The Shot CH1 domain recognises a distinct form of F-actin during Drosophila oocyte determination.

In Drosophila, only one cell in a multicellular female germline cyst is specified as an oocyte and a similar process occurs in mammals. The symmetry-breaking cue for oocyte selection is provided by the fusome, a tubular structure connecting all cells in the cyst. The Drosophila spectraplakin Shot localises to the fusome and translates its asymmetry into a polarised microtubule network that is essential for oocyte specification, but how Shot recognises the fusome is unclear. Here, we demonstrate that the actin-binding domain (ABD) of Shot is necessary and sufficient to localise Shot to the fusome and mediates Shot function in oocyte specification together with the microtubule-binding domains. The calponin homology domain 1 of the Shot ABD recognises fusomal F-actin and requires calponin homology domain 2 to distinguish it from other forms of F-actin in the cyst. By contrast, the ABDs of utrophin, Fimbrin, Filamin, Lifeact and F-tractin do not recognise fusomal F-actin. We therefore propose that Shot propagates fusome asymmetry by recognising a specific conformational state of F-actin on the fusome.

Knockdown of LRCH4 Remodels Tumor Microenvironment Through Inhibiting YAP and TGF-ß/Smad Signaling Pathway in Colorectal Cancer.

BACKGROUND: Colorectal cancer is one of the most common gastrointestinal malignancies worldwide. LRCH4 is the top 1 gene associated with an unfavorable prognosis in colorectal cancer. METHODS: Here, we reported that the knockdown of LRCH4 inhibited the proliferation, migration and invasion in HT29 cells. RESULTS: The activity of Yes-Associated Protein (YAP), a transcription factor in the Hppo-YAP signaling pathway, was significantly inhibited by LRCH4-siRNA. LRCH4 knockdown also reversed the EMT and regulated the expression of extracellular matrix (ECM) protein, Fibronectin and Collagen IV in HT29 cells. In addition, the TGF-ß/Smad signaling pathway, as the downstream pathway of Yap, was also inhibited by LRCH4 knockdown. CONCLUSION: Knockdown of LRCH4 involved in the regulation of ECM and EMT and inhibited YAP and the TGF-ß/Smad signaling pathway in colorectal cancer cells. Our study provided a mechanism of LRCH4 on colorectal cancer cells, and a new potential target for clinical tumor treatment.

Loss of Calponin 2 causes premature ovarian insufficiency in mice.

BACKGROUND: Premature ovarian insufficiency (POI) is a condition defined as women developing menopause before 40 years old. These patients display low ovarian reserve at young age and difficulties to conceive even with assisted reproductive technology. The pathogenesis of ovarian insufficiency is not fully understood. Genetic factors may underlie most of the cases. Actin cytoskeleton plays a pivotal role in ovarian folliculogenesis. Calponin 2 encoded by the Cnn2 gene is an actin associated protein that regulates motility and mechanical signaling related cellular functions. RESULTS: The present study compared breeding of age-matched calponin 2 knockout (Cnn2-KO) and wild type (WT) mice and found that Cnn2-KO mothers had significantly smaller litter sizes. Ovaries from 4 weeks old Cnn2-KO mice showed significantly lower numbers of total ovarian follicles than WT control with the presence of multi-oocyte follicles. Cnn2-KO mice also showed age-progressive earlier depletion of ovarian follicles. Cnn2 expression is detected in the cumulus cells of the ovarian follicles of WT mice and colocalizes with actin stress fiber, tropomyosin and myosin II in primary cultures of cumulus cells. CONCLUSIONS: The findings demonstrate that the loss of calponin 2 impairs ovarian folliculogenesis with premature depletion of ovarian follicles. The role of calponin 2 in ovarian granulosa cells suggests a molecular target for further investigations on the pathogenesis of POI and for therapeutic development.

Long non-coding RNA MEG3 acts as a suppressor in breast cancer by regulating miR-330/CNN1.

