Tropomodulin and tropomyosin are important components of sarcomeric thin filaments in striated muscles. Tropomyosin decorates the side of actin filaments and enhances tropomodulin capping at the ...pointed ends of the filaments. Their functional relationship has been extensively characterized in vitro, but in vivo and cellular studies in mammals are often complicated by the presence of functionally redundant isoforms. Here, we used the nematode Caenorhabditis elegans, which has a relatively simple composition of tropomodulin and tropomyosin genes, and demonstrated that tropomodulin (unc-94) and tropomyosin (lev-11) are mutually dependent on each other in their sarcomere localization and regulation of sarcomeric actin assembly. Mutation of tropomodulin caused sarcomere disorganization with formation of actin aggregates. However, the actin aggregation was suppressed when tropomyosin was depleted in the tropomodulin mutant. Tropomyosin was mislocalized to the actin aggregates in the tropomodulin mutants, while sarcomere localization of tropomodulin was lost when tropomyosin was depleted. These results indicate that tropomodulin and tropomyosin are interdependent in the regulation of organized sarcomeric assembly of actin filaments in vivo.
•Ablation of tropomodulin and tropomyosin cause distinct patterns of sarcomere disorganization.•Tropomyosin depletion reduces actin aggregates in tropomodulin mutants.•Tropomodulin mutation causes mislocalization of tropomyosin.•Tropomyosin depletion disrupts sarcomere localization of tropomodulin.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A copper-catalyzed aerobic 3-hydroxyisoindolinone synthesis was developed
via
the benzylic double C(sp
3
)-H functionalization of 2-alkylbenzamides. In this reaction, molecular oxygen was used as ...both an oxidant for C(sp
3
)-H functionalization and an oxygen source. Our method can be extended to diverse benzylic C(sp
3
)-H bonds and shows excellent functional group tolerance.
A copper-catalyzed aerobic 3-hydroxyisoindolinone synthesis was developed
via
the benzylic double C(sp
3
)-H functionalization of 2-alkylbenzamides.
Herein, we describe the copper-catalyzed aerobic C(sp
)-H functionalization of 2-alkylbenzamides for the synthesis of benzolactones. This reaction proceeds
1,5-hydrogen atom transfer of N-centered ...radicals directly generated by N-H bond cleavage and does not require the synthesis of pre-functionalized N-centered radical precursors or the use of strong stoichiometric oxidants.
A transition‐metal‐free intermolecular coupling reaction of halocompounds with styrenes in the presence of NaH and 1,10‐phenanthroline was developed. This reaction afforded hydrocarbonated products ...with complete anti‐Markovnikov selectivity. The method allows the use of a wide range of halocompounds, including aryl and alkyl halides, and good functional group tolerance. Detailed mechanistic studies indicated that an anilide anion generated in situ by the NaH‐mediated reduction of 1,10‐phenanthroline works as an electron donor and a hydrogen source.
A transition‐metal‐free intermolecular coupling strategy was developed, comprising the reaction of halocompounds with styrenes in the presence of NaH and 1,10‐phenanthroline, to afford hydrocarbonated products with complete anti‐Markovnikov selectivity. The developed strategy can be applied to a wide range of halocompounds, including aryl and alkyl halides, and shows good functional group tolerance.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Formalin‐fixed paraffin‐embedded (FFPE) tissues are promising biological resources for genetic research. Recent improvements in DNA extraction from FFPE samples allowed the use of these tissues for ...multiple sequencing methods. However, fundamental research addressing the application of FFPE‐derived DNA for targeted‐bisulfite sequencing (TB‐seq) is lacking. Here, we evaluated the suitability of FFPE‐derived DNA for TB‐seq. We conducted TB‐seq using FFPE‐derived DNA and corresponding fresh frozen (FF) tissues of patients with kidney cancer and compared the quality of DNA, libraries, and TB‐seq statistics between the two preservation methods. The approximately 600‐bp average fragment size of the FFPE‐derived DNA was significantly shorter than that of the FF‐derived DNA. The sequencing libraries constructed using FFPE‐derived DNA and the mapping ratio were approximately 10 times and 10% lower, respectively, than those constructed using FF‐derived DNA. In the mapped data of FFPE‐derived DNA, duplicated reads accounted for > 60% of the obtained sequence reads, with lower mean on‐target coverage. Therefore, the standard TB‐seq protocol is inadequate for obtaining high‐quality data for epigenetic analysis from FFPE‐derived DNA, and technical improvements are necessary for enabling the use of archived FFPE resources.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and ...obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B-induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.
