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Formin is an important player in promoting apical actin polymerization in pollen tubes, but the mechanism regulating its activity remains unknown. We here identify REN1, a Rho GTPase-activating protein, as a negative regulator of formins in Arabidopsis pollen tubes. Specifically, we found that depletion of REN1 promotes apical actin polymerization and increases the amount of filamentous actin in pollen tubes. Interestingly, the effect of REN1 loss of function phenocopies the effect of formin gain of function, as it causes the formation of supernumerary membrane-derived actin bundles, which leads to tube swelling and membrane deformation. Importantly, inhibition of formins suppresses the phenotypic defects in ren1 mutant pollen tubes. We further demonstrate that REN1 physically interacts with the Arabidopsis formin protein AtFH5, predominantly with the C terminus, and inhibits the ability of AtFH5 to nucleate and assemble actin in vitro. Depletion of AtFH5 partially suppresses the phenotype in ren1 mutant pollen tubes, demonstrating that REN1 regulates apical actin polymerization at least partially through inhibiting AtFH5. We thus uncover a novel mechanism regulating formins and actin polymerization in plants. [Display omitted] • Depletion of REN1 promotes apical actin polymerization in pollen tubes • REN1 inhibits the ability of AtFH5 to nucleate actin assembly in vitro • Depletion of AtFH5 partially suppresses the phenotype in ren1 mutant pollen tubes • AtFH5 interacts with REN1 predominantly through its C terminus Xu et al. demonstrate that REN1, a Rho GTPase-activating protein, regulates apical actin polymerization through inhibiting Arabidopsis formin protein AtFH5 in pollen tubes. This study uncovers a novel mechanism regulating formins and actin polymerization in plants. [ABSTRACT FROM AUTHOR]

Actin polymerization is a crucial process in cellular functions, impacting key processes like cell division, development, morphogenesis, motility, and polarity establishment. Abnormalities in actin polymerization can lead to various diseases, including cancer, highlighting its significance in both normal cell function and disease progression. While the amino acid sequences of actin are highly conserved across species, recent studies have revealed differences in their polymerization functions. The animal or plant actin can be used heterologously in yeast cells, but certain types like the rabbit skeletal muscle and maize pollen actin have unique polymerization properties. On the other hand, the yeast actin can interact with the animal or plant actin. This review explores the polymerization characteristics of the animal, plant, and yeast actin, highlighting functional disparities in polymerization both in vivo and in vitro. Additionally, we discuss drugs that target actin polymerization, aiming to offer potential avenues for treating human diseases and advancing our understanding of actin's molecular mechanisms. Further research in this area may uncover the underlying principles of actin diversity and functional evolution, providing valuable insights for related fields. [ABSTRACT FROM AUTHOR]

Form and function are often interdependent throughout biology. Inside cells, mitochondria have particularly attracted attention since both their morphology and functionality are altered under pathophysiological conditions. However, directly assessing their causal relationship has been beyond reach due to the limitations of manipulating mitochondrial morphology in a physiologically relevant manner. By engineering a bacterial actin regulator, ActA, we developed tools termed ActuAtor that inducibly trigger actin polymerization at arbitrary subcellular locations. The ActuAtor-mediated actin polymerization drives striking deformation and/or movement of target organelles, including mitochondria, Golgi apparatus, and nucleus. Notably, ActuAtor operation also disperses non-membrane-bound entities such as stress granules. We then implemented ActuAtor in functional assays, uncovering the physically fragmented mitochondria being slightly more susceptible to degradation, while none of the organelle functions tested are morphology dependent. The modular and genetically encoded features of ActuAtor should enable its application in studies of the form-function interplay in various intracellular contexts.

Although more difficult to detect than in the cytoplasm, it is now clear that actin polymerization occurs in the nucleus and that it plays a role in the specific processes of the nucleus such as transcription, replication, and DNA repair. A number of studies suggest that nuclear actin polymerization is promoting precise DNA repair by homologous recombination, which could potentially be of help for precise genome editing and gene therapy. This review summarizes the findings and describes the challenges and chances in the field. [ABSTRACT FROM AUTHOR]

Actin filaments, as key components of the cytoskeleton, have aroused great interest due to their numerous functional roles in eukaryotic cells, including intracellular electrical signaling. The aim of this research is to characterize the alternating current (AC) conduction characteristics of both globular and polymerized actin and quantitatively compare their values to those theoretically predicted earlier. Actin filaments have been demonstrated to act as conducting bionanowires, forming a signaling network capable of transmitting ionic waves in cells. We performed conductivity measurements for different concentrations of actin, considering both unpolymerized and polymerized actin to identify potential differences in their electrical properties. These measurements revealed two relevant characteristics: first, the polymerized actin, arranged in filaments, has a lower impedance than its globular counterpart; second, an increase in the actin concentration leads to higher conductivities. Furthermore, from the data collected, we developed a quantitative model to represent the electrical properties of actin in a buffer solution. We hypothesize that actin filaments can be modeled as electrical resistor–inductor–capacitor (RLC) circuits, where the resistive contribution is due to the viscous ion flows along the filaments; the inductive contribution is due to the solenoidal flows along and around the helix-shaped filament and the capacitive contribution is due to the counterion layer formed around each negatively charged filament. [ABSTRACT FROM AUTHOR]

Angiosarcoma is a rare refractory soft-tissue tumor with a poor prognosis and is treated by radiotherapy. The fibroblast growth factor 1 (FGF1) mutant, with enhanced thermostability due to several substituted amino acids, inhibits angiosarcoma cell metastasis, yet the mechanism of action is unclear. This study aims to clarify the FGF1 mutant mechanism of action using ISOS-1 mouse angiosarcoma cells. The wild-type FGF1 or FGF1 mutant was added to ISOS-1 cells and cultured, evaluating cell numbers over time. The invasive and migratory capacity of ISOS-1 cells was assessed by transwell analysis. ISOS-1 cell radiosensitivity was assessed by colony formation assay after X-ray irradiation. To examine whether mitogen-activated protein kinase (MEK) inhibitor counteracts the FGF1 mutant effects, a combination of MEK inhibitor and FGF1 mutant was added to ISOS-1 cells and cultured. The FGF1 mutant was observed to inhibit ISOS-1 cell proliferation, invasion and migration by sustained FGF1 signaling activation. A MEK inhibitor suppressed the FGF1 mutant-induced inhibition of proliferation, invasion and migration of ISOS-1 cells. Furthermore, the FGF1 mutant enhanced radiosensitivity of ISOS-1 cells, but MEK inhibition suppressed the increased radiosensitivity. In addition, we found that the FGF1 mutant strongly inhibits actin polymerization, suggesting that actin cytoskeletal dynamics are closely related to ISOS-1 cell radiosensitivity. Overall, this study demonstrated that in ISOS-1 cells, the FGF1 mutant inhibits proliferation, invasion and migration while enhancing radiosensitivity through sustained activation of the MEK-mediated signaling pathway. [ABSTRACT FROM AUTHOR]

