Biology subjects

Ko, F.

Publications and source records attributed to Ko, F..

3 recordsLinked to original sources

bioRxiv2024

Clearing-enabled light sheet microscopy as a novel method for three-dimensional mapping of the sensory innervation of the mouse knee

A major barrier that hampers our understanding of the precise anatomic distribution of pain sensing nerves in and around the joint is the limited view obtained from traditional two dimensional (D) histological approaches. Therefore, our objective was to develop a workflow that allows examination of the innervation of the intact mouse knee joint in 3D by employing clearing-enabled light sheet microscopy. We first surveyed existing clearing protocols (SUMIC, PEGASOS, and DISCO) to determine their ability to clear the whole mouse knee joint, and discovered that a DISCO protocol provided the most optimal transparency for light sheet microscopy imaging. We then modified the DISCO protocol to enhance binding and penetration of antibodies used for labeling nerves. Using the pan-neuronal PGP9.5 antibody, our protocol allowed 3D visualization of innervation in and around the mouse knee joint. We then implemented the workflow in mice intra-articularly injected with nerve growth factor (NGF) to determine whether changes in the nerve density can be observed. Both 3D and 2D analytical approaches of the light sheet microscopy images demonstrated quantifiable changes in midjoint nerve density following 4 weeks of NGF injection in the medial but not in the lateral joint compartment. We provide, for the first time, a comprehensive workflow that allows detailed and quantifiable examination of mouse knee joint innervation in 3D.

neuroscience↗

bioRxiv2023

A CRISPR activation screen identifies FBXO22 as an E3 ligase supporting targeted protein degradation

Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. This expansion will broaden the scope of potential protein targets, accommodating those with varying subcellular localizations and expression patterns. In this study, we describe a CRISPR-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. This approach allows us to address the limitations associated with investigating candidate degrader molecules in specific cell lines that either lack or have low levels of the desired E3 ligases. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FKBP12 when the FBXO22 gene transcription is activated. 22-SLF induced the degradation of endogenous FKBP12 in a FBXO22-dependent manner across multiple cancer cell lines. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in FBXO22 to achieve the target degradation. Finally, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading another endogenous protein BRD4. This study uncovers FBXO22 as an E3 ligase capable of supporting ligand-induced protein degradation through electrophilic PROTACs. The platform we have developed can readily be applied to elucidate protein degradation pathways by identifying E3 ligases that facilitate either small molecule-induced or endogenous protein degradation.

biochemistry↗

bioRxiv2021

Numb is required for optimal contraction of skeletal muscle

BackgroundThe role of Numb, a protein that is important for cell fate and development was investigated in adult skeletal muscle in mice using a conditional, inducible knockout (cKO) model. MethodsNumb expression was evaluated by Western blot. Numb localization was determined by confocal microscopy. The effects of cKO of Numb and the closely-related gene Numb-like in skeletal muscle fibers was evaluated by in-situ physiology; transmission and focused ion beam scanning electron microscopy; 3-dimensional reconstruction of mitochondrial; lipidomics; and bulk RNA-sequencing. Additional studies using primary mouse myotubes investigated the effects the effects of Numb knockdown on cell fusion, mitochondrial function and calcium transients. ResultsNumb protein expression was reduced by [~]70% (p < 0.01) at 24 as compared to 3 months of age. Numb was localized within muscle fibers as bands traversing fibers at regularly spaced intervals in close proximity to dihydropyridine receptors. The cKO of Numb and Numb-like reduced specific tetanic force by 36%, p < 0.01), altered mitochondrial spatial relationships to sarcomeric structures, increased Z-line spacing by 30% (p < 0.0001), perturbed sarcoplasmic reticulum organization and reduced mitochondrial volume by over 80% (p < 0.01). Only six genes were differentially expressed in cKO mice: Itga4, Sema7a, Irgm2, Vezf1, Mib1 and Tmem132a. Several lipid mediators derived from polyunsaturated fatty acid (PUFAs) through lipoxygenases were upregulated in Numb cKO skeletal muscle; 12-HEPE was increased by [~]250% (p < 0.05) and 17,18-EpETE by [~]240% (p < 0.05). In mouse primary myotubes, Numb knock-down reduced cell fusion ([~]20%, p < 0.01) and mitochondrial function and delayed the caffeine-induced rise in cytosolic calcium concentrations by more than 100% (p < 0.01). ConclusionsThese findings implicate Numb as a critical factor in skeletal muscle structure and function which appear to be critical for calcium release; we therefore speculate Numb plays critical roles in excitation-contraction coupling, one of the putative targets of aged skeletal muscles. These findings provide new insights into the molecular underpinnings of the loss of muscle function observed with sarcopenia.

physiology↗