Stage-dependent localization of F-actin and Na+ /K+ -ATPase in zebrafish embryos detected using optimized cryosectioning immunostaining protocol.

The increasing use of the zebrafish model in biomedical and (eco)toxicological studies aimed at understanding the function of various proteins highlight the importance of optimizing existing methods to study gene and protein expression and localization in this model. In this context, zebrafish cryosections are still underutilized compared with whole-mount preparations. In this study, we used zebrafish embryos (24-120 hpf) to determine key factors for the preparation of high-quality zebrafish cryosections and to determine the optimal protocol for (immuno)fluorescence analyses of Na+ /K+ -ATPase and F-actin, across developmental stages from 1 to 5 dpf. The results showed that the highest quality zebrafish cryosections were obtained after the samples were fixed in 4% paraformaldehyde (PFA) for 1 h, incubated in 2.5% bovine gelatin/25% sucrose mixture, embedded in OCT, and then sectioned to 8 µm thickness at -20°C. Fluorescence microscopy analysis of phalloidin-labeled zebrafish skeletal muscle revealed that 1-h-4% PFA-fixed samples allowed optimal binding of phalloidin to F-actin. Further immunofluorescence analyses revealed detailed localization of F-actin and Na+ /K+ -ATPase in various tissues of the zebrafish and a stage-dependent increase in their respective expression in the somitic muscles and pronephros. Finally, staining of zebrafish cryosections and whole-mount samples revealed organ-specific and zone-dependent localizations of the Na+ /K+ -ATPase α1-subunit. RESEARCH HIGHLIGHTS: This study brings optimization of existing protocols for preparation and use of zebrafish embryos cryosections in (immuno)histological analyses. It reveals stage-dependent localization/expression of F-actin and Na+ /K+ -ATPase in zebrafish embryos.

Molecular Mechanism of Mouse Uterine Smooth Muscle Regulation on Embryo Implantation.

Myometrium plays critical roles in multiple processes such as embryo spacing through peristalsis during mouse implantation, indicating vital roles of smooth muscle in the successful establishment and quality of implantation. Actin, a key element of cytoskeleton structure, plays an important role in the movement and contraction of smooth muscle cells (SMCs). However, the function of peri-implantation uterine smooth muscle and the regulation mechanism of muscle tension are still unclear. This study focused on the molecular mechanism of actin assembly regulation on implantation in smooth muscle. Phalloidin is a highly selective bicyclic peptide used for staining actin filaments (also known as F-actin). Phalloidin staining showed that F-actin gradually weakened in the CD-1 mouse myometrium from day 1 to day 4 of early pregnancy. More than 3 mice were studied for each group. Jasplakinolide (Jasp) used to inhibit F-actin depolymerization promotes F-actin polymerization in SMCs during implantation window and consequently compromises embryo implantation quality. Transcriptome analysis following Jasp treatment in mouse uterine SMCs reveals significant molecular changes associated with actin assembly. Tagln is involved in the regulation of the cell cytoskeleton and promotes the polymerization of G-actin to F-actin. Our results show that Tagln expression is gradually reduced in mouse uterine myometrium from day 1 to 4 of pregnancy. Furthermore, progesterone inhibits the expression of Tagln through the progesterone receptor. Using siRNA to knock down Tagln in day 3 SMCs, we found that phalloidin staining is decreased, which confirms the critical role of Tagln in F-actin polymerization. In conclusion, our data suggested that decreases in actin assembly in uterine smooth muscle during early pregnancy is critical to optimal embryo implantation. Tagln, a key molecule involved in actin assembly, regulates embryo implantation by controlling F-actin aggregation before implantation, suggesting moderate uterine contractility is conducive to embryo implantation. This study provides new insights into how the mouse uterus increases its flexibility to accommodate implanting embryos in the early stage of pregnancy.