BACKGROUND: The current study aimed to investigate the molecular mechanism of long non-coding RNA (lncRNA) MEG3 in the development of breast cancer. METHODS: The regulating relationships among lncRNA MEG3, miRNA-330 and CNN1 were predicted by bioinformatics analysis of breast cancer samples in the Cancer Genome Atlas database. The differential expression of lncRNA MEG3, miRNA-330 and CNN1 was first validated in breast cancer tissues and cells. The effects of lncRNA MEG3 on breast cancer malignant properties were evaluated by manipulating its expression in MCF-7 and BT-474 cells. Rescue experiments, dual-luciferase assays, and RNA immunoprecipitation (RIP) experiments were further used to validate the relationships among lncRNA MEG3, miRNA-330 and CNN1. RESULTS: Bioinformatics analysis showed that lncRNA MEGs and CNN1 were significantly downregulated in breast cancer tissues, while miR-330 was upregulated. These differential expressions were further validated in our cohort of breast cancer samples. High expression levels of lncRNA MEG3 and CNN1 as well as low expression of miR-330 were significantly associated with favorable overall survival. Overexpression of lncRNA MEG3 significantly inhibited cell viability, migration and invasion, decreased cells in S stage and promoted cell apoptosis. Dual-luciferase reporter gene assay and RIP experiments showed that lncRNA MEG3 could directly bind to miR-330. Moreover, miR-330 mimics on the basis of lncRNA MEG3 overexpression ameliorated the tumor-suppressing effects of lncRNA MEG3 in breast cancer malignant properties by decreasing CNN1 expression. CONCLUSION: Our study indicated lncRNA MEG3 is a breast cancer suppressor by regulating miR-330/CNN1 axis.

Segregated localization of two calponin-related proteins within sarcomeric thin filaments in Caenorhabditis elegans striated muscle.

The calponin family proteins are expressed in both muscle and non-muscle cells and involved in the regulation of cytoskeletal dynamics and cell contractility. In the nematode Caenorhabditis elegans, UNC-87 and CLIK-1 are calponin-related proteins with 42% identical amino acid sequences containing seven calponin-like motifs. Genetic studies demonstrated that UNC-87 and CLIK-1 have partially redundant function in regulating actin cytoskeletal organization in striated and non-striated muscle cells. However, biochemical studies showed that UNC-87 and CLIK-1 are different in their ability to bundle actin filaments. In this study, I extended comparison between UNC-87 and CLIK-1 and found additional differences in vitro and in vivo. Although UNC-87 and CLIK-1 bound to actin filaments similarly, UNC-87, but not CLIK-1, bound to myosin and inhibited actomyosin ATPase in vitro. In striated muscle, UNC-87 and CLIK-1 were segregated into different subregions within sarcomeric actin filaments. CLIK-1 was concentrated near the actin pointed ends, whereas UNC-87 was enriched toward the actin barbed ends. Restricted localization of UNC-87 was not altered in a clik-1-null mutant, suggesting that their segregated localization is not due to competition between the two related proteins. These results suggest that the two calponin-related proteins have both common and distinct roles in regulating actin filaments.

Calponin 1 inhibits agonist-induced ERK activation and decreases calcium sensitization in vascular smooth muscle.

Smooth muscle cell (SMC) contraction and vascular tone are modulated by phosphorylation and multiple modifications of the thick filament, and thin filament regulation of SMC contraction has been reported to involve extracellular regulated kinase (ERK). Previous studies in ferrets suggest that the actin-binding protein, calponin 1 (CNN1), acts as a scaffold linking protein kinase C (PKC), Raf, MEK and ERK, promoting PKC-dependent ERK activation. To gain further insight into this function of CNN1 in ERK activation and the regulation of SMC contractility in mice, we generated a novel Calponin 1 knockout mouse (Cnn1 KO) by a single base substitution in an intronic CArG box that preferentially abolishes expression of CNN1 in vascular SMCs. Using this new Cnn1 KO mouse, we show that ablation of CNN1 has two effects, depending on the cytosolic free calcium level: (1) in the presence of elevated intracellular calcium caused by agonist stimulation, Cnn1 KO mice display a reduced amplitude of stress and stiffness but an increase in agonist-induced ERK activation; and (2) during intracellular calcium depletion, in the presence of an agonist, Cnn1 KO mice exhibit increased duration of SM tone maintenance. Together, these results suggest that CNN1 plays an important and complex modulatory role in SMC contractile tone amplitude and maintenance.

Cardiomyocyte external mechanical unloading activates modifications of α-actinin differently from sarcomere-originated unloading.

