Formin-mediated nuclear actin at androgen receptors promotes transcription.

Steroid hormone receptors are ligand-binding transcription factors essential for mammalian physiology. The androgen receptor (AR) binds androgens mediating gene expression for sexual, somatic and behavioural functions, and is involved in various conditions including androgen insensitivity syndrome and prostate cancer1. Here we identified functional mutations in the formin and actin nucleator DAAM2 in patients with androgen insensitivity syndrome. DAAM2 was enriched in the nucleus, where its localization correlated with that of the AR to form actin-dependent transcriptional droplets in response to dihydrotestosterone. DAAM2 AR droplets ranged from 0.02 to 0.06 µm3 in size and associated with active RNA polymerase II. DAAM2 polymerized actin directly at the AR to promote droplet coalescence in a highly dynamic manner, and nuclear actin polymerization is required for prostate-specific antigen expression in cancer cells. Our data uncover signal-regulated nuclear actin assembly at a steroid hormone receptor necessary for transcription.

FAM110A promotes mitotic spindle formation by linking microtubules with actin cytoskeleton.

Precise segregation of chromosomes during mitosis requires assembly of a bipolar mitotic spindle followed by correct attachment of microtubules to the kinetochores. This highly spatiotemporally organized process is controlled by various mitotic kinases and molecular motors. We have recently shown that Casein Kinase 1 (CK1) promotes timely progression through mitosis by phosphorylating FAM110A leading to its enrichment at spindle poles. However, the mechanism by which FAM110A exerts its function in mitosis is unknown. Using structure prediction and a set of deletion mutants, we mapped here the interaction of the N- and C-terminal domains of FAM110A with actin and tubulin, respectively. Next, we found that the FAM110A-Δ40-61 mutant deficient in actin binding failed to rescue defects in chromosomal alignment caused by depletion of endogenous FAM110A. Depletion of FAM110A impaired assembly of F-actin in the proximity of spindle poles and was rescued by expression of the wild-type FAM110A, but not the FAM110A-Δ40-61 mutant. Purified FAM110A promoted binding of F-actin to microtubules as well as bundling of actin filaments in vitro. Finally, we found that the inhibition of CK1 impaired spindle actin formation and delayed progression through mitosis. We propose that CK1 and FAM110A promote timely progression through mitosis by mediating the interaction between spindle microtubules and filamentous actin to ensure proper mitotic spindle formation.

Nuclear actin dynamics and functions at a glance.

Actin is well known for its cytoskeletal functions, where it helps to control and maintain cell shape and architecture, as well as regulating cell migration and intracellular cargo transport, among others. However, actin is also prevalent in the nucleus, where genome-regulating roles have been described, including it being part of chromatin-remodeling complexes. More recently, with the help of advances in microscopy techniques and specialized imaging probes, direct visualization of nuclear actin filament dynamics has helped elucidate new roles for nuclear actin, such as in cell cycle regulation, DNA replication and repair, chromatin organization and transcriptional condensate formation. In this Cell Science at a Glance article, we summarize the known signaling events driving the dynamic assembly of actin into filaments of various structures within the nuclear compartment for essential genome functions. Additionally, we highlight the physiological role of nuclear F-actin in meiosis and early embryonic development.

To be or not to be assembled: progressing into nuclear actin filaments.

The paradigm states that cytoplasmic actin operates as filaments and nuclear actin is mainly monomeric, acting as a scaffold in transcription complexes. However, why should a powerful function of actin, namely polymerization, not be used in the nucleus? Recent progress in the field forces us to rethink this issue, as many actin filament assembly proteins have been linked to nuclear functions and new experimental approaches have provided the first direct visualizations of polymerized nuclear actin.

A tight grip on differentiation: Nuclear constriction by microtubules regulates hematopoietic stem cells.

Maintenance of the mature blood cells requires controlled cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). While our knowledge of the gene expression changes that facilitate differentiation has made a leap forward, less is known about the cellular triggers that induce them. Biedzinski et al (2020) now uncover a new intracellular mechanism that drives myeloid differentiation: Microtubule bundles squeeze the nucleus of HSPCs and form large invaginations, thus causing changes in chromatin organization. These microtubule-induced nuclear shape changes result in gene expression profiles that favor myeloid differentiation.

Extracellular MIF, but not its homologue D-DT, promotes fibroblast motility independently of its receptor complex CD74/CD44.