Pulmonary surfactant is essential for maintaining proper lung function. Alveolar epithelial type II (AE2) cells secrete surfactants via lamellar bodies (LBs). In tidal loading during each breath, the physiological cyclic stretching of AE2 cells promotes surfactant secretion. Excessive stretching inhibits surfactant secretion, which is considered to contribute to the development of lung damage. However, its precise mechanism remains unknown. This study tested whether actin polymerization and intracellular transport are required for pulmonary surfactant secretion and the association of actin polymerization and transport in identical human AE2-derived A549 cells using live-cell imaging, not in the bulk cells population. We found that overstretching approximately doubled actin polymerization into filaments (F-actin) and suppressed LB secretion by half in the fluorescent area ratio, compared with physiological stretching (F-actin: 1.495 vs 0.643 (P

C3G is a Rap1 GEF that plays a pivotal role in platelet-mediated processes such as angiogenesis, tumor growth, and metastasis by modulating the platelet secretome. Here, we explore the mechanisms through which C3G governs platelet secretion. For this, we utilized animal models featuring either overexpression or deletion of C3G in platelets, as well as PC12 cell clones expressing C3G mutants. We found that C3G specifically regulates α-granule secretion via PKCδ, but it does not affect δ-granules or lysosomes. C3G activated RalA through a GEF-dependent mechanism, facilitating vesicle docking, while interfering with the formation of the trans-SNARE complex, thereby restricting vesicle fusion. Furthermore, C3G promotes the formation of lamellipodia during platelet spreading on specific substrates by enhancing actin polymerization via Src and Rac1-Arp2/3 pathways, but not Rap1. Consequently, C3G deletion in platelets favored kiss-and-run exocytosis. C3G also controlled granule secretion in PC12 cells, including pore formation. Additionally, C3G-deficient platelets exhibited reduced phosphatidylserine exposure, resulting in decreased thrombin generation, which along with defective actin polymerization and spreading, led to impaired clot retraction. In summary, platelet C3G plays a dual role by facilitating platelet spreading and clot retraction through the promotion of outside-in signaling while concurrently downregulating α-granule secretion by restricting granule fusion. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) has been known as a common viral pathogen that can infect several parts of the body, leading to various clinical manifestations. According to this diverse manifestation, HSV-1 infection in many cell types was demonstrated. Besides the HSV-1 cell tropism, e.g., fibroblast, epithelial, mucosal cells, and neurons, HSV-1 infections can occur in human T lymphocyte cells, especially in activated T cells. In addition, several studies found that actin polymerization and filopodia formation support HSV-1 infection in diverse cell types. Hence, the goal of this review is to explore the mechanism of HSV-1 infection in various types of cells involving filopodia formation and highlight potential future directions for HSV-1 entry-related research. Moreover, this review covers several strategies for possible anti-HSV drugs focused on the entry step, offering insights into potential therapeutic interventions. [ABSTRACT FROM AUTHOR]

To acquire the capacity to fertilize the oocyte, mammalian spermatozoa must undergo a series of biochemical reactions in the female reproductive tract, which are collectively called capacitation. The capacitated spermatozoa subsequently interact with the oocyte zona-pellucida and undergo the acrosome reaction, which enables the penetration of the oocyte and subsequent fertilization. However, the spontaneous acrosome reaction (sAR) can occur prematurely in the sperm before reaching the oocyte cumulus oophorus, thereby jeopardizing fertilization. One of the main processes in capacitation involves actin polymerization, and the resulting F-actin is subsequently dispersed prior to the acrosome reaction. Several biochemical reactions that occur during sperm capacitation, including actin polymerization, protect sperm from sAR. In the present review, we describe the protective mechanisms that regulate sperm capacitation and prevent sAR. [ABSTRACT FROM AUTHOR]

Nemaline myopathy (NM) is one of the most common forms of congenital myopathy and it is identified by the presence of "nemaline bodies" (rods) in muscle fibers by histopathological examination. The most common forms of NM are caused by mutations in the Actin Alpha 1 (ACTA1) and Nebulin (NEB) genes. Clinical features include hypotonia and muscle weakness. Unfortunately, there is no curative treatment and the pathogenetic mechanisms remain unclear. In this manuscript, we examined the pathophysiological alterations in NM using dermal fibroblasts derived from patients with mutations in ACTA1 and NEB genes. Patients' fibroblasts were stained with rhodamine–phalloidin to analyze the polymerization of actin filaments by fluorescence microscopy. We found that patients' fibroblasts showed incorrect actin filament polymerization compared to control fibroblasts. Actin filament polymerization defects were associated with mitochondrial dysfunction. Furthermore, we identified two mitochondrial-boosting compounds, linoleic acid (LA) and L-carnitine (LCAR), that improved the formation of actin filaments in mutant fibroblasts and corrected mitochondrial bioenergetics. Our results indicate that cellular models can be useful to study the pathophysiological mechanisms involved in NM and to find new potential therapies. Furthermore, targeting mitochondrial dysfunction with LA and LCAR can revert the pathological alterations in NM cellular models. [ABSTRACT FROM AUTHOR]

Migrating cells must integrate multiple, competing external guidance cues. However, it is not well understood how cells prioritize among these cues. We investigate external cue integration by monitoring the response of wave-like, actin-polymerization dynamics, the driver of cell motility, to combinations of nanotopographies and electric fields in neutrophil-like cells. The electric fields provide a global guidance cue, and approximate conditions at wound sites in vivo. The nanotopographies have dimensions similar to those of collagen fibers, and act as a local esotactic guidance cue. We find that cells prioritize guidance cues, with electric fields dominating long-term motility by introducing a unidirectional bias in the locations at which actin waves nucleate. That bias competes successfully with the wave guidance provided by the bidirectional nanotopographies.

