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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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare that they have a provisional patent application related to the content of this manuscript. The patent application, titled “A Microfluidic Assay for the Isolation of Mechanoinsensitive Cells,” was filed with the United States Patent & Trademark Office on October 11, 2023. P.M. and F.A. are listed as the inventors on the patent application., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflicts of interest.

Aims: Protein kinase D (PKD), once considered an effector of protein kinase C (PKC), now plays many pathophysiological roles in various tissues. However, little is known about role of PKD in vascular function. We investigated the role of PKD in contraction of rat aorta and human aortic smooth muscle cells (HASMCs) and in haemodynamics in rats., Methods and Results: Isometric tension of rat aortic was measured to examine norepinephrine-induced contraction in the presence of PKD, PKC and Rho-kinase inhibitors. Phosphorylation of PKD1, myosin targeting subunit-1 (MYPT1), myosin light chain (MLC), CPI-17 and heat-shock protein 27 (HSP27), and actin polymerization were measured in the aorta. Phosphorylation of MYPT1 and MLC was also measured in HASMCs knocked down with specific siRNAs of PKD 1, 2 and 3. Intracellular calcium concentrations and cell shortening were measured in HASMCs. Norepinephrine-induced aortic contraction was accompanied by increased phosphorylation of PKD1, MYPT1 and MLC and actin polymerization, all of which were attenuated with PKD inhibitor CRT0066101. PKD1 phosphorylation was not inhibited by PKC inhibitor, chelerythrine or Rho kinase inhibitor, fasudil. In HASMCs, the phosphorylation of MYPT1 and MLC was attenuated by PKD1, but not PKD2, 3 knockdown. In HASMCs, CRT0066101 inhibited norepinephrine-induced cell shortening without affecting calcium concentration. Administration of CRT0066101 decreased systemic vascular resistance and blood pressure without affecting cardiac output in rats., Conclusions: PKD1 may play roles in aorta contraction and haemodynamics via phosphorylation of MYPT1 and actin polymerization in a calcium-independent manner., Competing Interests: Declaration of competing interest To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

The migratory and invasive potential of tumour cells relies on the actin cytoskeleton. We previously demonstrated that the tricyclic compound, TBE-31, inhibits actin polymerization and here we further examine the precise interaction between TBE-31 and actin. We demonstrate that iodoacetamide, a cysteine (Cys) alkylating agent, interferes with the ability of TBE-31 to interact with actin. In addition, in silico analysis identified Cys 217, Cys 272, Cys 285 and Cys 374 as potential binding sites for TBE-31. Using mass spectrometry analysis, we determined that TBE-31 associates with actin with a stoichiometric ratio of 1:1. We mutated the identified cysteines of actin to alanine and performed a pull-down analysis with a biotin labeled TBE-31 and demonstrated that by mutating Cys 374 to alanine the association between TBE-31 and actin was significantly reduced, suggesting that TBE-31 binds to Cys 374. A characterization of the NIH3T3 cells overexpressing eGFP-actin-C374A showed reduced stress fiber formation, suggesting Cys 374 is necessary for efficient incorporation into filamentous actin. Furthermore, migration of eGFP-Actin-WT expressing cells were observed to be inhibited by TBE-31, however fewer eGFP-Actin-C374A expressing cells were observed to migrate compared to the cells expressing eGFP-Actin-WT in the presence or absence of TBE-31. Taken together, our results suggest that TBE-31 binds to Cys 374 of actin to inhibit actin stress fiber formation and may potentially be a mechanism through which TBE-31 inhibits cell migration., Competing Interests: Declaration of competing interest The authors have no competing financial interests., (Copyright © 2023. Published by Elsevier B.V.)

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., Competing Interests: The authors declare no conflict of interest.

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.

The diaphanous-related formin, Diaphanous 1 (DIAPH1), is required for the assembly of Filamentous (F)-actin structures. DIAPH1 is an intracellular effector of the receptor for advanced glycation end products (RAGE) and contributes to RAGE signaling and effects such as increased cell migration upon RAGE stimulation. Mutations in DIAPH1, including those in the basic "RRKR" motif of its autoregulatory domain, diaphanous autoinhibitory domain (DAD), are implicated in hearing loss, macrothrombocytopenia, and cardiovascular diseases. The solution structure of the complex between the N-terminal inhibitory domain, DID, and the C-terminal DAD, resolved by NMR spectroscopy shows only transient interactions between DID and the basic motif of DAD, resembling those found in encounter complexes. Cross-linking studies placed the RRKR motif into the negatively charged cavity of DID. Neutralizing the cavity resulted in a 5-fold decrease in the binding affinity and 4-fold decrease in the association rate constant of DAD for DID, indicating that the RRKR interactions with DID form a productive encounter complex. A DIAPH1 mutant containing a neutralized RRKR binding cavity shows excessive colocalization with actin and is unresponsive to RAGE stimulation. This is the first demonstration of a specific alteration of the surfaces responsible for productive encounter complexation with implications for human pathology., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the content of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: KB, GH, MC, MB, BT No competing interests declared, KR KR is the founder and MD of KHR Biotec GmbH, (© 2023, Bogucka-Janczi et al.)

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.

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.

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.

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.