Permixon®, hexane-extracted Serenoa repens, inhibits human prostate and bladder smooth muscle contraction and exerts growth-related functions in human prostate stromal cells.

AIMS: Recently, the European Association of Urology recommended hexane-extracted fruit of Serenoa repens (HESr) in their guidelines on management of non-neurogenic male lower urinary tracts symptoms (LUTS). Despite previously lacking recommendations, Permixon® is the most investigated HESr in clinical trials, where it proved effective for male LUTS. In contrast, underlying mechanisms were rarely addressed and are only marginally understood. We therefore investigated effects of Permixon® on human prostate and detrusor smooth muscle contraction and on growth-related functions in prostate stromal cells. MAIN METHODS: Permixon® capsules were dissolved using n-hexane. Contractions of human prostate and detrusor tissues were induced in organ bath. Proliferation (EdU assay), growth (colony formation), apoptosis and cell death (flow cytometry), viability (CCK-8) and actin organization (phalloidin staining) were studied in cultured human prostate stromal cells (WPMY-1). KEY FINDINGS: Permixon® inhibited α1-adrenergic and thromboxane-induced contractions in prostate tissues, and methacholine-and thromboxane-induced contractions in detrusor tissues. Endothelin-1-induced contractions were not inhibited. Neurogenic contractions were inhibited in both tissues in a concentration-dependent manner. In WPMY-1 cells, Permixon® caused concentration-dependent breakdown of actin polymerization, inhibited colony formation, reduced cell viability, and proliferation, without showing cytotoxic or pro-apoptotic effects. SIGNIFICANCE: Our results provide a novel basis that allows, for the first time, to fully explain the ubiquitous beneficial effects of HESr in clinical trials. HESr may inhibit at least neurogenic, α1-adrenergic and thromboxane-induced smooth muscle contraction in the prostate and detrusor, and in parallel, prostate stromal cell growth. Together, this may explain symptom improvements by Permixon® in previous clinical trials.

Roles of H19/miR-29a-3p/COL1A1 axis in COE-induced lung cancer.

Occupational lung cancer caused by coke oven emissions (COE) has attracted increasing attention, but the mechanism is not clear. Many evidences show ceRNA (competing endogenous RNA) networks play important regulatory roles in cancers. In this study, we aimed to construct and verify the ceRNA regulatory network in the occurrence of COE-induced lung squamous cell carcinoma (LUSC). We performed RNA sequencing with lung bronchial epithelial cell (16HBE) and COE induced malignant transformed cell (Rf). Furthermore, we analyzed RNA sequencing data of LUSC and adjacent tissues in the cancer genome atlas (TCGA) database. Combined our data and TCGA data to determine the differentially expressed lncRNAs, miRNAs, mRNAs. lncBASE, miRDB and miRTarBase were used to predict the binding relationship between lncRNA and miRNA, miRNA and mRNA. Based on these, we construct the ceRNA network. FREMSA, dual-luciferase reporter assay, quantitative real-time PCR (qRT-PCR), western-blot were used to verify the regulatory axis. CCK8 assay, phalloidin staining, p53 detection were used to explore the roles of this axis in the COE induced malignant transformation. Results showed 7 lncRNAs, 7 miRNAs and 146 mRNAs were identified. Among these, we constructed a ceRNA network including 1 lncRNA, 2 miRNAs and 9 mRNAs. Further verification confirmed the trend of lncRNA H19, miR-29a-3p and COL1A1 were consistent with sequencing results. H19 and COL1A1 were significantly higher in Rf than in 16HBE and miR-29a-3p was reverse. Regulatory investigation revealed H19 increased COL1A1 expression by sponging miR-29a-3p. Knockdown of H19, COL1A1 or overexpression of miR-29a-3p in Rf cells could inhibit cell proliferation, increased cell adhesion and p53 level. However, knockdown of H19 while suppressing the miR-29a-3p partially rescue the malignant phenotype of Rf caused by H19. In conclusion, all these indicated H19 functioned as a ceRNA to increase COL1A1 by sponging miR-29a-3p and promoted COE-induced cell malignant transformation.