Although more difficult to detect than in the cytoplasm, it is now clear that actin polymerization occurs in the nucleus and that it plays a role in the specific processes of the nucleus such as transcription, replication, and DNA repair. A number of studies suggest that nuclear actin polymerization is promoting precise DNA repair by homologous recombination, which could potentially be of help for precise genome editing and gene therapy. This review summarizes the findings and describes the challenges and chances in the field.

ABSTRACT Dermatophytes invade and colonize host superficial tissues via hyphal growth. Although cytoskeletal reorganization and its regulation are essential for hyphal growth, the molecular mechanisms in dermatophytes and their applicability as antifungal drug targets remain poorly understood. The p21-activated kinase (PAK) is a downstream effector of the small GTPases Rac and CDC42, also known as p21, and is involved in various molecular and cellular functions, including actin polymerization and cell morphogenesis. In this study, we investigated the contribution of the PAK protein TrCla4 to morphogenesis and mycelial growth in Trichophyton rubrum, the most frequently isolated fungus in dermatophytosis (athlete’s foot). The actin polymerization inhibitor, cytochalasin A inhibited actin accumulation at the hyphal tip and mycelial growth of T. rubrum, suggesting the involvement of the actin cytoskeleton in mycelial growth. In the Trcla4 knockout strain (ΔTrcla4), we observed defects in mycelial growth, hyphal branching, and the accumulation of polymerized actin at the hyphal tip. Chemical inhibitors of TrRac-dependent TrCla4 kinase activity, FRAX486 and IPA-3, also inhibited spore germination and mycelial growth. Interestingly, ΔTrcla4 showed no additional inhibition of mycelial growth when treated with these inhibitors, indicating that their inhibitory effects are primarily mediated through TrCla4. In an invertebrate dermatophyte infection model, animals infected with ΔTrcla4 had higher survival rates than those infected with the wild-type, and IPA-3 and FRAX486 treatments both significantly improved animal survival rates. These results suggest that the dermatophyte PAK promotes mycelial growth by facilitating actin polymerization at the hyphal tip, making it a potential therapeutic target for dermatophytosis. IMPORTANCE Superficial fungal infections, such as athlete’s foot, affect more than 10% of the world’s population and have a significant impact on quality of life. Despite the fact that treatment-resistant fungi are a concern, there are just a few antifungal drug targets accessible, as opposed to the wide range of therapeutic targets found in bacterial infections. As a result, additional alternatives are sought. In this study, we generated a PAK TrCla4 deletion strain (∆Trcla4) of Trichophyton rubrum. The ∆Trcla4 strain exhibited deficiencies in mycelial growth, hyphal morphology, and polarized actin localization at the hyphal tip. IPA-3 and FRAX486, small chemical inhibitors of mammalian PAK, were discovered to limit fungal mycelial proliferation. According to our findings, fungal PAKs are interesting therapeutic targets for the development of new antifungal medicines.

Current studies on actin function primarily rely on cytoplasmic actin due to the absence of cellular models specifically expressing nuclear actin. Here, cell models capable of expressing varying levels of nuclear F/G‐actin are generated and a significant role of nuclear actin in the regulation of epithelial‐mesenchymal transition (EMT) is uncovered. Through immunoprecipitation and mass spectrometry analyses, distinct binding partners for nuclear F‐actin (β‐catenin, SMAD2, and SMAD3) and nuclear G‐actin (MYBBP1A, NKRF, and MYPOP) are investigated, which respectively modulate EMT‐promoting and EMT‐repressing transcriptional events. While nuclear F‐actin promotes EMT with enhanced cell migration, survival, and elongated mesenchymal morphology, nuclear G‐actin represses EMT and related cell activities. Mechanistically, nuclear F‐actin enhances β‐catenin, SMAD2, and SMAD3 expression and stability in the nuclei, while nuclear G‐actin increases MYBBP1A, NKRF, and MYPOP expression and stability in the nuclei. The association between nuclear F/G‐actin and N‐cadherin/E‐cadherin in the cell lines (in vitro), and increased nuclear actin polymerization in the wound healing cells (in vivo) affirm a significant role of nuclear actin in EMT regulation. With evidence of nuclear actin polymerization and EMT during development, and irregularities in disease states such as cancer and fibrosis, targeting nuclear actin dynamics to trigger dysregulated EMT warrants ongoing study. [ABSTRACT FROM AUTHOR]

Strumpellin/Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex subunit 5 (WASHC5) is a core component of the WASH complex, and its mutations confer pathogenicity for hereditary spastic paraplegia (HSP) type SPG8, a rare neurodegenerative gait disorder. WASH complex activates actin-related protein-2/3-mediated actin polymerization and plays a pivotal role in intracellular membrane trafficking in endosomes. In this study, we examined the role of strumpellin in the regulation of structural plasticity of cortical neurons involved in gait coordination. Administration of a lentivirus containing a strumpellin-targeting short hairpin RNA (shRNA) to cortical motor neurons lead to abnormal motor coordination in mice. Strumpellin knockdown using shRNA attenuated dendritic arborization and synapse formation in cultured cortical neurons, and this effect was rescued by wild-type strumpellin expression. Compared with the wild-type, strumpellin mutants N471D or V626F identified in patients with SPG8 exhibited no differences in rescuing the defects. Moreover, the number of F-actin clusters in neuronal dendrites was decreased by strumpellin knockdown and rescued by strumpellin expression. In conclusion, our results indicate that strumpellin regulates the structural plasticity of cortical neurons via actin polymerization. • Strumpellin/WASHC5 regulates the structural plasticity of cortical neurons via actin polymerization. • Administration of Lenti-Str-shRNA to cortical motor neurons lead to abnormal motor coordination in mice. • Mutations from SPG8 patients do not affect the role of strumpellin for the dendritic structure of cortical neurons. [ABSTRACT FROM AUTHOR]

