Phosphorylation of Synaptojanin Differentially Regulates Endocytosis of Functionally Distinct Synaptic Vesicle Pools

The rapid replenishment of synaptic vesicles through endocytosis is crucial for sustaining synaptic transmission during intense neuronal activity. Synaptojanin (Synj), a phosphoinositide phosphatase, is known to play an important role in vesicle recycling by promoting the uncoating of clathrin following synaptic vesicle uptake. Synj has been shown to be a substrate of the minibrain (Mnb) kinase, a fly homolog of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A); however, the functional impacts of Synj phosphorylation by Mnb are not well understood. Here we identify that Mnb phosphorylates Synj at S1029 in Drosophila. We find that phosphorylation of Synj at S1029 enhances Synj phosphatase activity, alters interaction between Synj and endophilin, and promotes efficient endocytosis of the active cycling vesicle pool (also referred to as exo-endo cycling pool) at the expense of reserve pool vesicle endocytosis. Dephosphorylated Synj, on the other hand, is deficient in the endocytosis of the active recycling pool vesicles but maintains reserve pool vesicle endocytosis to restore total vesicle pool size and sustain synaptic transmission. Together, our findings reveal a novel role for Synj in modulating reserve pool vesicle endocytosis and further indicate that dynamic phosphorylation and dephosphorylation of Synj differentially maintain endocytosis of distinct functional synaptic vesicle pools. SIGNIFICANCE STATEMENT Synaptic vesicle endocytosis sustains communication between neurons during a wide range of neuronal activities by recycling used vesicle membrane and protein components. Here we identify that Synaptojanin, a protein with a known role in synaptic vesicle endocytosis, is phosphorylated at S1029 in vivo by the Minibrain kinase. We further demonstrate that the phosphorylation status of Synaptojanin at S1029 differentially regulates its participation in the recycling of distinct synaptic vesicle pools. Our results reveal a new role for Synaptojanin in maintaining synaptic vesicle pool size and in reserve vesicle endocytosis. As Synaptojanin and Minibrain perturbations are associated with various neurological disorders, such as Parkinson's, autism, and Down syndrome, understanding mechanisms modulating Synaptojanin function provides valuable insights into processes affecting neuronal communication

Winter hardiness of \u3ci\u3eMiscanthus\u3c/i\u3e (III): Genome‐wide association and genomic prediction for overwintering ability in \u3ci\u3eMiscanthus sinensis\u3c/i\u3e

Overwintering ability is an important selection criterion for Miscanthus breeding in temperate regions. Insufficient overwintering ability of the currently leading Miscanthus biomass cultivar, M. ×giganteus (M×g) ‘1993–1780’, in regions where average annual minimum temperatures are −26.1°C (USDA hardiness zone 5) or lower poses a pressing need to develop new cultivars with superior cold tolerance. To facilitate breeding of Miscanthus, this study characterized phenotypic and genetic variation of overwintering ability in an M. sinensis germplasm panel consisting of 564 accessions, evaluated in field trials at three locations in North America and two in Asia. Genome‐wide association (GWA) and genomic prediction analyses were performed. The Korea/N China M. sinensis genetic group is a valuable gene pool for cold tolerance. The Yangtze‐Qinling, Southern Japan, and Northern Japan genetic groups were also potential sources of cold tolerance. A total of 73 marker–trait associations were detected for overwintering ability. Estimated breeding value for overwintering ability based on these 73 markers could explain 55% of the variation for first winter overwintering ability among M. sinensis. Average genomic prediction ability for overwintering ability across 50 fivefold cross‐validations was high (~0.73) after accounting for population structure. Common genomic regions for overwintering ability were detected by GWA analyses and a previous parallel QTL mapping study using three interconnected biparental F1 populations. One QTL on Miscanthus LG 8 encompassed five GWA hits and a known cold‐responsive gene, COR47. The other overwintering ability QTL on Miscanthus LG 11 contained two GWA hits and three known cold stress‐related genes, carboxylesterase 13 (CEX13), WRKY2 transcription factor, and cold shock domain (CSDP1). Miscanthus accessions collected from high latitude locations with cold winters had higher rates of overwintering, and more alleles for overwintering, than accessions collected from southern locations with mild winters

Realistic optical cell modeling and diffraction imaging simulation for study of optical and morphological parameters of nucleus

Coherent light scattering presents complex spatial patterns that depend on morphological and molecular features of biological cells. We present a numerical approach to establish realistic optical cell models for generating virtual cells and accurate simulation of diffraction images that are comparable to measured data of prostate cells. With a contourlet transform algorithm, it has been shown that the simulated images and extracted parameters can be used to distinguish virtual cells of different nuclear volumes and refractive indices against the orientation variation. These results demonstrate significance of the new approach for development of rapid cell assay methods through diffraction imaging.ECU Open Access Publishing Support Fun

