, 2014).

To assess the importance of cytoplasmic actin downstream from MAL, stable cell lines were derived from MDA-MB-231 that allowed the ORF of human β-actin, or an N-terminally Flag-tagged version of β-actin, to be induced from the strong CMV promoter by the TET-ON system. These cell lines showed some baseline expression of the transgenes and six-fold to 10-fold induction upon Tet addition. Under agarose, migration was similar to the parental cell line and not changed by Tet addition. Knockdown of mrtf-a and mrtf-b largely attenuated migration under agarose. Remarkably, ectopic induction of β-actin or Flag-β-actin in the mrtf-a/b-depleted cells rescued migration to control levels. This indicates that human cancer cells require MAL activity to perform invasive migration for the exact same reason that Drosophila border cells do: to regulate cytoplasmic actin gene expression. Regulation of any other potential MAL target genes may not be required for this cell behavior. These experiments rely on siRNA-mediated reduction of mrtf activity; thus, it remains possible that other target genes are important but require only low levels of mrtf activity (Salvany, 2014).

Elegant experiments have shown how activity of SRF and its transcriptional cofactor, MAL, is regulated by the level of free actin and thereby by the dynamics of the actin cytoskeleton. This study has shown that the key role of MAL is to regulate the expression level of cytoplasmic actin. It is suggested that this simple feedback system allows cells to produce sufficient free actin to meet the needs of their changing cellular cytoskeletal dynamics. The extensive MAL-decorated regulatory region displayed by Actin5C may serve to tune actin gene expression sensitively and accurately. This study provides evidence that cytoplasmic actin is the sole critical target gene for invasive migration in Drosophila and possibly also in human cells. It is therefore proposed that this regulation is the ancestral function of the MAL/SRF complex in animal cells. Additional target genes for MAL have been acquired in different species and are likely to contribute to cell fitness. Examples include other cytoskeletal proteins (Medjkane, 2009) as well as genes encoding heat-shock proteins, some of which may interact with actin. While some of these genes appear more dramatically regulated by MAL when considering relative mRNA levels, actin may be the 'most regulated' gene if considering the number of transcripts induced. In any case, identification of actin as the core, essential target of MAL reveals the core of this transcription regulatory 'network' to be a simple and logical feedback system (Salvany, 2014).

Why is the MAL-driven regulation of actin particularly critical for invasively migrating cells? The stimuli inducing a robust F-actin-based cytoskeleton when initiating migration into constrained space is likely to convert free G-actin into F-actin-rich structures. Maintaining the appropriate free actin pool for further cytoskeletal buildup or for other cellular functions then requires new production of actin. Other dramatic shape changes, such as cells rounding up in the stratified epidermal layer of the skin, may induce similar sudden actin pool depletion and therefore require MAL and SRF. It has been suggested that MAL forms part of a mechanical feedback system for invasive cells (Somogyi, 2004) whereby mechanical tension induces MAL activity in order to make 'robust' cells. Related concepts of tension-driven responsiveness have recently been indicated for mammalian MAL/SRF function as well (Connelly, 2010; McGee, 2011). The mechanical feedback logic is fully compatible with the simple molecular feedback system presented in this study. It can be regarded as an alternate point of perturbation impinging on actin homeostasis; namely, stretching or stressing cells to provoke a biomechanical cytoskeletal response. Robust feedback systems such as this one driven by MAL are likely to be well conserved through animal evolution, when the target is a crucial one. The ability of cells to occasionally migrate and invade is a characteristic of animal systems, and the dynamic actin cytoskeleton is central to this behavior (Salvany, 2014).


Search PubMed for articles about Drosophila

Cao, D., et al. (2005). Modulation of smooth muscle gene expression by association of histone acetyltransferases and deacetylases with myocardin. Mol. Cell. Biol. 25: 364-376. PubMed ID: 15601857

Cen, B., et al. (2003). Megakaryoblastic leukemia 1, a potent transcriptional coactivator for serum response factor (SRF), is required for serum induction of SRF target genes. Mol. Cell. Biol. 23: 6597-6608. PubMed ID: 12944485

Connelly, J. T., Gautrot, J. E., Trappmann, B., Tan, D. W., Donati, G., Huck, W. T. and Watt, F. M. (2010). Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions. Nat Cell Biol 12: 711-718. PubMed ID: 20581838

