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There are three main classes of actin nucleation factors: Arp2/3 complexes, Spire and Formin. Spire assembles microfilaments by nucleating stable longitudinal tetramers and binding actin to the growing end of the microfilament. As early as 1999, Wellington et al. identified Spire as an actin nucleating agent, however, over the years, most studies have focused on Arp2/3 and Formin proteins; there has been relatively less research on Spire as a member of the actin nucleating factors. Recent studies have shown that Spire is involved in the vesicular transport through the synthesis of actin and plays an important role in neural development. In this paper, we reviewed the structure, expression and function of Spire, and its association with disease in order to identify meaningful potential directions for studies on Spire.

Coronins play critical roles in actin network formation. The diverse functions of coronins are regulated by the structured N-terminal β propeller and the C-terminal coiled coil (CC). However, less is known about a middle "unique region" (UR), which is an intrinsically disordered region (IDR). The UR/IDR is an evolutionarily conserved signature in the coronin family. By integrating biochemical and cell biology experiments, coarse-grained simulations, and protein engineering, we find that the IDR optimizes the biochemical activities of coronins in vivo and in vitro. The budding yeast coronin IDR plays essential roles in regulating Crn1 activity by fine-tuning CC oligomerization and maintaining Crn1 as a tetramer. The IDR-guided optimization of Crn1 oligomerization is critical for F-actin cross-linking and regulation of Arp2/3-mediated actin polymerization. The final oligomerization status and homogeneity of Crn1 are contributed by three examined factors: helix packing, the energy landscape of the CC, and the length and molecular grammar of the IDR., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the developing cortex, excitatory neurons migrate along the radial fibers to their final destinations and build up synaptic connection with each other to form functional circuitry. The shaping of neuronal morphologies by actin cytoskeleton dynamics is crucial for neuronal migration. However, it is largely unknown how the distribution and assembly of the F-actin cytoskeleton are coordinated. In the present study, we found that an actin regulatory protein, coronin 2B, is indispensable for the transition from a multipolar to bipolar morphology during neuronal migration in ICR mice of either sex. Loss of coronin 2B led to heterotopic accumulation of migrating neurons in the intermediate zone along with reduced dendritic complexity and aberrant neuronal activity in the cortical plate. This was accompanied by increased seizure susceptibility, suggesting the malfunction of cortical development in coronin 2B-deficient brains. Coronin 2B knockdown disrupted the distribution of the F-actin cytoskeleton at the leading processes, while the migration defect in coronin 2B-deficient neurons was partially rescued by overexpression of Rac1 and its downstream actin-severing protein, cofilin. Our results collectively reveal the physiological function of coronin 2B during neuronal migration whereby it maintains the proper distribution of activated Rac1 and the F-actin cytoskeleton. SIGNIFICANCE STATEMENT Deficits in neuronal migration during cortical development result in various neurodevelopmental disorders (e.g., focal cortical dysplasia, periventricular heterotopia, epilepsy, etc.). Most signaling pathways that control neuronal migration process converge to regulate actin cytoskeleton dynamics. Therefore, it is important to understand how actin dynamics is coordinated in the critical processes of neuronal migration. Herein, we report that coronin 2B is a key protein that regulates neuronal migration through its ability to control the distribution of the actin cytoskeleton and its regulatory signaling protein Rac1 during the multipolar-bipolar transition in the intermediate zone, providing insights into the molecular machinery that drives the migration process of newborn neurons., (Copyright © 2023 the authors.)

Modulation of presynaptic actin dynamics is fundamental to synaptic growth and functional plasticity; yet the underlying molecular and cellular mechanisms remain largely unknown. At Drosophila NMJs, the presynaptic Rac1-SCAR pathway mediates BMP-induced receptor macropinocytosis to inhibit BMP growth signaling. Here, we show that the Rho-type GEF Vav acts upstream of Rac1 to inhibit synaptic growth through macropinocytosis. We also present evidence that Vav-Rac1-SCAR signaling has additional roles in tetanus-induced synaptic plasticity. Presynaptic inactivation of Vav signaling pathway components, but not regulators of macropinocytosis, impairs post-tetanic potentiation (PTP) and enhances synaptic depression depending on external Ca2+ concentration. Interfering with the Vav-Rac1-SCAR pathway also impairs mobilization of reserve pool (RP) vesicles required for tetanus-induced synaptic plasticity. Finally, treatment with an F-actin-stabilizing drug completely restores RP mobilization and plasticity defects in Vav mutants. We propose that actin-regulatory Vav-Rac1-SCAR signaling independently regulates structural and functional presynaptic plasticity by driving macropinocytosis and RP mobilization, respectively., (© 2022 Park et al.)

As a major co-factor of F-actin depolymerization, WD-repeat domain 1 (WDR1) affects the cellular microenvironment by cytoskeleton remodeling, thereby influencing cell molecular behavior. Our previous study showed that WDR1 activates YAP (Yes-associated protein) signaling in non-small-cell lung cancer (NSCLC) cells, but the mechanism remains unclear. We discovered that knockdown WDR1 in NSCLC cells decreased the expression of YAP and the nucleus-to-cytoplasm ratio. Disruption of cortical stress by drugs significantly inhibited YAP nuclear trafficking and enhanced YAP phosphorylation. In WDR1-knockdown NSCLC cells, inhibition of Hippo pathway reduced the nuclear exclusion of YAP and phosphorylated YAP. Our data suggest that WDR1-mediated cortical stress might be involved in regulating YAP signaling, thereby affecting the proliferation and migration of NSCLC cells.

The imbalance between osteogenic and adipogenic differentiation of Bone marrow-derived mesenchymal stem cells (BMSCs) is involved in the occurrence and development of osteoporosis (OP). Previous studies have indicated the potential of phosphatase and actin regulator 1 (Phactr1) in regulating osteogenic and adipogenic differentiation of BMSCs. The present study aims to investigate the function and mechanism of Phactr1 in regulating osteogenic and adipogenic differentiation of BMSCs. Herein, the expression of Phactr1 in bone and adipose tissue of OP rats was determined by immunohistochemical. BMSCs were subjected to osteogenic and adipogenic differentiation, and transfected with Phactr1 overexpression lentivirus, small interference RNA (siRNA) and KD025 (selective ROCK2 inhibitor). The relationship between Phactr1 and ROCK2 was detected by Co-IP experiment. The expression of Phactr1, Runx2, C/EBPα, RhoA and ROCK2 was detected by Western blot. Calcium nodule and lipid droplets were determined by alizarin red and Oil red O staining. Interestingly, Phactr1 increased in both bone and adipose tissue of OP rats. During osteogenic differentiation, Phactr1 decreased and active RhoA, ROCK2 increased, while overexpression Phactr1 inhibits the increase of Runx2. Phactr1 increased and active RhoA decreased, ROCK2 did not changed during adipogenic differentiation. While, Knockdown Phactr1 inhibits the increase of C/EBPα. Phactr1 and ROCK2 were combined in osteogenic differentiation, but not in adipogenic differentiation. By using KD025, the decrease of Phactr1 and increase of Runx2 were inhibited respectively in osteogenic differentiation. Meanwhile, when ROCK2 was inhibited, Phactr1, C/EBPα were significantly increased in adipogenic differentiation. These findings indicated that Phactr1 negatively regulates bone mass by inhibiting osteogenesis and promoting adipogenesis of BMSCs by activating RhoA/ROCK2., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Invadopodia are dynamic actin-rich membrane protrusions that have been implicated in cancer cell invasion and metastasis. In addition, invasiveness of cancer cells is strongly correlated with invadopodia formation, which are observed during extravasation and colonization of metastatic cancer cells at secondary sites. However, quantitative understanding of the interaction of invadopodia with extracellular matrix (ECM) is lacking, and how invadopodia protrusion speed is associated with the frequency of protrusion-retraction cycles remains unknown. Here, we present a computational framework for the characterization of invadopodia protrusions which allows two way interactions between intracellular branched actin network and ECM fibers network. We have applied this approach to predicting the invasiveness of cancer cells by computationally knocking out actin-crosslinking molecules, such as α-actinin, filamin and fascin. The resulting simulations reveal distinct invadopodia dynamics with cycles of protrusion and retraction. Specifically, we found that (1) increasing accumulation of MT1-MMP at tips of invadopodia as the duration of protrusive phase is increased, and (2) the movement of nucleus toward the leading edge of the cell becomes unstable as duration of the retractile phase (or myosin turnover time) is longer than 1 min., (© 2022. The Author(s).)