Impairment of endometrial decidual reaction in early pregnant mice fed with high fat diet.

OBJECTIVE: To investigate the effect of obesity induced by high fat diet on decidual reaction of endometrium in mice, and the effect of high fat treatment on decidual reaction of endometrial stromal cells. METHODS: Twelve 4-week-old healthy C57BL/6J female mice were randomly divided into high fat diet group and control group with 6 mice in each group. They were fed with high fat diet (22 kJ/g) or normal diet (16 kJ/g) for 12 weeks, respectively. The body weight of mice was measured every week. After feeding for 12 weeks, the body length and width of mice were measured, and the levels of fasting serum triglyceride and total cholesterol were determined. Then the mice were mated with healthy C57BL/6J male mice, and the uterine tissues were collected on the seventh day of pregnancy. The decidual cells and collagen fibers in mouse endometrium was observed by HE staining and Masson staining respectively. The expression of decidual reaction related proteins in mouse endometrium were detected by immunohistochemistry and Western blotting. Mouse endometrial stromal cells (mESCs) were isolated and treated with the oleic acid and palmitic acid in vitro, and the decidual reaction was induced with estradiol and progesterone. The accumulation of lipid droplets in mESCs was observed by oil red O and Bodipy staining. The cytoskeleton of mESCs was observed by phalloidin staining. The levels of decidual reaction related genes and proteins were detected by real-time fluorescence quantitative PCR and Western blotting. RESULTS: After feeding for 12 weeks, the body weight of mice in the high fat group was significantly higher than that in the control group ( P<0.01), and there was no significant difference in body length between two groups ( P>0.05), but the body width of mice in the high fat group was significantly larger than that in the control group ( P<0.01), and the levels of serum triglyceride and total cholesterol were significantly higher than those in the control group (Both P<0.05). The number of embryo implantation in the high fat group was significantly less than that in the control group ( P<0.01). The differentiation of mESCs to decidual cells in high fat group was slow and abnormal. The expression levels of decidual reaction markers bone morphogenetic protein (BMP)2 and homeobox A10 (HOXA10) were lower than those in the control group, and there was significant difference in the expression level of HOXA10 ( P<0.01). The results of oil red O and Bodipy staining in mESCs showed that after high fat treatment, the accumulation of lipid droplets increased significantly, phalloidin staining showed abnormal cytoskeleton morphology. The expression levels of decidual reaction related genes dtprp, HOXA10 and proteins BMP2, HOXA10 and cyclooxygenase (COX)2 were significantly lower than those in the control group ( P<0.05). CONCLUSION: Obesity induced by high fat diet and high fat treatment can impair the decidual reaction of endometrium and endometrial stromal cells in mice.

Characterization of phalloidin-negative nuclear actin filaments in U2OS cells expressing cytoplasmic actin-EGFP.

Actin is a major structural component of the cytoskeleton in eukaryotic cells, including fungi, plants, and animals, and exists not only in the cytoplasm as cytoskeleton but also in the nucleus. Recently, we developed a novel actin probe, ß-actin-EGFP fusion protein, which exhibited similar monomeric to filamentous ratio as that of endogenous actin, in contrast to the widely used EGFP-ß-actin fusion protein that over-assembles in cells. Unexpectedly, this novel probe visualized an interconnected meshwork of slightly curved beam-like bundles of actin filaments in the nucleus of U2OS cells. These structures were not labeled with rhodamine phalloidin, Lifeact-EGFP or anti-actin antibodies. In addition, immunofluorescence staining and expression of cofilin-EGFP revealed that this nuclear actin structures contained cofilin. We named these actin filaments as phalloidin-negative intranuclear (PHANIN) actin filaments. Since PHANIN actin filaments could not be detected by general detection methods for actin filaments, we propose that PHANIN actin filaments are different from previously reported nuclear actin structures.

