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Polystyrene microplastics (mic-PS) have become harmful pollutants that attracted substantial attention about their potential toxicity. Hydrogen sulfide (H2S) is the third reported endogenous gas transmitter with protective functions on numerous physiologic responses. Nevertheless, the roles for mic-PS on skeletal systems in mammals and the protective effects of exogenous H2S are still indistinct. Here, the proliferation of MC3T3-E1 cell was analyzed by CCK8. Gene changes between the control and mic-PS treatment groups were analyzed by RNA-seq. The mRNA expression of bone morphogenetic protein 4 (Bmp4), alpha cardiac muscle 1 (Actc1), and myosin heavy polypeptide 6 (Myh6) was analyzed by QPCR. ROS level was analyzed by 2 ′ ,7 ′ -dichlorofluorescein (DCFH-DA). The mitochondrial membrane potential (MMP) was analyzed by Rh123. Our results indicated after exposure for 24 h, 100 mg/L mic-PS induced considerable cytotoxicity in the osteoblastic cells of mice. There were 147 differentially expressed genes (DEGs) including 103 downregulated genes and 44 upregulated genes in the mic-PS-treated group versus the control. The related signaling pathways were oxidative stress, energy metabolism, bone formation, and osteoblast differentiation. The results indicate that exogenous H2S may relieve mic-PS toxicity by altering Bmp4, Actc1, and Myh6 mRNA expressions associated with mitochondrial oxidative stress. Taken together, this study demonstrated that the bone toxicity effects of mic-PS along with exogenous H2S have protective function in mic-PS-mediated oxidative damage and mitochondrial dysfunction in osteoblastic cells of mice.

SummaryRegulating cell proliferation and differentiation in CNS development requires both extraordinary complexity and precision. Neural progenitors receive graded overlapping signals from midline signaling centers, yet each makes a unique cell fate decision in a spatiotemporally restricted pattern. The Nde1-Lis1 complex regulates individualized cell fate decisions based on the geographical location with respect to the midline. While cells distant from the midline fail to self-renew in the Nde1-Lis1 double-mutant CNS, cells embedded in the signaling centers showed marked overproliferation. A direct interaction between Lis1 and Brap, a mitogen-activated protein kinase (MAPK) signaling threshold modulator, mediates this differential response to mitogenic signal gradients. Nde1-Lis1 deficiency resulted in a spatially dependent alteration of MAPK scaffold Ksr and hyperactivation of MAPK. Epistasis analyses supported synergistic Brap and Lis1 functions. These results suggest that a molecular complex composed of Nde1, Lis1, and Brap regulates the dynamic MAPK signaling threshold in a spatially dependent fashion.

Filamins are a family of actin-binding proteins responsible for diverse biological functions in the context of regulating actin dynamics and vesicle trafficking. Disruption of these proteins has been implicated in multiple human developmental disorders. To investigate the roles of different filamin isoforms, we focused on FlnA and FlnB interactions in the cartilage growth plate, since mutations in both molecules cause chondrodysplasias. Current studies show that FlnA and FlnB share a common function in stabilizing the actin cytoskeleton, they physically interact in the cytoplasm of chondrocytes, and loss of FlnA enhances FlnB expression of chondrocytes in the growth plate (and vice versa), suggesting compensation. Prolonged FlnB loss, however, promotes actin-stress fiber formation following plating onto an integrin activating substrate whereas FlnA inhibition leads to decreased actin formation. FlnA more strongly binds RhoA, although both filamins overlap with RhoA expression in the cell cytoplasm. FlnA promotes RhoA activation whereas FlnB indirectly inhibits this pathway. Moreover, FlnA loss leads to diminished expression of β1-integrin, whereas FlnB loss promotes integrin expression. Finally, fibronectin mediated integrin activation has been shown to activate RhoA and activated RhoA leads to stress fiber formation and cell spreading. Fibronectin stimulation in null FlnA cells impairs enhanced spreading whereas FlnB inhibited cells show enhanced spreading. While filamins serve a primary static function in stabilization of the actin cytoskeleton, these studies are the first to demonstrate a dynamic and antagonistic relationship between different filamin isoforms in the dynamic regulation of integrin expression, RhoGTPase activity and actin stress fiber remodeling.

