Actin Dynamics Research Articles

ANTXR1 deficiency promotes fibroblast senescence: implications for GAPO syndrome as a progeroid disorder

ANTXR1 is one of two cell surface receptors mediating the uptake of the anthrax toxin into cells. Despite substantial research on its role in anthrax poisoning and a proposed function as a collagen receptor, ANTXR1’s physiological functions remain largely undefined. Pathogenic variants in ANTXR1 lead to the rare GAPO syndrome, named for its four primary features: Growth retardation, Alopecia, Pseudoanodontia, and Optic atrophy. The disease is also associated with a complex range of other phenotypes impacting the cardiovascular, skeletal, pulmonary and nervous systems. Aberrant accumulation of extracellular matrix components and fibrosis are considered to be crucial components in the pathogenesis of GAPO syndrome, contributing to the shortened life expectancy of affected individuals. Nonetheless, the specific mechanisms connecting ANTXR1 deficiency to the clinical manifestations of GAPO syndrome are largely unexplored. In this study, we present evidence that ANTXR1 deficiency initiates a senescent phenotype in human fibroblasts, correlating with defects in nuclear architecture and actin dynamics. We provide novel insights into ANTXR1's physiological functions and propose GAPO syndrome to be reconsidered as a progeroid disorder highlighting an unexpected role for an integrin-like extracellular matrix receptor in human aging.

Proteome profile of Leishmania donovani Centrin1−/− parasite-infected human macrophage cell line and its implications in determining possible mechanisms of protective immunity

Our developed cell division-specific ‘centrin’ gene deleted Leishmania donovani (LdCen1−/−) the causative parasite of the fatal visceral-leishmaniasis (VL), exhibits a selective growth arrest at the intracellular stage and is anticipated as a live attenuated vaccine candidate against VL. LdCen1−/− immunization in animals has shown increased IFN-γ secreting CD4+ and CD8+ T cells along with protection conferred by a protective proinflammatory immune response. A label-free proteomics approach has been employed to understand the physiology of infection and predict disease interceptors during Leishmania-host interactions. Proteomic modulation after infection of human macrophage cell lines suggested elevated annexin A6, implying involvement in various biological processes such as membrane repair, transport, actin dynamics, cell proliferation, survival, differentiation, and inflammation, thereby potentiating its immunological protective capacity. Additionally, S100A8 and S100A9 proteins, known for maintaining homeostatic balance in regulating the inflammatory response, have been upregulated after infection. The inhibitory clade of serpins, known to inhibit cysteine proteases (CPs), was upregulated in host cells after 48 h of infection. This is reflected in the diminished expression of CPs in the parasites during infection. Such proteome analysis confirms LdCen1−/− efficacy as a vaccine candidate and predicts potential markers in future vaccine development strategies against infectious diseases.

CdSe/ZnS Quantum Dots' Impact on In Vitro Actin Dynamics.

Quantum dots (QDs) are a novel type of nanomaterial that has unique optical and physical characteristics. As such, QDs are highly desired because of their potential to be used in both biomedical and industrial applications. However, the mass adoption of QDs usage has raised concerns among the scientific community regarding QDs' toxicity. Although many papers have reported the negative impact of QDs on a cellular level, the exact mechanism of the QDs' toxicity is still unclear. In this investigation, we study the adverse effects of QDs by focusing on one of the most important cellular processes: actin polymerization and depolymerization. Our results showed that QDs act in a biphasic manner where lower concentrations of QDs stimulate the polymerization of actin, while high concentrations of QDs inhibit actin polymerization. Furthermore, we found that QDs can bind to filamentous actin (F-actin) and cause bundling of the filament while also promoting actin depolymerization. Through this study, we found a novel mechanism in which QDs negatively influence cellular processes and exert toxicity.