Actin filaments, as key components of the cytoskeleton, have aroused great interest due to their numerous functional roles in eukaryotic cells, including intracellular electrical signaling. The aim of this research is to characterize the alternating current (AC) conduction characteristics of both globular and polymerized actin and quantitatively compare their values to those theoretically predicted earlier. Actin filaments have been demonstrated to act as conducting bionanowires, forming a signaling network capable of transmitting ionic waves in cells. We performed conductivity measurements for different concentrations of actin, considering both unpolymerized and polymerized actin to identify potential differences in their electrical properties. These measurements revealed two relevant characteristics: first, the polymerized actin, arranged in filaments, has a lower impedance than its globular counterpart; second, an increase in the actin concentration leads to higher conductivities. Furthermore, from the data collected, we developed a quantitative model to represent the electrical properties of actin in a buffer solution. We hypothesize that actin filaments can be modeled as electrical resistor–inductor–capacitor (RLC) circuits, where the resistive contribution is due to the viscous ion flows along the filaments; the inductive contribution is due to the solenoidal flows along and around the helix-shaped filament and the capacitive contribution is due to the counterion layer formed around each negatively charged filament.

Herpes simplex virus type 1 (HSV-1) has been known as a common viral pathogen that can infect several parts of the body, leading to various clinical manifestations. According to this diverse manifestation, HSV-1 infection in many cell types was demonstrated. Besides the HSV-1 cell tropism, e.g., fibroblast, epithelial, mucosal cells, and neurons, HSV-1 infections can occur in human T lymphocyte cells, especially in activated T cells. In addition, several studies found that actin polymerization and filopodia formation support HSV-1 infection in diverse cell types. Hence, the goal of this review is to explore the mechanism of HSV-1 infection in various types of cells involving filopodia formation and highlight potential future directions for HSV-1 entry-related research. Moreover, this review covers several strategies for possible anti-HSV drugs focused on the entry step, offering insights into potential therapeutic interventions.

The intricate regulatory processes behind actin polymerization play a crucial role in cellular biology, including essential mechanisms such as cell migration or cell division. However, the self-organizing principles governing actin polymerization are still poorly understood. In this perspective article, we compare the Belousov-Zhabotinsky (BZ) reaction, a classic and well understood chemical oscillator known for its self-organizing spatiotemporal dynamics, with the excitable dynamics of polymerizing actin. While the BZ reaction originates from the domain of inorganic chemistry, it shares remarkable similarities with actin polymerization, including the characteristic propagating waves, which are influenced by geometry and external fields, and the emergent collective behavior. Starting with a general description of emerging patterns, we elaborate on single droplets or cell-level dynamics, the influence of geometric confinements and conclude with collective interactions. Comparing these two systems sheds light on the universal nature of self-organization principles in both living and inanimate systems.

Abstract Current studies on actin function primarily rely on cytoplasmic actin due to the absence of cellular models specifically expressing nuclear actin. Here, cell models capable of expressing varying levels of nuclear F/G‐actin are generated and a significant role of nuclear actin in the regulation of epithelial‐mesenchymal transition (EMT) is uncovered. Through immunoprecipitation and mass spectrometry analyses, distinct binding partners for nuclear F‐actin (β‐catenin, SMAD2, and SMAD3) and nuclear G‐actin (MYBBP1A, NKRF, and MYPOP) are investigated, which respectively modulate EMT‐promoting and EMT‐repressing transcriptional events. While nuclear F‐actin promotes EMT with enhanced cell migration, survival, and elongated mesenchymal morphology, nuclear G‐actin represses EMT and related cell activities. Mechanistically, nuclear F‐actin enhances β‐catenin, SMAD2, and SMAD3 expression and stability in the nuclei, while nuclear G‐actin increases MYBBP1A, NKRF, and MYPOP expression and stability in the nuclei. The association between nuclear F/G‐actin and N‐cadherin/E‐cadherin in the cell lines (in vitro), and increased nuclear actin polymerization in the wound healing cells (in vivo) affirm a significant role of nuclear actin in EMT regulation. With evidence of nuclear actin polymerization and EMT during development, and irregularities in disease states such as cancer and fibrosis, targeting nuclear actin dynamics to trigger dysregulated EMT warrants ongoing study.

Cell migration is an essential process that underlies many physiological processes, including the immune response, organogenesis in the embryo, and angiogenesis, as well as pathological processes such as cancer metastasis. Cells have at their disposal a variety of migratory behaviors and mechanisms that seem to be specific to cell type and the microenvironment. Research over the past two decades has elucidated the water channel protein family of aquaporins (AQPs) as a regulator of many cell migration-related processes, from physical phenomena to biological signaling pathways. The roles that AQPs play in cell migration are both cell type- and isoform-specific; thus, a large swath of information has accumulated as researchers seek to identify the responses across these distinct variables. There does not seem to be a universal role that AQPs play in cell migration; the complex interplay between AQPs and cell volume management, signaling pathway activation, and in a few identified circumstances, gene expression regulation, has shown the intricate, and perhaps paradoxical, role of AQPs in cell migration. The objective of this review is to provide an organized and integrated collection of recent work that has elucidated the many mechanisms by which AQPs regulate cell migration. [ABSTRACT FROM AUTHOR]

Ovarian cancer ranks fifth in terms of cancer mortality in women due to lack of early diagnosis and poor clinical management. Characteristics like high cellular proliferation, EMT and metabolic alterations contribute to oncogenicity. Cancer, being a “metabolic disorder,” is governed by various key regulatory factors like metabolic enzymes, oncogenes, and tumor suppressors. Sirtuins (SIRT1‐SIRT7) belong to the group of NAD+ deacetylase and ADP‐ribosylation enzymes that function as NAD+ sensors and metabolic regulators. Among sirtuin orthologs, SIRT6 emerges as an important oncogenic player, although its possible mechanistic involvement in ovarian cancer advancement is still elusive. Our data indicated a higher expression of SIRT6 in ovarian cancer tissues compared with the non‐malignant ovarian tissue. Further, we observed that overexpression of SIRT6 enhances glycolysis and oxidative phosphorylation in ovarian cancer cells. The energy derived from these processes facilitates migration and invasion through invadopodia formation by reorganization of actin fibers. Mechanistically, SIRT6 has been shown to promote ERK1/2‐driven activatory phosphorylation of DRP1 at serine‐616, which has an obligatory role in inducing mitochondrial fission. These fragmented mitochondria facilitate cell movement important for metastases. siRNA‐mediated downregulation of SIRT6 was found to decrease cellular invasion through compromised mitochondrial fragmentation and subsequent reduction in stress fiber formation in ovarian cancer cells. Thus, the present report establishes the impact of SIRT6 in the regulation of morphological and functional aspects of mitochondria that modulates invasion in ovarian cancer cells.

