Regulation Of Actin Polymerization Research Articles

Loss of cytoplasmic actin filaments raises nuclear actin levels to drive INO80C-dependent chromosome fragmentation

Loss of cytosolic actin filaments upon TORC2 inhibition triggers chromosome fragmentation in yeast, which results from altered base excision repair of Zeocin-induced lesions. To find the link between TORC2 kinase and this yeast chromosome shattering (YCS) we performed phosphoproteomics. YCS-relevant phospho-targets included plasma membrane-associated regulators of actin polymerization, such as Las17, the yeast Wiscott-Aldrich Syndrome protein. Induced degradation of Las17 was sufficient to trigger YCS in presence of Zeocin, bypassing TORC2 inhibition. In yeast, Las17 does not act directly at damage, but instead its loss, like TORC2 inhibition, raises nuclear actin levels. Nuclear actin, in complex with Arp4, forms an essential subunit of several nucleosome remodeler complexes, including INO80C, which facilitates DNA polymerase elongation. Here we show that the genetic ablation of INO80C activity leads to partial YCS resistance, suggesting that elevated levels of nuclear G-actin may stimulate INO80C to increase DNA polymerase processivity and convert single-strand lesions into double-strand breaks.

Characterization of Cytoskeletal Profilin Genes in Plasticity Elongation of Mesocotyl and Coleoptile of Maize Under Diverse Abiotic Stresses.

The plasticity elongation of mesocotyl (MES) and coleoptile (COL) largely determines the morphology of maize seedlings under abiotic stresses. The profilin (PRF) proteins play a pivotal role in cytoskeleton dynamics and plant development via regulating actin polymerization. However, little is known about whether and how the expression of the ZmPRF gene family regulates MES and COL elongation in maize under adverse abiotic stresses. Here, a total of eight ZmPRF gene members were identified in the maize genome. They were mainly located in the cytoplasm, chloroplast, and mitochondrion, and clearly divided into four classes, based on phylogenetic analysis. Segmental duplication was the main driver for the expansion of ZmPRF genes. Ka/Ks analysis indicated that most ZmPRF genes were intensely purified and selected. Promoter cis-element analysis suggested their potential roles in response to growth and development, stress adaption, hormone response, and light response. The protein-protein interaction network and two independent RNA-sequencing analyses revealed that eight ZmPRF genes and their thirty-seven interacting genes showed varied expression patterns in MES and COL of three maize genotypes under different sowing depths, 24-epibrassinolide application, and light spectral-quality treatments, of which ZmPRF3.3 was a potential core conserved gene for breeding application. Moreover, the quantitative real-time PCR (qRT-PCR) verified that the relative expression levels of most ZmPRF genes in MES and COL under above treatments were significantly correlated with the plasticity elongation of MES and COL in maize. Therefore, these results perform a comprehensive overview of the ZmPRF family and will provide valuable information for the validation of the function of ZmPRF genes in maize development under diverse abiotic stress.

Dennd2c Negatively Controls Multinucleation and Differentiation in Osteoclasts by Regulating Actin Polymerization and Protrusion Formation.

Osteoclasts are bone-resorbing multinucleated giant cells formed by the fusion of monocyte/macrophage lineages. Various small GTPases are involved in the multinucleation and differentiation of osteoclasts. However, the roles of small GTPases regulatory molecules in osteoclast differentiation remain unclear. In the present study, we examined the role of Dennd2c, a putative guanine nucleotide exchange factor for Rab GTPases, in osteoclast differentiation. Knockdown of Dennd2c promoted osteoclast differentiation, resorption, and expression of osteoclast markers. Morphologically, Dennd2c knockdown induced the formation of larger osteoclasts with several protrusions. In contrast, overexpression of Dennd2c inhibited the multinucleation and differentiation of osteoclasts, bone resorption, and the expression of osteoclast markers. Dennd2c-overexpressing macrophages exhibited spindle-shaped mononuclear cells and long thin protrusions. Treatment of Dennd2c-overexpressing cells with the Cdc42 inhibitor ML-141 or the Rac1 inhibitor 6-thio-GTP prevented protrusion formation. Moreover, treatment of Dennd2c-overexpressing cells with the actin polymerization inhibitor latrunculin B restored multinucleated and TRAP-positive osteoclast formation. These results indicate that Dennd2c negatively regulates osteoclast differentiation and multinucleation by modulating protrusion formation in macrophages.