Macrophage migration inhibitory factor (MIF) and its homologue D-dopachrome tautomerase (D-DT) are widely expressed pro-inflammatory cytokines with chemokine-like functions that coordinate a wide spectrum of biological activities, such as migration. Here, we biotin-tagged intracellular MIF/D-DT in vivo to identify important cytosolic interactors and found a plethora of actin cytoskeleton-associated proteins. Although the receptor complex between CD74 and CD44 (CD74/CD44) is essential for signalling transduction in fibroblasts via extracellular MIF/D-DT, our interactome data suggested direct effects. We, thus, investigated whether MIF/D-DT can modulate cell migration independently of CD74/CD44. To distinguish between receptor- and non-receptor-mediated motility, we used fibroblasts that are either deficient or that express CD74/CD44 proteins, and treated them with recombinant MIF/D-DT. Interestingly, only MIF could stimulate chemokinesis in the presence or absence of CD74/CD44. The pro-migratory effects of MIF depended on lipid raft/caveolae-mediated but not clathrin-mediated endocytosis, on its tautomerase activity and, probably, on its thiol protein oxidoreductase activity. As MIF treatment restrained actin polymerisation in vitro, our findings establish a new intracellular role for MIF/D-DT in driving cell motility through modulation of the actin cytoskeleton.

Actin chromobody imaging reveals sub-organellar actin dynamics.

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are challenging to detect with fluorescence microscopy, making it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed fluorescent-protein-tagged actin nanobodies, termed 'actin chromobodies' (ACs), targeted to organelle membranes to enable high-resolution imaging of sub-organellar actin dynamics.

Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.

Yersinia pseudotuberculosis cytotoxic necrotizing factor interacts with glycosaminoglycans.

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance, and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. Especially the C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to a hypothetical protein receptor may support the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface.

Postmitotic expansion of cell nuclei requires nuclear actin filament bundling by α-actinin 4.

The actin cytoskeleton operates in a multitude of cellular processes including cell shape and migration, mechanoregulation, and membrane or organelle dynamics. However, its filamentous properties and functions inside the mammalian cell nucleus are less well explored. We previously described transient actin assembly at mitotic exit that promotes nuclear expansion during chromatin decondensation. Here, we identify non-muscle α-actinin 4 (ACTN4) as a critical regulator to facilitate F-actin reorganization and bundling during postmitotic nuclear expansion. ACTN4 binds to nuclear actin filament structures, and ACTN4 clusters associate with nuclear F-actin in a highly dynamic fashion. ACTN4 but not ACTN1 is required for proper postmitotic nuclear volume expansion, mediated by its actin-binding domain. Using super-resolution imaging to quantify actin filament numbers and widths in individual nuclei, we find that ACTN4 is necessary for postmitotic nuclear actin reorganization and actin filament bundling. Our findings uncover a nuclear cytoskeletal function for ACTN4 to control nuclear size and chromatin organization during mitotic cell division.

Formins at the Junction.

The actin cytoskeleton and adhesion junctions are physically and functionally coupled at the cell-cell interface between epithelial cells. The actin regulatory complex Arp2/3 has an established role in the turnover of junctional actin; however, the role of formins, the largest group of actin regulators, is less clear. Formins dynamically shape the actin cytoskeleton and have various functions within cells. In this review we describe recent progress on how formins regulate actin dynamics at cell-cell contacts and highlight formin functions during polarized protein traffic necessary for epithelialization.

Actin visualization at a glance.

Actin functions in a multitude of cellular processes owing to its ability to polymerize into filaments, which can be further organized into higher-order structures by an array of actin-binding and regulatory proteins. Therefore, research on actin and actin-related functions relies on the visualization of actin structures without interfering with the cycles of actin polymerization and depolymerization that underlie cellular actin dynamics. In this Cell Science at a Glance and the accompanying poster, we briefly evaluate the different techniques and approaches currently applied to analyze and visualize cellular actin structures, including in the nuclear compartment. Referring to the gold standard F-actin marker phalloidin to stain actin in fixed samples and tissues, we highlight methods for visualization of actin in living cells, which mostly apply the principle of genetically fusing fluorescent proteins to different actin-binding domains, such as LifeAct, utrophin and F-tractin, as well as anti-actin-nanobody technology. In addition, the compound SiR-actin and the expression of GFP-actin are also applicable for various types of live-cell analyses. Overall, the visualization of actin within a physiological context requires a careful choice of method, as well as a tight control of the amount or the expression level of a given detection probe in order to minimize its influence on endogenous actin dynamics.

Mutant p53 promotes tumor progression and metastasis by the endoplasmic reticulum UDPase ENTPD5.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and are often associated with progression from benign to invasive stages with metastatic potential. Mutations inactivate tumor suppression by p53, and some endow the protein with novel gain of function (GOF) properties that actively promote tumor progression and metastasis. By comparative gene expression profiling of p53-mutated and p53-depleted cancer cells, we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene, which functions as a uridine 5'-diphosphatase (UDPase) in the endoplasmic reticulum (ER) to promote the folding of N-glycosylated membrane proteins. A comprehensive pan-cancer analysis revealed a highly significant correlation between p53 GOF mutations and ENTPD5 expression. Mechanistically, mutp53 is recruited by Sp1 to the ENTPD5 core promoter to induce its expression. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals folding of N-glycosylated membrane proteins in the ER as a mechanism underlying the metastatic progression of tumors with mutp53 that could provide new possibilities for cancer treatment.