Loss of myocardial mass in a neonatal rat cardiomyocyte culture is studied to determine whether there is a distinguishable cellular response based on the origin of mechano-signals. The approach herein compares the sarcomeric assembly and disassembly processes in heart cells by imposing mechano-signals at the interface with the extracellular matrix (extrinsic) and at the level of the myofilaments (intrinsic). Experiments compared the effects of imposed internal (inside/out) and external (outside/in) loading and unloading on modifications in neonatal rat cardiomyocytes. Unloading of the cellular substrate by myosin inhibition (1 µm mavacamten), or cessation of cyclic strain (1 Hz, 10% strain) after preconditioning, led to significant disassembly of sarcomeric α-actinin by 6 h. In myosin inhibition, this was accompanied by redistribution of intracellular poly-ubiquitin K48 to the cellular periphery relative to the poly-ubiquitin K48 reservoir at the I-band. Moreover, loading and unloading of the cellular substrate led to a three-fold increase in post-translational modifications (PTMs) when compared to the myosin-specific activation or inhibition. Specifically, phosphorylation increased with loading while ubiquitination increased with unloading, which may involve extracellular signal-regulated kinase 1/2 and focal adhesion kinase activation. The identified PTMs, including ubiquitination, acetylation, and phosphorylation, are proposed to modify internal domains in α-actinin to increase its propensity to bind F-actin. These results demonstrate a link between mechanical feedback and sarcomere protein homeostasis via PTMs of α-actinin that exemplify how cardiomyocytes exhibit differential responses to the origin of force. The implications of sarcomere regulation governed by PTMs of α-actinin are discussed with respect to cardiac atrophy and heart failure.

Evolution and function of calponin and transgelin.

Calponin and transgelin (originally named SM22) are homologous cytoskeleton proteins that regulate actin-activated myosin motor functions in smooth muscle contraction and non-muscle cell motility during adhesion, migration, proliferation, phagocytosis, wound healing, and inflammatory responses. They are abundant cytoskeleton proteins present in multiple cell types whereas their physiological functions remain to be fully established. This focused review summarizes the evolution of genes encoding calponin and transgelin and their isoforms and discusses the structural similarity and divergence in vertebrate and invertebrate species in the context of functions in regulating cell motility. As the first literature review focusing on the evolution of the calponin-transgelin family of proteins in relevance to their structure-function relationship, the goal is to outline a foundation of current knowledge for continued investigations to understand the biological functions of calponin and transgelin in various cell types during physiological and pathological processes.

Potential prognostic and immunotherapeutic value of calponin 1: A pan-cancer analysis.

Background: Emerging evidence has suggested a pro-oncogenic role of calponin 1 (CNN1) in the initiation of a variety of cancers. Despite this, CNN1 remains unknown in terms of its effects and mechanisms on angiogenesis, prognosis, and immunology in cancer. Materials and Methods: The expression of CNN1 was extracted and analyzed using the TIMER, UALCAN, and GEPIA databases. Meanwhile, we analyzed the diagnostic value of CNN1 by using PrognoScan and Kaplan-Meier plots. To elucidate the value of CNN1 in immunotherapy, we used the TIMER 2.0 database, TISIDB database, and Sangerbox database. Gene set enrichment analysis (GSEA) was used to analyze the expression pattern and bio-progression of CNN1 and the vascular endothelium growth factor (VEGF) in cancer. The expressions of CNN1 and VEGF in gastric cancer were confirmed using immunohistochemistry. We used Cox regression analysis to investigate the association between pathological characteristics, clinical prognosis, and CNN1 and VEGF expressions in patients with gastric cancer. Results: CNN1 expression was higher in normal tissues than it was in tumor tissues of most types of cancers. However, the expression level rebounds during the development of tumors. High levels of CNN1 indicate a poor prognosis for 11 tumors, which include stomach adenocarcinoma (STAD). There is a relationship between CNN1 and tumor-infiltrating lymphocytes (TILs), and the marker genes NRP1 and TNFRSF14 of TILs are significantly related to CNN1 expression in gastric cancers. The GSEA results confirmed the lower expression of CNN1 in tumors when compared to normal tissues. However, CNN1 again showed an increasing trend during tumor development. In addition, the results also suggest that CNN1 is involved in angiogenesis. The immunohistochemistry results validated the GSEA result (take gastric cancer as an example). Cox analysis suggested that high CNN1 expression and high VEGF expression are closely associated with poor clinical prognosis. Conclusion: Our study has shown that CNN1 expression is aberrantly elevated in various cancers and positively correlates with angiogenesis and the immune checkpoint, contributing to cancer progression and poor prognosis. These results suggest that CNN1 could serve as a promising candidate for pan-cancer immunotherapy.