Neurons in the cerebral cortex originate predominantly from asymmetrical divisions of polarized radial glial or neuroepithelial cells. Fate control of neural progenitors through regulating cell division asymmetry determines the final cortical neuronal number and organization. Haploinsufficiency of human LIS1 results in type I lissencephaly (smooth brain) with severely reduced surface area and laminar organization of the cerebral cortex. Here we show that LIS1 and its binding protein Nde1 (mNudE) regulate the fate of radial glial progenitors collaboratively. Mice with an allelic series of Lis1 and Nde1 double mutations displayed a striking dose-dependent size reduction and de-lamination of the cerebral cortex. The neocortex of the Lis1-Nde1 double mutant mice showed over 80% reduction in surface area and inverted neuronal layers. Dramatically increased neuronal differentiation at the onset of corticogenesis in the mutant led to overproduction and abnormal development of earliest-born preplate neurons and Cajal-Retzius cells at the expense of progenitors. While both Lis1 and Nde1 are known to regulate the mitotic spindle orientation, only a moderate alteration in mitotic cleavage orientation was detected in the Lis1-Nde1 double deficient progenitors. Instead, a striking change in the morphology of metaphase progenitors with reduced apical attachment to the ventricular surface and weakened lateral contacts to neighboring cells appear to hinder the accurate control of cell division asymmetry and underlie the dramatically increased neuronal differentiation. Our data suggest that maintaining the shape and cell-cell interactions of radial glial neuroepithelial progenitors by the Lis1-Nde1 complex is essential for their self renewal during the early phase of corticogenesis.

The doublecortin-like domains (DCX), which typically occur in tandem, are novel microtubule-binding modules. DCX tandems are found in doublecortin, a 360-residue protein expressed in migrating neurons; the doublecortin-like kinase (DCLK); the product of the RP1 gene that is responsible for a form of inherited blindness; and several other proteins. Mutations in the gene encoding doublecortin cause lissencephaly in males and the 'double-cortex syndrome' in females. We here report a solution structure of the N-terminal DCX domain of human doublecortin and a 1.5 A resolution crystal structure of the equivalent domain from human DCLK. Both show a stable, ubiquitin-like tertiary fold with distinct structural similarities to GTPase-binding domains. We also show that the C-terminal DCX domains of both proteins are only partially folded. In functional assays, the N-terminal DCX domain of doublecortin binds only to assembled microtubules, whereas the C-terminal domain binds to both microtubules and unpolymerized tubulin.

Mutations in the X-linked gene Filamin A (FLNA) lead to the human neurological disorder, periventricular heterotopia (PH). Although PH is characterized by a failure in neuronal migration into the cerebral cortex with consequent formation of nodules in the ventricular and subventricular zones, many neurons appear to migrate normally, even in males, suggesting compensatory mechanisms. Here we characterize expression patterns for FlnA and a highly homologous protein Filamin B (FlnB) within the nervous system, in order to better understand their potential roles in cortical development. FlnA mRNA was widely expressed in all cortical layers while FlnB mRNA was most highly expressed in the ventricular and subventricular zones during development. In adulthood, widespread but reduced expression of FlnA and FlnB persisted throughout the cerebral cortex. FlnA and FlnB proteins were highly expressed in both the leading processes and somata of migratory neurons during corticogenesis. Postnatally, FlnA immunoreactivity was largely localized to the cell body with FlnB in the soma and neuropil during neuronal differentiation. In adulthood, diminished expression of both proteins localized to the cell soma and nucleus. Moreover, the putative FLNB homodimerization domain strongly interacted with itself or the corresponding homologous region of FLNA by yeast two-hybrid interaction, the two proteins co-localized within neuronal precursors by immunocytochemistry and the existence of FLNA-FLNB heterodimers could be detected by co-immunoprecipitation. These results suggest that FLNA and FLNB may form both homodimers and heterodimers and that their interaction could potentially compensate for the loss of FLNA function during cortical development within PH individuals.

The Drosophila Na,K-ATPase (or sodium pump) alpha-subunit gene was found to contain 10 exons and span approx. 25 kb. Two nearly adjacent transcriptional initiation sites were identified, and the 2085-nucleotide sequence upstream of the first transcriptional start was analysed for promoter activity in transfected Drosophila SL2 cells. This region was found to contain many cis-acting elements that influence promoter activity, including elements that confer 2- to 3-fold higher activity in SL2 cells cultured at 30 degrees C versus 22 degrees C. Temperature-sensitive transcriptional regulation of the Na,K-ATPase alpha-subunit in Drosophila is a plausible mechanistic candidate for the factor driving temperature-dependent up-regulation of the Na,K-ATPase alpha-subunit described here for fly strains homozygous for single P-element insertions in the alpha-subunit gene. Four new P-element insertion strains were identified in this study, each insertion site lying within the first intron of the Na,K-ATPase alpha-subunit gene. The insertion in strain 0462 resulted in cold-sensitive recessive lethality; flies homozygous for the 0462 mutation could be rescued by growth at 29-30 degrees C, a condition that partially corrected a deficiency in the level of Na,K-ATPase alpha-subunit. The high-temperature rescue of homozygous 0462 flies appeared to result primarily from improved Na,K-ATPase expression rather than an increase in the rate of ion transport per Na,K-ATPase molecule. These observations point to a role for sodium-pump activity in determining the range of temperature tolerance in Drosophila and demonstrate that relatively subtle changes in sodium-pump expression can have major consequences in whole organisms.

