Kindlin-2 is required for myocyte elongation and is essential for myogenesis

<p>Abstract</p> <p>Background</p> <p>Integrins are required for normal muscle differentiation and disruptions in integrin signaling result in human muscle disease. The intracellular components that regulate integrin function during myogenesis are poorly understood. Unc-112 is an integrin-associated protein required for muscle development in C. elegans. To better understand the intracellular effectors of integrin signaling in muscle, we examined the mammalian homolog of Unc-112, kindlin-2.</p> <p>Results</p> <p>Kindlin-2 expression is upregulated during differentiation and highly enriched at sites of integrin localization. RNAi knockdown of kindlin-2 in C2C12 cells results in significant abnormalities during the early stages of myogenesis. Specifically, differentiating myocytes lacking kindlin-2 are unable to elongate and fail to fuse into multinucleated myotubes. These changes are correlated with decreased cell substratum adhesion and increased cell motility. They are also associated with redistribution of a known kindlin-2 binding partner, integrin linked kinase (ILK), to the membrane insoluble subcellular fraction.</p> <p>Conclusion</p> <p>In all, our study reveals kindlin-2 as a novel integrin adaptor protein important for muscle differentiation, and identifies it particularly as a critical regulator of myocyte elongation.</p

Increased Adhesive Potential of Antiphospholipid Syndrome Neutrophils Mediated by β2 Integrin Macâ 1

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153125/1/art41057.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153125/2/art41057_am.pd

Loss of Myotubularin Function Results in T-Tubule Disorganization in Zebrafish and Human Myotubular Myopathy

Myotubularin is a lipid phosphatase implicated in endosomal trafficking in vitro, but with an unknown function in vivo. Mutations in myotubularin cause myotubular myopathy, a devastating congenital myopathy with unclear pathogenesis and no current therapies. Myotubular myopathy was the first described of a growing list of conditions caused by mutations in proteins implicated in membrane trafficking. To advance the understanding of myotubularin function and disease pathogenesis, we have created a zebrafish model of myotubular myopathy using morpholino antisense technology. Zebrafish with reduced levels of myotubularin have significantly impaired motor function and obvious histopathologic changes in their muscle. These changes include abnormally shaped and positioned nuclei and myofiber hypotrophy. These findings are consistent with those observed in the human disease. We demonstrate for the first time that myotubularin functions to regulate PI3P levels in a vertebrate in vivo, and that homologous myotubularin-related proteins can functionally compensate for the loss of myotubularin. Finally, we identify abnormalities in the tubulo-reticular network in muscle from myotubularin zebrafish morphants and correlate these changes with abnormalities in T-tubule organization in biopsies from patients with myotubular myopathy. In all, we have generated a new model of myotubular myopathy and employed this model to uncover a novel function for myotubularin and a new pathomechanism for the human disease that may explain the weakness associated with the condition (defective excitation–contraction coupling). In addition, our findings of tubuloreticular abnormalities and defective excitation-contraction coupling mechanistically link myotubular myopathy with several other inherited muscle diseases, most notably those due to ryanodine receptor mutations. Based on our findings, we speculate that congenital myopathies, usually considered entities with similar clinical features but very disparate pathomechanisms, may at their root be disorders of calcium homeostasis

Molecular Evolutionary Trends and Feeding Ecology Diversification in the Hemiptera, Anchored by the Milkweed Bug Genome

Background: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. Results: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding. Conclusions: With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus’s strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes

Germline MBD4-deficiency causes a multi-tumor predisposition syndrome

We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5′-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management

Influenza Virus Ribonucleoprotein Complexes Gain Preferential Access to Cellular Export Machinery through Chromatin Targeting

In contrast to most RNA viruses, influenza viruses replicate their genome in the nucleus of infected cells. As a result, newly-synthesized vRNA genomes, in the form of viral ribonucleoprotein complexes (vRNPs), must be exported to the cytoplasm for productive infection. To characterize the composition of vRNP export complexes and their interplay with the nucleus of infected cells, we affinity-purified tagged vRNPs from biochemically fractionated infected nuclei. After treatment of infected cells with leptomycin B, a potent inhibitor of Crm1-mediated export, we isolated vRNP export complexes which, unexpectedly, were tethered to the host-cell chromatin with very high affinity. At late time points of infection, the cellular export receptor Crm1 also accumulated at the same regions of the chromatin as vRNPs, which led to a decrease in the export of other nuclear Crm1 substrates from the nucleus. Interestingly, chromatin targeting of vRNP export complexes brought them into association with Rcc1, the Ran guanine exchange factor responsible for generating RanGTP and driving Crm1-dependent nuclear export. Thus, influenza viruses gain preferential access to newly-generated host cell export machinery by targeting vRNP export complexes at the sites of Ran regeneration

Molecular evolutionary trends and feeding ecology diversification in the Hemiptera, anchored by the milkweed bug genome.