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. Actg2 R257C 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. Actg2 R257C 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 Actg2 R257C heterozygous variant impairs SMCs contraction by interfering with actin polymerization, leading to GI motility disorders., (© 2022 John Wiley & Sons Ltd.)

Background: Beauvericin (BEA) is a depsipeptide with antimicrobial, anti-inflammatory, and anti-cancer activities isolated from Beauveria bassiana. However, little is understood on its anti-cancer activities and mechanism., Purpose: Aim of this study was to explore the anti-cancer activity of BEA and its underlying molecular mechanism to provide a theoretical basis for its role as a candidate natural drug in cancer diseases., Study Design: Various cancer cells such as C6 glioma, U251, MDA-MB-231, HeLa, HCT-15, LoVo cells, and HEK293T cells were used to the anti-cancer activity of BEA., Methods: To evaluate the anti-cancer activity of BEA, cell viability test (MTT assay), morphological change check, confocal microscopy, actin polymerization assay, flow cytometry, and Western blotting analysis. To check the target enzyme of BEA, overexpression and site-directed mutagenesis was employed., Results: BEA inhibited the viability of cancer cells including C6, MDA-MB-231, HeLa, HCT-15, LoVo, and U251 cells. Treatment of BEA in C6 glioma cells induced cell membrane blebbing and apoptosis. Caspase-3 and -9 were dose-dependently activated by BEA, and the mRNA expression of Bcl-2 was inhibited by BEA. According to confocal microscopy, actin polymerization and actin-actin interaction were interrupted by BEA in C6 cells. BEA regulated the apoptosis of C6 cells depending on the protein phosphorylation of Src and Signal transducer and activator of transcription (STAT3). Moreover, c-terminal amino acids in Src directly interacted with BEA in C6 cells, and the binding of Src and BEA suppressed the kinase activity of Src., Conclusions: These results suggest that BEA may be a critical candidate or substitute drug for cancer treatment via suppression of the Src/STAT3 pathway., Competing Interests: Declaration of Competing Interest None of the authors have conflicts of interest to declare., (Copyright © 2022. Published by Elsevier GmbH.)

The use of artificial cells to mimic living tissues is beneficial for understanding the mechanism of interaction among cells. Artificial cells hold immense potential in the field of tissue engineering. Self-powered artificial cells capable of reversible deformation are developed by encapsulating living mitochondria, actins, and methylcellulose. Upon addition of pyruvate molecules, the mitochondria produce adenosine triphosphate (ATP), which acts as an energy source to trigger actin polymerization. The reversible deformation of artificial cells occurs with a spindle shape resulting from the polymerization of actins to form filaments adjacent to the lipid bilayer that subsequently returns to a spherical shape resulting from the depolymerization of actin filaments upon laser irradiation. The linear colonies composed of these artificial cells exhibit collective contraction and relaxation to mimic muscle tissues. At maximum contraction, the long axis of each giant unilamellar vesicle (GUV) is parallel to each other. All the colonies are synchronized in the contraction phase. The deformation of each GUV in the colonies is influenced by its adjacent GUVs. The muscle-like artificial cell colonies described here pave the way to develop sustainably self-powered artificial tissues., (© 2022 Wiley-VCH GmbH.)

The abLIM1 is a nonerythroid actin-binding protein critical for stable plasma membrane-cortex interactions under mechanical tension. Its depletion by RNA interference results in sparse, poorly interconnected cortical actin networks and severe blebbing of migrating cells. Its isoforms, abLIM-L, abLIM-M, and abLIM-S, contain, respectively four, three, and no LIM domains, followed by a C terminus entirely homologous to erythroid cortex protein dematin. How abLIM1 functions, however, remains unclear. Here we show that abLIM1 is a liquid-liquid phase separation (LLPS)-dependent self-organizer of actin networks. Phase-separated condensates of abLIM-S-mimicking ΔLIM or the major isoform abLIM-M nucleated, flew along, and cross-linked together actin filaments (F-actin) to produce unique aster-like radial arrays and interconnected webs of F-actin bundles. Interestingly, ΔLIM condensates facilitated actin nucleation and network formation even in the absence of Mg 2+ . Our results suggest that abLIM1 functions as an LLPS-dependent actin nucleator and cross-linker and provide insights into how LLPS-induced condensates could self-construct intracellular architectures of high connectivity and plasticity.

Single molecule imaging has shown that part of actin disassembles within a few seconds after incorporation into the dendritic filament network in lamellipodia, suggestive of frequent destabilization near barbed ends. To investigate the mechanisms behind network remodeling, we created a stochastic model with polymerization, depolymerization, branching, capping, uncapping, severing, oligomer diffusion, annealing, and debranching. We find that filament severing, enhanced near barbed ends, can explain the single molecule actin lifetime distribution, if oligomer fragments reanneal to free ends with rate constants comparable to in vitro measurements. The same mechanism leads to actin networks consistent with measured filament, end, and branch concentrations. These networks undergo structural remodeling, leading to longer filaments away from the leading edge, at the +/-35° orientation pattern. Imaging of actin speckle lifetimes at sub-second resolution verifies frequent disassembly of newly-assembled actin. We thus propose a unified mechanism that fits a diverse set of basic lamellipodia phenomenology., Competing Interests: DH, AH, EU, SY, NW, DV No competing interests declared, (© 2022, Holz et al.)

