Project description:As the biology of hematopoietic stem cells (HSCs) has been predominantly studied under transplantation conditions, it has been challenging to study dynamic HSC behaviors in their native niche given under steady state conditions. Here, we describe the generation of a double knock-in fluorescent reporter that restricts the reporter labeling exclusively to the LT-HSC compartment. Single cell RNAseq comparing previously published LSK signatures (GEO: GSE90742) to our sorted fluorescently labeled cells (sorted without the use of any other cell surface markers) demonstrates that the fluorescently marked cells in our unique mouse model are exclusively found in the LT-HSC compartment in terms of their overal RNA content. This confirms that the generated mice label LT-HSCs in a highly specific manner.
Project description:We introduce CALIPERS, a Fluorescence Ubiquitin Cell Cycle Indicator enabling cell cycle-aware phenotyping based on multiplexed live-cell imaging. We validate CALIPERS in three- and four-color reporter lines (HaCaT) and human induced pluripotent stem cells (WTC-11), co-expressing genetically encoded structural and functional fluorescent sensors. To exemplify the broad applications of CALIPERS, we show cell cycle-aware phenotyping in live-cell imaging applications ranging from proliferation and migration to cardiac-specific maturation and drug testing.
Project description:Here we directly compare for the first time how the longstanding static model of mouse Dentate Gyrus (DG) development compares with a comprehensive high-resolution live-cell multiphoton (live-MPM) imaging approach. We took advantage of multiple fluorescent protein-based cell-type specific reporters to identify Neural Stem Cells (NSC), Intermediate Neurogenic Progenitors (INPs), and Granule Neurons (GNs) to generate live 4D cellular datasets across embryonic, postnatal and adult ages. Live-MPM revealed that INPs and NSCs migrated long distances along multiple routes to seed the SGZ from multiple directions, and from mosaic progenitor zones along the septo-temporal axis of the hippocampus. We found that dynamic INPs processes and interactions contributed to the architecture of both transient and permanent NSC niches during embryonic development, and that INP cellular plasticity is maintained in the adult SGZ NSC niche. We also used a Molecular Systems (MS) approach to determine the basis for maintained INP cellular plasticity that revealed an overlapping signaling network infrastructure based largely on Rho-family mediated regulation of cytoskeletal dynamics. Our combined strategies revealed that dynamic INPs are a major molecular signaling transition state in the adult SGZ, and that Tbr2 expression defines the initial stage of GN commitment. Our novel findings reveal fundamental new insight into one of the most well studied brain regions key for normal cognitive function, and the importance of analyzing the development of live stem cell niches in vivo. In concert with live-cell imaging, we used microarray analysis to identify genes that may be involved in the development of the Dentate Gyrus NSC niche.
Project description:Eukaryotic cells regulate higher-order chromatin architecture, gene expression, and gene recombination via compaction of the genome into chromatin loops and topologically associating domains (TADs). While chromatin architecture has been thoroughly characterized for many eukaryotic genomes using cell-destructive techniques such as 3C-based methods, live-cell biosensing tools that can probe three-dimensional chromatin contacts in real-time are lacking. Using a dual dCas9 DNA biosensor based on a split NanoLuc luciferase reporter, we directly detected chromatin loops in live cells using luminescence quantification in a luminometer. We were able to see signal above background ratios of up to 10-fold. In addition, we directly visualized chromatin looping at the MYC TAD in live cells using high-resolution, low light live-cell imaging. Our biosensing platform therefore provides a useful methodology for live-cell, real-time detection of known or novel loops and for monitoring looping dynamics upon alterations in cell state.
Project description:We present a spatiotemporal m6A imaging system (SMIS) that can monitor the m6A modification and the translation of mRNAs with high specificity and sensitivity in a single live cell. The SMIS system contains a m6A-modified reporter mRNA and fluorescence resonance energy transfer (FRET) biosensors. To quantify the methylation viariation achieved at m6A sites in the reporter mRNA after STM2457 treatment, a classical METTL3 inhibitor, we performed m6ACE-seq to measure the relative m6A level with single-base-resolusion.