Investigating human monocyte adhesion, migration and transmigration and their modulation by Zika virus

Human circulating monocytes are established targets for Zika virus (ZIKV) infection. Because of their important migratory properties toward any tissues, including the central nervous system (CNS), a better understanding of the mechanisms underlying monocyte transmigration upon ZIKV infection is required. Here, we monitored adhesion, migration and transmigration properties of monocytes exposed to ZIKV. We found that ZIKV enhanced monocyte adhesion on collagen compared to mock-exposed samples, and that pharmacological inhibition of mDia and Cdc42 function induced a significant decrease of adhesion in both mock- and ZIKV-exposed monocytes. In contrast, monocyte migration through collagen was inhibited by most of the tested small molecules targeting regulators of actin polymerization, including Rac1, ROCK, Cdc42, mDia, Arp2/3, Myosin-II and LFA-1. ZIKV-exposed monocyte migration showed a very similar profile to that of their mock-exposed counterparts. Finally, assessment of monocyte transmigration through human cerebral microvascular endothelial cells (hCMEC/D3) showed dependency on Rac1, ROCK, and Cdc42, independently of their infection status. In contrast, we identified that BIRT377, an antagonist of LFA-1, significantly inhibited transmigration of ZIKV-exposed but not mock-exposed monocytes. As BIRT377 increased adhesion of ZIKV-exposed monocytes, we propose that LFA-1 might be involved in a post-adhesion step to enhance viro-induced transmigration. These data suggest that ZIKV exposure triggers specific migratory properties of monocytes that are not exploited under physiological conditions. This work provides further insights on virus-host interactions important for viral neuroinvasion and offers novel targets to specifically inhibit the infiltration of infected cells to the CNS. Summary sentenceMonocyte transmigration involves massive actin cytoskeleton reorganization regulated by small Rho GTPases and integrins, which can be subverted by viruses.

Neurodevelopmental disorder-associated CYFIP2 regulates membraneless organelles and eIF2α phosphorylation via protein interactors and actin cytoskeleton.

De novo variants in the Cytoplasmic FMR1-interacting protein 2 (CYFIP2) have been repeatedly associated with neurodevelopmental disorders and epilepsy, underscoring its critical role in brain development and function. While CYFIP2's role in regulating actin polymerization as part of the WAVE regulatory complex (WRC) is well-established, its additional molecular functions remain relatively unexplored. In this study, we performed unbiased quantitative proteomic analysis, revealing 278 differentially expressed proteins (DEPs) in the forebrain of Cyfip2 knock-out embryonic mice compared to wild-type mice. Unexpectedly, these DEPs, in conjunction with previously identified CYFIP2 brain interactors, included not only other WRC components but also numerous proteins associated with membraneless organelles (MLOs) involved in mRNA processing and translation within cells, including the nucleolus, stress granules, and processing bodies. Additionally, single-cell transcriptomic analysis of the Cyfip2 knock-out forebrain revealed gene expression changes linked to cellular stress responses and MLOs. We also observed morphological changes in MLOs in Cyfip2 knock-out brains and CYFIP2 knock-down cells under basal and stress conditions. Lastly, we demonstrated that CYFIP2 knock-down in cells, potentially through WRC-dependent actin regulation, suppressed the phosphorylation levels of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α), thereby enhancing protein synthesis. These results suggest a physical and functional connection between CYFIP2 and various MLO proteins and also extend CYFIP2's role within the WRC from actin regulation to influencing eIF2α phosphorylation and protein synthesis. With these dual functions, CYFIP2 may fine-tune the balance between MLO formation/dynamics and protein synthesis, a crucial aspect of proper mRNA processing and translation.

Synthetic control of actin polymerization and symmetry breaking in active protocells.

Nonlinear biomolecular interactions on membranes drive membrane remodeling crucial for biological processes including chemotaxis, cytokinesis, and endocytosis. The complexity of biomolecular interactions, their redundancy, and the importance of spatiotemporal context in membrane organization impede understanding of the physical principles governing membrane mechanics. Developing a minimal in vitro system that mimics molecular signaling and membrane remodeling while maintaining physiological fidelity poses a major challenge. Inspired by chemotaxis, we reconstructed chemically regulated actin polymerization inside vesicles, guiding membrane self-organization. An external, undirected chemical input induced directed actin polymerization and membrane deformation uncorrelated with upstream biochemical cues, suggesting symmetry breaking. A biophysical model incorporating actin dynamics and membrane mechanics proposes that uneven actin distributions cause nonlinear membrane deformations, consistent with experimental findings. This protocellular system illuminates the interplay between actin dynamics and membrane shape during symmetry breaking, offering insights into chemotaxis and other cell biological processes.

Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein.

Cell processes require precise regulation of actin polymerization that is mediated by plus-end regulatory proteins. Detailed mechanisms that explain plus-end dynamics involve regulators with opposing roles, including factors that enhance assembly, e.g., the formin mDia1, and others that stop growth (capping protein, CP). We explore IQGAP1's roles in regulating actin filament plus-ends and the consequences of perturbing its activity in cells. We confirm that IQGAP1 pauses elongation and interacts with plus ends through two residues (C756 and C781). We directly visualize the dynamic interplay between IQGAP1 and mDia1, revealing that IQGAP1 displaces the formin to influence actin assembly. Using four-color TIRF, we show that IQGAP1's displacement activity extends to formin-CP "decision complexes," promoting end-binding protein turnover at plus-ends. Loss of IQGAP1 or its plus-end activities disrupts morphology and migration, emphasizing its essential role. These results reveal a new role for IQGAP1 in promoting protein turnover on filament ends and provide new insights into how plus-end actin assembly is regulated in cells.

IQGAP1 Regulates Actin Polymerization and Contributes to Bleomycin-Induced Lung Fibrosis.

We previously found IQ motif containing GTPase activating protein (IQGAP1) to be consistently elevated in lung fibroblasts (LF) isolated from patients with scleroderma (systemic sclerosis, SSc)-associated interstitial lung disease (ILD) and reported that IQGAP1 contributed to SSc by regulating expression and organization of α-smooth muscle actin (SMA) in LF. The aim of this study was to compare the development of ILD in the presence and absence of IQGAP1. Pulmonary fibrosis was induced in IQGAP1 knockout (KO) and wild-type (WT) mice by a single-intratracheal instillation of bleomycin. Two and three weeks later, mice were euthanized and investigated. We observed that the IQGAP1 KO mouse was characterized by a reduced rate of actin polymerization with reduced accumulation of actin in the lung compared to the WT mouse. After exposure to bleomycin, the IQGAP1 KO mouse demonstrated decreased contractile activity of LF, reduced expression of SMA, TGFβ, and collagen, and lowered overall fibrosis scores compared to the WT mouse. The numbers of inflammatory cells and expression of pro-inflammatory cytokines in lung tissue were not significantly different between IQGAP1 KO and WT mice. We conclude that IQGAP1 plays an important role in the development of lung fibrosis induced by bleomycin, and the absence of IQGAP1 reduces the contractile activity of lung fibroblast and bleomycin-induced pulmonary fibrosis. Thus, IQGAP1 may be a potential target for novel anti-fibrotic therapies for lung fibrosis.

Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption

Bone resorption is highly dependent on the dynamic rearrangement of the osteoclast actin cytoskeleton to allow formation of actin rings and a functional ruffled border. Hem1 is a hematopoietic-specific subunit of the WAVE-complex which regulates actin polymerization and is crucial for lamellipodia formation in hematopoietic cell types. However, its role in osteoclast differentiation and function is still unknown. Here, we show that although the absence of Hem1 promotes osteoclastogenesis, the ability of Hem1-/- osteoclasts to degrade bone was severely impaired. Global as well as osteoclast-specific deletion of Hem1 in vivo revealed increased femoral trabecular bone mass despite elevated numbers of osteoclasts in vivo. We found that the resorption defect derived from the morphological distortion of the actin-rich sealing zone and ruffled border deformation in Hem1-deficient osteoclasts leading to impaired vesicle transport and increased intracellular acidification. Collectively, our data identify Hem1 as a yet unknown key player in bone remodeling by regulating ruffled border formation and consequently the resorptive capacity of osteoclasts.

