Biology subjects

Chen, J. A.

Publications and source records attributed to Chen, J. A..

3 recordsLinked to original sources

bioRxiv2025

Biosynthesis of minimal C-phycocyanin chromophore assemblies in E. coli provides a platform to dissect protein-mediated tuning of exciton transfer

Cyanobacteria are arguably among the most evolutionarily successful organisms on Earth, inhabiting a wide range of ocean, fresh water, soil, and even desert environments on every continent. The cyanobacterial phycobilisome consists of stacks of disk-like light-collecting moieties, allophycocyanin (APC) and phycocyanin (CPC), with covalently bound phycocyanobilin (PCB) pigments. The ways in which the energies of the specific chromophores in these complexes are tuned by the protein to achieve its highly efficient and directional energy transfer are not fully understood, as complex combinations of decay pathways are occurring simultaneously and competitively through this elaborate light-harvesting system. This makes it difficult to extract information about isolated protein-pigment interactions. We provide herein a description of a useful new experimental platform in which we have recombinantly expressed a fully functioning CPC complex and selectively created minimal chromophore sets to study their individual contributions to the overall CPC spectra. Structural and computational analysis of this protein system have provided a greater understanding of how the protein environment serves to alter the photophysics of each of these chromophores. Introduction of a quencher into various positions within CPC confirmed the ability of the protein environment to tune the directionality of energy transport in this assembly. Further mutational analysis suggested the roles of key amino acids surrounding the chromophores, showcasing the utility of heterologous expression techniques for understanding the effects of structure on EET mechanisms in the phycobilisome.

bioengineering↗

bioRxiv2023

YAP and TAZ differentially regulate postnatal cortical progenitor proliferation and astrocyte differentiation

WW domain-containing transcription regulator 1 (TAZ) and Yes-associated protein (YAP) are transcriptional co-activators traditionally studied together as a part of the Hippo pathway and best known for their roles in stem cell proliferation and differentiation. Despite their similarities, TAZ and YAP can exert divergent cellular effects by differentially interacting with other signaling pathways that regulate stem cell maintenance or differentiation. In the developing central nervous system, In this study, we show that TAZ regulates astrocytic differentiation and maturation of postnatal neural stem and progenitor cells (NPCs), and that TAZ mediates some but not all of the effects of bone morphogenetic protein (BMP) signaling on astrocytic development. By contrast, TAZ and YAP both mediate effects on NPC fate of {beta}1-integrin and integrin-linked kinase (ILK) signaling, and these effects are dependent on extracellular matrix (ECM) cues. These findings demonstrate that TAZ and YAP perform divergent functions in the regulation of astrocyte differentiation, where YAP regulates cell cycle states of astrocytic progenitors and TAZ regulates differentiation and maturation from astrocytic progenitors into astrocytes. Summary StatementAstrocytes are accounts for nearly half of the cells in the central nervous system, where they perform a diverse array of physiological functions. During development, astrocytes are primarily generated after neuronal differentiation in a stepwise manner from multiple glial committed progenitor subtypes. How gliogenic progenitors maintain proliferative properties versus differentiate into astrocytes is not fully understood. This work aims to elucidate how environmental signals utilizes molecularly similar intracellular components to achieve distinct developmental outcomes. In addition, many of the cell types that are involved in glial development are also present in brain tumors including glioblastoma. Knowledge on mechanisms regulating proliferation and differentiation of glial progenitors will provide insights into differences and similarities between normal and malignant cells.

neuroscience↗

bioRxiv2017

Precision genome-editing with CRISPR/Cas9 in human induced pluripotent stem cells

Genome engineering in human induced pluripotent stem cells (iPSCs) represent an opportunity to examine the contribution of pathogenic and disease modifying alleles to molecular and cellular phenotypes. However, the practical application of genome-editing approaches in human iPSCs has been challenging. We have developed a precise and efficient genome-editing platform that relies on allele-specific guideRNAs (gRNAs) paired with a robust method for culturing and screening the modified iPSC clones. By applying an allele-specific gRNA design strategy, we have demonstrated greatly improved editing efficiency without the introduction of additional modifications of unknown consequence in the genome. Using this approach, we have modified nine independent iPSC lines at five loci associated with neurodegeneration. This genome-editing platform allows for efficient and precise production of isogenic cell lines for disease modeling. Because the impact of CRISPR/Cas9 on off-target sites remains poorly understood, we went on to perform thorough off-target profiling by comparing the mutational burden in edited iPSC lines using whole genome sequencing. The bioinformatically predicted off-target sites were unmodified in all edited iPSC lines. We also found that the numbers of de novo genetic variants detected in the edited and unedited iPSC lines were similar. Thus, our CRISPR/Cas9 strategy does not specifically increase the mutational burden. Furthermore, our analyses of the de novo genetic variants that occur during iPSC culture and genome-editing indicate an enrichment of de novo variants at sites identified in dbSNP. Taken together, we propose that this enrichment represents regions of the genome more susceptible to mutation. Herein, we present an efficient and precise method for allele-specific genome-editing in iPSC and an analyses pipeline to distinguish off-target events from de novo mutations occurring with culture.

genetics↗