Low-dose ionizing radiation attenuates mast cell migration through suppression of monocyte chemoattractant protein-1 (MCP-1) expression by Nr4a2.

Purpose: The aim of this study was to investigate whether low-dose ionizing radiation attenuates mast cell migration by modulating migration-associated signaling pathways and the expression of chemotactic cytokines.Materials and methods: IgE-sensitized RBL-2H3 mast cells were exposed with ionizing radiation at 0.01, 0.05, 0.1, or 0.5 Gy using a 137Cs γ-irradiator and stimulated with 2,4-dinitrophenol-human serum albumin. Cell migration was determined using a transwell assay system, F-actin distribution using Alex Fluor 488-conjugated phalloidin, expression of various signaling proteins by Western blotting, mRNA expression by RT-PCR.Results: Low-dose ionizing radiation significantly suppressed mast cell migration induced by IgE-mediated mast cell activation. Furthermore, low-dose ionizing radiation altered cell morphology, as reflected by changes in F-actin distribution, and inhibited the activation of PI3K, Btk, Rac1, and Cdc42. These effects were mediated by Nr4a2, an immune-modulating factor. Knockdown of Nr4a2 reduced mast cell migration, inhibited the PI3K and Btk signaling pathways, and reduced expression of the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). We further demonstrated that direct blockade of MCP-1 using neutralizing antibodies inhibits mast cell migration.Conclusion: Low-dose ionizing radiation inhibits mast cell migration through the regulation production of MCP-1 by Nr4a2 in the activated mast cell system.

High-resolution cryo-EM structures of actin-bound myosin states reveal the mechanism of myosin force sensing.

Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to a range of motile challenges. Here, we reveal the structural basis for force-sensing based on near-atomic resolution structures of one rigor and two ADP-bound states of myosin-IB (myo1b) bound to actin, determined by cryo-electron microscopy. The two ADP-bound states are separated by a 25° rotation of the lever. The lever of the first ADP state is rotated toward the pointed end of the actin filament and forms a previously unidentified interface with the N-terminal subdomain, which constitutes the upper half of the nucleotide-binding cleft. This pointed-end orientation of the lever blocks ADP release by preventing the N-terminal subdomain from the pivoting required to open the nucleotide binding site, thus revealing how myo1b is inhibited by mechanical loads that restrain lever rotation. The lever of the second ADP state adopts a rigor-like orientation, stabilized by class-specific elements of myo1b. We identify a role for this conformation as an intermediate in the ADP release pathway. Moreover, comparison of our structures with other myosins reveals structural diversity in the actomyosin binding site, and we reveal the high-resolution structure of actin-bound phalloidin, a potent stabilizer of filamentous actin. These results provide a framework to understand the spectrum of force-sensing capacities among the myosin superfamily.

Versatility of Prolyl Oligopeptidase B in Peptide Macrocyclization.

Cyclic peptides are promising compounds for new chemical biological tools and therapeutics due to their structural diversity, resistance to proteases, and membrane permeability. Amatoxins, the toxic principles of poisonous mushrooms, are biosynthesized on ribosomes as 35mer precursor peptides, which are ultimately converted to hydroxylated bicyclic octapeptides. The initial cyclization steps, catalyzed by a dedicated prolyl oligopeptidase (POPB), involves removal of the 10-amino acid leader sequence from the precursor peptide and transpeptidation to produce a monocyclic octapeptide intermediate. The utility of POPB as a general catalyst for peptide cyclization was systematically characterized using a range of precursor peptide substrates produced either in E. coli or chemically. Substrates produced in E. coli were expressed either individually or in mixtures produced by codon mutagenesis. A total of 127 novel peptide substrates were tested, of which POPB could cyclize 100. Peptides of 7-16 residues were cyclized at least partially. Synthetic 25mer precursor peptide substrates containing modified amino acids including d-Ala, ß-Ala, N-methyl-Ala, and 4-hydroxy-Pro were also successfully cyclized. Although a phalloidin heptapeptide with all L amino acids was not cyclized, partial cyclization was seen when l-Thr at position #5 was replaced with the naturally occurring D amino acid. POPB should have broad applicability as a general catalyst for macrocyclization of peptides containing 7 to at least 16 amino acids, with an optimum of 8-9 residues.