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol., Competing Interests: Conflicts of interest The authors declare that they have no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: It was suggested that group V secretory phospholipase A 2 (sPLA 2 -V) existed in the nucleus. This study examined whether nuclear sPLA 2 -V plays a role in endocytosis of acetylated low-density lipoprotein (AcLDL) in monocyte/macrophage-like cell line RAW264.7 cells., Methods: RAW264.7 cells were transfected with shRNA vector targeting sPLA 2 -V (sPLA 2 -V-knockdown [KD] cells) or empty vector (sPLA 2 -V-wild-type [WT] cells). AcLDL endocytosis was assessed by incubation with 125 I-AcLDL or AcLDL conjugated with pHrodo. Actin polymerization was assessed by flow cytometry using Alexa Fluor 546-phalloidin., Results: In immunofluorescence microscopic studies, sPLA 2 -V was detected in the nucleus. ChIP-Seq and ChIP-qPCR analyses showed binding of sPLA 2 -V to the promoter region of the phosphoglycerate kinase 1 (Pgk1) gene. In the promoter assay, sPLA 2 -V-KD cells had lower promoter activity of the Pgk1 gene than sPLA 2 -V-WT cells, and this decrease could be reversed by transfection with a vector encoding sPLA 2 -V-H48Q that lacks enzymatic activity. Compared with sPLA 2 -V-WT cells, sPLA 2 -V-KD cells had decreased PGK1 protein expression, beclin 1 (Beclin1) phosphorylation at S30, and class III PI3-kinase activity that could also be restored by transfection with sPLA 2 -V-H48Q. sPLA 2 -V-KD cells had impaired actin polymerization and endocytosis, which was reversed by introduction of sPLA 2 -V-H48Q or PGK1 overexpression. In sPLA 2 -V-WT cells, siRNA-mediated depletion of PGK1 suppressed Beclin1 phosphorylation and impaired actin polymerization and intracellular trafficking of pHrodo-conjugated AcLDL., Conclusions: Nuclear sPLA 2 -V binds to the Pgk1 gene promoter region and increases its transcriptional activity. sPLA 2 -V regulates AcLDL endocytosis through PGK1-Beclin1 in a manner that is independent of its enzymatic activity in RAW264.7 cells.

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.

Taurine, one of the most abundant amino acids, is ubiquitously distributed in mammalian tissues and is known to react with myeloperoxidase-derived hypochlorous acid (HOCl/OCl - ) to produce taurine chloramine (Tau-Cl), which prevents inflammation by both suppressing pro-inflammatory mediators and increasing antioxidant levels. The migration of inflammatory cells, including neutrophils and macrophages, to infection sites is critical to the development of inflammation. In the present study, we investigated whether Tau-Cl suppresses the migration of inflammatory cells. Tau-Cl inhibited thioglycollate-induced leukocyte migration to the peritoneal cavity, as well as both fMLP-induced neutrophil migration and LPS-stimulated macrophage migration in a transwell system. Tau-Cl also inhibited LPS-induced actin polymerization, adhesion, and ERK phosphorylation in macrophages. Together, these findings suggest that Tau-Cl inhibits the infiltration of inflammatory cells into infection sites by inhibiting ERK activation, thereby preventing actin polymerization, and thus, the excessive infiltration of inflammatory cells, which can cause chronic inflammation., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Neural-derived 17β-estradiol (E2) plays an important role in the synaptic plasticity of the hippocampus and prefrontal cortex, but the mechanism is not well defined. This study was designed to explore the effect and mechanism of neural-derived E2 on synaptic plasticity of the hippocampus and prefrontal cortex. Primary cultured hippocampal and prefrontal cells in mice were randomly divided into the DMSO (D), aromatase (Rate-limiting enzymes for E2 synthesizes) inhibitor letrozole (L), and ERs antagonist (MPG) treated groups. After intervention for 48 h, the cell was collected, and then, the expressions of AMPA-receptor subunit GluR1 (GluR1), synaptophysin (SYN), p-21-Activated kinase (PAK) phosphorylation, Rho kinase (ROCK), p-Cofilin, F-actin, and G-actin proteins were detected. Letrozole or ER antagonists inhibited the expression of GluR1, F-actin/G-actin, p-PAK and p-Cofilin proteins in prefrontal cells significantly. And the expressions of GluR1 and F-actin/G-actin proteins were declined in hippocampal cells markedly after adding letrozole or ERs antagonists. In conclusion, neural-derived E2 and ERs regulated the synaptic plasticity, possibly due to promoting actin polymerization in prefrontal and hippocampal cells. The regional specificity in the effect of neural-derived E2 and ERs on the actin polymerization-related pathway may provide a theoretical basis for the functional differences between the hippocampus and prefrontal cortex., (Copyright © 2021 Elsevier Inc. All rights reserved.)