Ischemic stroke is a major cause of death and long-term disability. We demonstrate that middle cerebral artery occlusion (MCAO) in mice leads to a strong decline in dendritic arborization of penumbral neurons. These defects were subsequently repaired by an ipsilateral recovery process requiring the actin nucleator Cobl. Ischemic stroke and excitotoxicity, caused by calpain-mediated proteolysis, significantly reduced Cobl levels. In an apparently unique manner among excitotoxicity-affected proteins, this Cobl decline was rapidly restored by increased mRNA expression and Cobl then played a pivotal role in poststroke dendritic arbor repair in peri-infarct areas. In Cobl knockout (KO) mice, the dendritic repair window determined to span day 2 to 4 poststroke in wild-type (WT) strikingly passed without any dendritic regrowth. Instead, Cobl KO penumbral neurons of the primary motor cortex continued to show the dendritic impairments caused by stroke. Our results thereby highlight a powerful poststroke recovery process and identified causal molecular mechanisms critical during poststroke repair., Competing Interests: The authors have declared that no competing interests exist.

Alterations in the balance between excitation and inhibition, especially in the brain's critical developmental periods, are considered an integral part of the pathophysiology of autism. However, the precise mechanisms have not yet been established. SH3 and multiple Ankyrin repeat domains 3 (Shank3) deficient mice represent a well-established transgenic model of a neurodevelopmental disorder with autistic symptomatology. In this study, we characterize the consequences of Shank3 deficiency according to (1) expression of specific markers of different neuronal populations in pups and adult mice and (2) social behaviour and anxiety in adult mice. Our research found enhanced expression of serotonin transporter and choline acetyltransferase in the hippocampus and hypothalamus in Shank3-deficient pups. We demonstrated marked brain region differences in expression of excitatory glutamatergic markers in pups and adult Shank3 deficient mice. We also observed reduced expression of inhibitory GABAergic markers and GABA receptor subunits in several brain areas in both pups and adult Shank3 deficient mice. Further analysis of dopaminergic brain areas (nucleus accumbens, ventral tegmental area) revealed lower expression levels of GABAergic markers in adult Shank3 deficient mice. Adult Shank3 - deficient mice exhibited excessive repetitive behaviour, a higher level of anxiety, and lower locomotor activity. Our data support the theory of an imbalance between excitatory and inhibitory neurotransmission in conditions of abnormal SHANK3 protein. We therefore suggest that autism-like conditions are accompanied by reduced expression of GABAergic markers in the brain during early development as well as in the adult age, which could be associated with long-lasting behavioural abnormalities., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

FRG1 has a role in tumorigenesis and angiogenesis. Our preliminary analysis showed that FRG1 mRNA expression is associated with overall survival (OS) in certain cancers, but the effect varies. In cervix and gastric cancers, we found a clear difference in the OS between the low and high FRG1 mRNA expression groups, but the difference was not prominent in breast, lung, and liver cancers. We hypothesized that FRG1 expression level could affect the functionality of the correlated genes or vice versa, which might mask the effect of a single gene on the OS analysis in cancer patients. We used the multivariate Cox regression, risk score, and Kaplan Meier analyses to determine OS in a multigene model. STRING, Cytoscape, HIPPIE, Gene Ontology, and DAVID (KEGG) were used to deduce FRG1 associated pathways. In breast, lung, and liver cancers, we found a distinct difference in the OS between the low and high FRG1 mRNA expression groups in the multigene model, suggesting an independent role of FRG1 in survival. Risk scores were calculated based upon regression coefficients in the multigene model. Low and high-risk score groups showed a significant difference in the FRG1 mRNA expression level and OS. HPF1, RPL34, and EXOSC9 were the most common genes present in FRG1 associated pathways across the cancer types. Validation of the effect of FRG1 mRNA expression level on these genes by qRT-PCR supports that FRG1 might be an upstream regulator of their expression. These genes may have multiple regulators, which also affect their expression, leading to the masking effect in the survival analysis. In conclusion, our study highlights the role of FRG1 in the survivability of cancer patients in tissue-specific manner and the use of multigene models in prognosis., (© 2021. The Author(s).)

The formation of the branched actin networks is essential for cell polarity, but it remains unclear how the debranching activity of actin filaments contributes to this process. Here, we showed that an evolutionarily conserved coronin family protein, the Caenorhabditis elegans POD-1, debranched the Arp2/3-nucleated actin filaments in vitro. By fluorescence live imaging analysis of the endogenous POD-1 protein, we found that POD-1 colocalized with Arp2/3 at the leading edge of the migrating C. elegans neuroblasts. Conditional mutations of POD-1 in neuroblasts caused aberrant actin assembly, disrupted cell polarity, and impaired cell migration. In C. elegans one-cell-stage embryos, POD-1 and Arp2/3, moved together during cell polarity establishment, and inhibition of POD-1 blocked Arp2/3 motility and affected the polarized cortical flow, leading to symmetric segregation of cell fate determinants. Together, these results indicate that F-actin debranching organizes actin network and cell polarity in migrating neuroblasts and asymmetrically dividing embryos., Competing Interests: The authors declare no competing interest.

The aim of the present study was to investigate the role and potential regulatory mechanisms of fascin in the invasion and epithelial-to-mesenchymal transition of pituitary adenoma cells. A total of 30 specimens were assessed in the present study. The expression levels of fascin in the invasive pituitary adenoma group and non-invasive pituitary adenoma group were determined by immunochemistry. Fascin was downregulated via small interfering RNA in mouse pituitary AtT-20 cells. The proliferation, cell cycle and apoptosis of AtT-20 cells were assessed using Cell Counting Kit‑8 and flow cytometry. The invasion of AtT-20 cells was detected using a Transwell assay. Transmission electron microscopy was utilized to observe the ultrastructure of AtT-20 cells. Real-time quantitative PCR, Western blotting and immunofluorescence staining were utilized to detect the expression levels of fascin and EMT markers. In the present study, fascin expression and clinical characteristics were not significantly correlated in pituitary adenoma. The protein expression level of fascin in invasive pituitary adenoma was higher than that in non-invasive pituitary adenoma, as assessed by immunochemistry. Downregulation of fascin resulted in significant decreases in cell viability, proliferation and invasion, arrested the cell cycle at the G1 phase and increased apoptosis. In addition, downregulation of fascin significantly decreased the expression levels of N-cadherin, the mesenchymal cell marker vimentin and the transcription factor Twist but significantly increased the expression levels of the epithelial cell marker E-cadherin. Further experiments revealed that overexpression of E-cadherin resulted in significant decreases in cell viability, proliferation, invasion, and the expression of fascin and transcription factor Twist and also arrested the cell cycle at the G2 phase. The results of the present study suggest that suppressing the expression level of fascin could regulate the invasion, proliferation and apoptosis of pituitary tumour cells and alter the expression level of various EMT markers. The present study identified that fascin effectively promotes the invasion, proliferation and apoptosis of pituitary tumour cells partially via the EMT pathway., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Recent studies have focused on capillary pruning in various organs and species. However, the way in which large-diameter vessels are pruned remains unclear. Here we show that pruning of the zebrafish caudal vein (CV) from ventral capillaries of the CV plexus in different transgenic embryos is driven by endothelial cell (EC) rearrangement, which involves EC nucleus migration, junction remodeling, and actin cytoskeleton remodeling. Further observation reveals a growing difference in blood flow velocity between the two vessels in CV pruning in zebrafish embryos. With this model, we identify the critical role of Kruppel-like factor 6a (klf6a) in CV pruning. Disruption of klf6a functioning impairs CV pruning in zebrafish. klf6a is required for EC nucleus migration, junction remodeling, and actin cytoskeleton dynamics in zebrafish embryos. Moreover, actin-related protein transgelin 2 (tagln2) is a direct downstream target of klf6a in CV pruning in zebrafish embryos. Together these results demonstrate that the klf6a-tagln2 axis regulates CV pruning by promoting EC rearrangement., Competing Interests: The authors have declared that no competing interests exist.

