BackgroundCell migration plays an important role in many physiological and pathological processes, including immune cell chemotaxis and cancer metastasis. It is a coordinated process that involves dynamic changes in the actin cytoskeleton and its interplay with focal adhesions. At the leading edge of a migrating cell, it is the re-arrangement of actin and its attachment to focal adhesions that generates the driving force necessary for movement. However, the mechanisms involved in the attachment of actin filaments to focal adhesions are still not fully understood.ResultsSignaling by the FAK-Src complex plays a crucial role in regulating the formation of protein complexes at focal adhesions to which the actin filaments are attached. Cortactin, an F-actin associated protein and a substrate of Src kinase, was found to interact with FAK through its SH3 domain and the C-terminal proline-rich regions of FAK. We found that the autophosphorylation of Tyr397 in FAK, which is necessary for FAK activation, was not required for the interaction with cortactin, but was essential for the tyrosine phosphorylation of the associated cortactin. At focal adhesions, cortactin was phosphorylated at tyrosine residues known to be phosphorylated by Src. The tyrosine phosphorylation of cortactin and its ability to associate with the actin cytoskeleton were required in tandem for the regulation of cell motility. Cell motility could be inhibited by truncating the N-terminal F-actin binding domains of cortactin or by blocking tyrosine phosphorylation (Y421/466/475/482F mutation). In addition, the mutant cortactin phosphorylation mimic (Y421/466/475/482E) had a reduced ability to interact with FAK and promoted cell motility. The promotion of cell motility by the cortactin phosphorylation mimic could also be inhibited by truncating its N-terminal F-actin binding domains.ConclusionsOur results suggest that cortactin acts as a bridging molecule between actin filaments and focal adhesions. The cortactin N-terminus associates with F-actin, while its C-terminus interacts with focal adhesions. The tyrosine phosphorylation of cortactin by the FAK-Src complex modulates its interaction with FAK and increases its turnover at focal adhesions to promote cell motility.

BackgroundCell migration plays an important role in many physiological and pathological processes, including immune cell chemotaxis and cancer metastasis. It is a coordinated process that involves dynamic changes in the actin cytoskeleton and its interplay with focal adhesions. At the leading edge of a migrating cell, it is the re-arrangement of actin and its attachment to focal adhesions that generates the driving force necessary for movement. However, the mechanisms involved in the attachment of actin filaments to focal adhesions are still not fully understood.ResultsSignaling by the FAK-Src complex plays a crucial role in regulating the formation of protein complexes at focal adhesions to which the actin filaments are attached. Cortactin, an F-actin associated protein and a substrate of Src kinase, was found to interact with FAK through its SH3 domain and the C-terminal proline-rich regions of FAK. We found that the autophosphorylation of Tyr397 in FAK, which is necessary for FAK activation, was not required for the interaction with cortactin, but was essential for the tyrosine phosphorylation of the associated cortactin. At focal adhesions, cortactin was phosphorylated at tyrosine residues known to be phosphorylated by Src. The tyrosine phosphorylation of cortactin and its ability to associate with the actin cytoskeleton were required in tandem for the regulation of cell motility. Cell motility could be inhibited by truncating the N-terminal F-actin binding domains of cortactin or by blocking tyrosine phosphorylation (Y421/466/475/482F mutation). In addition, the mutant cortactin phosphorylation mimic (Y421/466/475/482E) had a reduced ability to interact with FAK and promoted cell motility. The promotion of cell motility by the cortactin phosphorylation mimic could also be inhibited by truncating its N-terminal F-actin binding domains.ConclusionsOur results suggest that cortactin acts as a bridging molecule between actin filaments and focal adhesions. The cortactin N-terminus associates with F-actin, while its C-terminus interacts with focal adhesions. The tyrosine phosphorylation of cortactin by the FAK-Src complex modulates its interaction with FAK and increases its turnover at focal adhesions to promote cell motility.

BackgroundIncreased disease resistance through improved general immune capacity would be beneficial for the welfare and productivity of farm animals. T lymphocyte subpopulations in peripheral blood play an important role in immune capacity and disease resistance in animals. However, very little research to date has focused on quantitative trait loci (QTL) for T lymphocyte subpopulations in peripheral blood in swine.ResultsIn the study, experimental animals consist of 446 piglets from three different breed populations. To identify QTL for T lymphocyte subpopulations in peripheral blood in swine, the proportions of CD4+, CD8+, CD4+CD8+, CD4+CD8-, CD4-CD8+, and CD4-CD8- T cells and the ratio of CD4+:CD8+ T cells were measured for all individuals before and after challenge with modified live CSF (classical swine fever) vaccine. Based on the combined data of individuals from three breed populations, genome-wide scanning of QTL for these traits was performed based on a variance component model, and the genome wide significance level for declaring QTL was determined via permutation tests as well as FDR (false discovery rate) correction. A total of 27 QTL (two for CD4+CD8+, one for CD4+CD8-, three for CD4-CD8+, two for CD4-CD8-, nine for CD4+, two for CD8+, and eight for CD4+:CD8+ ratio) were identified with significance level of FDR < 0.10, of which 11 were significant at the level of FDR < 0.05, including the five significant at FDR < 0.01.ConclusionsWithin these QTL regions, a number of known genes having potential relationships with the studied traits may serve as candidate genes for these traits. Our findings herein are helpful for identification of the causal genes underlying these immune-related trait and selection for immune capacity of individuals in swine breeding in the future.