Abstract LB433: Targeting of ABI1 via DNA-based PROTACs

Abstract Breast cancer is the most commonly diagnosed non-cutaneous cancer in American women. Although current therapies reduce initial tumor burden, patients often relapse with aggressive tumors which can metastasize. Abelson Interactor 1 (ABI1) is an adaptor protein which is traditionally seen as a WAVE complex component- regulating actin polymerization, cell migration and adhesion. In this role, ABI1 serves as a driver of breast cancer metastasis. Recent studies have discovered that ABI1 contains a homeobox homology region (HHR) which is able to bind DNA and regulate transcription. In order to specifically target and degrade ABI1, we designed a novel PROteolysis-TArgeting Chimera (PROTAC) system using DNA as the bait for ABI1. We hypothesized that by linking ABI1 DNA targets to the ubiquitin system, we would be able to effectively target ABI1 for degradation. Lenalidomide, an E3 ubiquitin ligase ligand, was attached to an oligonucleotide via click chemistry. Cells were then treated with PROTAC systems via direct addition to media or via lipofection reagents, and levels of ABI1 were detected using western blotting. Using this DNA-based PROTAC system in cell-culture models of both breast and ABI1-elevated prostate cancer suggested that the system is able to target and degrade ABI1. Usage of lipofection reagents were able to improve drug delivery and effectiveness. Further research is needed to identify off-target effects and effectiveness in vivo models of disease to reduce metastasis. In conclusion, PROTACs targeting ABI1 may prove to be an effective and novel method to reduce metastasis and improve outcomes for cancer treatment. Citation Format: Kevin M. Lin, Xiang Li, Leszek Kotula. Targeting of ABI1 via DNA-based PROTACs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB433.

Abstract 6992: Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma

Abstract Background:Oral squamous cell carcinoma (OSCC) stands as a prevalent malignancy worldwide. Profilin 2 (PFN2), an actin-binding protein regulating actin polymerization dynamics and associated with cell motility, has recently gained attention as a significant regulator in various cancers. However, the clinical significance and biological function of PFN2 in OSCC remain debatable. This study aims to elucidate the role of PFN2 in OSCC. Methods:We assessed PFN2 expression in OSCC and adjacent normal oral mucosa tissues through Q-RT-PCR and immunohistochemistry analysis. Associations between PFN2 expression, clinicopathological features, and OSCC prognosis were analyzed. Cell proliferation, invasion, and migration effects were examined via MTT and Transwell assays. Western blotting assessed the protein expression related to epithelial–mesenchymal transition (EMT). Additionally, we established OSCC xenografts to determine tumorigenicity in vivo using female Nude mice. Results:PFN2 RNA and protein expression were significantly increased in OSCC compared to normal oral mucosa. PFN2 expression positively correlated with lymphovascular invasion and cervical lymph node metastasis. MTT and Transwell assays demonstrated that PFN2 promoted OSCC cell proliferation, migration, invasion, and endothelial invasion. Downregulation of PFN2 induced an EMT phenotype, including an increase in the expression of the epithelial marker E-cadherin and a decrease in the expression of mesenchymal markers Vimentin, Snail, and Slug in OSCC cells in vitro. Similarly, OSCC cells with reduced PFN2 expression exhibited weaker tumorigenicity in vivo. Conclusions:Our findings unveil a novel role for PFN2 in promoting OSCC progression and metastasis, suggesting its potential as an early biomarker for high-risk populations. Targeting PFN2 may offer a promising therapeutic strategy for OSCC treatment. Citation Format: Chung Ji LIu, Li-Han Lin, Kuo-Wei Chang, Hui-Wen Cheng. Profilin 2 (PFN2) promotes the proliferation, migration, invasion and epithelial-to-mesenchymal transition of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6992.

Arpc2 integrates ecdysone and juvenile hormone metabolism to influence metamorphosis and reproduction in Tribolium castaneum.

Actin-related protein 2/3 complex regulates actin polymerization and the formation of branched actin networks. However, the function and evolutionary relationship of this complex subunit 2 (Arpc2) has been poorly understood in insects. To address these issues, we performed comprehensive analysis of Arpc2 in Tribolium castaneum. Phylogenetic analysis revealed that Arpc2 was originated from one ancestral gene in animals but evolved independently between vertebrates and insects after species differentiation. T. castaneum Arpc2 has a 906-bp coding sequence and consists of 4 exons. Arpc2 transcripts were abundantly detected in embryos and pupae but less so in larvae and adults, while it had high expression in the gut, fat body and head but low expression in the epidermis of late-stage larvae. Knockdown of it at the late larval stage inhibited the pupation and resulted in arrested larvae. Silencing it in 1-day pupae impaired eclosion, which caused adult wings to fail to close. Injection of Arpc2 dsRNAs into 5-day pupae made adults have smaller testis and ovary and could not lay eggs. The expression of vitellogenin 1 (Vg1), Vg2 and Vg receptor (VgR) was downregulated after knocking down Arpc2 5 days post-adult emergence. Arpc2 silencing reduced 20-hydroxyecdysone titer by affecting the enzymes of its biosynthesis and catabolism but increased juvenile biosynthesis via upregulating JHAMT3 expression. Our results indicate that Arpc2 is associated with the metamorphosis and reproduction by integrating ecdysone and juvenile hormone metabolism in T. castaneum. This study provides theoretical basis for developing Arpc2 as a potential RNA interference target for pest control. © 2024 Society of Chemical Industry.