Fascin-1 (FSCN1) is an actin-bundling protein associated with an invasive and aggressive phenotype of several solid carcinomas, as it is involved in cell cytoskeleton rearrangement and filopodia formation. Adrenocortical carcinoma (ACC) is a rare endocrine malignancy characterized by poor prognosis, particularly when metastatic at diagnosis. Radical resection is the only therapeutic option for ACC patients in addition to the adjuvant treatment with mitotane. Novel specific biomarkers suggestive of tumor progression to refine diagnosis and prognosis of patients with advanced ACC are urgently needed. ACC intratumoral FSCN1 has previously been suggested as a valid prognostic marker. In the present study, we identified FSCN1 in the bloodstream of a small cohort of ACC patients (n = 27), through a specific ELISA assay for human FSCN1. FSCN1 can be detected in the serum, and its circulating levels were evaluated in pre-surgery samples, which resulted to be significantly higher in ACC patients from stage I/II and stage III/IV compared with nontumoral healthy controls (HC, n = 4, FI: 5.5 ± 0.8, P<0.001, and 8.0 ± 0.5, P < 0.001 for stage I/II and stage III/IV group vs HC, respectively). In particular, FSCN1 levels were significantly higher in advanced stage versus stage I/II (22.8 ± 1.1 vs 15.8 ± 1.8 ng/ml, P < 0.005, respectively). Interestingly, circulating levels of pre-surgical FSCN1 can significantly predict tumor progression/recurrence (Log rank = 0.013), but not the overall survival (Log rank=0.317), in patients stratified in high/low PreS FSCN1. In conclusion, these findings-though very preliminary-suggest that circulating FSCN1 may represent a new minimally-invasive prognostic marker in advanced ACC, in particular when measured before surgery enables histological diagnosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cantini, Fei, Canu, De Filpo, Ercolino, Nesi, Mannelli and Luconi.)

Mechanical forces control cell behavior, including cancer progression. Cells sense forces through actomyosin to activate YAP. However, the regulators of F-actin dynamics playing relevant roles during mechanostransduction in vitro and in vivo remain poorly characterized. Here we identify the Fascin1 F-actin bundling protein as a factor that sustains YAP activation in response to ECM mechanical cues. This is conserved in the mouse liver, where Fascin1 regulates YAP-dependent phenotypes, and in human cholangiocarcinoma cell lines. Moreover, this is relevant for liver tumorigenesis, because Fascin1 is required in the AKT/NICD cholangiocarcinogenesis model and it is sufficient, together with AKT, to induce cholangiocellular lesions in mice, recapitulating genetic YAP requirements. In support of these findings, Fascin1 expression in human intrahepatic cholangiocarcinomas strongly correlates with poor patient prognosis. We propose that Fascin1 represents a pro-oncogenic mechanism that can be exploited during intrahepatic cholangiocarcinoma development to overcome a mechanical tumor-suppressive environment.

Accumulation of vascular smooth muscle cells (VSMCs) is a hallmark of multiple vascular pathologies, including following neointimal formation after injury and atherosclerosis. However, human VSMCs in advanced atherosclerotic lesions show reduced cell proliferation, extensive and persistent DNA damage, and features of premature cell senescence. Here, we report that stress-induced premature senescence (SIPS) and stable expression of a telomeric repeat-binding factor 2 protein mutant (TRF2 T188A ) induce senescence of human VSMCs, associated with persistent telomeric DNA damage. VSMC senescence is associated with formation of micronuclei, activation of cGAS-STING cytoplasmic sensing, and induction of multiple pro-inflammatory cytokines. VSMC-specific TRF2 T188A expression in a multicolor clonal VSMC-tracking mouse model shows no change in VSMC clonal patches after injury, but an increase in neointima formation, outward remodeling, senescence and immune/inflammatory cell infiltration or retention. We suggest that persistent telomere damage in VSMCs inducing cell senescence has a major role in driving persistent inflammation in vascular disease.

Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid-liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.

Intercellular adhesion is essential for tissue integrity and homeostasis. Desmosomes are abundant in the epidermis and the myocardium-tissues, which are under constantly changing mechanical stresses. Yet, it is largely unclear whether desmosomal adhesion can be rapidly adapted to changing demands, and the mechanisms underlying desmosome turnover are only partially understood. In this study we show that the loss of the actin-binding protein α-adducin resulted in reduced desmosome numbers and prevented the ability of cultured keratinocytes or murine epidermis to withstand mechanical stress. This effect was not primarily caused by decreased levels or impaired adhesive properties of desmosomal molecules but rather by altered desmosome turnover. Mechanistically, reduced cortical actin density in α-adducin knockout keratinocytes resulted in increased mobility of the desmosomal adhesion molecule desmoglein 3 and impaired interactions with E-cadherin, a crucial step in desmosome formation. Accordingly, the loss of α-adducin prevented increased membrane localization of desmoglein 3 in response to cyclic stretch or shear stress. Our data demonstrate the plasticity of desmosomal molecules in response to mechanical stimuli and unravel a mechanism of how the actin cytoskeleton indirectly shapes intercellular adhesion by restricting the membrane mobility of desmosomal molecules., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cells actively position their nuclei within the cytoplasm for multiple cellular and physiological functions. 1-3 Consequently, nuclear mispositioning is usually associated with cell dysfunction and disease, from muscular disorders to cancer metastasis. 4-7 Different cell types position their nuclei away from the leading edge during cell migration. 8-11 In migrating fibroblasts, nuclear positioning is driven by an actin retrograde flow originated at the leading edge that drives dorsal actin cables away from the leading edge. The dorsal actin cables connect to the nuclear envelope by the linker of nucleoskeleton and cytoskeleton (LINC) complex on transmembrane actin-associated nuclear (TAN) lines. 12-14 Dorsal actin cables are required for the formation of TAN lines. How dorsal actin cables are organized to promote TAN lines formation is unknown. Here, we report a role for Ctdnep1/Dullard, a nuclear envelope phosphatase, 15-22 and the actin regulator Eps8L2 23-25 on nuclear positioning and cell migration. We demonstrate that Ctdnep1 and Eps8L2 directly interact, and this interaction is important for nuclear positioning and cell migration. We also show that Ctdnep1 and Eps8L2 are involved in the formation and thickness of dorsal actin cables required for TAN lines engagement during nuclear movement. We propose that Ctdnep1-Eps8L2 interaction regulates dorsal actin cables for nuclear movement during cell migration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nasopharyngeal carcinoma is a type of otolaryngological malignancy with high incidence. Long non-coding RNAs (lncRNAs) are closely related to nasopharyngeal carcinoma. LncRNA AFAP1-AS1 (AFAP1-AS1) has been found to play important roles in nasopharyngeal carcinoma progression and poor prognosis. However, the mechanism underlying AFAP1-AS1 in regulating nasopharyngeal carcinoma is still unclear. In current study, AFAP1-AS1 was found to be up-regulated in nasopharyngeal carcinoma tissues and cells. AFAP1-AS1 overexpression and knockdown were conducted in nasopharyngeal carcinoma cells. The results proved that AFAP1-AS1 promoted the survival and migration of nasopharyngeal carcinoma cells. Additionally, specificity protein 1 (SP1) was enhanced in nasopharyngeal carcinoma tissues and cells, and induced AFAP1-AS1 expression. The interaction between AFAP1-AS1 and miR-497-5p was confirmed. AFAP1-AS1 was demonstrated to regulate CELF1, a target gene of miR-497-5p. Further functional analysis revealed that AFAP1-AS1 knockdown attenuated SP1-induced nasopharyngeal carcinoma progression. These results indicate that SP1-induced AFAP1-AS1 facilitates nasopharyngeal carcinoma progression by regulating miR-497-5p/CELF1 pathway, which provides a new target for nasopharyngeal carcinoma treatment.