SET domain protein 3 (SETD3) is an actin-specific methyltransferase, a rare post-translational modification with limited known biological functions. Till now, the function of SETD3 in cerebral ischemia-reperfusion (I/R)-induced injury remains unknown. Here, we show that the protein level of SETD3 is decreased in rat neurons after cerebral I/R injury. SETD3 promotes neuronal survival after both glucose and oxygen deprivation/reoxygenation (OGD/R) and cerebral I/R injury, and knockdown of SETD3 increases OGD/R-induced neuronal death. We further show that OGD/R-induced downregulation of SETD3 leads to the decrease of cellular ATP level, the reduction of mitochondrial electric potential and the increase of ROS production, thereby promoting mitochondrial dysfunction. We found that SETD3 reduction-induced mitochondrial dysfunction is mediated by the suppression of actin polymerization after OGD/R. Furthermore, we demonstrate that I/R-induced upregulation of PTEN leads to the downregulation of SETD3, and suppressing PTEN protects against ischemic neuronal death through downregulation of SETD3 and enhancement of actin polymerization. Together, this study provides the first evidence suggesting that I/R-induced downregulation of SETD3 mediates PTEN upregulation-induced ischemic neuronal death through downregulation of SETD3 and subsequent suppression of actin polymerization. Thus, upregulating SETD3 is a potential approach for the development of ischemic stroke therapy., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

GBM is a high-grade cancer that originates from glial cells and has a poor prognosis. Although a combination of surgery, radiotherapy, and chemotherapy is prescribed to patients, GBM is highly resistant to therapies, and surviving cells show increased aggressiveness. In this study, we investigated the molecular mechanism underlying GBM progression after radiotherapy by establishing a GBM orthotopic xenograft mouse model. Based on transcriptomic analysis, we found that the expression of BEX1 and BEX4 was upregulated in GBM cells surviving radiotherapy. We also found that upregulated expression of BEX1 and BEX4 was involved in the formation of the filamentous cytoskeleton and altered mechanotransduction, which resulted in the activation of the YAP/TAZ signaling pathway. BEX1- and BEX4-mediated YAP/TAZ activation enhanced the tumor formation, growth, and radioresistance of GBM cells. Additionally, latrunculin B inhibited GBM progression after radiotherapy by suppressing actin polymerization in an orthotopic xenograft mouse model. Taken together, we suggest the involvement of cytoskeleton formation in radiation-induced GBM progression and latrunculin B as a GBM radiosensitizer.

Chemokines play an important role in various diseases as signaling molecules for immune cells. Therefore, the inhibition of the chemokine-receptor interaction and the characterization of potential inhibitors are important steps in the development of new therapies. Here, we present a new cell-based assay for chemokine-receptor interaction, using chemokine-dependent actin polymerization as a readout. We used interleukin-8 (IL-8, CXCL8) as a model chemokine and measured the IL-8-dependent actin polymerization with Atto565-phalloidin by monitoring the fluorescence intensity in the cell layer after activation with IL-8. This assay needs no transfection, is easy to perform and requires only a few working steps. It can be used to confirm receptor activation and to characterize the effect of chemokine receptor antagonists. Experiments with the well-known CXCR1/2 inhibitor reparixin confirmed that the observed increase in fluorescence intensity is a result of chemokine receptor activation and can be inhibited in a dose-dependent manner. With optimized parameters, the difference between positive and negative control was highly significant and statistical Z´-factors of 0.4 were determined on average., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Actin exists in monomeric globular (G-) and polymerized filamentous (F-) forms and the dynamics of its polymerization/depolymerization are tightly regulated in both the cytoplasm and the nucleus. Various essential functions of nuclear actin have been identified including regulation of gene expression and involvement in the repair of DNA double-strand breaks (DSB). Small G-actin-binding molecules affect F-actin formation and can be utilized for analysis and manipulation of actin in living cells. However, these G-actin-binding molecules are obtained by extraction from natural sources or through complex chemical synthesis procedures, and therefore, the generation of their derivatives for analytical tools is underdeveloped. In addition, their effects on nuclear actin cannot be separately evaluated from those on cytoplasmic actin. Previously, we have generated synthetic bicyclic peptides, consisting of two macrocyclic rings, which bind to G-actin but not to F-actin. Here, we describe the introduction of these bicyclic peptides into living cells. Furthermore, by conjugation to a nuclear localization signal (NLS), the bicyclic peptides accumulated in the nucleus. The NLS-bicyclic peptides repress the formation of nuclear F-actin, and impair transcriptional regulation and DSB repair. These observations highlight a potential role for NLS-linked bicyclic peptides in the manipulation of dynamics and functions of nuclear actin., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Background: Actin is an essential cellular protein that assembles into microfilaments and regulates numerous processes such as cell migration, maintenance of cell shape, and material transport., Methods: In this study, we explored the effect of actin polymerization state on the osteogenic differentiation of human adipose-derived stem cells (hASCs). The hASCs were treated for 7 days with different concentrations (0, 1, 5, 10, 20, and 50 nM) of jasplakinolide (JAS), a reagent that directly polymerizes F-actin. The effects of the actin polymerization state on cell proliferation, apoptosis, migration, and the maturity of focal adhesion-related proteins were assessed. In addition, western blotting and alizarin red staining assays were performed to assess osteogenic differentiation., Results: Cell proliferation and migration in the JAS (0, 1, 5, 10, and 20 nM) groups were higher than in the control group and the JAS (50 nM) group. The FAK, vinculin, paxillin, and talin protein expression levels were highest in the JAS (20 nM) group, while zyxin expression was highest in the JAS (50 nM) group. Western blotting showed that osteogenic differentiation in the JAS (0, 1, 5, 10, 20, and 50 nM) group was enhanced compared with that in the control group, and was strongest in the JAS (50 nM) group., Conclusions: In summary, our data suggest that the actin polymerization state may promote the osteogenic differentiation of hASCs by regulating the protein expression of focal adhesion-associated proteins in a concentration-dependent manner. Our findings provide valuable information for exploring the mechanism of osteogenic differentiation in hASCs.

