Functional Analysis Of Tubgcp4 In Embryogenesis Of Early Stage And Retinal Degeneration In Mice

Microtubules are very important in a number of cellular processes.They form the cytoskeleton,together with microfilaments and intermediate filaments,which play important roles in maintaining of the cell structure and functions.They are involved in a variety of cellular processes,including movement of secretory vesicles,organelles,and intracellular macromolecular assemblies.In metazoan,?-Tubulin Small Complexes(?-TuSCs)composed of ?-tubulin and two other proteins(TUBGCP2 and TUBGCP3)are essential to the microtubules nucleation.The ?-Tubulin Ring Complexes(?-TuRCs)are formed of y-TuSCs associated with three additional proteins(TUBGCP4 and TUBGCP5 and TUBGCP6).?-TuSCs-specific proteins are essential for embryo development,however,the function of ?-TuRCs-specific proteins in embryogenesis are largely unknown.TUBGCP4 variants have been identified in patients with autosomal-recessive microcephaly and chorioretinopathy(MCCRP),but the mechanism causing the disease remains to be explored.In this study,a line of TUBGCP4 heterozygous mutant(Tubgcp4+/-)mice by homologous recombination was generated.These Tubgcp4+/-mice appeared indistinguishable from wild-type(Tubgcp4+/+)mice.Although heterozygous mice can produce offspring,the ratio of wild type and heterozygous was close to 2:1 and no homozygous mutant(Tubgcp4-/-)mice were born,indicating that Tubgcp4-/-mice died before birth.To investigate Tubgcp4-/-embryonic lethality,embryos of different stages were analyzed for their genotypes,which showed Tubgcp4-/-embryos death at 6.5 day after fertilization and development disorders of primitive streak.This data suggested that Tubgcp4 play an important role in the formation of primitive streak during embryonic development.In order to explore how Tubgcp4 lead to autosomal-recessive microcephaly with chorioretinopathy,the head circumference and retinal function of Tubgcp4+/' mice were further analyzed.Electroretinography(ERG)showed that both rods reponse and cones reponse were significantly lower in Tubgcp4+/-mice retina than those in Tubgcp4+/+ mice retina,indicating that there was dysfunction in Tubgcp4+/-mice retina.Transmission electron microscopy showed that Tubgcp4 mutation resulted in degeneration and structure disturbance in retinal photoreceptor outer segment in Tubgcp4+/-mice.In addition,autophagosomes were observed in retinal photoreceptor iner segment and retinal pigment epithelium(RPE)cell.y-TuRCs had depolymerized in the retina of heterozygous mice.This study will be helpful for our understanding of the function of microtubules minus binding protein in maintaining the structure and function of photoreceptor cells.Tubgcp4 can be used as a new target for the treatment of human illness associated with retinal degeneration.Autophagy is a cellular catabolic process that relies on the cooperation of autophagosomes and lysosomes,which functions in support metabolism in response to starvation and to clean damaged proteins and organelles in response to stress.Genetic evidences have indicated that autophagy plays an important role in the renewing process of photoreceptor outer segment.The expression levels of LC3-II in retina were analyzed using Western blots,which showed that LC3-II protein in the heterozygous mice was higher than in wild-type mice.In addition,autophagosomes were observed mainly in the photoreceptor inner segment and RPE cells by staining of frozen section of retina with LC3 antibody.Besides,Further Co-IP showed that Tubgcp4 can interact with a autophagy protein Atg7.These results suggested that the y-TuRCs-specific protein Tubgcp4 may play an important role in maintaining retinal structure and functions in mammals.