Linking actin dynamics and gene transcription to drive cellular motile functions

Abstract

Numerous physiological and pathological stimuli promote the rearrangement of the actin cytoskeleton, thereby modulating cellular motile functions. Although it seems intuitively obvious that cell motility requires coordinated protein biosynthesis, until recently the linkage between cytoskeletal actin dynamics and correlated gene activities remained unknown. This knowledge gap was filled in part by the discovery that globular actin polymerization liberates myocardin-related transcription factor (MRTF) cofactors, thereby inducing the nuclear transcription factor serum response factor (SRF) to modulate the expression of genes encoding structural and regulatory effectors of actin dynamics. This insight stimulated research to better understand the actin-MRTF-SRF circuit and to identify alternative mechanisms that link cytoskeletal dynamics and genome activity.

Figure 1. receptors affecting actin dynamics and MrTF-mediated regulation of SrF target genes

a | Cytoskeletal actin microfilament dynamics are affected by the activation of six classes of plasma membrane receptor: receptor Tyr kinases (RTKs), G protein-coupled receptors (GPCRs; with α-subunits of Gα_12/13, Gα_q/11 or Gα_i/0), integrins as structural mediators of focal adhesions, transforming growth factor-β receptors (TGFβRs), E-cadherins at adherens junctions and Frizzled, which mediates the non-canonical Wnt–planar cell polarity (PCP) pathway involving Dishevelled (DVL). These receptors modulate the activity of Rho GTPases through Rho guanine nucleotide exchange factors (GEFs). Effectors of Rho GTPases, including Rho-associated kinases (ROCKs), formins (such as Diaphanous-related formins (DRFs)), Wiskott–Aldrich syndrome protein (WASP), WASP-family verprolin homologues (WAVEs) and the actin-related protein 2/3 (ARP2/3) complex and other actin-binding proteins (ABPs), orchestrate actin polymerization by incorporating globular actin (G-actin) into the filamentous actin (F-actin) polymer. High levels of cytoplasmic G-actin retain serum response factor (SRF) cofactor proteins, myocardin-related transcription factors (MRTFs), in the cytoplasm. Incorporation of G-actin into the F-actin filament liberates MRTFs to enter the nucleus and interact with the transcription factor SRF. This triggers expression of a subset of SRF target genes, namely cytoskeletal genes. b | Activation of SRF class II target genes. Nuclear MRTF can be complexed by nuclear G-actin, which inhibits MRTF-mediated stimulation of SRF-dependent transcription and facilitates MRTF nuclear export. SRF class II target genes that are transcribed as a result of MRTF–SRF activation include actin itself and many genes that modulate actin dynamics, such as gelsolin and vinculin. These newly made proteins, with increasing time and concentration, might stimulate cytoplasmic actin polymerization, complex cytoplasmic MRTF or elevate levels of nuclear G-actin to downregulate MRTF-mediated transcription and stimulate nuclear export of MRTF. FAK, focal adhesion kinase; ILK, integrin-linked protein kinase; LIMK, LIM domain kinase.

Figure 2. Actin-binding proteins as microfilament messengers

A model summarizing the nucleus–cytoplasm shuttling of three different types of actin-binding proteins (ABPs): globular actin (G-actin)-binding proteins (G-ABPs), filamentous actin (F-actin) binding proteins (F-ABPs) and F-actin complex-associated proteins (F-ACAPs). Examples of G-ABPs include myocardin-related transcription factors (MRTFs), striated muscle activator of Rho-dependent signalling (STARS, also known as ABRA), junction-mediating and regulatory protein (JMY), β-thymosin, profilin, neural Wiskott–Aldrich syndrome protein (N-WASP), the actin-related protein 2/3 (ARP2/3) complex and spire. Examples of F-ABPs include the actin-binding LIM proteins (ABLIMs) cofilin, gelsolin, filamin, α-actinin, supervillin and LIM and SH3 domain protein 1 (LASP1)^,, and examples of F-ACAPs include ABL1, integrin cytoplasmic domain-associated protein 1 (ICAP1α), LIM domain proteins and p120-catenin. The shuttling LIM domain proteins zyxin, lipoma-preferred partner (LPP), Cys-rich proteins (CRPs), Hic-5 (also known as TGFB1I1), antileukoproteinase (ALP), paxilin and LIM and SH3 domain protein 1 (LASP1) were shown to directly bind F-actin, whereas LIM kinase, particularly interesting new Cys-His protein 1 (PINCH; also known as LIMS1) and four and a half LIM domains protein 2 (FHL2) are probably indirectly linked to F-ACs^,. F-ACs assemble at the cytoplasmic sides of focal adhesions, cadherin-mediated cell–cell adherens junctions and cell–cell tight junctions. TF, transcription factor.