Numerous studies have assembled a complex picture, in which extracellular stimuli and intracellular signaling pathways modulate the chondrocyte phenotype. Because many diseases are mechanobiology-related, this review asked to what extent phenotype regulators control chondrocyte function through the cytoskeleton and cytoskeleton-regulating signaling processes. Such information would generate leverage for advanced articular cartilage repair. Serial passaging, pro-inflammatory cytokine signaling (TNF-α, IL-1α, IL-1β, IL-6, and IL-8), growth factors (TGF-α), and osteoarthritis not only induce dedifferentiation but also converge on RhoA/ROCK/Rac1/mDia1/mDia2/Cdc42 to promote actin polymerization/crosslinking for stress fiber (SF) formation. SF formation takes center stage in phenotype control, as both SF formation and SOX9 phosphorylation for COL2 expression are ROCK activity-dependent. Explaining how it is molecularly possible that dedifferentiation induces low COL2 expression but high SF formation, this review theorized that, in chondrocyte SOX9, phosphorylation by ROCK might effectively be sidelined in favor of other SF-promoting ROCK substrates, based on a differential ROCK affinity. In turn, actin depolymerization for redifferentiation would "free-up" ROCK to increase COL2 expression. Moreover, the actin cytoskeleton regulates COL1 expression, modulates COL2/aggrecan fragment generation, and mediates a fibrogenic/catabolic expression profile, highlighting that actin dynamics-regulating processes decisively control the chondrocyte phenotype. This suggests modulating the balance between actin polymerization/depolymerization for therapeutically controlling the chondrocyte phenotype.

Cardiac fibrosis is a common pathological feature of cardiac hypertrophy. This study was designed to investigate a novel function of Yes-associated protein (YAP) circular RNA, circYap, in modulating cardiac fibrosis and the underlying mechanisms. By circular RNA sequencing, we found that three out of fifteen reported circYap isoforms were expressed in nine human heart tissues, with the isoform hsa_circ_0002320 being the highest. The levels of this isoform in the hearts of patients with cardiac hypertrophy were found to be significantly decreased. In the pressure overload mouse model, the levels of circYap were reduced in mouse hearts with transverse aortic constriction (TAC). Upon circYap plasmid injection, the cardiac fibrosis was attenuated, and the heart function was improved along with the elevation of cardiac circYap levels in TAC mice. Tropomyosin-4 (TMP4) and gamma-actin (ACTG) were identified to bind with circYap in cardiac cells and mouse heart tissues. Such bindings led to an increased TPM4 interaction with ACTG, resulting in the inhibition of actin polymerization and the following fibrosis. Collectively, our study uncovered a novel molecule that could regulate cardiac remodeling during cardiac fibrosis and implicated a new function of circular RNA. This process may be targeted for future cardio-therapy., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe the function of Smoothelin-like 2 (SMTNL2), a member of the smooth-muscle-related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during development in multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of coronin-1B. Although coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular cortex., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Actin remodeling is at the heart of the response of cells to external or internal stimuli, allowing a variety of membrane protrusions to form. Fifteen years ago, tunneling nanotubes (TNTs) were identified, bringing a novel addition to the family of actin-supported cellular protrusions. Their unique property as conduits for cargo transfer between distant cells emphasizes the unique nature of TNTs among other protrusions. While TNTs in different pathological and physiological scenarios have been described, the molecular basis of how TNTs form is not well understood. In this review, we discuss the role of several actin regulators in the formation of TNTs and suggest potential players based on their comparison with other actin-based protrusions. New perspectives for discovering a distinct TNT formation pathway would enable us to target them in treating the increasing number of TNT-involved pathologies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A method to generate small amount of reactive oxygen species (ROSs) at intracellular targeted region has great potential to manipulate the function of particular proteins. The present protocol introduces a fusion protein that consisted of firefly luciferase (FLuc), photosensitizer protein KillerRed and F-actin-targeting peptide Lifeact (Lifeact-KillerFirefly) to generate ROSs in the vicinity of F-actin and found that morphological change in F-actin structure was induced by the fusion protein after luciferin treatment. This manipulating and imaging method is of use to analyze the role of the locally generated ROSs on the function of intracellular proteins.