Macrophages and monocytes are important for clearance of Leishmania infections. However, immune evasion tactics employed by the parasite results in suppressed inflammatory responses, marked by deficient macrophage functions and increased accumulation of monocytes. This results in an ineffective ability to clear parasite loads. Allograft Inflammatory Factor-1 (AIF1) is expressed in myeloid cells and serves to promote immune responses. However, AIF1 involvement in monocyte and macrophage functions during parasitic infections has not been explored. This study now shows that Leishmania donovani inhibits AIF1 expression in macrophages to block pro-inflammatory responses. Mice challenged with the parasite had markedly reduced AIF1 expression in splenic macrophages. Follow-up studies using in vitro approaches confirmed that L. donovani infection in macrophages suppresses AIF1 expression, which correlated with reduction in pro-inflammatory cytokine production and increased parasite load. Ectopic overexpression of AIF1 in macrophages provided protection from infection, marked by robust pro-inflammatory cytokine production and efficient pathogen clearance. Further investigations found that inhibiting AIF1 expression in bone marrow cells or monocytes impaired differentiation into functional macrophages. Collectively, results show that AIF1 is a critical regulatory component governing monocyte and macrophage immune functions and that L. donovani infection can suppress the gene as an immune evasion tactic.

As structural and signaling platform in plant cell, the actin cytoskeleton is regulated by diverse actin binding proteins (ABPs). Villins are one type of major ABPs responsible for microfilament bundling, which have proved to play important roles in plant growth and development. However, the function of villins in stress tolerance is poorly understood. Here, we report the function of cotton GhVLN4 in Verticillium wilt resistance and abiotic stress tolerance. The expression of GhVLN4 was up-regulated by gibberellin, ethylene, ABA, salicylic acid, jasmonate, NaCl, PEG, and Verticillium dahliae treatment, suggesting the involvement of GhVLN4 in multiple stress and hormone responses and signaling. Virus-induced gene silencing GhVLN4 made cotton more susceptible to V. dahliae characterized by the preferential colonization and rapid growth of the fungus in both phloem and xylem of the infected stems. Arabidopsis overexpressing GhVLN4 exhibited higher resistance to V. dahliae, salt and drought than the wild-type plants. The enhanced resistance to V. dahliae is likely related to the upregulated components in SA signaling pathway; the improved tolerance to salt and drought is characterized by upregulation of the components both in ABA- related and ABA-independent signal pathways, along with altered stomatal aperture under drought. Our findings demonstrate that GhVLN4 may play important roles in regulating plant tolerance to both biotic and abiotic stresses., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The actin cytoskeleton drives cell motility and is essential for neuronal development and function. LIM and SH3 protein 1 (LASP1) is a unique actin-binding protein that is expressed in a wide range of cells including neurons, but its roles in cellular motility and neuronal development are not well understood. We report that LASP1 is expressed in rat hippocampus early in development, and this expression is maintained through adulthood. High-resolution imaging reveals that LASP1 is selectively concentrated at the leading edge of lamellipodia in migrating cells and axonal growth cones. This local enrichment of LASP1 is dynamically associated with the protrusive activity of lamellipodia, depends on the barbed ends of actin filaments, and requires both the LIM domain and the nebulin repeats of LASP1. Knockdown of LASP1 in cultured rat hippocampal neurons results in a substantial reduction in axonal outgrowth and arborization. Finally, loss of the Drosophila homologue Lasp from a subset of commissural neurons in the developing ventral nerve cord produces defasciculated axon bundles that do not reach their targets. Together, our data support a novel role for LASP1 in actin-based lamellipodial protrusion and establish LASP1 as a positive regulator of both in vitro and in vivo axon development.

Subcellular lumen formation by single-cells involves complex cytoskeletal remodelling. We have previously shown that centrosomes are key players in the initiation of subcellular lumen formation in Drosophila melanogaster , but not much is known on the what leads to the growth of these subcellular luminal branches or makes them progress through a particular trajectory within the cytoplasm. Here, we have identified that the spectraplakin Short-stop (Shot) promotes the crosstalk between MTs and actin, which leads to the extension and guidance of the subcellular lumen within the tracheal terminal cell (TC) cytoplasm. Shot is enriched in cells undergoing the initial steps of subcellular branching as a direct response to FGF signalling. An excess of Shot induces ectopic acentrosomal luminal branching points in the embryonic and larval tracheal TC leading to cells with extra-subcellular lumina. These data provide the first evidence for a role for spectraplakins in single-cell lumen formation and branching., Competing Interests: DR, SA No competing interests declared, (© 2020, Ricolo and Araujo.)