BACKGROUND: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. RESULTS: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins. The discovery of intron gain and turnover specific to the Hemiptera also prompted the evaluation of lineage and genome size as predictors of gene structure evolution. Furthermore, we identify enzymatic gains and losses that correlate with feeding biology, particularly for reductions associated with derived, fluid nutrition feeding. CONCLUSIONS: With the milkweed bug, we now have a critical mass of sequenced species for a hemimetabolous insect order and close outgroup to the Holometabola, substantially improving the diversity of insect genomics. We thereby define commonalities among the Hemiptera and delve into how hemipteran genomes reflect distinct feeding ecologies. Given Oncopeltus's strength as an experimental model, these new sequence resources bolster the foundation for molecular research and highlight technical considerations for the analysis of medium-sized invertebrate genomes

Kindlin-2 is required for myocyte elongation and is essential for myogenesis-7

Ay 4 (D4) transfected with scrambled (scr) or kindlin-2 (kind2) RNAi. Blots were probed with anti-ILK and anti-GAPDH. No difference by densitometry was detected between scrambled and kindlin-2 RNAi cells. Dot indicates ILK band. B. Immunoblot on subcellular fractions from day 2 differentiated C2C12 cells probed with anti-ILK. ILK localizes to the cytosolic (cyto) fraction in scrambled RNAi cells and to the membrane/insoluble (mem) fraction in kindlin-2 RNAi cells. No expression was seen with either condition in the cytoskeletal or nuclear fractions (data not shown).<p><b>Copyright information:</b></p><p>Taken from "Kindlin-2 is required for myocyte elongation and is essential for myogenesis"</p><p>http://www.biomedcentral.com/1471-2121/9/36</p><p>BMC Cell Biology 2008;9():36-36.</p><p>Published online 8 Jul 2008</p><p>PMCID:PMC2478659.</p><p></p

Kindlin-2 is required for myocyte elongation and is essential for myogenesis-9

On change to differentiation media, control cells withdraw from the cell cycle and elongate. In cells with reduced levels of kindlin-2, cells fail to elongate, and instead maintain a "pro-migratory" phenotype. C. By differentiation day 4, elongated control cells fuse into multinucleated myotubes. Reduced levels of kindlin-2 result in failure of myotube formation, likely as a result of decreased elongation, inadequate cell adhesion and impaired myoblast fusion. One mechanism underlying these alterations is a failure of redistribution of ILK containing focal adhesions.<p><b>Copyright information:</b></p><p>Taken from "Kindlin-2 is required for myocyte elongation and is essential for myogenesis"</p><p>http://www.biomedcentral.com/1471-2121/9/36</p><p>BMC Cell Biology 2008;9():36-36.</p><p>Published online 8 Jul 2008</p><p>PMCID:PMC2478659.</p><p></p

Kindlin-2 is required for myocyte elongation and is essential for myogenesis-6

Ectin coated cell culture dishes and incubated for 1 hour at 370C. Cells were fixed and then examined for morphologic changes consistent with adhesion and spreading. On non coated dishes, percent spreading was 71.6% (+/-0.02%) for scrambled and 41.8% (+/-0.03%) for kindlin-2 RNAi (p < 0.0001, n = 5). On fibronectin, percent spreading was 93. 8% (+/-0.01%) for scrambled and 47.2% (+/-0.03) for kindlin-2 RNAi (P < 0.0001, n = 5). B. Bright field photomicrograph taken at 10× of representative cells from the assay described in A. The arrow points to a spread cell while the arrowhead points to a non-spread cell in kindlin-2 RNAi transfectents. C. RNAi transfected C2C12 cells were replated onto transwell and allowed to migrate through the wells to a lower chamber filled with media. Migrated cells were counted after 12 hours. Significantly more migration was observed with kindlin-2 knockdown (scrambled v kindlin-2 RNAi, 0.11+/-0.01 v 0.16+/-0.01; p = 0.019; n = 3).<p><b>Copyright information:</b></p><p>Taken from "Kindlin-2 is required for myocyte elongation and is essential for myogenesis"</p><p>http://www.biomedcentral.com/1471-2121/9/36</p><p>BMC Cell Biology 2008;9():36-36.</p><p>Published online 8 Jul 2008</p><p>PMCID:PMC2478659.</p><p></p