A bang-sensitive enhancer trap line was isolated in a behavioral screen. The flies show a weak bang-sensitive paralysis, recovering after about 7 s. The P element insert is localized at 93131-2 on the salivary chromosomes, the site of the (Na + ,K + )ATPase a subunit gene. Molecular characterization demonstrates that the transposon is inserted into the first intron of this gene. This insertion leads to normal-sized transcripts, but reduced levels of expression. This change is also reflected in lower amounts of a normal-sized a subunit protein. Mutant flies show a much greater sensitivity to ouabain, likewise indicating, on a functional level, a reduction in Na + pump activity. Furthermore, the bang-sensitive behavior can also be mimicked by injecting sublethal doses of ouabain into wild-type flies. The molecular and functional evidence indicates that the insertion has produced a hypomorphic mutation of the (Na + ,K + )ATPase α subunit gene, opening the way to future studies of the regulation of the Na + pump.

Cells in mechanically challenged environments must cope with high amplitude forces to maintain cell viability and tissue homeostasis. Currently, force-induced cell death and the identity of mechanoprotective factors are not defined. We examined death in cultured periodontal fibroblasts, connective tissue cells that are exposed to heavy applied forces in vivo. Static tensile forces (0.48 piconewtons/microm2 cell area) were applied through magnetite beads coated with collagen or bovine serum albumin. There was a time-dependent increase of the percentage of propidium iodide-permeable cells in force-loaded cultures incubated with collagen but not bovine serum albumin beads, indicating a role for integrins. Cells exhibited reduced mitochondrial membrane potential, increased caspase-3 activation, nuclear condensation, terminal deoxynucleotidyl transferase nick end labeling staining, and detachment from the culture dish. The caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde reduced detachment 3-fold. There was a rapid (

How mitotic cell fate is regulated in the developing mammalian CNS is an important but largely unanswered question. Recently reporting in Neuron, Godin et al. showed that Huntingtin, the protein mutated in Huntington's disease, is required for both cerebral cortical neurogenesis and mitotic spindle function in neural progenitors.

The inv(16) chromosomal aberration is found in 10% of the cases with AML. The resultant CBFB-MYH11 fusion gene that plays a key-role in the leukemia development, but for full transformation, additional genetic hits are necessary. We report that the MYH11 gene overlaps with the NDE1 gene. At their 3 prime ends the genes overlap at the genomic, transcript as well as coding level and as a consequence, the inv(16) disrupts NDE1 in addition to CBFB and MYH11 in 90% of CBFB-MYH11 positive cases. To determine the relevance of Nde1 function in hematopoiesis leukemia development, hematopoiesis was studied in Nde1-/− and Nde1+/− mutant mice. A significant percentage of the Nde1 null mutant mice developed a myeloproliferative (MPD) phenotype at 6–12 months of age. This phenotype was observed in 6 out of 15 (40%) Nde1−/− mice, 2 out of 15 (13%) Nde1+/− mice and none of 4 wild type mice. All mice with the MPD phenotype exhibited a profound splenomegaly with spleen sizes ranging from 5 to 20-fold larger than normal wild type controls. Histopathological analysis demonstrated hypercellular bone marrow, effacement of normal splenic architecture with expansion of a myeloid cells, and infiltration of proliferating myeloid cells in various organs . A 10-fold increase in Gr1+/Mac1+ double positive mature myeloid cells in the spleen was confirmed by flow-cytometric analysis. Peripheral blood analyses showed that the white blood cell counts were slightly elevated in Nde1 mutant mice that had not yet developed a MPD phenotype compared to wild type mice. A somewhat higher white blood cell count was observed in Nde1 mutant mice with MPD phenotype. In the Nde1 mutant mice with or without splenomegaly we did not observe an increase in the number of B- and T-cells as measured by flowcytometer using various lymphoid cell surface markers. Based on these data we conclude that loss of Nde1 function and Nde1 haploinsufficiency results in the onset of a MPD phenotype. Earlier studies showed that Nde1 is a centrosomal protein. To test whether Nde1 is involved in spindle pole formation and chromosome segregation, we analyzed the mitotic behaviour of mouse embryo fibroblasts (MEFs). From passage 3 to passage 10, aneuploid metaphases increased much more rapidly in Nde1−/− MEFs than in the wild type controls. In addition, a broader spectrum of abnormal chromosome numbers was seen in Nde1−/− MEFs. Thus, Nde1 deficiency results in chromosomal instability. To study whether the numerical chromosome abnormalities were associated with aberrant spindle formation we stained Nde1−/− MEFs with a monoclonal antibody against tubulin. Multipolar spindles and misassembled spindles were more frequently observed in Nde1−/− MEFs compared to wild type cells. We conclude that the abnormal chromosomal segregation of Nde1−/− MEFs is associated with defective mitotic spindle structure. Since Nde1 is disrupted in the majority of inv(16) positive AML patients and the disruption in mice resulted in genetic instability and the onset of myeloproliferative disease, we suggest that the disruption of Nde1 in inv(16)+ AML may serve as additional genetic defect that contributes to leukemogenesis.