Association of Cell Adhesion Kinase bata (CAKbata/PYK2) with Cytoskeleton

Cell adhesion kinase bata (CAKbata/PYK2), which we identified by cDNA cloning, is the second protein-tyrosine kinase of the focal adhesion kinase subfamily and and has large N- and C-terminal domains in addition to the central kinase domain,In this paper, we report that CAKbata is present in association with both microtubules and microfilaments. The intracellular localization of CAKbata was studied in epithelial cells (A-431, Caco-2, and MDCK) and in nonepithelial cells (PC-12h and WFB), The immunocytochemical staining of epithelial cells revealed that CAKbata was mainly found at the cell-to-cell borders and at the perinuclear regions, although focal adhesions were also stained to some extent, The cell-to-cell immunostained in fine dotted lines and a significant portion of CAKbata was found in association with the microtubules, Moreever, an association of CAKbata with microfilaments was found in the cytoplasm, Among WFB cells only a limited number were positive for the CAKbata immunostaining at the focal contacts, although the protein was present in the tips of lamelipodia and microspikes in many cells, Furthermore, CAKbata was concentrated at the edge of ruffling membranes, When A431 cells were stimulated with epidermal growth factor, CAKbata rapidly moved to the edges of the ruffling membranes from the sites of cell-cell contacts where the protein had been present before the stimulation, Anti-actin or ati-alpha-tubulin antibodies coimmunoprecipitated CAKbata from a cytoskeleton-rich fraction of WFB cells. Moreover,the amount of CAKbata increased when the immunoprecipitation was carried out with the fraction prepared from the cells after stabilizing the tubular structure of the cytoskeleton.Imunoelectron microscopy showed that CAKbata was present along the microfilaments. In the WFB cells transfected with recombinant adenovirus expressing CAKbata, the protein was found at the focal adhesions of many cells and at the perinuclear region of all cells. Thus, CAKbata may associate with both microfilaments and microtubules, and play important roles for cellular movements such as membrane ruffling, cellular attachments, and cytokinesi

A rat model for LGI1-related epilepsies.

Mutations of the leucine-rich glioma-inactivated 1 (LGI1) gene cause an autosomal dominant partial epilepsy with auditory features also known as autosomal-dominant lateral temporal lobe epilepsy. LGI1 is also the main antigen present in sera and cerebrospinal fluids of patients with limbic encephalitis and seizures, highlighting its importance in a spectrum of epileptic disorders. LGI1 encodes a neuronal secreted protein, whose brain function is still poorly understood. Here, we generated, by ENU (N-ethyl-N-nitrosourea) mutagenesis, Lgi1-mutant rats carrying a missense mutation (L385R). We found that the L385R mutation prevents the secretion of Lgi1 protein by COS7 transfected cells. However, the L385R-Lgi1 protein was found at low levels in the brains and cultured neurons of Lgi1-mutant rats, suggesting that mutant protein may be destabilized in vivo. Studies on the behavioral phenotype and intracranial electroencephalographic signals from Lgi1-mutant rats recalled several features of the human genetic disorder. We show that homozygous Lgi1-mutant rats (Lgi1(L385R/L385R)) generated early-onset spontaneous epileptic seizures from P10 and died prematurely. Heterozygous Lgi1-mutant rats (Lgi1(+/L385R)) were more susceptible to sound-induced, generalized tonic-clonic seizures than control rats. Audiogenic seizures were suppressed by antiepileptic drugs such as carbamazepine, phenytoin and levetiracetam, which are commonly used to treat partial seizures, but not by the prototypic absence seizure drug, ethosuximide. Our findings provide the first rat model with a missense mutation in Lgi1 gene, an original model complementary to knockout mice. This study revealed that LGI1 disease-causing missense mutations might cause a depletion of the protein in neurons, and not only a failure of Lgi1 secretion

Disruption of Genetic Interaction Between Two Autosomal Regions and the X Chromosome Causes Reproductive Isolation Between Mouse Strains Derived From Different Subspecies

Reproductive isolation that initiates speciation is likely caused by incompatibility among multiple loci in organisms belonging to genetically diverging populations. Laboratory C57BL/6J mice, which predominantly originated from Mus musculus domesticus, and a MSM/Ms strain derived from Japanese wild mice (M. m. molossinus, genetically close to M. m. musculus) are reproductively isolated. Their F(1) hybrids are fertile, but successive intercrosses result in sterility. A consomic strain, C57BL/6J-ChrX(MSM), which carries the X chromosome of MSM/Ms in the C57BL/6J background, shows male sterility, suggesting a genetic incompatibility of the MSM/Ms X chromosome and other C57BL/6J chromosome(s). In this study, we conducted genomewide linkage analysis and subsequent QTL analysis using the sperm shape anomaly that is the major cause of the sterility of the C57BL/6J-ChrX(MSM) males. These analyses successfully detected significant QTL on chromosomes 1 and 11 that interact with the X chromosome. The introduction of MSM/Ms chromosomes 1 and 11 into the C57BL/6J-ChrX(MSM) background failed to restore the sperm-head shape, but did partially restore fertility. This result suggests that this genetic interaction may play a crucial role in the reproductive isolation between the two strains. A detailed analysis of the male sterility by intracytoplasmic sperm injection and zona-free in vitro fertilization demonstrated that the C57BL/6J-ChrX(MSM) spermatozoa have a defect in penetration through the zona pellucida of eggs