Simvastatin activates the spindle assembly checkpoint and causes abnormal cell division by modifying small GTPases

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which is a rate-limiting enzyme of the cholesterol synthesis pathway. It has been used clinically as a lipid-lowering agent to reduce low-density lipoprotein (LDL) cholesterol levels. In addition, antitumor activity has been demonstrated. Although simvastatin attenuates the prenylation of small GTPases, its effects on cell division in which small GTPases play an important role, have not been examined as a mechanism underlying its cytostatic effects. In this study, we determined its effect on cell division. Cell cycle synchronization experiments revealed a delay in mitotic progression in simvastatin-treated cells at concentrations lower than the IC50. Time-lapse imaging analysis indicated that the duration of mitosis, especially from mitotic entry to anaphase onset, was prolonged. In addition, simvastatin increased the number of cells exhibiting misoriented anaphase/telophase and bleb formation. Inhibition of the spindle assembly checkpoint (SAC) kinase Mps1 canceled the mitotic delay. Additionally, the number of cells exhibiting kinetochore localization of BubR1, an essential component of SAC, was increased, suggesting an involvement of SAC in the mitotic delay. Enhancement of F-actin formation and cell rounding at mitotic entry indicates that cortical actin dynamics were affected by simvastatin. The cholesterol removal agent methyl-β-cyclodextrin (MβCD) accelerated mitotic progression differently from simvastatin, suggesting that cholesterol loss from the plasma membrane is not involved in the mitotic delay. Of note, the small GTPase RhoA, which is a critical factor for cortical actin dynamics, exhibited upregulated expression. In addition, Rap1 was likely not geranylgeranylated. Our results demonstrate that simvastatin affects actin dynamics by modifying small GTPases, thereby activating the spindle assembly checkpoint and causing abnormal cell division.

Elevated α-synuclein levels inhibit mitophagic flux

The pathogenic effect of SNCA gene multiplications indicates that elevation of wild-type α-synuclein levels is sufficient to cause Parkinson’s disease (PD). Mitochondria have been proposed to be a major target of α-synuclein-induced damage. PINK1/parkin/DJ-1-mediated mitophagy is a defense strategy that allows cells to selectively eliminate severely damaged mitochondria. Here, we quantified mitophagic flux and non-mitochondrial autophagic flux in three models of increased α-synuclein expression: 1/Drosophila melanogaster that transgenically express human wild-type and mutant α-synuclein in flight muscle; 2/human skin fibroblasts transfected with α-synuclein or β-synuclein; and 3/human induced pluripotent stem cell (iPSC)-derived neurons carrying an extra copy of wild-type SNCA under control of a doxycycline-inducible promoter, allowing titratable α-synuclein upregulation. In each model, elevated α-synuclein levels potently suppressed mitophagic flux, while non-mitochondrial autophagy was preserved. In human neurons, a twofold increase in wild-type α-synuclein was already sufficient to induce this effect. PINK1 and parkin activation and mitochondrial translocation of DJ-1 after mitochondrial depolarization were not affected by α-synuclein upregulation. Overexpression of the actin-severing protein cofilin or treatment with CK666, an inhibitor of the actin-related protein 2/3 (Arp2/3) complex, rescued mitophagy in neurons with increased α-synuclein, suggesting that excessive actin network stabilization mediated the mitophagy defect. In conclusion, elevated α-synuclein levels inhibit mitophagic flux. Disruption of actin dynamics may play a key role in this effect.

Abstract 193: Melanoma cytoskeletal dynamics, membrane remodeling, and motility are regulated by TRIM9