The Impact of Non-Lethal Single-Dose Radiation on Tumor Invasion and Cytoskeletal Properties.

Irradiation is the standard therapy for glioblastoma multiforme. Glioblastoma are highly resistant to radiotherapy and the underlying mechanisms remain unclear. To better understand the biological effects of irradiation on glioblastoma cells, we tested whether nonlethal irradiation influences the invasiveness, cell stiffness, and actin cytoskeleton properties. Two different glioblastoma cell lines were irradiated with 2 Gy and changes in mechanical and migratory properties and alterations in the actin structure were measured. The invasiveness of cell lines was determined using a co-culture model with organotypic hippocampal slice cultures. Irradiation led to changes in motility and a less invasive phenotype in both investigated cell lines that were associated with an increase in a "generalized stiffness" and changes in the actin structure. In this study we demonstrate that irradiation can induce changes in the actin cytoskeleton and motility, which probably results in reduced invasiveness of glioblastoma cell lines. Furthermore, "generalized stiffness" was shown to be a profound marker of the invasiveness of a tumor cell population in our model.

Analysis of the cytoskeleton organization and its possible functions in male earthworm germ-line cysts equipped with a cytophore.

We studied the organization of F-actin and the microtubular cytoskeleton in male germ-line cysts in the seminal vesicles of the earthworm Dendrobaena veneta using light, fluorescent and electron microscopy along with both chemically fixed tissue and life cell imaging. Additionally, in order to follow the functioning of the cytoskeleton, we incubated the cysts in colchicine, nocodazole, cytochalasin D and latrunculin A. The male germ-line cells of D. veneta are interconnected via stable intercellular bridges (IB), and form syncytial cysts. Each germ cell has only one IB that connects it to the anuclear central cytoplasmic mass, the cytophore. During the studies, we analyzed the cytoskeleton in spermatogonial, spermatocytic and spermatid cysts. F-actin was detected in the cortical cytoplasm and forms distinct rings in the IBs. The arrangement of the microtubules changed dynamically during spermatogenesis. The microtubules are distributed evenly in whole spermatogonial and spermatocytic cysts; however, they primarily accumulate within the IBs in spermatogonia. In early spermatids, microtubules pass through the IBs and are present in whole cysts. During spermatid elongation, the microtubules form a manchette while they are absent in the cytophore and in the IBs. Use of cytoskeletal drugs did not alter the general morphology of the cysts. Detectable effects-the occurrence of nuclei in the late spermatids and manchette fragments in the cytophore-were observed only after incubation in nocodazole. Our results suggest that the microtubules are responsible for cytoplasmic/organelle transfer between the germ cells and the cytophore during spermatogenesis and for the positioning of the spermatid nuclei.

NF-κB-dependent increase in tissue factor expression is responsible for hypoxic podocyte injury.