Tyrosinase regulates the motility of human melanoma cell line A375 through its hydroxylase enzymatic activity

Melanoma, originating from melanocytes, is a highly aggressive tumor. Tyrosinase is involved in melanin production in melanocytes, and its overexpression is noted in malignant melanomas. However, the role of tyrosinase in melanomas remains unclear. Therefore, this study aimed to evaluate the potential functions of tyrosinase in the human melanoma cell line A375. The expression level of tyrosinase in A375 cells was undetectable. However, markedly increased expression level was observed in the mouse melanoma cell line B16F10 and the human melanoma cell line WM266-4. Subsequently, we investigated the effect of ectopic tyrosinase expression on A375 cell motility using wound-healing assay. The overexpression of tyrosinase resulted in enhanced cell migration in both stable and transient tyrosinase expression cells. The levels of filamentous actin were decreased in tyrosinase-expressing A375 cells, suggesting that tyrosinase regulates cell motility by modulating actin polymerization. Histidine residues in tyrosinase are important for its enzymatic activity for synthesizing melanin. Substitution of these histidine residues to alanine residues mitigated the promotion of tyrosinase-induced A375 cell metastasis. Furthermore, melanin treatment enhanced A375 cell metastasis and phosphorylation of Cofilin. Thus, our findings suggest that tyrosinase increases the migration of A375 cells by regulating actin polymerization through its enzymatic activity.

Transcriptional mechanism of E2F1/TFAP2C/NRF1 in regulating KANK2 gene in nephrotic syndrome

The mortality rate linked with nephrotic syndrome (NS) is quite high. The renal tubular injury influences the response of NS patients to steroid treatment. KN motif and ankyrin repeat domains 2 (KANK2) regulates actin polymerization, which is required for renal tubular cells to maintain their function. In this study, we found that the levels of KANK2 in patients with NS were considerably lower than those in healthy controls, especially in NS patients with acute kidney injury (AKI). To get a deeper understanding of the KANK2 transcriptional control mechanism, the core promoter region of the KANK2 gene was identified. KANK2 was further found to be positively regulated by E2F Transcription Factor 1 (E2F1), Transcription Factor AP-2 Gamma (TFAP2C), and Nuclear Respiratory Factor 1 (NRF1), both at mRNA and protein levels. Knocking down E2F1, TFAP2C, or NRF1 deformed the cytoskeleton of renal tubular cells and reduced F-actin content. EMSA and ChIP assays confirmed that all three transcription factors could bind to the upstream promoter transcription site of KANK2 to transactivate KANK2 in renal tubular epithelial cells. Our study suggests that E2F1, TFAP2C, and NRF1 play essential roles in regulating the KANK2 transcription, therefore shedding fresh light on the development of putative therapeutic options for the treatment of NS patients.

Different dysregulations of CYFIP1 and CYFIP2 in distinct types of dementia

In humans, the cytoplasmic FMR1 interacting protein (CYFIP) family consists of two members, namely CYFIP1 and CYFIP2. Both CYFIP1 and CYFIP2 function in the WAVE regulatory complex (WRC), which regulates actin polymerization. Additionally, these two proteins form a posttranscriptional regulatory complex with the fragile X mental retardation protein (FMRP), which suppresses mRNA translation. Thus, CYFIP1 and CYFIP2 are important signalling regulators at synapses, and mutations in their genes are associated with neurodevelopmental and neuropsychiatric disorders, including intellectual disabilities. Moreover, dysregulation of the CYFIP protein family is involved in Alzheimer’s disease (AD). However, the relevance of the CYFIP family in other dementias is largely unknown. Here, we compared CYFIP1/2 protein levels in the post-mortem hippocampus from patients with AD, dementia with Lewy bodies (DLB), vascular dementia (VaD) and frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP). Consistent with previous findings, CYFIP2 was reduced in AD hippocampus. In DLB and VaD hippocampus, the protein level of CYFIP2 and CYFIP1 was unaltered. Finally, an increase in the protein level of both CYFIP1 and CYFIP2 was noted in FTLD-TDP hippocampus. These findings reveal that the protein levels of the CYFIP family is distinct in different types of dementia, suggesting that the pathogenesis of these neurodegenerative disorders has divergent impacts on hippocampal synaptic function.