Abstract Cell shape change and motility are essential in metastasis and involve remodeling of the actin cytoskeleton, cell adhesions, and plasma membrane. How these cytoskeletal and membrane remodeling are regulated and coordinated remains largely unknown. We previously identified TRIM9 as a regulator of actin dynamics and exocytosis in developing neurons. Deletion of murine Trim9 impairs neuronal migration, netrin-induced axon turning, and axonal and dendritic branching, and increased exocytosis and filopodial stability. In addition, we have shown TRIM9 alters dynamics of the actin polymerase VASP at filopodia tips via non-degrative mono-ubiquitination. TRIM9 is expressed in other motile cells, but the non-neuronal role of TRIM9 remains unknown. TRIM9 was identified as a possible prognostic biomarker in melanoma and high expression correlates with low patient survival. Melanomas undergo phenotype switching, where three distinct phenotypes exist that are associated with differences in gene expression. The three phenotypes consist of a melanocytic proliferative state, a neural crest-like invasive state, an intermediate state. Single cell RNAseq (scRNASeq) data from patient-derived melanoma indicate TRIM9 is highly expressed in the proliferative melanocytic state, which correlates with poor prognosis. We hypothesize that TRIM9 coordinates actin dynamics, adhesion, and exocytosis in melanoma. We show that TRIM9 protein is enriched in several human melanoma lines. Here we examine the role of TRIM9 in regulation of focal adhesions, exocytosis, migration, and invasion in two of these lines. Genetic loss of TRIM9 increased random migration velocity but reduced directional persistence. Interestingly, we find that TRIM9 plays a role in promoting bleb-like morphology and inhibits the ability to durotax on shallow gradients on soft substrate. Fluorescence recovery after photobleaching (FRAP), Total internal reflection fluorescence microscopy (TIRF), and widefield microscopy revealed that loss of TRIM9 results in increased focal adhesions, cell size, and altered dynamics of focal adhesion proteins VASP, zyxin, and paxillin dynamics. In addition, TRIM9 knockout cells exhibit reduced filopodial length and density and altered filopodial localization of VASP. In-gel zymography indicates that TRIM9 knockout cells also display an increased degradative capacity. Current studies are investigating how loss of TRIM9 alters parameters of cell contractility, lamellipodia, invadopodia, and exocytosis to define the role of TRIM9 in melanoma motility. Together these findings suggest TRIM9 reduces adhesion and migration, increases proliferation and blebbing and may be an important regulator of phenotype switching in melanoma. Citation Format: Kimberly Lukasik, Jordan Brooks, Sam Redinbo, James Bear, Stephanie Gupton. Melanoma cytoskeletal dynamics, membrane remodeling, and motility are regulated by TRIM9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 193.

The Hippo kinase cascade regulates a contractile cell behavior and cell density in a close unicellular relative of animals.

The genomes of close unicellular relatives of animals encode orthologs of many genes that regulate animal development. However, little is known about the function of such genes in unicellular organisms or the evolutionary process by which these genes came to function in multicellular development. The Hippo pathway, which regulates cell proliferation and tissue size in animals, is present in some of the closest unicellular relatives of animals, including the amoeboid organism Capsaspora owczarzaki. We previously showed that the Capsaspora ortholog of the Hippo pathway nuclear effector Yorkie/YAP/TAZ (coYki) regulates actin dynamics and the three-dimensional morphology of Capsaspora cell aggregates, but is dispensable for cell proliferation control (Phillips et al., 2022). However, the function of upstream Hippo pathway components, and whether and how they regulate coYki in Capsaspora, remained unknown. Here, we analyze the function of the upstream Hippo pathway kinases coHpo and coWts in Capsaspora by generating mutant lines for each gene. Loss of either kinase results in increased nuclear localization of coYki, indicating an ancient, premetazoan origin of this Hippo pathway regulatory mechanism. Strikingly, we find that loss of either kinase causes a contractile cell behavior and increased density of cell packing within Capsaspora aggregates. We further show that this increased cell density is not due to differences in proliferation, but rather actomyosin-dependent changes in the multicellular architecture of aggregates. Given its well-established role in cell density-regulated proliferation in animals, the increased density of cell packing in coHpo and coWts mutants suggests a shared and possibly ancient and conserved function of the Hippo pathway in cell density control. Together, these results implicate cytoskeletal regulation but not proliferation as an ancestral function of the Hippo pathway kinase cascade and uncover a novel role for Hippo signaling in regulating cell density in a proliferation-independent manner.

The Hippo kinase cascade regulates a contractile cell behavior and cell density in a close unicellular relative of animals