Linusorbs (LOs) are natural peptides found in flaxseed oil that exert various biological activities. Of LOs, LOB3 ([1-9-NαC]-linusorb B3) was reported to have antioxidative and anti-inflammatory activities; however, its anti-cancer activity has been poorly understood. Therefore, this study investigated the anti-cancer effect of LOB3 and its underlying mechanism in glioblastoma cells. LOB3 induced apoptosis and suppressed the proliferation of C6 cells by inhibiting the expression of anti-apoptotic genes, B cell lymphoma 2 (Bcl-2) and p53, as well as promoting the activation of pro-apoptotic caspases, caspase-3 and -9. LOB3 also retarded the migration of C6 cells, which was achieved by suppressing the formation of the actin cytoskeleton critical for the progression, invasion, and metastasis of cancer. Moreover, LOB3 inhibited the activation of the proto-oncogene, Src, and the downstream effector, signal transducer and activator of transcription 3 (STAT3), in C6 cells. Taken together, these results suggest that LOB3 plays an anti-cancer role by inducing apoptosis and inhibiting the migration of C6 cells through the regulation of apoptosis-related molecules, actin polymerization, and proto-oncogenes.

Heterodimeric capping protein (CP) binds the rapidly growing barbed ends of actin filaments and prevents the addition (or loss) of subunits. Capping activity is generally considered to be essential for actin-based motility induced by Arp2/3 complex nucleation. By stopping barbed end growth, CP favors nucleation of daughter filaments at the functionalized surface where the Arp2/3 complex is activated, thus creating polarized network growth, which is necessary for movement. However, here using an in vitro assay where Arp2/3 complex-based actin polymerization is induced on bead surfaces in the absence of CP, we produce robust polarized actin growth and motility. This is achieved either by adding the actin polymerase Ena/VASP or by boosting Arp2/3 complex activity at the surface. Another actin polymerase, the formin FMNL2, cannot substitute for CP, showing that polymerase activity alone is not enough to override the need for CP. Interfering with the polymerase activity of Ena/VASP, its surface recruitment or its bundling activity all reduce Ena/VASP's ability to maintain polarized network growth in the absence of CP. Taken together, our findings show that CP is dispensable for polarized actin growth and motility in situations where surface-directed polymerization is favored by whatever means over the growth of barbed ends in the network., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Abou-Ghali et al.)

Membrane repair is essential for cell and organism survival. Exocytosis and endocytosis facilitate membrane repair in small wounds within a single cell; however, it remains unclear how large wounds in the plasma membrane are repaired in metazoans. Here, we show that wounding triggers rapid transcriptional upregulation and dynamic recruitment of the fusogen EFF-1 to the wound site in C. elegans epidermal cells. EFF-1 recruitment at the wounded membrane depends on the actin cytoskeleton and is important for membrane repair. We identified syntaxin-2 (SYX-2) as an essential regulator of EFF-1 recruitment. SYX-2 interacts with the C terminus of EFF-1 to promote its recruitment, facilitating both endoplasmic and exoplasmic membrane repair. Furthermore, we show that SYX-2-EFF-1 repair machinery acts downstream of the ESCRT III signal. Together, our findings identify a key pathway underlying membrane repair and provide insights into tissue repair and regenerative medicine after injury., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

During the last decade, intracellular actin waves have attracted much attention due to their essential role in various cellular functions, ranging from motility to cytokinesis. Experimental methods have advanced significantly and can capture the dynamics of actin waves over a large range of spatio-temporal scales. However, the corresponding coarse-grained theory mostly avoids the full complexity of this multi-scale phenomenon. In this perspective, we focus on a minimal continuum model of activator-inhibitor type and highlight the qualitative role of mass conservation, which is typically overlooked. Specifically, our interest is to connect between the mathematical mechanisms of pattern formation in the presence of a large-scale mode, due to mass conservation, and distinct behaviors of actin waves.

The actomyosin cytoskeleton network plays a key role in a variety of fundamental cellular processes such as cell division, migration, and cell adhesion. The functions of cytoskeleton rely on its capability to receive, generate, respond to and transmit mechanical signals throughout the cytoskeleton network within the cells and throughout the tissue via cell-extracellular matrix and cell-cell adhesions. Crucial to the cytoskeleton's functions is actin polymerization that is regulated by many cellular factors. Among these factors, the formin family proteins, which bind the barbed end of an actin filament (F-actin), are known to be a major actin polymerization promoting factor. Mounting evidence from single-molecule mechanical manipulation experiments have suggested that formin-dependent actin polymerization is sensitively regulated by the force and torque applied to the F-actin, making the formin family an emerging mechanosensing factor that selectively promotes elongation of the F-actin under tensile forces. In this review, we will focus on the current understanding of the mechanical regulation of formin-mediated actin polymerization, the key technologies that have enabled quantification of formin-mediated actin polymerization under mechanical constraints, and future perspectives and studies on molecular mechanisms involved in the mechanosensing of actin dynamics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Although best known from its functions in the cytoplasm, actin also localizes to the cell nucleus, where it has been linked to many essential functions from regulation of gene expression to maintenance of genomic integrity. While majority of cytoplasmic functions of actin depend on controlled actin polymerization, in the nucleus both actin monomers and filaments have their own specific roles. Actin monomers are core components of several chromatin remodeling and modifying complexes and can also regulate the activity of specific transcription factors, while actin filaments have been linked to DNA damage response and cell cycle progression. Consequently the balance between monomeric and filamentous actin must be precise controlled also in the nucleus, since their effects are dynamically coupled. In this review, we discuss the recent data on how actin dynamics is regulated within the nucleus and how this influences the different nuclear processes dependent on actin., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