A census of actin-associated proteins in humans.

Actin filaments help in maintaining the cell structure and coordinating cellular movements and cargo transport within the cell. Actin participates in the interaction with several proteins and also with itself to form the helical filamentous actin (F-actin). Actin-binding proteins (ABPs) and actin-associated proteins (AAPs) coordinate the actin filament assembly and processing, regulate the flux between globular G-actin and F-actin in the cell, and help maintain the cellular structure and integrity. We have used protein-protein interaction data available through multiple sources (STRING, BioGRID, mentha, and a few others), functional annotation, and classical actin-binding domains to identify actin-binding and actin-associated proteins in the human proteome. Here, we report 2482 AAPs and present an analysis of their structural and sequential domains, functions, evolutionary conservation, cellular localization, abundance, and tissue-specific expression patterns. This analysis provides a base for the characterization of proteins involved in actin dynamics and turnover in the cell.

Low-grade adenosquamous carcinoma of the breast: a clinical, morphological and immunohistochemical analysis of 25 patients.

AIMS: Due to its rarity and non-specific clinical and pathological features, low-grade adenosquamous carcinoma (LGASC) of the breast continues to pose diagnostic challenges. Unlike other triple-negative breast carcinomas, LGASC tends to have an indolent clinical behaviour. It is essential to recognise this lesion for accurate diagnosis and appropriate management. METHODS AND RESULTS: Twenty-five cases of LGASC were identified in our archives and collaborating institutes. Cases of LGASC with dominant coexisting other type carcinomas were excluded. We studied the clinical presentation, morphological features, patterns of the commonly used immunohistochemical stains and follow-up. In our cohort, LGASC was commonly located at the outer aspect of the breast and associated with intraductal papilloma. The morphology of LGASC is characterised by infiltrating small glands and nests with variable squamous differentiation. We also found cuffing desmoplastic (fibrolamellar) stromal change in 75% of patients and peripheral lymphocytic aggregates in 87.5% of patients. P63 and smooth muscle myosin (SMM) were the most common myoepithelial markers used to assist in diagnosis. P63 often stained peripheral tumour cells surrounding invasive glands (circumferential staining in 80% of the cases), mimicking myoepithelial cells. It also stained the small nests with squamous differentiation. However, SMM was negative in 63% of the cases. The vast majority of our cases were triple-negative; only a few had focal and weak expressions of ER and PR. One patient who did not have excision developed lymph node metastasis. Most patients underwent excision or mastectomy with negative margins as surgical treatment; there were no recurrences or metastases in these patients with clinical follow-ups up to 108 months. CONCLUSIONS: LGASC has some unique, although not entirely specific, morphological features and immunohistochemical staining patterns. Fibrolamellar stromal change, peripheral lymphocytic aggregates and variable staining of p63 and SMM are valuable features to facilitate the diagnosis.

Calponin 2 harnesses metabolic reprogramming to determine kidney fibrosis.

OBJECTIVE: In the fibrotic kidneys, the extent of a formed deleterious microenvironment is determined by cellular mechanical forces. This process requires metabolism for energy. However, how cellular mechanics and metabolism are connected remains unclear. METHODS: A multi-disciplinary approach was employed: the fibrotic kidney disease models were induced by renal ischemia-reperfusion injury and unilateral ureteral obstruction in Calponin 2 (CNN2) knockdown mice. Proteomics, bioinformatics, and in vivo and in vitro molecular experimental pathology studies were performed. RESULT: Our proteomics revealed that actin filament binding and cell metabolism are the two most dysregulated events in the fibrotic kidneys. As a prominent actin stabilizer, CNN2 was predominantly expressed in fibroblasts and pericytes. In CKD patients, CNN2 levels was markedly induced in blood. In mice, CNN2 knockdown preserves kidney function and alleviates fibrosis. Global proteomics profiled that CNN2 knockdown enhanced the activities of the key rate-limiting enzymes and regulators of fatty acid oxidation (FAO) in the diseased kidneys. Inhibiting carnitine palmitoyltransferase 1α in the FAO pathway resulted in lipid accumulation and extracellular matrix deposition in the fibrotic kidneys, which were restored after CNN2 knockdown. Bioinformatics and chromatin immunoprecipitation showed that CNN2 interactor, estrogen receptor 2 (ESR2), binds peroxisome proliferator-activated receptor-α (PPARα) to transcriptionally regulate FAO downstream target genes expression amid kidney fibrosis. In vitro, ESR2 knockdown repressed the mRNA levels of PPARα and the key genes in the FAO pathway. Conversely, activation of PPARα reduced CNN2-induced matrix inductions. CONCLUSIONS: Our results suggest that balancing cell mechanics and metabolism is crucial to develop therapeutic strategies to halt kidney fibrosis.