BACKGROUND: Fibrin deposition within glomeruli is commonly seen in kidney biopsy specimens, suggesting enhanced coagulant activity. Tissue factor (TF) is a coagulation factor which is also related to various biological effects, and TF is upregulated by hypoxia in cancer cells. Recently, hypoxic podocyte injury has been proposed, therefore, we investigated TF expression in hypoxia. METHODS: Conditionally immortalized human podocytes were differentiated and treated under hypoxic or normoxic conditions. mRNA expressions of TF and tissue factor pathway inhibitor (TFPI) were analyzed by quantitative RT-PCR. Protein levels of TF and TFPI were tested by enzyme-linked immunosorbent assay. We employed small interfering RNA (siRNA) to temporary knockdown early growth response protein 1 (Egr-1), hypoxia-inducible factor-1α (HIF-1α) and TF. The expression of CD2-associated protein (CD2AP) mRNA and phalloidin staining was examined to assess podocyte injury. RESULTS: Hypoxia increased mRNA expression of TF (6 h: 2.3 ± 0.05 fold, p < 0.001, 24 h: 5.6 ± 2.4 fold, p < 0.05) and suppressed TFPI (6 h: 0.54 ± 0.04 fold, p < 0.05, 24 h: 0.24 ± 0.06 fold, p < 0.001) compared with normoxia. Similarly, protein levels of TF were increased and TFPI were decreased. Egr-1 siRNA did not change TF mRNA expression. Pyrrolidine dithiocarbamate (PDTC), a nuclear factor kappa B (NF-κB) inhibitor, significantly reduced hypoxia induced TF expression, and HIF-1α knockdown further increased TF. Hypoxia resulted in decreased CD2AP and actin reorganization in podocytes, and these changes were attenuated by TF siRNA. CONCLUSION: Hypoxia increased the expression of TF in human podocytes NF-κB dependently. TF may have a critical role in the hypoxic podocyte injury.

Intranuclear Actin Regulates Osteogenesis.

Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and global effects on gene transcription. We here show that in mesenchymal stem cells (MSCs), cytochalasin D treatment causes rapid cofilin-/importin-9-dependent transfer of G-actin into the nucleus. The continued presence of intranuclear actin, which forms rod-like structures that stain with phalloidin, is associated with induction of robust expression of the osteogenic genes osterix and osteocalcin in a Runx2-dependent manner, and leads to acquisition of osteogenic phenotype. Adipogenic differentiation also occurs, but to a lesser degree. Intranuclear actin leads to nuclear export of Yes-associated protein (YAP); maintenance of nuclear YAP inhibits Runx2 initiation of osteogenesis. Injection of cytochalasin into the tibial marrow space of live mice results in abundant bone formation within the space of 1 week. In sum, increased intranuclear actin forces MSC into osteogenic lineage through controlling Runx2 activity; this process may be useful for clinical objectives of forming bone.

Impact of C24:0 on actin-microtubule interaction in human neuronal SK-N-BE cells: evaluation by FRET confocal spectral imaging microscopy after dual staining with rhodamine-phalloidin and tubulin tracker green.

Disorganization of the cytoskeleton of neurons has major consequences on the transport of neurotransmitters via the microtubule network. The interaction of cytoskeleton proteins (actin and tubulin) was studied in neuronal SK-N-BE cells treated with tetracosanoic acid (C24:0), which is cytotoxic and increased in Alzheimer's disease patients. When SK-N-BE cells were treated with C24:0, mitochondrial dysfunctions and a non-apoptotic mode of cell death were observed. Fluorescence microscopy revealed shrunken cells with perinuclear condensation of actin and tubulin. Impact of C24:0 on actin-microtubule interaction in human neuronal SK-N-BE cells: evaluation by FRET confocal spectral imaging microscopy after dual staining with rhodamine-phalloidin and tubulin tracker green After staining with rhodamine-phalloidin and with an antibody raised against α-/ß-tubulin, modifications of F-actin and α-/ß-tubulin levels were detected by flow cytometry. Lower levels of α-tubulin were found by Western blotting. In C24:0-treated cells, spectral analysis and fluorescence recovery after photobleaching (FRAP) measured by confocal microscopy proved the existence of fluorescence resonance energy transfer (FRET) when actin and tubulin were stained with tubulin tracker and rhodamine-phalloidin demonstrating actin and tubulin co-localization/interaction. In control cells, no FRET was observed. Our data demonstrate quantitative changes in actin and tubulin, and modified interactions between actin and tubulin in SK-N-BE cells treated with C24:0. They also show that FRET confocal imaging microscopy is an interesting method for specifying the impact of cytotoxic compounds on cytoskeleton proteins.