CXCL8 is the principal human neutrophil-attracting chemokine and a major mediator of inflammation. The chemokine exerts its neutrophil-chemotactic and neutrophil-activating activities via interaction with glycosaminoglycans (GAGs) and activation of the G protein-coupled receptors (GPCRs) CXCR1 and CXCR2. Natural CXCL8 displays an exceptional degree of amino (NH 2 )-terminal heterogeneity. Most CXCL8 forms result from proteolytic processing of authentic CXCL8(1-77). Here, we compared the potencies to activate and recruit neutrophils of the 3 most abundant natural CXCL8 forms: full-length 77 amino acid CXCL8 and the 2 major natural truncated forms lacking 5 or 8 NH 2 -terminal amino acids. NH 2 -terminal truncation hardly affected the capacity of CXCL8 to induce shedding of CD62L or to up-regulate the expression of the adhesion molecules CD11a, CD11b, or CD15 on human neutrophils. In addition, the potency of CXCL8 to induce neutrophil degranulation and its effect on phagocytosis remained unaltered upon removal of 5 or 8 NH 2 -terminal residues. However, NH 2 -terminal truncation strongly potentiated CXCL8-induced actin polymerization. CXCL8(6-77) and CXCL8(9-77) showed a comparable capacity to induce Ca 2+ signaling in human neutrophils and to direct in vitro neutrophil migration. Strikingly, the ability of CXCL8(9-77) to recruit neutrophils into the peritoneal cavity of mice was significantly enhanced compared to CXCL8(6-77). These results suggest that NH 2 -terminal truncation influences specific biological activities of CXCL8 and indicate that CXCL8(9-77) may be the most potent neutrophil-attracting CXCL8 form in vivo., (©2020 Society for Leukocyte Biology.)

Numerous studies have shown that alteration of actin remodeling plays a pivotal role in the regulation of morphologic and phenotypic changes leading to malignancy. In the present study, we searched for drugs that can regulate actin polymerization and reverse the malignant phenotype in cancer cells. We developed a cell-free high-throughput screening assay for the identification of compounds that induce the actin polymerization in vitro, by fluorescence anisotropy. Then, the potential of the hit compound to restore the actin cytoskeleton and reverse the malignant phenotype was checked in EWS-Fli1-transformed fibroblasts and in B16-F10 melanoma cells. A β-carboline extracted from Peganum harmala (i.e., harmine) is identified as a stimulator of actin polymerization through a mechanism independent of actin binding and requiring intracellular factors involved in a process that regulates actin kinetics. Treatment of malignant cells with non-cytotoxic concentrations of harmine induces the recovery of a non-malignant cell morphology accompanied by reorganization of the actin cytoskeleton, rescued cell-cell adhesion, inhibition of cell motility and loss of anchorage-independent growth. In conclusion, harmine induces the reversion of the malignant phenotype by a process involving the modulation of actin dynamics and is a potential anti-tumor agent acting principally through a non-cytotoxic process.

In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase-activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein-expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by-products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells., (© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

Scope: Urolithin A (UA) is a gut-derived bacterial metabolite from ellagic acid found in pomegranates, berries, and nuts can downregulate cell proliferation and migration. Cell proliferation and cell motility require actin reorganization, which is under control of ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1). The present study explores whether UA can modify actin cytoskeleton in cancer cells., Methods: The effect of UA on globular over filamentous actin ratio is determined utilizing Western blotting, immunofluorescence, and flow cytometry. Rac1 and PAK1 levels are measured by quantitative RT-PCR and immunoblotting. As a result, a 24 h treatment with UA (20 µm) significantly decreased Rac1 and PAK1 transcript levels and activity, depolymerized actin and wound healing. The effect of UA on actin polymerization is mimicked by pharmacological inhibition of Rac1 and PAK1. The effect is also mirrored by knock down using siRNA., Conclusion: UA leads to disruption of Rac1 and Pak1 activity with subsequent actin depolymerization and migration. Thus, use of dietary UA in cancer prevention or as adjuvant therapy is promising., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Following T-cell antigen receptor (TCR) engagement, rearrangement of the actin cytoskeleton supports intracellular signal transduction and T-cell activation. The non-catalytic region of the tyrosine kinase (Nck) molecule is an adapter protein implicated in TCR-induced actin polymerization. Further, Nck is recruited to the CD3ε subunit of the TCR upon TCR triggering. Here we examine the role of actin polymerization in the recruitment of Nck to the TCR. To this end, Nck binding to CD3ε was quantified in Jurkat cells using the proximity ligation assay. We show that inhibition of actin polymerization using cytochalasin D delayed the recruitment of Nck1 to the TCR upon TCR triggering. Interestingly, CD3ε phosphorylation was also delayed. These findings suggest that actin polymerization promotes the recruitment of Nck to the TCR, enhancing downstream signaling, such as phosphorylation of CD3ε., (© 2019 John Wiley & Sons Ltd.)