Routine Use of a Pocket-Sized Handheld Echoscopic Device Plus a Biomarker by Emergency Medicine Residents with an Early Screening Algorithm for Suspected Type A Acute Aortic Syndrome.

(1) Background: The early screening strategy for type A acute aortic syndrome (A-AAS) patients has always been challenging. (2) Methods: From September 2020-31 March 2022, 179 consecutive patients with suspected A-AAS were retrospectively reviewed. We assessed the diagnostic value of the use of handheld echocardiographic devices (PHHEs) by emergency medicine (EM) residents either alone or in combination with serum acidic calponin in this patient group. (3) Results: The direct sign of PHHE had a specificity (SP) of 97.7%. The sign of ascending aortic dilatation showed SE = 77.6%, SP = 68.5%, PPV = 48.1% and NPV = 89%. SE, SP, PPV and NPV of a positive PHHE direct sign were 55.6%, 100%, 100% and 71.4% in 19 hypotension/shock patients with suspected A-AAS, respectively. The area under curve (AUC) of acidic calponin combined with an ascending aorta diameter >40 mm was 0.927, with an SE and SP of 83.7% and 89.2%, respectively. These two combined indicators significantly improved the diagnostic efficiency of A-AAS compared with either of them alone (p = 0.017; standard error 0.016, Z value 2.39; p = 0.001, standard error 0.028, Z value 3.29). (4) Conclusion: EM resident-performed PHHE was highly indicative of A-AAS in patients presenting with shock or hypotension. An ascending aorta diameter > 40 mm combined with acidic calponin demonstrated acceptable diagnostic accuracy as a rapid first-line triage tool to identify patients with suspected A-AAS.

Promoting effects of calponin 3 on the growth of diffuse large B­cell lymphoma cells.

Diffuse large B­cell lymphoma (DLBCL) is one of the most common types of lymphoma. Calponin 3 (CNN3) is a thin filament­associated protein previously known to regulate smooth muscle contraction. Recent evidence illustrates its involvement in carcinogenesis; however, its roles in DLBCL remain unknown. CNN3 was found to be highly expressed in DLBCL specimens according to the online Gene Expression Profiling Interactive Analysis data. The aim of the present study was to investigate the roles of CNN3 in the progression of DLBCL. In vitro, the ectopic expression of CNN3 promoted the proliferation and G1/S transition of DLBCL cells, while its silencing led to opposite alterations. A similar tumor­promoting role of CNN3 was also demonstrated by injecting nude mice with DLBCL cells over­ or underexpressing CNN3. The results of dual­luciferase reporter and chromatin immunoprecipitation assays revealed that forkhead box O3 (FOXO3), a known tumor suppressor in DLBCL, bound to the CNN3 promoter at ­1955/­1948 and ­1190/­1183, and suppressed the transcription of CNN3. The alterations induced by FOXO3 were partly blocked by CNN3 overexpression. On the whole, the present study demonstrates that CNN3, whose transcriptional activity is negatively regulated by FOXO3, contributes to the malignant behavior of DLBCL cells. The findings of the present study may provide novel diagnostic or therapeutic insight for DLBCL in clinical practice.

Calponin 1 increases cancer-associated fibroblasts-mediated matrix stiffness to promote chemoresistance in gastric cancer.