Guanine nucleotide-binding proteins of the G12 family shape immune functions by controlling CD4+ T cell adhesiveness and motility.

Integrin-mediated adhesion plays a central role in T cell trafficking and activation. Genetic inactivation of the guanine nucleotide-binding (G) protein alpha-subunits Galpha(12) and Galpha(13) resulted in an increased activity of integrin leukocyte-function-antigen-1 in murine CD4(+) T cells. The interaction with allogeneic dendritic cells was enhanced, leading to an abnormal proliferative response in vitro. In vivo, T cell-specific inactivation of Galpha(12) and Galpha(13) caused lymphadenopathy due to increased lymph node entry and enhanced T cell proliferation, and the susceptibility toward T cell-mediated diseases was enhanced. Mechanistically, we show that in the absence of Galpha(12) and Galpha(13) the activity of the small GTPases Rac1 and Rap1 was increased, whereas signaling of the small GTPase RhoA was strongly reduced. Our data indicate that locally produced mediators signal through Galpha(12)- and Galpha(13)-coupled receptors to negatively regulate cell polarization and adhesiveness, thereby fine-tuning T cell trafficking, proliferation, and susceptibility toward T cell-mediated diseases.

Nuclear F-actin formation and reorganization upon cell spreading.

We recently discovered signal-regulated nuclear actin network assembly. However, in contrast to cytoplasmic actin regulation, polymeric nuclear actin structures and functions remain only poorly understood. Here we describe a novel molecular tool to visualize real-time nuclear actin dynamics by targeting the Actin-Chromobody-TagGFP to the nucleus, thus establishing a nuclear Actin-Chromobody. Interestingly, we observe nuclear actin polymerization into dynamic filaments upon cell spreading and fibronectin stimulation, both of which appear to be triggered by integrin signaling. Furthermore, we show that nucleoskeletal proteins such as the LINC (linker of nucleoskeleton and cytoskeleton) complex and components of the nuclear lamina couple cell spreading or integrin activation by fibronectin to nuclear actin polymerization. Spreading-induced nuclear actin polymerization results in serum response factor (SRF)-mediated transcription through nuclear retention of myocardin-related transcription factor A (MRTF-A). Our results reveal a signaling pathway, which links integrin activation by extracellular matrix interaction to nuclear actin polymerization through the LINC complex, and therefore suggest a role for nuclear actin polymerization in the context of cellular adhesion and mechanosensing.

Pharmacological inhibition of actin assembly to target tumor cell motility.

Tumor metastasis remains an unsolved clinical problem. An initial and essential step in this process is active migration of tumor cells, which critically depends on reorganization of the actin cytoskeleton. Factors regulating actin assembly are just beginning to emerge as potential targets for preventing dissemination and invasion of tumor cells. Recent studies have shown that actin-dependent cellular processes, including tumor invasion, can be pharmacologically modulated by small-molecule inhibitors of actin assembly. In this chapter, we summarize reports on newly identified small-molecule inhibitors that target a growing number of actin nucleation and assembly factors relevant for human disease.

Dynamics of Gαq-protein-p63RhoGEF interaction and its regulation by RGS2.

Some G-protein-coupled receptors regulate biological processes via Gα12/13- or Gαq/11-mediated stimulation of RhoGEFs (guanine-nucleotide-exchange factors). p63RhoGEF is known to be specifically activated by Gαq/11 and mediates a major part of the acute response of vascular smooth muscle cells to angiotensin II treatment. In order to gain information about the dynamics of receptor-mediated activation of p63RhoGEF, we developed a FRET-based assay to study interactions between Gαq-CFP and Venus-p63RhoGEF in single living cells. Upon activation of histaminergic H1 or muscarinic M3 receptors, a robust FRET signal occurred that allowed for the first time the analysis of the kinetics of this interaction in detail. On- and off-set kinetics of Gαq-p63RhoGEF interactions closely resembled the kinetics of Gαq activity. Analysis of the effect of RGS2 (regulator of G-protein signalling 2) on the dynamics of Gαq activity and their interaction with p63RhoGEF showed that RGS2 is able to accelerate both deactivation of Gαq proteins and dissociation of Gαq and p63RhoGEF to a similar extent. Furthermore, we were able to detect activation-dependent FRET between RGS2 and p63RhoGEF and observed a reduced p63RhoGEF-mediated downstream signalling in the presence of RGS2. In summary, these observations support the concept of a functional activation-dependent p63RhoGEF-Gαq-RGS2 complex.