Cellular sensing of most environmental cues involves receptors that affect a signal-transduction excitable network (STEN), which is coupled to a cytoskeletal excitable network (CEN). We show that the mechanism of sensing of nanoridges is fundamentally different. CEN activity occurs preferentially on nanoridges, whereas STEN activity is constrained between nanoridges. In the absence of STEN, waves disappear, but long-lasting F-actin puncta persist along the ridges. When CEN is suppressed, wave propagation is no longer constrained by nanoridges. A computational model reproduces these experimental observations. Our findings indicate that nanotopography is sensed directly by CEN, whereas STEN is only indirectly affected due to a CEN-STEN feedback loop. These results explain why texture sensing is robust and acts cooperatively with multiple other guidance cues in complex, in vivo microenvironments. [ABSTRACT FROM AUTHOR]

Our recent live-cell single-molecule analysis revealed the force-velocity relationship of actin polymerization at the lamellipodium tip. Locally-applied traction force enhanced actin polymerization exclusively at the tip, followed by gradual cell protrusion. The results are consistent with the force-velocity curve predicted by the Brownian ratchet theory. In the lamellipodium, the load on each actin barbed end is kept low by the constant retrograde actin flow and densely-packed filaments. This enables fast reactivity of cell edge protrusion against subtle force changes. Cells appear to use the Brownian ratchet mechanism to harness ‘information’ for their force-directed morphogenesis and migration. [ABSTRACT FROM AUTHOR]

Golgi-derived PI4P-containing vesicles play important roles in mitochondrial division, which is essential for maintaining cellular homeostasis. However, the mechanism of the PI4P-containing vesicle effect on mitochondrial division is unclear. Here, we found that actin appeared to polymerize at the contact site between PI4P-containing vesicles and mitochondria, causing mitochondrial division. Increasing the content of PI4P derived from the Golgi apparatus increased actin polymerization and reduced the length of the mitochondria, suggesting that actin polymerization through PI4P-containing vesicles is involved in PI4P vesicle-related mitochondrial division. Collectively, our results support a model in which PI4P-containing vesicles derived from the Golgi apparatus cooperate with actin filaments to participate in mitochondrial division by contributing to actin polymerization, which regulates mitochondrial dynamics. This study enriches the understanding of the pathways that regulate mitochondrial division and provides new insight into mitochondrial dynamics. [ABSTRACT FROM AUTHOR]

Modulation of presynaptic short-term plasticity induced by actin polymerization was studied in rat hippocampal slices using the paired-pulse paradigm. Schaffer collaterals were stimulated with paired pulses with a 70-ms interstimulus interval every 30 s before and during perfusion with jasplakinolide, an activator of actin polymerization. Jasplakinolide application resulted in the increase in the amplitudes of CA3-CA1 responses (potentiation) accompanied by a decrease in the paired-pulse facilitation, suggesting induction of presynaptic modifications. Jasplakinolide-induced potentiation depended on the initial paired-pulse rate. These data indicate that the jasplakinolide-mediated changes in actin polymerization increased the probability of neurotransmitter release. Less typical for CA3-CA1 synapses responses, such as a very low paired-pulse ratio (close to 1 or even lower) or even paired-pulse depression, were affected differently. Thus, jasplakinolide caused potentiation of the second, but not the first response to the paired stimulus, which increased the paired-pulse ratio from 0.8 to 1.0 on average, suggesting a negative impact of jasplakinolide on the mechanisms promoting paired-pulse depression. In general, actin polymerization facilitated potentiation, although the patterns of potentiation differed depending on the initial synapse characteristics. We conclude that in addition to the increase in the neurotransmitter release probability, jasplakinolide induced other actin polymerization-dependent mechanisms, including those involved in the paired-pulse depression. [ABSTRACT FROM AUTHOR]

Surface topography profoundly influences cell adhesion, differentiation, and stem cell fate control. Numerous studies using a variety of materials demonstrate that nanoscale topographies change the intracellular organization of actin cytoskeleton and therefore a broad range of cellular dynamics in live cells. However, the underlying molecular mechanism is not well understood, leaving why actin cytoskeleton responds to topographical features unexplained and therefore preventing researchers from predicting optimal topographic features for desired cell behavior. Here we demonstrate that topography-induced membrane curvature plays a crucial role in modulating intracellular actin organization. By inducing precisely controlled membrane curvatures using engineered vertical nanostructures as topographies, we find that actin fibers form at the sites of nanostructures in a curvature-dependent manner with an upper limit for the diameter of curvature at ∼400 nm. Nanotopography-induced actin fibers are branched actin nucleated by the Arp2/3 complex and are mediated by a curvature-sensing protein FBP17. Our study reveals that the formation of nanotopography-induced actin fibers drastically reduces the amount of stress fibers and mature focal adhesions to result in the reorganization of actin cytoskeleton in the entire cell. These findings establish the membrane curvature as a key linkage between surface topography and topography-induced cell signaling and behavior.

The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding-proteins and nucleation-promoting factors to perform fundamental cellular functions. We have previously shown that ERK3, an atypical MAPK, controls IL-8 production and chemotaxis (Bogueka et al., 2020). Here, we show in human cells that ERK3 directly acts as a guanine nucleotide exchange factor for CDC42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent RAC1 and CDC42 activation, maintenance of F-actin content, filopodia formation, and epithelial cell migration. Further, ERK3 protein bound directly to the purified ARP2/3 complex and augmented polymerization of actin in vitro. ERK3 kinase activity was required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions.