Huntingtin-interacting protein family members are evolutionarily conserved from yeast to humans, and they are known to be key factors in clathrin-mediated endocytosis. Here we identified the Caenorhabditis elegans protein huntingtin-interacting protein-related 1 (HIPR-1) as a host factor essential for Orsay virus infection of C. elegans Ablation of HIPR-1 resulted in a greater than 10,000-fold reduction in viral RNA, which could be rescued by ectopic expression of HIPR-1. Viral RNA replication from an endogenous transgene replicon system was not affected by lack of HIPR-1, suggesting that HIPR-1 plays a role during an early, prereplication virus life-cycle stage. Ectopic expression of HIPR-1 mutants demonstrated that neither the clathrin light chain-binding domain nor the clathrin heavy chain-binding motif were needed for virus infection, whereas the inositol phospholipid-binding and F-actin-binding domains were essential. In human cell culture, deletion of the human HIP orthologs HIP1 and HIP1R led to decreased infection by Coxsackie B3 virus. Finally, ectopic expression of a chimeric HIPR-1 harboring the human HIP1 ANTH (AP180 N-terminal homology) domain rescued Orsay infection in C. elegans , demonstrating conservation of its function through evolution. Collectively, these findings further our knowledge of cellular factors impacting viral infection in C. elegans and humans., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Background: The molecular signature underlying pancreatic ductal adenocarcinoma (PDAC) progression may include key proteins affecting the malignant phenotypes. Here, we aimed to identify the proteins implicated in PDAC with different tumour-node-metastasis (TNM) stages., Methods: Eight-plex isobaric tags coupled with two-dimensional liquid chromatography-tandem mass spectrometry were used to analyse the proteome of PDAC tissues with different TNM stages. A loss-of-function study was performed to evaluate the oncogenic roles of WD repeat-containing protein 1 (WDR1) in PDAC. The molecular mechanism by which WDR1 promotes PDAC progression was studied by real-time qPCR, Western blotting, proximity ligation assay and co-immunoprecipitation., Results: A total of 5036 proteins were identified, and 4708 proteins were quantified with high confidence. Compared with normal pancreatic tissues, 37 proteins were changed significantly in PDAC tissues of different stages. Moreover, 64 proteins were upregulated or downregulated in a stepwise manner as the TNM stages of PDAC increased, and 10 proteins were related to tumorigenesis. The functionally uncharacterised protein, WDR1, was highly expressed in PDAC and predicted a poor prognosis. WDR1 knockdown suppressed PDAC tumour growth and metastasis in vitro and in vivo. Moreover, WDR1 knockdown repressed the activity of the Wnt/β-Catenin pathway; ectopic expression of a stabilised form of β-Catenin restored the suppressive effects of WDR1 knockdown. Mechanistically, WDR1 interacted with USP7 to prevent ubiquitination-mediated degradation of β-Catenin., Conclusion: Our study identifies several previous functional unknown proteins implicated in the progression of PDAC, and provides new insight into the oncogenic roles of WDR1 in PDAC development.

The shapes of many eukaryotic cells depends on the actin cytoskeleton, and changes in actin assembly dynamics underlie many changes in cell shape. Ena/VASP-family actin polymerases, for example, modulate cell shape by accelerating actin filament assembly locally and slowing filament capping. When concentrated into discrete foci at the leading edge, VASP promotes filopodia assembly and forms part of a poorly understood molecular complex that remains associated with growing filopodia tips. Here we identify precursors of this filopodia tip complex in migrating B16F1 cells: small leading-edge clusters of the adaptor protein lamellipodin (Lpd) that subsequently recruit VASP and initiate filopodia formation. Dimerization, membrane association, and VASP binding are all required for lamellipodin to incorporate into filopodia tip complexes, and overexpression of monomeric, membrane--targeted lamellipodin mutants disrupts tip complex assembly. Once formed, tip complexes containing VASP and lamellipodin grow by fusing with each other, but their growth is limited by a size-dependent dynamic instability. Our results demonstrate that assembly and disassembly dynamics of filopodia tip complexes are determined, in part, by a network of multivalent interactions between Ena/VASP proteins, EVH1 ligands, and actin filaments.

BACKGROUND The dishevelled associated activator of morphogenesis (DAAM) family, consisting of DAAM1 and DAAM2, is an important component of the Wnt signal pathway. Previous studies have suggested that DAAM2 reduces Von Hippel-Lindau (VHL) expression by promoting its ubiquitination, but the correlation between DAAM and HIF-1alpha in hepatocellular carcinoma (HCC) has not been studied. MATERIAL AND METHODS In our study, expression of DAAM1 and DAAM2 in HCCs and tumor-adjacent liver tissues was assessed with qRT-PCR and immunohistochemistry. Correlations between DAAM1/2 and the clinicopathologic variables were evaluated with the Chi-square test. With univariate and multivariate analysis, we further evaluated the prognostic significance of DAAM1 and DAAM2. Using in vitro experiments, we assessed the functions of DAAM1 and DAAM2 in invasion and proliferation in different HCC cell lines and investigated their underlying mechanisms. RESULTS DAAM1 and 2 overexpression were 18.8% and 48.7%, respectively, of the whole cohort. mRNAs of DAAM2 in HCCs were substantially higher than mRNAs in liver tissues, while DAAM1 mRNA had no marked difference. High DAAM2 expression was notably associated with advanced T stage (P=0.032), TNM stage (P=0.032), and overall survival (OS) rate (P=0.004). DAAM 2 knockdown promoted VHL accumulation and subsequent HIF-1alpha down-regulation in HCC cells. In HCC specimens, DAAM2 expression was also negatively correlated with VHL and positively associated with HIF-1alpha. Moreover, HIF-1alpha was required in DAAM2-induced invasion of HCC cells. CONCLUSIONS DAAM2, rather than DAAM1, was able to predict prognosis of HCC. DAAM2 decreased VHL expression and consequently upregulated HIF-1alpha, eventually facilitating invasion of HCC.

Villin is a major actin-bundling protein that assembles the brush border of intestinal and renal epithelial cells. The villin "headpiece" domain and the actin-binding residues within it regulate its actin-bundling function. Substantial experimental and theoretical information about the three-dimensional structure of the isolated villin headpiece, including a description of the actin-binding residues within the headpiece, is available. Despite that, the actin-bundling site in the full-length (FL) villin protein remains unidentified. We used this existing villin headpiece nuclear magnetic resonance data and performed mutational analysis and functional assays to identify the actin-bundling site in FL human villin protein. By careful evaluation of these conserved actin-binding residues in human advillin protein, we demonstrate their functional significance in the over 30 proteins that contain a villin-type headpiece domain. Our study is the first that combines the available structural data on villin headpiece with functional assays to identify the actin-binding residues in FL villin that regulate its filament-bundling activity. Our findings could have wider implications for other actin-bundling proteins that contain a villin-type headpiece domain.

Browning of white adipose tissue (WAT) has been shown to reduce obesity and obesity-related complications, suggesting that factors that promote WAT browning may have applications in the development of therapeutic strategies for treating obesity. Here, we show that ablation of spinophilin (SPL), a ubiquitously expressed, multidomain scaffolding protein, increases metabolism and improves energy balance. Male and female SPL knockout (KO) and wild-type (WT) littermate controls were fed a chow diet or a high-fat diet (HFD). Body weight, hepatic steatosis, glucose and insulin tolerance, physical activity, and expression of browning genes in adipose tissues were measured and compared. Male SPL knockout (KO) mice fed a chow diet were significantly leaner, had lower body weights, and exhibited better glucose tolerance and insulin sensitivity than wild-type (WT) littermate controls. When fed an HFD, SPL KO mice were protected from increased body fat, weight gain, hepatic steatosis, hyperinsulinemia, and insulin resistance. Physical activity of SPL KO mice was markedly increased compared with WT controls. Furthermore, expression of the brown adipocyte marker, uncoupling protein-1 (UCP-1), and the mitochondrial activity markers, cd137 and c-idea, were significantly increased in visceral WAT (vWAT) of SPL KO mice, suggesting that SPL knockout protected the mice from HFD-induced obesity and its metabolic complications, at least in part, by promoting the browning of white adipocytes in vWAT. Our data identify a critical role of SPL in regulating glucose homeostasis, obesity, and adipocyte browning. These results suggest SPL may serve as a drug target for obesity and diabetes.