T cell migration requires ion and water influx to regulate actin polymerization

Migration of T cells is essential for their ability to mount immune responses. Chemokine-induced T cell migration requires WNK1, a kinase that regulates ion influx into the cell. However, it is not known why ion entry is necessary for T cell movement. Here we show that signaling from the chemokine receptor CCR7 leads to activation of WNK1 and its downstream pathway at the leading edge of migrating CD4+ T cells, resulting in ion influx and water entry by osmosis. We propose that WNK1-induced water entry is required to swell the membrane at the leading edge, generating space into which actin filaments can polymerize, thereby facilitating forward movement of the cell. Given the broad expression of WNK1 pathway proteins, our study suggests that ion and water influx are likely to be essential for migration in many cell types, including leukocytes and metastatic tumor cells.

The amount of Nck rather than N-WASP correlates with the rate of actin-based motility of Vaccinia virus.

Vaccinia virus is a large double-stranded DNA virus and a close relative of Mpox and Variola virus, the causative agent of smallpox. During infection, Vaccinia hijacks its host's transport systems and promotes its spread into neighboring cells by recruiting a signaling network that stimulates actin polymerization. Over the years, Vaccinia has provided a powerful model to understand how signaling networks regulate actin polymerization. Nevertheless, we still lack important quantitative information about the system, including the precise number of viral and host molecules required to induce actin polymerization. Using quantitative fluorescence microscopy techniques, we have determined the number of viral and host signaling proteins accumulating on virions during their egress. Our analysis has uncovered two unexpected new aspects of this process: the number of viral proteins in the virion is not fixed and the velocity of virus movement depends on the level of a single adaptor within the signaling network.

Sfxn5 Regulation of Actin Polymerization for Neutrophil Spreading Depends on a Citrate-Cholesterol-PI(4,5)P2 Pathway.

Cell spreading is an initial and critical step in neutrophil adhesion and migration, leading to neutrophil recruitment to inflammatory tissues. Sideroflexin (Sfxn) family proteins are metabolite transporters located in the mitochondrial membrane. Recombinant SFXN5 protein is a citrate transporter invitro; however, whether Sfxn5 regulates any cellular behavior or function remains unknown. In this study, we found that small interfering RNA transfection or morpholino injection achieving Sfxn5 deficiency in neutrophils significantly decreased neutrophil recruitment in mice and zebrafish, respectively. Sfxn5 deficiency impaired neutrophil spreading and spreading-associated cellular phenotypes, such as cell adhesion, chemotaxis, and ROS production. Actin polymerization is critical for neutrophil spreading, and we found that actin polymerization in spreading neutrophils was partially inhibited by Sfxn5 deficiency. Mechanistically, we observed that the levels of cytosolic citrate and its downstream metabolic products, acetyl-CoA and cholesterol, were decreased in Sfxn5-deficient neutrophils. The levels of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a mediator for the regulation of actin polymerization by cholesterol, were reduced in the plasma membrane of Sfxn5-deficient neutrophils. Exogenous supplementation with citrate or cholesterol partially reversed the reduction in PI(4,5)P2 levels, defective neutrophil actin polymerization, and cell spreading. Altogether, we demonstrated that Sfxn5 maintains cytosolic citrate levels and ensures the synthesis of sufficient cholesterol to promote actin polymerization in a PI(4,5)P2-dependent manner during neutrophil spreading, which is essential for the eventual inflammatory recruitment of neutrophils. Our study revealed the importance of Sfxn5 in neutrophil spreading and migration, thus identifying, to our knowledge, for the first time, the physiological cellular functions of the Sfxn5 gene.

Nucleophosmin 1 associating with engulfment and cell motility protein 1 regulates hepatocellular carcinoma cell chemotaxis and metastasis.

