Regulation of nuclear actin dynamics in development and disease

Actin has essential functions both in the cytoplasm and in the nucleus, where it has been linked to key nuclear processes, from transcription to DNA damage response. The multifunctional nature of actin suggests that the cell must contain mechanisms to accurately control the cellular actin balance. Indeed, recent results have demonstrated that nuclear actin levels fluctuate to regulate the transcriptional activity of the cell and that controlled nuclear actin polymerization is required for transcription activation, cell cycle progression, and DNA repair. Intriguingly, aberrant nuclear actin regulation has been observed, for example, in cancer, signifying the importance of this process for cellular homeostasis. This review discussed the latest research on how nuclear actin is regulated, and how this influences actin-dependent nuclear processes.Peer reviewe

Diverse functions for different forms of nuclear actin

In addition to its essential roles as part of the cytoskeleton, actin has also been linked to many processes in the nucleus. Recent data has demonstrated the presence of both monomeric and polymeric actin in the nucleus, and implied distinct functional roles for these actin pools. Monomeric actin seems to be involved in regulation of gene expression through transcription factors, chromatin regulating complexes and RNA polymerases. In addition to cytoplasmic actin regulators, nuclear proteins, such as emerin, can regulate actin polymerization properties specifically in this compartment. Besides of structural roles, nuclear actin filaments may be required for organizing the nuclear contents and for the maintenance of genomic integrity.Peer reviewe

Regulation of nuclear actin levels and MRTF/SRF target gene expression during PC6.3 cell differentiation

Actin has important functions in both cytoplasm and nucleus of the cell, with active nuclear transport mechanisms maintaining the cellular actin balance. Nuclear actin levels are subject to regulation during many cellular processes from cell differentiation to cancer. Here we show that nuclear actin levels increase upon dif-ferentiation of PC6.3 cells towards neuron-like cells. Photobleaching experiments demonstrate that this increase is due to decreased nuclear export of actin during cell differentiation. Increased nuclear actin levels lead to decreased nuclear localization of MRTF-A, a well-established transcription cofactor of SRF. In line with MRTF-A localization, transcriptomics analysis reveals that MRTF/SRF target gene expression is first transiently activated, but then substantially downregulated during PC6.3 cell differentiation. This study therefore describes a novel cellular context, where regulation of nuclear actin is utilized to tune MRTF/SRF target gene expression during cell differentiation.Peer reviewe

An actin-regulated importin alpha/beta-dependent extended bipartite NLS directs nuclear import of MRTF-A

Peer reviewe

Lamin A/C and emerin regulate MKL1/SRF activity by modulating actin dynamics

Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss Muscular Dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome (HGPS).1 The majority of LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and disturbed interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes.1 We report here that lamin A/C-deficient (Lmna−/−) and Lmna N195K mutant cells have impaired nuclear translocation and downstream signaling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function.2 Disturbed nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna−/− and N195K mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease etiology for the cardiac phenotype in many laminopathies, whereby lamins A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization

Myosin-1C uses a novel phosphoinositide-dependent pathway for nuclear localization

Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin-dependent motor protein Myosin-1C (Myo1C) resembles the diffusion–retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms.Peer reviewe

Lamina-associated polypeptide 2 alpha is required for intranuclear MRTF-A activity

Myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), regulates the expression of many cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Here we describe a novel mechanism to regulate MRTF-A activity within the nucleus by showing that lamina-associated polypeptide 2 alpha (Lap2 alpha), the nucleoplasmic isoform of Lap2, is a direct binding partner of MRTF-A, and required for the efficient expression of MRTF-A/SRF target genes. Mechanistically, Lap2 alpha is not required for MRTF-A nuclear localization, unlike most other MRTF-A regulators, but is required for efficient recruitment of MRTF-A to its target genes. This regulatory step takes place prior to MRTF-A chromatin binding, because Lap2 alpha neither interacts with, nor specifically influences active histone marks on MRTF-A/SRF target genes. Phenotypically, Lap2 alpha is required for serum-induced cell migration, and deregulated MRTF-A activity may also contribute to muscle and proliferation phenotypes associated with loss of Lap2 alpha. Our studies therefore add another regulatory layer to the control of MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2 alpha in transcriptional regulation.Peer reviewe

Twinfilin is required for actin-dependent developmental processes in Drosophila

The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer–binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A mutation in the twf gene leads to a number of developmental defects, including aberrant bristle morphology. This results from uncontrolled polymerization of actin filaments and misorientation of actin bundles in developing bristles. In wild-type bristles, twinfilin localizes diffusively to cytoplasm and to the ends of actin bundles, and may therefore be involved in localization of actin monomers in cells. We also show that twinfilin and the ADF/cofilin encoding gene twinstar interact genetically in bristle morphogenesis. These results demonstrate that the accurate regulation of size and dynamics of the actin monomer pool by twinfilin is essential for a number of actin-dependent developmental processes in multicellular eukaryotes

Nuclear translocation of vitellogenin in the honey bee (Apis mellifera)

Vitellogenin (Vg) is a conserved protein used by nearly all oviparous animals to produce eggs. It is also pleiotropic and performs functions in oxidative stress resistance, immunity, and, in honey bees, behavioral development of the worker caste. It has remained enigmatic how Vg affects multiple traits. Here, we asked whether Vg enters the nucleus and acts via DNA-binding. We used cell fractionation, immunohistology, and cell culture to show that a structural subunit of honey bee Vg translocates into cell nuclei. We then demonstrated Vg-DNA binding theoretically and empirically with prediction software and chromatin immunoprecipitation with sequencing (ChIP-seq), finding binding sites at genes influencing immunity and behavior. Finally, we investigated the immunological and enzymatic conditions affecting Vg cleavage and nuclear translocation and constructed a 3D structural model. Our data are the first to show Vg in the nucleus and suggest a new fundamental regulatory role for this ubiquitous protein.Peer reviewe

Nuclear actin modulates cell motility via transcriptional regulation of adhesive and cytoskeletal genes

The actin cytoskeleton is a classic biomechanical mediator of cell migration. While it is known that actin also shuttles in and out of the nucleus, its functions within this compartment remain poorly understood. In this study, we investigated how nuclear actin regulates keratinocyte gene expression and cell behavior. Gene expression profiling of normal HaCaT keratinocytes compared to HaCaTs over-expressing wild-type beta-actin or beta-actin tagged with a nuclear localization sequence (NLS-actin), identified multiple adhesive and cytoskeletal genes, such as MYL9, ITGB1, and VCL, which were significantly down-regulated in keratinocytes with high levels of nuclear actin. In addition, genes associated with transcriptional regulation and apoptosis were up-regulated in cells over expressing NLS-actin. Functionally, accumulation of actin in the nucleus altered cytoskeletal and focal adhesion organization and inhibited cell motility. Exclusion of endogenous actin from the nucleus by knocking down Importin 9 reversed this phenotype and enhanced cell migration. Based on these findings, we conclude that the level of actin in the nucleus is a transcriptional regulator for tuning keratinocyte migration.Peer reviewe