Cells have many types of actin structures, which must assemble from a common monomer pool. Yet, it remains poorly understood how monomers are distributed to and shared between different filament networks. Simplified model systems suggest that monomers are limited and heterogeneous, which alters actin network assembly through biased polymerization and internetwork competition. However, less is known about how monomers influence complex actin structures, where different networks competing for monomers overlap and are functionally interdependent. One example is the leading edge of migrating cells, which contains filament networks generated by multiple assembly factors. The leading edge dynamically switches between the formation of different actin structures, such as lamellipodia or filopodia, by altering the balance of these assembly factors' activities. Here, we sought to determine how the monomer-binding protein profilin 1 (PFN1) controls the assembly and organization of actin in mammalian cells. Actin polymerization in PFN1 knockout cells was severely disrupted, particularly at the leading edge, where both Arp2/3 and Mena/VASP-based filament assembly was inhibited. Further studies showed that in the absence of PFN1, Arp2/3 no longer localizes to the leading edge and Mena/VASP is non-functional. Additionally, we discovered that discrete stages of internetwork competition and collaboration between Arp2/3 and Mena/VASP networks exist at different PFN1 concentrations. Low levels of PFN1 caused filopodia to form exclusively at the leading edge, while higher concentrations inhibited filopodia and favored lamellipodia and pre-filopodia bundles. These results demonstrate that dramatic changes to actin architecture can be made simply by modifying PFN1 availability., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Brush borders of intestinal epithelial cells are mandatory for nutrient uptake. Yet, which actin nucleators are crucial for forming the F-actin bundles supporting microvilli and the actin filaments of the terminal web, in which microvilli are rooted, is unknown. We show that mice lacking the actin nucleator Cobl surprisingly did not display reduced microvilli densities or changes in microvillar F-actin bundles or microvilli diameter but particularly in the duodenum displayed increased microvillar length. Interestingly, Cobl-deficient mice furthermore showed a significant widening of the terminal web. Quantitative analyses of high-resolution cryo-scanning electron microscopy (EM) of deep-etched duodenum samples revealed that Cobl is specifically important for the formation of fine filaments in the central terminal web that connect the apical structure of the terminal web underlying the plasma membrane, the microvilli rootlets and the basal structure of the terminal web with each other. Thus, the actin nucleator Cobl is critically involved in generating one of the cellular structures of the brush border-decorated apical cortex of enterocytes representing the absorptive intestinal surface.

Background: Malformation of blood vessels represents a hallmark of cancers, but the role and regulation of vascular mural cells (vMCs), including vascular smooth muscle cells (vSMCs) and pericytes, in tumors has not been fully understood. SM22α has been identified as a marker of vSMCs. This study aims at elucidating the function and regulation of SM22α + mural cells (SM22-MCs) in tumor stroma., Methods: Gene-modified mice with a SM22α-CreER T2 transgene were employed to deplete SM22-MCs or activate/block Notch signaling in these cells. vSMCs from mouse dorsal aorta (vSMCs-DA) were cultured in vitro. RNA-seq was used to compare gene expression profiles. qRT-PCR and western blotting were used to determine gene expression level. Immunofluorescence was used to observe morphological alterations in tumors., Results: SM22-MCs are essential for stabilizing tumor vasculature. Notch signaling was downregulated in tumor-derived SM22-MCs and vSMCs-DA treated with cancer cell-derived conditioned medium. Notch activation in SM22-MCs normalized tumor vasculature and repressed tumor growth. On the other hand, Notch disruption aggravated abnormal tumor vasculature and promoted growth and metastasis. Gene expression profiling of vSMCs-DA showed that Notch activation enhances their contractile phenotype and suppresses their secretory phenotype, further attenuating the invasion and proliferation of tumor cells. In contrast, Notch blockade in vSMCs-DA mitigated their contractile phenotype while strengthened the secretory phenotype., Conclusion: SM22-MCs facilitate vessel stability in tumors, and they gain a secretory phenotype and promote tumor malignancy in the absence of Notch signaling.

Background: NEXN (nexilin) is a protein of the junctional membrane complex required for development of cardiac T-tubules. Global and cardiomyocyte-specific loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy and premature death. Therefore, little is known as to the role of NEXN in adult cardiomyocytes. Transverse-axial tubular system remodeling are well-known features in heart failure. Although NEXN is required during development for T-tubule formation, its role, if any, in mature T-tubules remains to be addressed., Methods: Nexn inducible adult cardiomyocyte-specific KO mice were generated. Comprehensive morphological and functional analyses were performed. Heart samples (n>3) were analyzed by molecular, biochemical, and electron microscopy analyses. Isolated single adult cardiomyocytes were analyzed by confocal microscopy, and myocyte shortening/re-lengthening and Ca 2+ transient studies were conducted., Results: Inducible cardiomyocyte-specific loss of Nexn in adult mice resulted in a dilated cardiomyopathy with reduced cardiac function (13% reduction in percentage fractional shortening; P <0.05). In vivo and in vitro analyses of adult mouse heart samples revealed that NEXN was essential for optimal contraction and calcium handling and was required for maintenance of T-tubule network organization (transverse tubular component in Nexn inducible adult cardiomyocyte-specific KO mice reduced by 40% with respect to controls, P <0.05)., Conclusions: Results here reported reveal NEXN to be a pivotal component of adult junctional membrane complexes required for maintenance of transverse-axial tubular architecture. These results demonstrate that NEXN plays an essential role in the adult cardiomyocyte and give further understanding of pathological mechanisms responsible for cardiomyopathy in patients carrying mutations in the NEXN gene.

Spermatogenesis is a highly complex physiological process which contains spermatogonia proliferation, spermatocyte meiosis and spermatid morphogenesis. In the past decade, actin binding proteins and signaling pathways which are critical for regulating the actin cytoskeleton in testis had been found. In this review, we summarized 5 actin-binding proteins that have been proven to play important roles in the seminiferous epithelium. Lack of them perturbs spermatids polarity and the transport of spermatids. The loss of Arp2/3 complex, Formin1, Eps8, Palladin and Plastin3 cause sperm release failure suggesting their irreplaceable role in spermatogenesis. Actin regulation relies on multiple signal pathways. The PI3K/Akt signaling pathway positively regulate the mTOR pathway to promote actin reorganization in seminiferous epithelium. Conversely, TSC1/TSC2 complex, the upstream of mTOR, is activated by the LKB1/AMPK pathway to inhibit cell proliferation, differentiation and migration. The increasing researches focus on the function of actin binding proteins (ABPs), however, their collaborative regulation of actin patterns and potential regulatory signaling networks remains unclear. We reviewed ABPs that play important roles in mammalian spermatogenesis and signal pathways involved in the regulation of microfilaments. We suggest that more relevant studies should be performed in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Multinucleate cells can be produced in Dictyostelium by electric pulse-induced fusion. In these cells, unilateral cleavage furrows are formed at spaces between areas that are controlled by aster microtubules. A peculiarity of unilateral cleavage furrows is their propensity to join laterally with other furrows into rings to form constrictions. This means cytokinesis is biphasic in multinucleate cells, the final abscission of daughter cells being independent of the initial direction of furrow progression. Myosin-II and the actin filament cross-linking protein cortexillin accumulate in unilateral furrows, as they do in the normal cleavage furrows of mononucleate cells. In a myosin-II-null background, multinucleate or mononucleate cells were produced by cultivation either in suspension or on an adhesive substrate. Myosin-II is not essential for cytokinesis either in mononucleate or in multinucleate cells but stabilizes and confines the position of the cleavage furrows. In fused wild-type cells, unilateral furrows ingress with an average velocity of 1.7 µm × min -1 , with no appreciable decrease of velocity in the course of ingression. In multinucleate myosin-II-null cells, some of the furrows stop growing, thus leaving space for the extensive broadening of the few remaining furrows.