Discrimination of bladder cancer cells from normal urothelial cells with high specificity and sensitivity: combined application of atomic force microscopy and modulated Raman spectroscopy.

Atomic force microscopy (AFM) and modulated Raman spectroscopy (MRS) were used to discriminate between living normal human urothelial cells (SV-HUC-1) and bladder tumour cells (MGH-U1) with high specificity and sensitivity. MGH-U1 cells were 1.5-fold smaller, 1.7-fold thicker and 1.4-fold rougher than normal SV-HUC-1 cells. The adhesion energy was 2.6-fold higher in the MGH-U1 cells compared to normal SV-HUC-1 cells, which possibly indicates that bladder tumour cells are more deformable than normal cells. The elastic modulus of MGH-U1 cells was 12-fold lower than SV-HUC-1 cells, suggesting a higher elasticity of the bladder cancer cell membranes. The biochemical fingerprints of cancer cells displayed a higher DNA and lipid content, probably due to an increase in the nuclear to cytoplasm ratio. Normal cells were characterized by higher protein contents. AFM studies revealed a decrease in the lateral dimensions and an increase in thickness of cancer cells compared to normal cells; these studies authenticate the observations from MRS. Nanostructural, nanomechanical and biochemical profiles of bladder cells provide qualitative and quantitative markers to differentiate between normal and cancerous cells at the single cellular level. AFM and MRS allow discrimination between adhesion energy, elasticity and Raman spectra of SV-HUC-1 and MGH-U1 cells with high specificity (83, 98 and 95%) and sensitivity (97, 93 and 98%). Such single-cell-level studies could have a pivotal impact on the development of AFM-Raman combined methodologies for cancer profiling and screening with translational significance.

Gly126Arg substitution causes anomalous behaviour of α-skeletal and ß-smooth tropomyosins during the ATPase cycle.

To investigate how TM stabilization induced by the Gly126Arg mutation in skeletal α-TM or in smooth muscle ß-TM affects the flexibility of TMs and their position on troponin-free thin filaments, we labelled the recombinant wild type and mutant TMs with 5-IAF and F-actin with FITC-phalloidin, incorporated them into ghost muscle fibres and studied polarized fluorescence at different stages of the ATPase cycle. It has been shown that in the myosin- and troponin-free filaments the Gly126Arg mutation causes a shift of TM strands towards the outer domain of actin, reduces the number of switched on actin monomers and decreases the rigidity of the C-terminus of α-TM and increases the rigidity of the N-terminus of ß-TMs. The binding of myosin subfragment-1 to the filaments shifted the wild type TMs towards the inner domain of actin, decreased the flexibility of both terminal parts of TMs, and increased the number of switched on actin monomers. Multistep alterations in the position of α- and ß-TMs and actin monomers in the filaments and in the flexibility of TMs and F-actin during the ATPase cycle were observed. The Gly126Arg mutation uncouples a correlation between the position of TM and the number of the switched on actin monomers in the filaments.

Competitive displacement of cofilin can promote actin filament severing.

Cofilin is an essential actin filament severing protein that functions in the dynamic remodeling of the actin cytoskeleton. Filament severing activity is most efficient at sub-stoichiometric cofilin binding densities (i.e. <1 cofilin per actin filament subunit), and peaks when the number density of boundaries (i.e. junctions) between bare and cofilin-decorated segments is maximal. A model in which local topological and mechanical discontinuities lead to preferential fragmentation at boundaries accounts for available experimental data, including direct visualization of cofilin and actin during real-time severing events. The boundary-severing model predicts that ligands (e.g. other actin-binding proteins) that compete with cofilin for actin filament binding and modulate cofilin occupancy on filaments will alter the bare-decorated segment boundary density, and thus, the filament severing activity of cofilin. Here, we directly test this model prediction by evaluating the effects of phalloidin and myosin, two ligands that compete with cofilin for filament binding, on the actin filament binding and severing activities of cofilin. Our experiments demonstrate that competitive displacement of cofilin lowers cofilin occupancy and promotes severing when initial cofilin occupancy is high (i.e. >50%). Even in the presence of competitive ligands, maximum severing activity occurs when cofilin-decorated boundary density is highest, consistent with preferential fragmentation at boundaries. We propose a general "severodyne" framework for the modulation of cofilin-mediated actin filament severing by small molecule or actin-binding protein ligands that compete with cofilin for actin filament binding.