To date, it has been shown that the phenomenon of liquid–liquid phase separation (LLPS) underlies many seemingly completely different cellular processes. This provided a new idea of the spatiotemporal organization of the cell. The new paradigm makes it possible to provide answers to many long-standing, but still unresolved questions facing the researcher. In particular, spatiotemporal regulation of the assembly/disassembly of the cytoskeleton, including the formation of actin filaments, becomes clearer. To date, it has been shown that coacervates of actin-binding proteins that arise during the phase separation of the liquid–liquid type can integrate G-actin and thereby increase its concentration to initiate polymerization. It has also been shown that the activity intensification of actin-binding proteins that control actin polymerization, such as N-WASP and Arp2/3, can be caused by their integration into liquid droplet coacervates formed by signaling proteins on the inner side of the cell membrane. [ABSTRACT FROM AUTHOR]

Dilated cardiomyopathy (DCM) with left ventricular non-compaction (LVNC) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure, and excessive risk of sudden cardiac death. Using whole-exome sequencing to investigate a possible genetic cause of DCM with LVNC in a consanguineous child, a homozygous nucleotide change c.1532G>A causing p.Arg511His in PHACTR2 was found. The missense change can affect the binding of PHACTR2 to actin by eliminating the hydrogen bonds between them. The amino acid change does not change PHACTR2 localization to the cytoplasm. The patient's fibroblasts showed a decreased globular to fibrillary actin ratio compared to the control fibroblasts. The re-polymerization of fibrillary actin after treatment with cytochalasin D, which disrupts the actin filaments, was slower in the patient's fibroblasts. Finally, the patient's fibroblasts bridged a scar gap slower than the control fibroblasts because of slower and indirect movement. This is the first report of a human variation in this PHACTR family member. The knock-out mouse model presented no significant phenotype. Our data underscore the importance of PHACTR2 in regulating the monomeric actin pool, the kinetics of actin polymerization, and cell movement, emphasizing the importance of actin regulation for the normal function of the human heart. [ABSTRACT FROM AUTHOR]

Background: Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a rare and serious congenital disorder with poor outcomes, where a heterozygous missense mutation is present in the ACTG2 gene. Here, we aimed to investigate the pathogenesis of ACTG2 in MMIHS. Methods: A cohort with 20 patients with MMIHS was screened. Actg2R257C heterozygous mutant mice were generated using the CRISPR/Cas9 system. Gastrointestinal (GI) motility, voluntary urination, collagen gel contraction, and G‐actin/F‐actin analysis were performed. Key Results: The R257C variant of ACTG2 most frequently occurred in patients with MMIHS and demonstrated the typical symptoms of MMIHS. Actg2R257C heterozygous mutant mice had dilated intestines and bladders. The functional assay showed a prolonged total time of GI transit and decreased urine spot area. Collagen gel contraction assay and G‐actin/F‐actin analysis indicated that mutant mice showed reduced area of contraction of smooth muscle cells (SMCs) and impaired actin polymerization. Conclusions & Inferences: A mouse model demonstrating MMIHS‐like symptoms was generated. The Actg2R257C heterozygous variant impairs SMCs contraction by interfering with actin polymerization, leading to GI motility disorders. [ABSTRACT FROM AUTHOR]

Glycoprotein (GP) Ibα shedding mediated by ADAM17 (a disintegrin and metalloproteinase 17) plays an important role in negatively regulating platelet function and thrombus formation. However, the mechanism of GPIbα shedding remains elusive. Here, we show that jasplakinolide (an actin-polymerizing peptide)-induced actin polymerization results in GPIbα shedding and impairs platelet function. Thrombin and A23187-induced GPIbα shedding is increased by jasplakinolide; in contrast, GPIbα shedding is reduced by a depolymerization regent (cytochalasin B). We find that actin polymerization activates calpain leading to filamin A hydrolyzation. We further demonstrate that the interaction of filamin A with the cytoplasmic domain of GPIbα plays a critical role in regulating actin polymerization-induced GPIbα shedding. Taken together, these data demonstrate that actin polymerization regulates ADAM17-mediated GPIbα shedding, suggesting a novel strategy to negatively regulate platelet function.

Our understanding of how fluid forces influence cell migration in confining environments remains limited. By integrating microfluidics with live-cell imaging, we demonstrate that cells in tightly—but not moderately—confined spaces reverse direction and move upstream upon exposure to fluid forces. This fluid force-induced directional change occurs less frequently when cells display diminished mechanosensitivity, experience elevated hydraulic resistance, or sense a chemical gradient. Cell reversal requires actin polymerization to the new cell front, as shown mathematically and experimentally. Actin polymerization is necessary for the fluid force-induced activation of NHE1, which cooperates with calcium to induce upstream migration. Calcium levels increase downstream, mirroring the subcellular distribution of myosin IIA, whose activation enhances upstream migration. Reduced lamin A/C levels promote downstream migration of metastatic tumor cells by preventing cell polarity establishment and intracellular calcium rise. This mechanism could allow cancer cells to evade high-pressure environments, such as the primary tumor. [Display omitted] • Tight confinement promotes upstream migration of cancerous and non-cancerous cells • High hydraulic resistance and chemotaxis oppose confinement-induced upstream motility • Upstream migration entails the polarization and activation of migration machinery components • Low mechanosensitivity enables metastatic tumor cells to escape high-pressure environments Amiri et al. report that fluid forces cause upstream migration of confined cells via mechanisms involving the cytoskeleton, nucleus, and ion channels. This migration is less likely under conditions of chemotaxis or high hydraulic resistance. Metastatic cells with low levels of lamin A/C display reduced mechanosensitivity, resulting in downstream migration. [ABSTRACT FROM AUTHOR]

Nemaline myopathy (NM) is one of the most common forms of congenital myopathy and it is identified by the presence of “nemaline bodies” (rods) in muscle fibers by histopathological examination. The most common forms of NM are caused by mutations in the Actin Alpha 1 (ACTA1) and Nebulin (NEB) genes. Clinical features include hypotonia and muscle weakness. Unfortunately, there is no curative treatment and the pathogenetic mechanisms remain unclear. In this manuscript, we examined the pathophysiological alterations in NM using dermal fibroblasts derived from patients with mutations in ACTA1 and NEB genes. Patients’ fibroblasts were stained with rhodamine–phalloidin to analyze the polymerization of actin filaments by fluorescence microscopy. We found that patients’ fibroblasts showed incorrect actin filament polymerization compared to control fibroblasts. Actin filament polymerization defects were associated with mitochondrial dysfunction. Furthermore, we identified two mitochondrial-boosting compounds, linoleic acid (LA) and L-carnitine (LCAR), that improved the formation of actin filaments in mutant fibroblasts and corrected mitochondrial bioenergetics. Our results indicate that cellular models can be useful to study the pathophysiological mechanisms involved in NM and to find new potential therapies. Furthermore, targeting mitochondrial dysfunction with LA and LCAR can revert the pathological alterations in NM cellular models.