Processing of and responding to various signals is an essential cellular function that influences survival, homeostasis, development, and cell death. Extra- or intracellular signals are perceived via specific receptors and transduced in a particular signalling pathway that results in a precise response. Reversible post-translational redox modifications of cysteinyl and methionyl residues have been characterised in countless signal transduction pathways. Due to the low reactivity of most sulfur-containing amino acid side chains with hydrogen peroxide, for instance, and also to ensure specificity, redox signalling requires catalysis, just like phosphorylation signalling requires kinases and phosphatases. While reducing enzymes of both cysteinyl- and methionyl-derivates have been characterised in great detail before, the discovery and characterisation of MICAL proteins evinced the first examples of specific oxidases in signal transduction. This article provides an overview of the functions of MICAL proteins in the redox regulation of cellular functions., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The mechanisms that control intrinsic axon growth potential, and thus axon regeneration following injury, are not well understood. Developmental axon regrowth of Drosophila mushroom body γ-neurons during neuronal remodeling offers a unique opportunity to study the molecular mechanisms controlling intrinsic growth potential. Motivated by the recently uncovered developmental expression atlas of γ-neurons, we here focus on the role of the actin-severing protein cofilin during axon regrowth. We show that Twinstar (Tsr), the fly cofilin, is a crucial regulator of both axon growth and branching during developmental remodeling of γ-neurons. tsr mutant axons demonstrate growth defects both in vivo and in vitro , and also exhibit actin-rich filopodial-like structures at failed branch points in vivo Our data is inconsistent with Tsr being important for increasing G-actin availability. Furthermore, analysis of microtubule localization suggests that Tsr is required for microtubule infiltration into the axon tips and branch points. Taken together, we show that Tsr promotes axon growth and branching, likely by clearing F-actin to facilitate protrusion of microtubules., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Efficient generation of antibodies by B cells is one of the prerequisites of protective immunity. B cell activation by cognate antigens via B cell receptors (BCRs), or pathogen-associated molecules through pattern-recognition receptors, such as Toll-like receptors (TLRs), leads to transcriptional and metabolic changes that ultimately transform B cells into antibody-producing plasma cells or memory cells. BCR signaling and a number of steps downstream of it rely on coordinated action of cellular membranes and the actin cytoskeleton, tightly controlled by concerted action of multiple regulatory proteins, some of them exclusive to B cells. Here, we dissect the role of Missing-In-Metastasis (MIM), or Metastasis suppressor 1 (MTSS1), a cancer-associated membrane and actin cytoskeleton regulating protein, in B cell-mediated immunity by taking advantage of MIM knockout mouse strain. We show undisturbed B cell development and largely normal composition of B cell compartments in the periphery. Interestingly, we found that MIM -/- B cells are defected in BCR signaling in response to surface-bound antigens but, on the other hand, show increased metabolic activity after stimulation with LPS or CpG. In vivo , MIM knockout animals exhibit impaired IgM antibody responses to immunization with T cell-independent antigen. This study provides the first comprehensive characterization of MIM in B cells, demonstrates its regulatory role for B cell-mediated immunity, as well as proposes new functions for MIM in tuning receptor signaling and cellular metabolism, processes, which may also contribute to the poorly understood functions of MIM in cancer., (Copyright © 2020 Sarapulov, Petrov, Hernández-Pérez, Šuštar, Kuokkanen, Cords, Samuel, Vainio, Fritzsche, Carrasco and Mattila.)

HIP-55 (HPK1 [hematopoietic progenitor kinase 1] -interacting protein of 55 kDa) contains an actin-depolymerizing factor homology (ADF-H) domain at the N-terminus and a src homology 3 (SH3) domain at the C-terminus, which plays an important role in the T cell receptor (TCR) and B-cell receptor (BCR) signaling and immune system. In our previous studies, HIP-55 was found to be highly expressed in several types of tumors and function as a novel oncogenic signaling hub that regulates tumor progression and metastasis through defined functional domains, actin-binding and SH3 modules. However, the wider functions and mechanisms of HIP-55 are still unclear. Here, multi-omic analysis revealed that one of the main biofunctions of HIP-55 is the regulation of cytokines release. Furthermore, to investigate the role of HIP-55 in the cytokine production, a series Cytokine Antibody Arrays were performed to detect differentially expressed cytokines between control and HIP-55 knockdown cells. A total of 97 differentially expressed cytokines were identified from 300 cytokines in A549 cell. Bioinformatics analysis showed these differentially cytokines were mainly enriched in cancer signal pathways and IL-6 is the most critical hub in the integrated network. Analysis of RNAseq data from lung cancer patients showed that there is a strong negative correlation between HIP-55 and interleukin-6 (IL-6) in samples from lung adenocarcinoma patients. Our data indicated that HIP-55 may participate in cancer progression and metastasis via regulating cytokines release., (© 2020 The Author(s).)

Self-incompatibility (SI) in the poppy Papaver rhoeas triggers dramatic alterations in actin within pollen tubes. However, how these actin alterations are mechanistically achieved remains largely unexplored. Here, we used treatment with the Ca 2+ ionophore A23187 to mimic the SI-induced elevation in cytosolic Ca 2+ and trigger formation of the distinctive F-actin foci. Live-cell imaging revealed that this remodeling involves F-actin fragmentation and depolymerization, accompanied by the rapid formation of punctate actin foci and subsequent increase in their size. We established that actin foci are generated and enlarged from crosslinking of fragmented actin filament structures. Moreover, we show that villins associate with actin structures and are involved in this actin reorganization process. Notably, we demonstrate that Arabidopsis VILLIN5 promotes actin depolymerization and formation of actin foci by fragmenting actin filaments, and controlling the enlargement of actin foci via bundling of actin filaments. Our study thus uncovers important novel insights about the molecular players and mechanisms involved in forming the distinctive actin foci in pollen tubes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Epidermal morphogenesis and hair follicle (HF) development depend on the ability of keratinocytes to adhere to the basement membrane (BM) and migrate along the extracellular matrix. Integrins are cell-matrix receptors that control keratinocyte adhesion and migration, and are recognized as major regulators of epidermal homeostasis. How integrins regulate the behavior of keratinocytes during epidermal morphogenesis remains insufficiently understood. Here, we show that α-parvin (α-pv), a focal adhesion protein that couples integrins to actin cytoskeleton, is indispensable for epidermal morphogenesis and HF development. Inactivation of the murine α-pv gene in basal keratinocytes results in keratinocyte-BM detachment, epidermal thickening, ectopic keratinocyte proliferation and altered actin cytoskeleton polarization. In vitro, α-pv-null keratinocytes display reduced adhesion to BM matrix components, aberrant spreading and stress fibers formation, and impaired directed migration. Together, our data demonstrate that α-pv controls epidermal homeostasis by facilitating integrin-mediated adhesion and actin cytoskeleton organization in keratinocytes., Competing Interests: The authors have declared that no competing interests exist.