Han, Z., Li, X., Wu, J. and Olson, E. N. (2004). A myocardin-related transcription factor regulates activity of serum response factor in Drosophila. Proc. Natl. Acad. Sci. 101: 12567-12572. PubMed ID: 15314239

Li, S., Wang, D.-Z., Richardson, J. A. and Olson, E. N. (2003). The serum response factor coactivator myocardin is required for vascular smooth muscle development. Proc. Natl. Acad. Sci. 100: 9366-9370. PubMed ID: 12867591

Long, X., et al. (2007). Myocardin is a bifunctional switch for smooth versus skeletal muscle differentiation. Proc. Natl. Acad. Sci. 104(42): 16570-5. PubMed ID: 17940050

Ma, Z., et al., (2001). Fusion of two novel genes, RBM15 and MKL1, in the t(1;22)(p13;q13) of acute megakaryoblastic leukemia. Nat. Genet. 28: 220-221. PubMed ID: 11431691

McGee, K. M., Vartiainen, M. K., Khaw, P. T., Treisman, R. and Bailly, M. (2011). Nuclear transport of the serum response factor coactivator MRTF-A is downregulated at tensional homeostasis. EMBO Rep 12: 963-970. PubMed ID: 21799516

Salvany, L., Muller, J., Guccione, E. and Rorth, P. (2014). The core and conserved role of MAL is homeostatic regulation of actin levels. Genes Dev 28: 1048-1053. PubMed ID: 24831700

Medjkane, S., Perez-Sanchez, C., Gaggioli, C., Sahai, E. and Treisman, R. (2009). Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis. Nat Cell Biol 11: 257-268. PubMed ID: 19198601

Mercher, T., et al. (2001). Involvement of a human gene related to the Drosophila spen gene in the recurrent t(1;22) translocation of acute megakaryocytic leukemia. Proc. Natl. Acad. Sci. 98: 5776-5779. PubMed ID: 11344311

Miralles, F. et al. (2003). Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell 113: 329-342. PubMed ID: 12732141

Mokalled, M. H., Johnson, A., Kim, Y., Oh, J. and Olson, E. N. (2010). Myocardin-related transcription factors regulate the Cdk5/Pctaire1 kinase cascade to control neurite outgrowth, neuronal migration and brain development. Development 137(14): 2365-74. PubMed ID: 20534669

Pinheiro, E. M., Xie, Z., Norovich, A. L., Vidaki, M., Tsai, L. H. and Gertler, F. B. (2011). Lpd depletion reveals that SRF specifies radial versus tangential migration of pyramidal neurons. Nat Cell Biol 13: 989-995. PubMed ID: 21785421

Pipes, G. C. T., Creemers, E. E. and Olson, E. N. (2006). The myocardin family of transcriptional coactivators: versatile regulators of cell growth, migration, and myogenesis. Genes Dev. 20: 1545-1556. PubMed ID: 16778073

Sartorelli, V., Huang, J., Hamamori, Y. and Kedes, L. (1997). Molecular mechanisms of myogenic coactivation by p300: direct interaction with the activation domain of MyoD and with the MADS box of MEF2C. Mol. Cell. Biol. 17: 1010-1026. PubMed ID: 9001254

Sasazuki, T., et al. (2002). Identification of a novel transcriptional activator, BSAC, by a functional cloning to inhibit tumor necrosis factor-induced cell death. J. Biol. Chem. 277: 28853-28860. PubMed ID: 12019265

Selvaraj, A. and Prywes, P. (2003). Megakaryoblastic leukemia-1/2, a transcriptional co-activator of serum response factor, is required for skeletal myogenic differentiation. J. Biol. Chem. 278: 41977-41987. PubMed ID: 14565952

Somogyi, K. and Rorth, P. (2014). Evidence for tension-based regulation of Drosophila MAL and SRF during invasive cell migration. Dev Cell 7: 85-93. PubMed ID: 15239956

Wang, D., et al. (2001). Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor. Cell 105: 851-862. PubMed ID: 11439182

Wang, D. Z., et al. (2002). Potentiation of serum response factor activity by a family of myocardin-related transcription factors. Proc. Natl. Acad. Sci. 99: 14855-14860. PubMed ID: 12397177

Biological Overview

date revised: 20 November 2010

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