Profilin is a major regulator of actin dynamics in multiple specific processes localized in different cellular compartments. This specificity is not only meditated by its binding to actin but also its interaction with phospholipids such as phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) at the membrane and a plethora of proteins containing poly-L-proline (PLP) stretches. These interactions are fine-tuned by posttranslational modifications such as phosphorylation. Several phospho-sites have already been identified for profilin1, the ubiquitously expressed isoform. However, little is known about the phosphorylation of profilin2a. Profilin2a is a neuronal isoform important for synapse function. Here, we identified several putative profilin2a phospho-sites in silico and tested recombinant phospho-mimetics with regard to their actin-, PLP-, and PIP 2 -binding properties. Moreover, we assessed their impact on actin dynamics employing a pyrene-actin polymerization assay. Results indicate that distinct phospho-sites modulate specific profilin2a functions. We could identify a molecular switch site at serine residue 71 which completely abrogated actin binding-as well as other sites important for fine-tuning of different functions, for example, tyrosine 29 for PLP binding. Our findings suggest that differential profilin2a phosphorylation is a sensitive mechanism for regulating its neuronal functions. Moreover, the dysregulation of profilin2a phosphorylation may contribute to neurodegeneration., (© 2019 The Authors. The FASEB Journal published by Wiley Periodicals, Inc. on behalf of Federation of American Societies for Experimental Biology.)

Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, or abortion. Listeria induces its internalization (entry) into some human cells through interaction of the bacterial surface protein InlB with its host receptor, the Met tyrosine kinase. InlB and Met promote entry through stimulation of localized actin polymerization and exocytosis. How actin cytoskeletal changes and exocytosis are controlled during entry is not well understood. Here, we demonstrate important roles for the host GTPase Arf1 and its effectors AP1 and PICK1 in actin polymerization and exocytosis during InlB-dependent uptake. Depletion of Arf1 by RNA interference (RNAi) or inhibition of Arf1 activity using a dominant-negative allele impaired InlB-dependent internalization, indicating an important role for Arf1 in this process. InlB stimulated an increase in the GTP-bound form of Arf1, demonstrating that this bacterial protein activates Arf1. RNAi and immunolocalization studies indicated that Arf1 controls exocytosis and actin polymerization during entry by recruiting the effectors AP1 and PICK1 to the plasma membrane. In turn, AP1 and PICK1 promoted plasma membrane translocation of both Filamin A (FlnA) and Exo70, two host proteins previously found to mediate exocytosis during InlB-dependent internalization (M. Bhalla, H. Van Ngo, G. C. Gyanwali, and K. Ireton, Infect Immun 87:e00689-18, 2018, https://doi.org/10.1128/IAI.00689-18). PICK1 mediated recruitment of Exo70 but not FlnA. Collectively, these results indicate that Arf1, AP1, and PICK1 stimulate exocytosis by redistributing FlnA and Exo70 to the plasma membrane. We propose that Arf1, AP1, and PICK1 are key coordinators of actin polymerization and exocytosis during infection of host cells by Listeria ., (Copyright © 2020 American Society for Microbiology.)

Cellular organization through cytoskeletal trafficking is a process of fundamental importance. Highly specialized systems evolved that enable motors to identify and select the optimal tracks for motility. In this chapter, we examine the profound effect of actin filament networks on myosin motility patterns. We argue that the myosin classes have adaptations that allow them to detect local structural and chemical cues on actin. These cues are often arranged in a coherent manner on actin filament networks, allowing for directed transport over long distances. We identify a number of potentially important cues, ranging from the biochemical states of actin subunits all the way to multi-filament networks and bundles.

Alzheimer's disease (AD) is the most common form of dementia in the elderly, characterized by amyloid-beta (Aβ) plaques and tau neurofibrillary tangles (NFTs). Synaptic plasticity impairment is one of the early pathological events in AD. Transgenic APP/PS1 mice that overproduce Aβ are one of the most extensively used AD animal models. Many studies have investigated the roles of NTF-related p-Tau, non-amyloidogenic ADAM10, amyloidogenic BACE1, Aβ proteolytic NEP and IDE in certain ages of APP/PS1 mice as well as dendritic spine-related Rictor and Profilin-1 in normal mice, but there are few studies exploring the age-related changes of these molecules in the hippocampus of APP/PS1 mice. Furthermore, current studies regarding when memory impairment occurs in these mice are controversial. Thus, we examined the changes of these molecules in APP/PS1 and control mice using Western blot in mice 2-month-old (2 m) to 10 m of age and behavior changes using the Morris water maze from 4 m to 8 m. The results showed that in APP/PS1 mice, significant changes of hippocampal p-Tau, Aβ, ADAM10, BACE1 and Rictor occurred at 6 m, NEP at 8 m, and IDE and Profilin-1 at 10 m. In control mice, changes of p-Tau, ADAM10, and BACE1 occurred at 8 m and NEP at 10 m, while IDE, Rictor and Profilin-1 remained unchanged. Importantly, the Morris water maze test revealed that spatial memory impairment was detected at 8 m but not 4 or 6 m. The above findings clearly evidence that neurochemical changes overtly precede cognitive dysfunctions in this AD model and provide novel knowledge for a better understanding of the molecular events driving AD., (Copyright © 2019 Elsevier B.V. All rights reserved.)