The genomes of close unicellular relatives of animals encode orthologs of many genes that regulate animal development. However, little is known about the function of such genes in unicellular organisms or the evolutionary process by which these genes came to function in multicellular development. The Hippo pathway, which regulates cell proliferation and tissue size in animals, is present in some of the closest unicellular relatives of animals, including the amoeboid organism Capsaspora owczarzaki. We previously showed that the Capsaspora ortholog of the Hippo pathway nuclear effector Yorkie/YAP/TAZ (coYki) regulates actin dynamics and the three-dimensional morphology of Capsaspora cell aggregates, but is dispensable for cell proliferation control (Phillips et al., 2022). However, the function of upstream Hippo pathway components, and whether and how they regulate coYki in Capsaspora, remained unknown. Here, we analyze the function of the upstream Hippo pathway kinases coHpo and coWts in Capsaspora by generating mutant lines for each gene. Loss of either kinase results in increased nuclear localization of coYki, indicating an ancient, premetazoan origin of this Hippo pathway regulatory mechanism. Strikingly, we find that loss of either kinase causes a contractile cell behavior and increased density of cell packing within Capsaspora aggregates. We further show that this increased cell density is not due to differences in proliferation, but rather actomyosin-dependent changes in the multicellular architecture of aggregates. Given its well-established role in cell density-regulated proliferation in animals, the increased density of cell packing in coHpo and coWts mutants suggests a shared and possibly ancient and conserved function of the Hippo pathway in cell density control. Together, these results implicate cytoskeletal regulation but not proliferation as an ancestral function of the Hippo pathway kinase cascade and uncover a novel role for Hippo signaling in regulating cell density in a proliferation-independent manner.

Kisspeptin/KISS1R Signaling Modulates Human Airway Smooth Muscle Cell Migration.

Airway remodeling is a cardinal feature of asthma, associated with increased airway smooth muscle (ASM) cell mass and upregulation of extracellular matrix deposition. Exaggerated ASM cell migration contributes to excessive ASM mass. Previously, we demonstrated the alleviating role of Kp (kisspeptin) receptor (KISS1R) activation by Kp-10 in mitogen (PDGF [platelet-derived growth factor])-induced human ASM cell proliferation invitro and airway remodeling invivo in a mouse model of asthma. Here, we examined the mechanisms by which KISS1R activation regulates mitogen-induced ASM cell migration. KISS1R activation using Kp-10 significantly inhibited PDGF-induced ASM cell migration, further confirmed using KISS1R shRNA. Furthermore, KISS1R activation modulated F/G actin dynamics and the expression of promigration proteins like CDC42 (cell division control protein 42) and cofilin. Mechanistically, we observed reduced ASM RhoA-GTPAse with KISS1R activation. The antimigratory effect of KISS1R was abolished by PKA (protein kinase A)-inhibitory peptide. Conversely, KISS1R activation significantly increased cAMP and phosphorylation of CREB (cAMP-response element binding protein) in PDGF-exposed ASM cells. Overall, these results highlight the alleviating properties of Kp-10 in the context of airway remodeling.

Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

CD56/NCAM mediates cell migration of human NK cells by promoting integrin-mediated adhesion turnover.

Natural killer (NK) cells patrol tissue to mediate lysis of virally infected and tumorigenic cells. Human NK cells are typically identified by their expression of neural cell adhesion molecule (NCAM, CD56), yet despite its ubiquitous expression on NK cells, CD56 remains a poorly understood protein on immune cells. CD56 has been previously demonstrated to play roles in NK cell cytotoxic function and cell migration. Specifically, CD56-deficient NK cells have impaired cell migration on stromal cells and CD56 is localized to the uropod of NK cells migrating on stroma. Here, we show that CD56 is required for NK cell migration on ICAM-1 and is required for the establishment of persistent cell polarity and unidirectional actin flow. The intracellular domain of CD56 (NCAM-140) is required for its function and the loss of CD56 leads to enlarged actin foci and sequestration of phosphorylated Pyk2 accompanied by increased size and frequency of activated LFA-1 clusters. Together, these data identify a role for CD56 in regulating human NK cell migration through modulation of actin dynamics and integrin turnover.

Collective dynamics of actin and microtubule and its crosstalk mediated by FHDC1.

The coordination between actin and microtubule network is crucial, yet this remains a challenging problem to dissect and our understanding of the underlying mechanisms remains limited. In this study, we used travelling waves in the cell cortex to characterize the collective dynamics of cytoskeletal networks. Our findings show that Cdc42 and F-BAR-dependent actin waves in mast cells are mainly driven by formin-mediated actin polymerization, with the microtubule-binding formin FH2 domain-containing protein 1 (FHDC1) as an early regulator. Knocking down FHDC1 inhibits actin wave formation, and this inhibition require FHDC1's interaction with both microtubule and actin. The phase of microtubule depolymerization coincides with the nucleation of actin waves and microtubule stabilization inhibit actin waves, leading us to propose that microtubule shrinking and the concurrent release of FHDC1 locally regulate actin nucleation. Lastly, we show that FHDC1 is crucial for multiple cellular processes such as cell division and migration. Our data provided molecular insights into the nucleation mechanisms of actin waves and uncover an antagonistic interplay between microtubule and actin polymerization in their collective dynamics.