The chemokine, C-X-C motif chemokine ligand 12 (CXCL12) and its G-protein-coupled receptor (GPCR) and C-X-C chemokine receptor type 4 (CXCR4), are closely associated with promoting hepatocellular carcinoma (HCC) chemotaxis and metastasis. The binding of CXCL12 and CXCR4 depends on the heterotrimeric Gi proteins to regulate actin polymerisation and mobility in HCC. Although the role of GPCR/Gi signalling in carcinogenesis migration has been intensively studied, the detailed mechanism remains largely unknown. In this study, a small interfering RNA technique was used to knock down the Nucleophosmin 1 (NPM1) gene expression. Through the chemotaxis and invasion assays, wound healing, proliferation, filamentous-actin, immunofluorescence, immunohistochemical assays, and co-immunoprecipitation assays, we investigated the specific biological role and underlying mechanisms of the NPM1 in HCC. Additionally, dimethyl fumarate (DMF), a fumaric acid ester, was used to inhibit the HCC cell chemokines and metastasis by regulating ELMO1 and NPM1. Therefore, this study reported that NPM1 gene expression was upregulated in the HCC tissues and cell lines. The NPM1 knockdown significantly inhibited the proliferation, migration, and chemotaxis of the HepG2 cells in vitro. Further mechanistic studies suggested that the NPM1 interacts with ELMO1 and the CXCL12/CXCR4 pathway activates NPM1-dependent regulation of the ELMO1 localisation. Furthermore, the DMF significantly inhibited tumour metastasis induced by the NPM1/ELMO1 signalling pathway, as observed in in vitro cell functional experiments. These data suggested that as a potentially novel therapeutic approach, the simultaneous targeting of NPM1 and ELMO1 could effectively be used to treat HCC.

Visfatin-Induced Smooth Muscle Cell Stiffness Syndrome during Lysosomal Acid Ceramidase Deficiency

Recent studies have reported that intrinsic stiffness of smooth muscle cells (SMCs) plays an important role in contributing to arterial wall stiffness, which led to the concept of smooth muscle cell stiffness syndrome (SMCSS). However, the molecular mechanisms regulating the intrinsic stiffness of SMCs are still poorly understood. The present study tested whether lysosomal acid ceramidase (AC) determines intrinsic stiffness of mouse aortic SMCs via regulation of actin polymerization and integrin recycling. Atom force microscopy was used to study mechanical properties of SMCs. As an injurious adipokine, visfatin was found to reduce SMC height, but elevate cortical stiffness and focal adhesion in SMCs, indicating the augmentation of intrinsic stiffness. Moreover, visfatin-induced elevation of cortical stiffness was associated with increased actin polymerization in peripheral areas of SMCs. These pathological changes were much more remarkable in SMCs from smooth muscle-specific Asah1 gene (gene code of AC) knockout (Asah1fl/fl/SMcre) mice compared to SMCs from WT/WT mice. Super-resolution microscopy showed that multivesicular bodies (MVBs) as integrin degrading organelles were much less interacted or fused with lysosomes in SMCs from Asah1fl/fl/SMcre mice than WT/WT mice upon visfatin stimulation. In contrast, MVB-α5 integrin encounters increased more in SMCs from Asah1fl/fl/SMcre mice compared to SMCs from control littermates, suggesting that AC deficiency increases α5 integrin accumulation in MVB, which may enhance recycling endosome moving to cell membrane. Given the vital role of lysosomal transient receptor potential mucolipin 1 (TRPML1) channel in the regulation of lysosome trafficking and its interaction with MVB, we tested whether this lysosomal channel is disturbed by visfatin. By GCaMP3 Ca2+ imaging and whole-lysosome patch clamp recording, we found that ML-SA5 (TRPML1 channel agonist) induced remarkable lysosomal Ca2+ release in WT/WT SMCs, which was attenuated by visfatin. In SMCs lacking Asah1 gene, however, ML-SA5 induced much less Ca2+ release from lysosomes, which was almost abolished by visfatin. Lysosome trafficking analysis showed that ML-SA5 activation of lysosomal TRPML1 channels markedly increased lysosome movement in SMCs, which was significantly inhibited by visfatin and Asah1 gene deletion. Based on these results, we concluded that lysosomal AC deficiency leads to dysregulation of actin polymerization and integrin recycling in SMCs, which may be a novel molecular mechanism of SMCSS and ultimate arterial wall stiffening during obesity. This study was supported by NIH grants HL057244 and HL075316. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.