The mechanical microenvironment regulated by cancer-associated fibroblasts (CAFs) influence tumor progression. Chemotherapeutic interventions including 5-Fluorouracil (5-Fu) are commonly used for primary treatment of patients with advanced gastric cancer (GC), and the development of acquired resistance to 5-Fu limits the clinical efficacy of these chemotherapies. However, if and how the interplay between CAFs and the mechanical microenvironment regulates GC response to 5-Fu is poorly understood. In this study, we demonstrate that high-level expression of calponin 1(CNN1) in gastric CAFs predicts poor clinical outcomes of GC patients, especially for those treated with 5-Fu. CNN1 knockdown in CAFs improves the effectiveness of 5-Fu in reducing tumor growth in a mouse GC model and confers increased sensitivity to 5-Fu in a 3D culture system. Furthermore, CNN1 knockdown impairs CAF contraction and reduces matrix stiffness without affecting the expression of matrix proteins. Mechanistically, CNN1 interacts with PDZ and LIM Domain 7 (PDLIM7) and prevents its degradation by the E3 ubiquitin ligase NEDD4-1, which leads to activation of the ROCK1/MLC pathway. The increased matrix stiffness, in turn, contributes to 5-Fu resistance in GC cells by activating YAP. Taken together, our data reveal a critical role of the mechanical microenvironment in 5-Fu resistance, which is modulated by CNN1hi CAFs-mediated matrix stiffening, indicating that targeting CAFs may provide a novel option for overcoming drug resistance in GC.

Loss of Calponin 2 causes age-progressive proteinuria in mice.

Proteinuria is a major manifestation of kidney disease, reflecting injuries of glomerular podocytes. Actin cytoskeleton plays a pivotal role in stabilizing the foot processes of podocytes against the hydrostatic pressure of filtration. Calponin is an actin associated protein that regulates mechanical tension-related cytoskeleton functions and its role in podocytes has not been established. Here we studied the kidney phenotypes of calponin isoform 2 knockout (KO) mice. Urine samples were examined to quantify the ratio of albumin and creatinine. Kidney tissue samples were collected for histology and ultrastructural studies. A mouse podocyte cell line (E11) was used to study the expression and cellular localization of calponin 2. In comparison with wild-type (WT) controls, calponin 2 KO mice showed age-progressive high proteinuria and degeneration of renal glomeruli. High levels of calponin 2 are expressed in E11 podocytes and colocalized with actin stress fibers, tropomyosin and myosin IIA. Electron microscopy showed that aging calponin 2 KO mice had effacement of the podocyte foot processes and increased thickness of the glomerular basement membrane as compared to that of WT control. The findings demonstrate that deletion of calponin 2 aggravates age-progressive degeneration of the glomerular structure and function as filtration barrier. The critical role of calponin 2 in podocytes suggests a molecular target for understanding the pathogenesis of proteinuria and therapeutic development.

A Preparative Method for the Isolation of Calponin from Molluscan Catch Muscle.

We describe the development of a preparative method to isolate molluscan catch muscle, calponin. This method is based on the ability of calponin to interact with actin in a temperature-dependent manner. After extracting thin filaments, as previously described, the extract was ultracentrifuged at 2 °C. While other surface proteins of thin filaments co-precipitated with actin, calponin, along with some minor contaminants, remained in the supernatant. Calponin was purified through cation-exchange chromatography. The yield of pure protein was four-fold higher than that achieved through high-temperature extraction. To evaluate functionally isolated proteins, we determined the effect of calponin on Mg2+-ATPase activity of hybrid and non-hybrid actomyosin. The degree of ATPase inhibition was consistent with previously published data but strongly dependent on the environmental conditions and source of actin and myosin used. Furthermore, at low concentrations, calponin could induce the ATPase activity of hybrid actomyosin. This result was consistent with data indicating that calponin can modulate actin conformation to increase the relative content of "switched on" actin monomers in thin filaments. We assume that calponin obtained by the isolation method proposed herein is a fully functional protein that can both inhibit and induce the ATPase activity.

Ubiquitin profiling of lysophagy identifies actin stabilizer CNN2 as a target of VCP/p97 and uncovers a link to HSPB1.