BackgroundIncreased disease resistance through improved general immune capacity would be beneficial for the welfare and productivity of farm animals. T lymphocyte subpopulations in peripheral blood play an important role in immune capacity and disease resistance in animals. However, very little research to date has focused on quantitative trait loci (QTL) for T lymphocyte subpopulations in peripheral blood in swine.ResultsIn the study, experimental animals consist of 446 piglets from three different breed populations. To identify QTL for T lymphocyte subpopulations in peripheral blood in swine, the proportions of CD4+, CD8+, CD4+CD8+, CD4+CD8-, CD4-CD8+, and CD4-CD8- T cells and the ratio of CD4+:CD8+ T cells were measured for all individuals before and after challenge with modified live CSF (classical swine fever) vaccine. Based on the combined data of individuals from three breed populations, genome-wide scanning of QTL for these traits was performed based on a variance component model, and the genome wide significance level for declaring QTL was determined via permutation tests as well as FDR (false discovery rate) correction. A total of 27 QTL (two for CD4+CD8+, one for CD4+CD8-, three for CD4-CD8+, two for CD4-CD8-, nine for CD4+, two for CD8+, and eight for CD4+:CD8+ ratio) were identified with significance level of FDR < 0.10, of which 11 were significant at the level of FDR < 0.05, including the five significant at FDR < 0.01.ConclusionsWithin these QTL regions, a number of known genes having potential relationships with the studied traits may serve as candidate genes for these traits. Our findings herein are helpful for identification of the causal genes underlying these immune-related trait and selection for immune capacity of individuals in swine breeding in the future.

BackgroundHeavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants. Heavy-ion beams show high linear energy transfer (LET) and thus more effectively induce DNA double-strand breaks than other mutagenic techniques. Previously, we determined the most effective heavy-ion LET (LETmax: 30.0 keV μm-1) for Arabidopsis mutagenesis by analysing the effect of LET on mutation induction. However, the molecular structure of mutated DNA induced by heavy ions with LETmax remains unclear. Knowledge of the structure of mutated DNA will contribute to the effective exploitation of heavy-ion beam mutagenesis.ResultsDry Arabidopsis thaliana seeds were irradiated with carbon (C) ions with LETmax at a dose of 400 Gy and with LET of 22.5 keV μm-1 at doses of 250 Gy or 450 Gy. The effects on mutation frequency and alteration of DNA structure were compared. To characterise the structure of mutated DNA, we screened the well-characterised mutants elongated hypocotyls (hy) and glabrous (gl) and identified mutated DNA among the resulting mutants by high-resolution melting curve, PCR and sequencing analyses. The mutation frequency induced by C ions with LETmax was two-fold higher than that with 22.5 keV μm-1 and similar to the mutation frequency previously induced by ethyl methane sulfonate. We identified the structure of 22 mutated DNAs. Over 80% of the mutations caused by C ions with both LETs were base substitutions or deletions/insertions of less than 100 bp. The other mutations involved large rearrangements.ConclusionsThe C ions with LETmax showed high mutation efficiency and predominantly induced base substitutions or small deletions/insertions, most of which were null mutations. These small alterations can be determined by single-nucleotide polymorphism (SNP) detection systems. Therefore, C ions with LETmax might be useful as a highly efficient reverse genetic system in conjunction with SNP detection systems, and will be beneficial for forward genetics and plant breeding.

BackgroundHeavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants. Heavy-ion beams show high linear energy transfer (LET) and thus more effectively induce DNA double-strand breaks than other mutagenic techniques. Previously, we determined the most effective heavy-ion LET (LETmax: 30.0 keV μm-1) for Arabidopsis mutagenesis by analysing the effect of LET on mutation induction. However, the molecular structure of mutated DNA induced by heavy ions with LETmax remains unclear. Knowledge of the structure of mutated DNA will contribute to the effective exploitation of heavy-ion beam mutagenesis.ResultsDry Arabidopsis thaliana seeds were irradiated with carbon (C) ions with LETmax at a dose of 400 Gy and with LET of 22.5 keV μm-1 at doses of 250 Gy or 450 Gy. The effects on mutation frequency and alteration of DNA structure were compared. To characterise the structure of mutated DNA, we screened the well-characterised mutants elongated hypocotyls (hy) and glabrous (gl) and identified mutated DNA among the resulting mutants by high-resolution melting curve, PCR and sequencing analyses. The mutation frequency induced by C ions with LETmax was two-fold higher than that with 22.5 keV μm-1 and similar to the mutation frequency previously induced by ethyl methane sulfonate. We identified the structure of 22 mutated DNAs. Over 80% of the mutations caused by C ions with both LETs were base substitutions or deletions/insertions of less than 100 bp. The other mutations involved large rearrangements.ConclusionsThe C ions with LETmax showed high mutation efficiency and predominantly induced base substitutions or small deletions/insertions, most of which were null mutations. These small alterations can be determined by single-nucleotide polymorphism (SNP) detection systems. Therefore, C ions with LETmax might be useful as a highly efficient reverse genetic system in conjunction with SNP detection systems, and will be beneficial for forward genetics and plant breeding.