Non-centrosomal microtubule-organizing centres (ncMTOCs) have a variety of roles that are presumed to serve the diverse functions of the range of cell types in which they are found. ncMTOCs are diverse in their composition, subcellular localization and function. Here we report a perinuclear MTOC in Drosophila fat body cells that is anchored by the Nesprin homologue Msp300 at the cytoplasmic surface of the nucleus. Msp300 recruits the microtubule minus-end protein Patronin, a calmodulin-regulated spectrin-associated protein (CAMSAP) homologue, which functions redundantly with Ninein to further recruit the microtubule polymerase Msps-a member of the XMAP215 family-to assemble non-centrosomal microtubules and does so independently of the widespread microtubule nucleation factor γ-Tubulin. Functionally, the fat body ncMTOC and the radial microtubule arrays that it organizes are essential for nuclear positioning and for secretion of basement membrane components via retrograde dynein-dependent endosomal trafficking that restricts plasma membrane growth. Together, this study identifies a perinuclear ncMTOC with unique architecture that regulates microtubules, serving vital functions.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous chronic destructive airway disease. PCD is traditionally diagnosed by nasal nitric oxide measurement, analysis of ciliary beating, transmission electron microscopy (TEM), and/or genetic testing. In most genetic PCD variants, laterality defects can occur. However, it is difficult to establish a diagnosis in individuals with PCD and central pair (CP) defects, and alternative strategies are required because of very subtle ciliary beating abnormalities, a normal ciliary ultrastructure, and normal situs composition. Mutations in HYDIN are known to cause CP defects, but the genetic analysis of HYDIN variants is confounded by the pseudogene HYDIN2 , which is almost identical in terms of intron/exon structure. We have previously shown that several types of PCD can be diagnosed via immunofluorescence (IF) microscopy analyses. Here, using IF microscopy, we demonstrated that in individuals with PCD and CP defects, the CP-associated protein SPEF2 is absent in HYDIN -mutant cells, revealing its dependence on functional HYDIN. Next, we performed IF analyses of SPEF2 in respiratory cells from 189 individuals with suspected PCD and situs solitus . Forty-one of the 189 individuals had undetectable SPEF2 and were subjected to a genetic analysis, which revealed one novel loss-of-function mutation in SPEF2 and three reported and 13 novel HYDIN mutations in 15 individuals. The remaining 25 individuals are good candidates for new, as-yet uncharacterized PCD variants that affect the CP apparatus. SPEF2 mutations have been associated with male infertility but have not previously been identified to cause PCD. We identified a mutation of SPEF2 that is causative for PCD with a CP defect. We conclude that SPEF2 IF analyses can facilitate the detection of CP defects and evaluation of the pathogenicity of HYDIN variants, thus aiding the molecular diagnosis of CP defects.

Dendrites develop elaborate morphologies in concert with surrounding glia, but the molecules that coordinate dendrite and glial morphogenesis are mostly unknown. C. elegans offers a powerful model for identifying such factors. Previous work in this system examined dendrites and glia that develop within epithelia, similar to mammalian sense organs. Here, we focus on the neurons BAG and URX, which are not part of an epithelium but instead form membranous attachments to a single glial cell at the nose, reminiscent of dendrite-glia contacts in the mammalian brain. We show that these dendrites develop by retrograde extension, in which the nascent dendrite endings anchor to the presumptive nose and then extend by stretching during embryo elongation. Using forward genetic screens, we find that dendrite development requires the adhesion protein SAX-7/L1CAM and the cytoplasmic protein GRDN-1/CCDC88C to anchor dendrite endings at the nose. SAX-7 acts in neurons and glia, while GRDN-1 acts in glia to non-autonomously promote dendrite extension. Thus, this work shows how glial factors can help to shape dendrites, and identifies a novel molecular mechanism for dendrite growth by retrograde extension., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Neuronal dendrites have specialized actin-rich structures called dendritic spines that receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic morphology and stability are largely mediated by regulation of the actin cytoskeleton; however, the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important role in the postsynaptic development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 are enriched in dendritic spines, and their knockdown impairs spine development and synapse formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine stabilization. Importantly, the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stability and dendritic arbor complexity. These findings identify LASP1 and LASP2 as novel regulators of neuronal circuitry. SIGNIFICANCE STATEMENT Proper regulation of the actin cytoskeleton is essential for the structural stability of dendrites and dendritic spines. Consequently, the malformation of dendritic structures accompanies numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are best known for their role in regulating the stabilization and function of actin thin filaments in muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly expressed in the brain, little is currently known about their function in the nervous system. In this study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation and long-term maintenance of dendrites and dendritic spines., (Copyright © 2020 the authors.)

During organogenesis, precise control of spindle orientation balances proliferation and differentiation. In the developing murine epidermis, planar and perpendicular divisions yield symmetric and asymmetric fate outcomes, respectively. Classically, division axis specification involves centrosome migration and spindle rotation, events occurring early in mitosis. Here, we identify a novel orientation mechanism which corrects erroneous anaphase orientations during telophase. The directionality of reorientation correlates with the maintenance or loss of basal contact by the apical daughter. While the scaffolding protein LGN is known to determine initial spindle positioning, we show that LGN also functions during telophase to reorient oblique divisions toward perpendicular. The fidelity of telophase correction also relies on the tension-sensitive adherens junction proteins vinculin, α-E-catenin, and afadin. Failure of this corrective mechanism impacts tissue architecture, as persistent oblique divisions induce precocious, sustained differentiation. The division orientation plasticity provided by telophase correction may enable progenitors to adapt to local tissue needs., Competing Interests: KL, KB, CD, DS, AB, GB, LR, SW No competing interests declared, (© 2019, Lough et al.)

Membrane curvature has recently been recognized as an active regulator of cellular function, with several protein families identified as sensors and generators of membrane curvature. Amongst them, the inverse Bin/Amphiphysin/Rvs (I-BAR) domain family has been implicated in the sensing and generation of membrane structures with negative membrane curvature e.g. filopodia or dendritic spines. However, to date, quantitative biophysical investigations of I-BAR domains have mostly taken place in reconstitution. Here, we use fluorescence microscopy to quantitatively investigate membrane curvature sensing and generation by I-BARs in filopodia of living cells. As a model system, we selected two prototypic members of the I-BAR family, the insulin receptor substrate p53 and missing-in-metastasis. Our data demonstrated how I-BARs sense negative membrane curvature in the complex environment of live cells by revealing a dependence on membrane curvature for both their binding affinity to membranes and their saturation density. The non-monotonic dependence of protein sorting with negative membrane curvature allowed us to apply previously developed thermodynamic models to provide estimates of the effective intrinsic curvature and bending rigidity of the two I-BARs bound at the plasma membrane. Our results agree with studies performed on the insulin receptor substrate p53 in reconstitution. To quantitate membrane curvature generation by I-BARs we measured how their overexpression reduces the peak and the width of the size distribution of filopodia, resulting in filopodia populations with smaller and more uniform diameters. Our findings provide a quantitative biophysical insight in the ability of I-BARs to sense and generate negative membrane curvature in the crowded environment of living cells.

The membrane composition modulates membrane fusion by altering membrane physical properties and the structure, organization and dynamics of fusion proteins and peptides. The journey of developing peptide-based viral fusion inhibitors is often stalled by the change in lipid composition of viral and target membranes. This makes it important to study the role of membrane composition on the organization, dynamics and fusion inhibiting abilities of the peptide-based fusion inhibitors. Cholesterol, an important constituent of mammalian cell membrane, modulates bilayer properties in multiple ways and impart its effect on the membrane fusion. We have previously shown that TG-23 peptide derived from phagosomal coat protein, coronin 1, shows significant inhibition of fusion between membranes without cholesterol. In this work, we have studied the effect of the TG-23 peptide on the polyethylene glycol-mediated membrane fusion in presence of different concentrations of membrane cholesterol. Our results show that the inhibitory effect of TG-23 is being completely reversed in cholesterol containing membranes. We have evaluated the structure, organization, dynamics and depth of penetration of TG-23 in membranes having different lipid compositions and its effect on membrane properties. Our results demonstrate that cholesterol does not affect the secondary structure of the peptide, however, alters the depth of penetration of the peptide and modifies peptide organization and dynamics. The cholesterol dependent change in organization and dynamics of the peptide influences its efficacy in membrane fusion. Therefore, we envisage that the study of peptide organization and dynamics is extremely important to determine the effect of peptide on the membrane fusion., (Copyright © 2019 Elsevier B.V. All rights reserved.)