To acquire the capacity to fertilize the oocyte, mammalian spermatozoa must undergo a series of biochemical reactions in the female reproductive tract, which are collectively called capacitation. The capacitated spermatozoa subsequently interact with the oocyte zona-pellucida and undergo the acrosome reaction, which enables the penetration of the oocyte and subsequent fertilization. However, the spontaneous acrosome reaction (sAR) can occur prematurely in the sperm before reaching the oocyte cumulus oophorus, thereby jeopardizing fertilization. One of the main processes in capacitation involves actin polymerization, and the resulting F-actin is subsequently dispersed prior to the acrosome reaction. Several biochemical reactions that occur during sperm capacitation, including actin polymerization, protect sperm from sAR. In the present review, we describe the protective mechanisms that regulate sperm capacitation and prevent sAR.

Liver sinusoidal endothelial cells (LSECs) are ideally situated to sense stiffness and generate angiocrine programs that potentially regulate liver fibrosis and portal hypertension. We explored how specific focal adhesion (FA) proteins parlay LSEC mechanotransduction into stiffness-induced angiocrine signaling in vitro and in vivo. Primary human and murine LSECs were placed on gels with incremental stiffness (0.2 kPa vs. 32 kPa). Cell response was studied by FA isolation, actin polymerization assay, RNA-sequencing and electron microscopy. Glycolysis was assessed using radioactive tracers. Epigenetic regulation of stiffness-induced genes was analyzed by chromatin-immunoprecipitation (ChIP) analysis of histone activation marks, ChIP sequencing and circularized chromosome conformation capture (4C). Mice with LSEC-selective deletion of glycolytic enzymes (Hk2 fl/fl/ Cdh5 cre-ERT2) or treatment with the glycolysis inhibitor 3PO were studied in portal hypertension (partial ligation of the inferior vena cava, pIVCL) and early liver fibrosis (CCl 4) models. Glycolytic enzymes, particularly phosphofructokinase 1 isoform P (PFKP), are enriched in isolated FAs from LSECs on gels with incremental stiffness. Stiffness resulted in PFKP recruitment to FAs, which paralleled an increase in glycolysis. Glycolysis was associated with expansion of actin dynamics and was attenuated by inhibition of integrin β1. Inhibition of glycolysis attenuated a stiffness-induced CXCL 1 -dominant angiocrine program. Mechanistically, glycolysis promoted CXCL1 expression through nuclear pore changes and increases in NF-kB translocation. Biochemically, this CXCL1 expression was mediated through spatial re-organization of nuclear chromatin resulting in formation of super-enhancers, histone acetylation and NF-kB interaction with the CXCL1 promoter. Hk2 fl/fl/ Cdh5 cre-ERT2 mice showed attenuated neutrophil infiltration and portal hypertension after pIVCL. 3PO treatment attenuated liver fibrosis in a CCl 4 model. Glycolytic enzymes are involved in stiffness-induced angiocrine signaling in LSECs and represent druggable targets in early liver disease. Treatment options for liver fibrosis and portal hypertension still represent an unmet need. Herein, we uncovered a novel role for glycolytic enzymes in promoting stiffness-induced angiocrine signaling, which resulted in inflammation, fibrosis and portal hypertension. This work has revealed new targets that could be used in the prevention and treatment of liver fibrosis and portal hypertension. [Display omitted] • Increased stiffness leads to recruitment of glycolytic enzymes to focal adhesions in LSECs, paralleling an increase in glycolysis. • Inhibition of glycolysis blocks a stiffness-induced CXCL1-dominant angiocrine program. • Glycolysis promotes CXCL1 expression through nuclear pore changes and increases in NF-kB translocation. • LSEC-selective deletion of glycolytic enzymes results in attenuation of portal hypertension in vivo. • Treatment with the glycolysis inhibitor 3PO attenuates early liver fibrosis in vivo. [ABSTRACT FROM AUTHOR]

Ovarian cancer ranks fifth in terms of cancer mortality in women due to lack of early diagnosis and poor clinical management. Characteristics like high cellular proliferation, EMT and metabolic alterations contribute to oncogenicity. Cancer, being a "metabolic disorder," is governed by various key regulatory factors like metabolic enzymes, oncogenes, and tumor suppressors. Sirtuins (SIRT1‐SIRT7) belong to the group of NAD+ deacetylase and ADP‐ribosylation enzymes that function as NAD+ sensors and metabolic regulators. Among sirtuin orthologs, SIRT6 emerges as an important oncogenic player, although its possible mechanistic involvement in ovarian cancer advancement is still elusive. Our data indicated a higher expression of SIRT6 in ovarian cancer tissues compared with the non‐malignant ovarian tissue. Further, we observed that overexpression of SIRT6 enhances glycolysis and oxidative phosphorylation in ovarian cancer cells. The energy derived from these processes facilitates migration and invasion through invadopodia formation by reorganization of actin fibers. Mechanistically, SIRT6 has been shown to promote ERK1/2‐driven activatory phosphorylation of DRP1 at serine‐616, which has an obligatory role in inducing mitochondrial fission. These fragmented mitochondria facilitate cell movement important for metastases. siRNA‐mediated downregulation of SIRT6 was found to decrease cellular invasion through compromised mitochondrial fragmentation and subsequent reduction in stress fiber formation in ovarian cancer cells. Thus, the present report establishes the impact of SIRT6 in the regulation of morphological and functional aspects of mitochondria that modulates invasion in ovarian cancer cells. [ABSTRACT FROM AUTHOR]