Genetic structure of Miscanthus sinensis and Miscanthus sacchariflorus in Japan indicates a gradient of bidirectional but asymmetric introgression

Unilateral introgression from diploids to tetraploids has been hypothesized to be an important evolutionary mechanism in plants. However, few examples have been definitively identified, perhaps because data of sufficient depth and breadth were difficult to obtain prior to the advent of affordable high-density genotyping. Throughout Japan, tetraploid Miscanthus sacchariflorus and diploid M. sinensis are common, and occasionally hybridize. In this study, we characterized 667 M. sinensis and 78 M. sacchariflorus genotypes from Japan using 20,704 SNPs and ten plastid microsatellites. Similarity of SNP genotypes between diploid and tetraploid M. sacchariflorus indicated that the tetraploids originated via autopolyploidy. Structure analysis indicated a gradient of introgression from diploid M. sinensis into tetraploid M. sacchariflorus throughout Japan; most tetraploids had some M. sinensis DNA. Among phenotypically M. sacchariflorus tetraploids, M. sinensis ancestry averaged 7% and ranged from 1-39%, with introgression greatest in southern Japan. Unexpectedly, rare (~1%) diploid M. sinensis individuals from northern Japan were found with 6-27% M. sacchariflorus ancestry. Population structure of M. sinensis in Japan included three groups, and was driven primarily by distance, and secondarily by geographic barriers such as mountains and straits. Miscanthus speciation is a complex and dynamic process. In contrast to limited introgression between diploid M. sacchariflorus and M. sinensis in northern China, selection for adaptation to a moderate maritime climate likely favored cross-ploidy introgressants in southern Japan. Our results will help guide the selection of Miscanthus accessions for the breeding of biomass cultivars.Office of Science - Biological and Environmental Research, U.S. Department of Energy [Project ID 0017582]Energy Biosciences InstituteOpe

Genome biology of the paleotetraploid perennial biomass crop Miscanthus

Miscanthus is a perennial wild grass that is of global importance for paper production, roofing, horticultural plantings, and an emerging highly productive temperate biomass crop. We report a chromosome-scale assembly of the paleotetraploid M. sinensis genome, providing a resource for Miscanthus that links its chromosomes to the related diploid Sorghum and complex polyploid sugarcanes. The asymmetric distribution of transposons across the two homoeologous subgenomes proves Miscanthus paleo-allotetraploidy and identifies several balanced reciprocal homoeologous exchanges. Analysis of M. sinensis and M. sacchariflorus populations demonstrates extensive interspecific admixture and hybridization, and documents the origin of the highly productive triploid bioenergy crop M. x giganteus. Transcriptional profiling of leaves, stem, and rhizomes over growing seasons provides insight into rhizome development and nutrient recycling, processes critical for sustainable biomass accumulation in a perennial temperate grass. The Miscanthus genome expands the power of comparative genomics to understand traits of importance to Andropogoneae grasses

Isolation and Characterization of a Metastatic Hybrid Cell Line Generated by ER Negative and ER Positive Breast Cancer Cells in Mouse Bone Marrow

BACKGROUND: The origin and the contribution of breast tumor heterogeneity to its progression are not clear. We investigated the effect of a growing orthotopic tumor formed by an aggressive estrogen receptor (ER)-negative breast cancer cell line on the metastatic potential of a less aggressive ER-positive breast cancer cell line for the elucidation of how the presence of heterogeneous cancer cells might affect each other's metastatic behavior. METHODS: ER positive ZR-75-1/GFP/puro cells, resistant to puromycin and non-tumorigenic/non-metastatic without exogenous estrogen supplementation, were injected intracardiacally into mice bearing growing orthotopic tumors, formed by ER negative MDA-MB-231/GFP/Neo cells resistant to G418. A variant cell line B6, containing both estrogen-dependent and -independent cells, were isolated from GFP expressing cells in the bone marrow and re-inoculated in nude mice to generate an estrogen-independent cell line B6TC. RESULTS: The presence of ER negative orthotopic tumors resulted in bone metastasis of ZR-75-1 without estrogen supplementation. The newly established B6TC cell line was tumorigenic without estrogen supplementation and resistant to both puromycin and G418 suggesting its origin from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. Compared to parental cells, B6TC cells were more metastatic to lung and bone after intracardiac inoculation. More significantly, B6TC mice also developed brain metastasis, which was not observed in the MDA-MB-231/GFP/Neo cell-inoculated mice. Low expression of ERα and CD24, and high expression of EMT-related markers such as Vimentin, CXCR4, and Integrin-β1 along with high CD44 and ALDH expression indicated stem cell-like characteristics of B6TC. Gene microarray analysis demonstrated a significantly different gene expression profile of B6TC in comparison to those of parental cell lines. CONCLUSIONS: Spontaneous generation of the novel hybrid cell line B6TC, in a metastatic site with stem cell-like properties and propensity to metastasize to brain, suggest that cell fusion can contribute to tumor heterogeneity