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BFBNIB, NUK, PNG, UL, UM, UPUK
The use of heated tobacco products (HTP) has increased exponentially in Japan since 2016; however, their effects on health remain a major concern.
Tsuruoka Metabolome Cohort Study participants (
= ...11,002) were grouped on the basis of their smoking habits as never smokers (NS), past smokers (PS), combustible tobacco smokers (CS), and HTP users for <2 years. Peripheral blood mononuclear cells were collected from 52 participants per group matched to HTP users using propensity scores, and DNA and RNA were purified from the samples. DNA methylation (DNAm) analysis of the 17 smoking-associated DNAm biomarker genes (such as
,
,
, and
), as well as whole transcriptome analysis, was performed.
Ten of the 17 genes were significantly hypomethylated in CS and HTP users compared with NS, among which
,
, and
showed intermediate characteristics between CS and NS; nonetheless,
expression was significantly higher in CS than in the other three groups. Conversely,
and
were more hypomethylated in HTP users than in NS, and
expression was markedly upregulated in all the groups when compared with that in NS.
HTP users (switched from CS <2 years) display abnormal DNAm and transcriptome profiles, albeit to a lesser extent than the CS. However, because the molecular genetic effects of long-term HTP use are still unknown, long-term molecular epidemiologic studies are needed.
This study provides new insights into the molecular genetic effects on DNAm and transcriptome profiles in HTP users who switched from CS.
Tropomyosin is generally known as an actin-binding protein that regulates actomyosin interaction and actin filament stability. In metazoans, multiple tropomyosin isoforms are expressed, and some of ...them are involved in generating subpopulations of actin cytoskeleton in an isoform-specific manner. However, functions of many tropomyosin isoforms remain unknown. Here, we report identification of a novel alternative exon in the
Caenorhabditis elegans
tropomyosin gene and characterization of the effects of alternative splicing on the properties of tropomyosin isoforms. Previous studies have reported six tropomyosin isoforms encoded by the
C. elegans lev-11
tropomyosin gene. We identified a seventh isoform, LEV-11U, that contained a novel alternative exon, exon 7c (E7c). LEV-11U is a low-molecular-weight tropomyosin isoform that differs from LEV-11T only at the exon 7-encoded region.
In silico
analyses indicated that the E7c-encoded peptide sequence was unfavorable for coiled-coil formation and distinct from other tropomyosin isoforms in the pattern of electrostatic surface potentials.
In vitro
, LEV-11U bound poorly to actin filaments, whereas LEV-11T bound to actin filaments in a saturable manner. When these isoforms were transgenically expressed in the
C. elegans
striated muscle, LEV-11U was present in the diffuse cytoplasm with tendency to form aggregates, whereas LEV-11T co-localized with sarcomeric actin filaments. Worms with a mutation in E7c showed reduced motility and brood size, suggesting that this exon is important for the optimal health. These results indicate that alternative splicing of a single exon can produce biochemically diverged tropomyosin isoforms and suggest that a tropomyosin isoform with poor actin affinity has a novel biological function.
Assembly of contractile apparatuses in striated muscle requires precisely regulated reorganization of the actin cytoskeletal proteins into sarcomeric organization. Regulation of actin filament ...dynamics is one of the essential processes of myofibril assembly, but the mechanism of actin regulation in striated muscle is not clearly understood. Actin depolymerizing factor (ADF)/cofilin is a key enhancer of actin filament dynamics in striated muscle in both vertebrates and nematodes. Here, we report that CAS-1, a cyclase-associated protein in Caenorhabditis elegans, promotes ADF/cofilin-dependent actin filament turnover in vitro and is required for sarcomeric actin organization in striated muscle. CAS-1 is predominantly expressed in striated muscle from embryos to adults. In vitro, CAS-1 binds to actin monomers and enhances exchange of actin-bound ATP/ADP even in the presence of UNC-60B, a muscle-specific ADF/cofilin that inhibits the nucleotide exchange. As a result, CAS-1 and UNC-60B cooperatively enhance actin filament turnover. The two proteins also cooperate to shorten actin filaments. A cas-1 mutation is homozygous lethal with defects in sarcomeric actin organization. cas-1-mutant embryos and worms have aggregates of actin in muscle cells, and UNC-60B is mislocalized to the aggregates. These results provide genetic and biochemical evidence that cyclase-associated protein is a critical regulator of sarcomeric actin organization in striated muscle.
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