Cancer invasion through basement membranes represents the initial step of tumor dissemination and metastasis. However, little is known about how human cancer cells breach basement membranes. Here, we used a three-dimensional in vitro invasion model consisting of cancer spheroids encapsulated by a basement membrane and embedded in 3D collagen gels to visualize the early events of cancer invasion by confocal microscopy and live-cell imaging. Human breast cancer cells generated large numbers of basement membrane perforations, or holes, of varying sizes that expanded over time during cell invasion. We used a wide variety of small molecule inhibitors to probe the mechanisms of basement membrane perforation and hole expansion. Protease inhibitor treatment (BB94), led to a 63% decrease in perforation size. After myosin II inhibition (blebbistatin), the basement membrane perforation area decreased by only 15%. These treatments produced correspondingly decreased cellular breaching events. Interestingly, inhibition of actin polymerization dramatically decreased basement membrane perforation by 80% and blocked invasion. Our findings suggest that human cancer cells can primarily use proteolysis and actin polymerization to perforate the BM and to expand perforations for basement membrane breaching with a relatively small contribution from myosin II contractility. [ABSTRACT FROM AUTHOR]

The formin protein Diaph3 is an actin nucleator that regulates numerous cytoskeleton-dependent cellular processes through the activation of actin polymerization. Expression and activity of Diaph3 is tightly regulated: lack of Diaph3 results in developmental defects and embryonic lethality in mice, while overexpression of Diaph3 causes auditory neuropathy. It is known that Diaph3 homophilic interactions include the intramolecular interaction of its Dia-inhibitory domain (DID)-diaphanous autoregulatory domain (DAD) domains and the intermolecular interactions of DD-DD domains or FH2-FH2 domains. However, the physiological significance of these interactions in Diaph3 protein stability and activity is not fully understood. In this study, we show that FH2-FH2 interaction promotes Diaph3 activity, while DID-DAD and DD-DD interactions inhibit Diaph3 activity through distinct mechanisms. DID-DAD interaction is responsible for the autoinhibition of Diaph3 protein, which is disrupted by binding of Rho GTPases. Interestingly, we find that DID-DAD interaction stabilizes the expression of each DID or DAD domain against proteasomal-mediated degradation. Disruption of DID-DAD interaction by RhoA binding or M1041A mutation causes increased Diaph3 activity and accelerated degradation of the activated Diaph3 protein. Further, the activated Diaph3 is ubiquitinated at K1142/1143/1144 lysine residues by the E3 ligase Stub1. Expression of Stub1 is causally related to the stability and activity of Diaph3. Knockdown of Stub1 in mouse cochlea results in hair cell stereocilia defects, neuronal degeneration, and hearing loss, resembling the phenotypes of mice overexpressing Diaph3. Thus, our study reports a novel regulatory mechanism of Diaph3 protein expression and activity whereby the active but not inactive Diaph3 is readily degraded to prevent excessive actin polymerization., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hydrogels are pivotal in tissue engineering, regenerative medicine, and drug delivery applications. Existing hydrogel platforms are not easily customizable and often lack precise programmability, making them less suited for 3D tissue culture and programming of cells. DNA molecules stand out among other promising biomaterials due to their unparalleled precision, programmability, and customization. In this study, we introduced a palette of novel cellular scaffolding platforms made of pure DNA-based hydrogel systems while improving the shortcomings of the existing platforms. We showed a quick and easy one step synthesis of DNA hydrogels using thermal annealing based on sequence specific hybridization strategy. We also demonstrated the formation of multi-armed branched supramolecular scaffolds with custom mechanical stiffness, porosity, and network density by increasing or decreasing the number of branching arms. These mechanically tuneable DNA hydrogels proved to be a suitable suitable platform for modulating the physiological processes of retinal pigment epithelial cells (RPE1). In-vitro studies showed dynamic changes at multiple levels, ranging from a change in morphology to protein expression patterns, enhanced membrane traffic, and proliferation. The soft DNA hydrogels explored here are mechanically compliant and pliable, thus excellently suited for applications in cellular programming and reprogramming. This research lays the groundwork for developing a DNA hydrogel system with a higher dynamic range of stiffness, which will open exciting avenues for tissue engineering and beyond., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Dhiraj Bhatia reports financial support was provided by Indian Institute of Technology Gandhinagar. Dhiraj Bhatia reports a relationship with Indian Institute of Technology Gandhinagar that includes: employment. None If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Profilin proteins (PRFs) are small (12–15 kD) actin-binding protein, which play a significant role in cytoskeleton dynamics and plant development via regulating actin polymerization. Profilins have been well documented in Arabidopsis, Zea mays L. as well as Phaseolus vulgaris, however no such fully characterization of rice (Oryza sativa L.) profilin gene family has been reported thus far. Result: In the present study, a comprehensive genome-wide analysis of rice PRF genes was completed and three members were identified. OsPRF1 and OsPRF2 shared 98.5% similarity (6 nucleotide divergence), but the deduced amino acid sequences of OsPRF1 and OsPRF2 are fully identical. In contrast, the OsPRF3 presents relatively lower similarity with OsPRF1 and OsPRF2. Phylogenetic analysis also support that OsPRF1 has a closer relationship with OsPRF2. Expression pattern analysis revealed the differential expression of OsPRFs in tissues of mature plant, which suggested the potential spatial functional specificity for rice profilin genes. Subcellular localization analysis revealed the OsPRFs were localized in cytoplasm and nucleus and all of them could bind actin monomers. Furthermore, abiotic stresses and hormones treatments assay indicated that the three OsPRF genes could be differentially regulated, suggesting that OsPRF genes might participate in different stress processes in rice. Conclusions: Taken together, our study provides a comprehensive analysis of the OsPRF gene family and will provide a basis for further studies on their roles in rice development and in response to abiotic stresses.How to cite: Zhang Y, Dong G, Wu L, et al. Identification and characterization of profilin gene family in rice. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.08.004.