Effects of rpl1001 Gene Deletion on Cell Division of Fission Yeast and Its Molecular Mechanism.

The rpl1001 gene encodes 60S ribosomal protein L10, which is involved in intracellular protein synthesis and cell growth. However, it is not yet known whether it is involved in the regulation of cell mitosis dynamics. This study focuses on the growth, spore production, cell morphology, the dynamics of microtubules, chromosomes, actin, myosin, and mitochondria of fission yeast (Schizosaccharomyces pombe) to investigate the impact of rpl1001 deletion on cell mitosis. RNA-Seq and bioinformatics analyses were also used to reveal key genes, such as hsp16, mfm1 and isp3, and proteasome pathways. The results showed that rpl1001 deletion resulted in slow cell growth, abnormal spore production, altered cell morphology, and abnormal microtubule number and length during interphase. The cell dynamics of the rpl1001Δ strain showed that the formation of a monopolar spindle leads to abnormal chromosome segregation with increased rate of spindle elongation in anaphase of mitosis, decreased total time of division, prolonged formation time of actin and myosin loops, and increased expression of mitochondrial proteins. Analysis of the RNA-Seq sequencing results showed that the proteasome pathway, up-regulation of isp3, and down-regulation of mfm1 and mfm2 in the rpl1001Δ strain were the main factors underpinning the increased number of spore production. Also, in the rpl1001Δ strain, down-regulation of dis1 caused the abnormal microtubule and chromosome dynamics, and down-regulation of hsp16 and pgk1 were the key genes affecting the delay of actin ring and myosin ring formation. This study reveals the effect and molecular mechanism of rpl1001 gene deletion on cell division, which provides the scientific basis for further clarifying the function of the Rpl1001 protein in cell division.

Circulating small extracellular vesicles mediate vascular hyperpermeability in diabetes

Aims/hypothesisA hallmark chronic complication of type 2 diabetes mellitus is vascular hyperpermeability, which encompasses dysfunction of the cerebrovascular endothelium and the subsequent development of associated cognitive impairment. The present study tested the hypothesis that during type 2 diabetes circulating small extracellular vesicles (sEVs) exhibit phenotypic changes that facilitate pathogenic disruption of the vascular barrier.MethodssEVs isolated from the plasma of a mouse model of type 2 diabetes and from diabetic human individuals were characterised for their ability to disrupt the endothelial cell (EC) barrier. The contents of sEVs and their effect on recipient ECs were assessed by proteomics and identified pathways were functionally interrogated with small molecule inhibitors.ResultsUsing intravital imaging, we found that diabetic mice (Leprdb/db) displayed hyperpermeability of the cerebrovasculature. Enhanced vascular leakiness was recapitulated following i.v. injection of sEVs from diabetic mice into non-diabetic recipient mice. Characterisation of circulating sEV populations from the plasma of diabetic mice and humans demonstrated increased quantity and size of sEVs compared with those isolated from non-diabetic counterparts. Functional experiments revealed that sEVs from diabetic mice or humans induced the rapid and sustained disruption of the EC barrier through enhanced paracellular and transcellular leak but did not induce inflammation. Subsequent sEV proteome and recipient EC phospho-proteome analysis suggested that extracellular vesicles (sEVs) from diabetic mice and humans modulate the MAPK/MAPK kinase (MEK) and Rho-associated protein kinase (ROCK) pathways, cell–cell junctions and actin dynamics. This was confirmed experimentally. Treatment of sEVs with proteinase K or pre-treatment of recipient cells with MEK or ROCK inhibitors reduced the hyperpermeability-inducing effects of circulating sEVs in the diabetic state.Conclusions/interpretationDiabetes is associated with marked increases in the concentration and size of circulating sEVs. The modulation of sEV-associated proteins under diabetic conditions can induce vascular leak through activation of the MEK/ROCK pathway. These data identify a new paradigm by which diabetes can induce hyperpermeability and dysfunction of the cerebrovasculature and may implicate sEVs in the pathogenesis of cognitive decline during type 2 diabetes.Graphical

Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells.