Figure 3. Structure of myocardin family members

Functional domains of homology among the myocardin family proteins are shown and the numbers of amino acids are indicated. Myocardin-related transcription factors (MRTFs) are potent transcriptional coactivators that associate with serum response factor (SRF) through a basic region (++) and an adjacent Glu-rich domain (Q). Between these domains is a short α-helical region with similar secondary structure to a domain in the ternary complex factor protein ELK1, known as a B box, which mediates their interaction with SRF^,,. Myocard in family proteins contain Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs, which mediate their interact ion with globular actin (G-actin). Members of the myocardin family have a homologous SAP domain, named after SAFA or SAFB, acinus and PIAS, which participates in different kinds of chromosomal DNA metabolism. Deletion of this region disrupts the ability of myocardin to activate a subset of SRF-dependent genes. Myocardin and MRTFs contain powerful transcriptional activation domains (TADs) required for the stimulation of SRF activity. A dimerization motif resembling a Leu zipper mediates homo- and heterodimerization of myocardin and MRTFs. Alternative usage of 5′ exons in the myocardin gene gives rise to proteins with different amino termini. A cardiac-specific splice variant of myocard in contains a unique amino-terminal sequence that confers the ability to interact with the myocyte-specific enhancer factor 2 (MEF2) transcription factor, a MADS-boxtranscription factor related to SRF. This MEF2-interaction domain is also contained in a divergent member of the myocard in family called MEF2-activating SAP transcriptional regulator (MASTR). MASTR lacks the SRF-interaction domain.The OTT (also known as RBM1B)-MAL (also known as MRTF-A) fusion protein of AMKL leukaemia cells contains MRTF-A sequences.

Figure 4. SRF-mediated regulation of miRNAs

a | Regulation of microRNA-1 (miR-1) and miR-133 by sereum response factor (SRF). SRF activates transcription of a bicistronic miRNA cluster encoding miR-1 and miR-133 (REFS ^,). miR-133a inhibits SRF expression, establishing a precisely titrated feedback loop to modulate SRF activity. There are two clusters of miR-1 and miR-133a genes in mammalian genomes, which are expressed specifically in cardiac and skeletal muscle cells. A third homologous pair of miRNAs, miR-206 and miR-133b, is expressed specifically in skeletal muscle independently of SRF. Genetic deletion of miR-1 results in phentoypes that suggest it has a role in mesoderm formation. Genetic deletion of the two miR-133a genes results in perinatal lethality owing to cardiac defects. miR-1 and miR-133a also repress neuroectoderm and endoderm genes and promote mesoderm gene expression. miR-1 can substitute for SRF to regulate downstream genes involved in mesoderm development by an undefined mechanism. b | Regulation of miR-143 and miR-145 by SRF. SRF activates transcription of a bicistronic miRNA cluster encoding miR-143 and miR-145, which are expressed specifically in cardiac and smooth muscle cells^,. These miRNAs, regulate the expression of numerous mRNAs encoding regulators of actin signalling and myocardin-related transcription factor (MRTF)–SRF activity, thereby establishing an elaborate series of feedback loops to modulate the actin–MRTF–SRF signalling pathway. Targets of miR-143 or miR-145 include the zinc-finger proteins krueppel-like factor 4 (KLF4) and KLF5 (which inhibit SRF), MRTF-B, slingshot 2 phosphatase (SHH2; which controls actin polymerization by cofilin phosphorylation), adducin 3 (which promotes actin polymerization) and Slit-Robo Rho GTPase-activating protein 1 (SRGAP1) and SRGAP2 (which inhibit Rho signalling). This pathway has been shown to be essential for vascular remodelling in response to injury. In the absence of miR-143 and miR-145, actin stress fibre formation is disrupted, rendering smooth muscle cells insensitive to mechanical stimuli that typically cause vascular stenosis.