Transplantation of human umbilical mesenchymal stem cells cures the corneal defects of mucopolysaccharidosis VII mice.

Mucopolysaccharidosis (MPS) are a family of related disorders caused by a mutation in one of the lysosomal exoglycosidases which leads to the accumulation of glycosaminoglycans (GAGs). MPS VII, caused by a mutation in ß-glucuronidase, manifests hepatomegaly, skeletal dysplasia, short stature, corneal clouding, and developmental delay. Current treatment regimens for MPS are not effective for treating corneal clouding and impaired mental development. We hypothesized that human umbilical mesenchymal stem cells (UMSCs) transplanted into the corneal stroma could participate in the catabolism of GAGs providing a means of cell therapy for MPS. For such treatment, human UMSCs were intrastromally transplanted into corneas of MPS VII mice. UMSC transplantation restored the dendritic and hexagonal morphology of host keratocytes and endothelial cells, respectively, and in vivo confocal microscopy (HRT-II) revealed reduced corneal haze. Immunohistochemistry using antibodies against heparan sulfate and chondroitin sulfate chains as well as lysosomal-associated membrane protein 2 revealed a decrease in GAG content and both lysosomal number and size in the treated corneas. Labeling UMSC intracellular compartments prior to transplantation revealed the distribution of UMSC vesicles throughout the corneal stroma and endothelium. An in vitro coculture assay between skin fibroblasts isolated from MPS VII mice and UMSC demonstrated that neutral vesicles released by the UMSC are taken up by the fibroblasts and proceed to fuse with the acidic lysosomes. Therefore, transplanted UMSCs participate both in extracellular GAG turnover and enable host keratocytes to catabolize accumulated GAG products, suggesting that UMSC could be a novel alternative for treating corneal defects associated with MPS and other congenital metabolic disorders.

The nuclear F-actin interactome of Xenopus oocytes reveals an actin-bundling kinesin that is essential for meiotic cytokinesis.

Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-ß and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis.

Visualization of actin filaments and monomers in somatic cell nuclei.

In addition to its long-studied presence in the cytoplasm, actin is also found in the nuclei of eukaryotic cells. The function and form (monomer, filament, or noncanonical oligomer) of nuclear actin are hotly debated, and its localization and dynamics are largely unknown. To determine the distribution of nuclear actin in live somatic cells and evaluate its potential functions, we constructed and validated fluorescent nuclear actin probes. Monomeric actin probes concentrate in nuclear speckles, suggesting an interaction of monomers with RNA-processing factors. Filamentous actin probes recognize discrete structures with submicron lengths that are excluded from chromatin-rich regions. In time-lapse movies, these actin filament structures exhibit one of two types of mobility: 1) diffusive, with an average diffusion coefficient of 0.06-0.08 µm(2)/s, or (2) subdiffusive, with a mobility coefficient of 0.015 µm(2)/s. Individual filament trajectories exhibit features of particles moving within a viscoelastic mesh. The small size of nuclear actin filaments is inconsistent with a role in micron-scale intranuclear transport, and their localization suggests that they do not participate directly in chromatin-based processes. Our results instead suggest that actin filaments form part of a large, viscoelastic structure in the nucleoplasm and may act as scaffolds that help organize nuclear contents.