The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and adaptation to external stimuli. The accurate and unbiased visualization of the diverse actin assemblies within cells is an ongoing challenge. We describe here the identification and use of designed ankyrin repeat proteins (DARPins) as synthetic actin binders. Actin-binding DARPins were identified through ribosome display and validated biochemically. When introduced or expressed inside living cells, fluorescently labeled DARPins accumulated at actin filaments, validated through phalloidin colocalization on fixed cells. Nevertheless, different DARPins displayed different actin labeling patterns: some DARPins labeled efficiently dynamic structures, such as filopodia, lamellipodia, and blebs, while others accumulated primarily in stress fibers. This differential intracellular distribution correlated with DARPin-actin binding kinetics, as measured by fluorescence recovery after photobleaching experiments. Moreover, the rapid arrest of actin dynamics induced by pharmacological treatment led to the fast relocalization of DARPins. Our data support the hypothesis that the localization of actin probes depends on the inherent dynamic movement of the actin cytoskeleton. Compared to the widely used LifeAct probe, one DARPin exhibited enhanced signal-to-background ratio while retaining a similar ability to label stress fibers. In summary, we propose DARPins as promising actin-binding proteins for labeling or manipulation in living cells.

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.

IntAct: A nondisruptive internal tagging strategy to study the organization and function of actin isoforms.

Mammals have 6 highly conserved actin isoforms with nonredundant biological functions. The molecular basis of isoform specificity, however, remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoforms in fixed and living cells. We identified a residue pair in β-actin that permits tag integration and used knock-in cell lines to demonstrate that IntAct β-actin expression and filament incorporation is indistinguishable from wild type. Furthermore, IntAct β-actin remains associated with common actin-binding proteins (ABPs) and can be targeted in living cells. We demonstrate the usability of IntAct for actin isoform investigations by showing that actin isoform-specific distribution is maintained in human cells. Lastly, we observed a variant-dependent incorporation of tagged actin variants into yeast actin patches, cables, and cytokinetic rings demonstrating cross species applicability. Together, our data indicate that IntAct is a versatile tool to study actin isoform localization, dynamics, and molecular interactions.

Transcriptional responses to salinity-induced changes in cell wall morphology of the euryhaline diatom Pleurosira laevis.

Diatoms are unicellular algae with morphologically diverse silica cell walls, which are called frustules. The mechanism of frustule morphogenesis has attracted attention in biology and nanomaterials engineering. However, the genetic regulation of the morphology remains unclear. We therefore used transcriptome sequencing to search for genes involved in frustule morphology in the centric diatom Pleurosira laevis, which exhibits morphological plasticity between flat and domed valve faces in salinity 2 and 7, respectively. We observed differential expression of transposable elements (TEs) and transporters, likely due to osmotic response. Up-regulation of mechanosensitive ion channels and down-regulation of Ca2+-ATPases in cells with flat valves suggested that cytosolic Ca2+ levels were changed between the morphologies. Calcium signaling could be a mechanism for detecting osmotic pressure changes and triggering morphological shifts. We also observed an up-regulation of ARPC1 and annexin, involved in the regulation of actin filament dynamics known to affect frustule morphology, as well as the up-regulation of genes encoding frustule-related proteins such as BacSETs and frustulin. Taken together, we propose a model in which salinity-induced morphogenetic changes are driven by upstream responses, such as the regulation of cytosolic Ca2+ levels, and downstream responses, such as Ca2+-dependent regulation of actin dynamics and frustule-related proteins. This study highlights the sensitivity of euryhaline diatoms to environmental salinity and the role of active cellular processes in controlling gross valve morphology under different osmotic pressures.

The non-muscle actinopathy-associated mutation E334Q in cytoskeletal γ-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins.

Various heterozygous cytoskeletal γ-actin mutations have been shown to cause Baraitser-Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal γ-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin β4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation, and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover, contribute to the observed disease phenotype.

The non-muscle actinopathy-associated mutation E334Q in cytoskeletal γ-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins

Various heterozygous cytoskeletal γ-actin mutations have been shown to cause Baraitser–Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal γ-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin β4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation, and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover, contribute to the observed disease phenotype.