Lysosomal membrane permeabilization (LMP) is an underlying feature of diverse conditions including neurodegeneration. Cells respond by extensive ubiquitylation of membrane-associated proteins for clearance of the organelle through lysophagy that is facilitated by the ubiquitin-directed AAA-ATPase VCP/p97. Here, we assessed the ubiquitylated proteome upon acute LMP and uncovered a large diversity of targets and lysophagy regulators. They include calponin-2 (CNN2) that, along with the Arp2/3 complex, translocates to damaged lysosomes and regulates actin filaments to drive phagophore formation. Importantly, CNN2 needs to be ubiquitylated during the process and removed by VCP/p97 for efficient lysophagy. Moreover, we identified the small heat shock protein HSPB1 that assists VCP/p97 in the extraction of CNN2 and show that other membrane regulators including SNAREs, PICALM, AGFG1, and ARL8B are ubiquitylated during lysophagy. Our data reveal a framework of how ubiquitylation and two effectors, VCP/p97 and HSPB1, cooperate to protect cells from the deleterious effects of LMP.

Calponin 3 Acts as a Potential Diagnostic and Prognostic Marker and Promotes Glioma Cell Proliferation, Migration, and Invasion.

BACKGROUND: Calponin 3 (CNN3) is involved in the proliferation and invasion of cervical cancer and osteosarcoma cells. However, the role of CNN3 in glioma tumorigenesis remains to be elucidated. METHODS: CNN3 mRNA expression in normal brain tissue and gliomas, including glioblastoma multiforme and lower-grade glioma, was analyzed using GEPIA 2 and Oncomine. CNN3 levels in glioma tissues were identified using immunohistochemical data provided by the Human Protein Atlas website. The relationship between CNN3 mRNA expression and clinical characteristics of patients with glioma was analyzed using the Oncomine database and The Cancer Genome Atlas. The diagnostic value of CNN3 expression in glioma was analyzed using receiver operating characteristic analysis according to The Cancer Genome Atlas and Genotype-Tissue Expression data. The relationship between CNN3 and prognosis was analyzed using GEPIA 2. The function of CNN3 knockdown in glioma cell lines was analyzed using cell proliferation, Transwell, and Western blot assays. RESULTS: Both mRNA and protein levels of CNN3 were distinctly higher in lower-grade glioma and glioblastoma multiforme tissues than those in normal brain tissues, particularly glioblastoma. A higher CNN3 mRNA level had significant relationship with higher World Health Organization grade, isocitrate dehydrogenase wild-type status, and 1p/19q noncodeletion. CNN3 mRNA expression is a highly accurate marker for the diagnosis of glioma. Patients with glioma in the high-CNN3 group had significantly lower disease-free survival and survival rates. In addition, CNN3 silencing significantly inhibited cell proliferation, migration, invasion, and the phosphorylation of P65 NF-κB. CONCLUSIONS: CNN3 expression is increased in glioma, particularly glioblastoma. Silencing CNN3 expression inhibited the proliferation, migration, and invasion of glioma cell lines.

Photorhabdus luminescens TccC3 Toxin Targets the Dynamic Population of F-Actin and Impairs Cell Cortex Integrity.

Due to its essential role in cellular processes, actin is a common target for bacterial toxins. One such toxin, TccC3, is an effector domain of the ABC-toxin produced by entomopathogenic bacteria of Photorhabdus spp. Unlike other actin-targeting toxins, TccC3 uniquely ADP-ribosylates actin at Thr-148, resulting in the formation of actin aggregates and inhibition of phagocytosis. It has been shown that the fully modified F-actin is resistant to depolymerization by cofilin and gelsolin, but their effects on partially modified actin were not explored. We found that only F-actin unprotected by tropomyosin is the physiological TccC3 substrate. Yet, ADP-ribosylated G-actin can be produced upon cofilin-accelerated F-actin depolymerization, which was only mildly inhibited in partially modified actin. The affinity of TccC3-ADP-ribosylated G-actin for profilin and thymosin-ß4 was weakened moderately but sufficiently to potentiate spontaneous polymerization in their presence. Interestingly, the Arp2/3-mediated nucleation was also potentiated by T148-ADP-ribosylation. Notably, even partially modified actin showed reduced bundling by plastins and α-actinin. In agreement with the role of these and other tandem calponin-homology domain actin organizers in the assembly of the cortical actin network, TccC3 induced intense membrane blebbing in cultured cells. Overall, our data suggest that TccC3 imposes a complex action on the cytoskeleton by affecting F-actin nucleation, recycling, and interaction with actin-binding proteins involved in the integration of actin filaments with each other and cellular elements.