Biology subjects

Ler, S. G.

Publications and source records attributed to Ler, S. G..

3 recordsLinked to original sources

bioRxiv2026

Ovarian extracellular matrix mechanics regulate oocyte-follicle interactions during female reproductive aging

Female reproductive aging is associated with ovarian functional decline, leading to infertility. During aging, biochemical and biophysical changes in the ovarian extracellular matrix (ECM) occur, yet how these properties affect follicle growth and oocyte quality remains poorly understood. Here we describe spatiotemporal changes in the ovarian ECM with age using mass spectrometry, immunohistochemistry, and nanoindentation. While follicle stiffness remains unchanged, stromal matrix remodeling is associated with a [~]2.5-fold increase in stiffness. To understand how this increase in stromal stiffness affects age-related follicular dysfunction, isolated young follicles were cultured in soft and stiff hydrogels mimicking young and aged ovarian stromal stiffness, respectively. Higher stiffness leads to a decrease in granulosa cell (GC) proliferation, oocyte quality, and GC-oocyte interactions mediated via transzonal projections (TZPs). RNA-seq revealed TGF-{beta} signaling as a major pathway affected by stiffness, and activation of TGF-{beta} signaling through Mongersen treatment rescued TZP formation and oocyte quality in stiff matrix. These findings provide mechanistic insight into how changes in ECM mechanics contribute to ovarian aging functional decline and reveal potential therapeutic targets to counter fertility loss associated with tissue aging and fibrosis.

cell biology↗

bioRxiv2025

Optogenetic control of Protein Kinase C-epsilon activity reveals its intrinsic signaling properties with spatiotemporal resolution

The regulation of PKC epsilon (PKC{varepsilon}) and its downstream effects is still not fully understood, making it challenging to develop targeted therapies or interventions. A more precise tool that enables spatiotemporal control of PKC{varepsilon} activity is thus required. Here, we describe a photo-activatable optogenetic PKC{varepsilon} probe (Opto-PKC{varepsilon}) consisting of an engineered PKC{varepsilon} catalytic domain and a blue-light inducible dimerization domain. Molecular dynamics and AlphaFold simulations enable rationalization of the dark-light activity of the optogenetic probe. We first characterize the binding partners of Opto-PKC{varepsilon}, which are similar to those of PKC{varepsilon}. Subsequent validation of the Opto-PKC{varepsilon} tool is performed with phosphoproteome analysis, which reveals that only PKC{varepsilon} substrates are phosphorylated upon light activation. Opto-PKC{varepsilon} could be engineered for recruitment to specific subcellular locations. Activation of Opto-PKC{varepsilon} in isolated hepatocytes reveals its sustained activation at the plasma membrane is required for its phosphorylation of the insulin receptor at Thr1160. In addition, Opto-PKC{varepsilon} recruitment to the mitochondria results in its lowering of the spare respiratory capacity through phosphorylation of complex I NDUFS4. These results demonstrate that Opto-PKC{varepsilon} may have broad applications for the studies of PKC{varepsilon} signaling with high specificity and spatiotemporal resolution. SummaryWe have developed a photo-activatable optogenetic PKC{varepsilon} probe, which demonstrates differential activity in light versus dark. The tool is subsequently validated with protein association studies and phosphoproteome analysis. It enables dissection of signaling events arising from its activation at defined subcellular locations. For instance, sustained activation of PKC{varepsilon} at the plasma membrane is required for its phosphorylation of the insulin receptor at Thr1160.

molecular biology↗

bioRxiv2024

Spatiotemporal control of subcellular O-GlcNAc signaling using Opto-OGT

The posttranslational modification of intracellular proteins through O-linked {beta}-N-acetylglucosamine (O-GlcNAc) is a conserved regulatory mechanism in multicellular organisms. Catalyzed by O-GlcNAc transferase (OGT), this dynamic modification plays an essential role in signal transduction, gene expression, organelle function, and systemic physiology. Here we present Opto-OGT, an optogenetic probe that allows for precise spatiotemporal control of OGT activity through light stimulation. By fusing a photosensitive cryptochrome protein to OGT, Opto-OGT can be robustly and reversibly activated with high temporal resolution by blue light and exhibits minimal background activity without illumination. Transient activation of Opto-OGT results in mTORC activation and AMPK suppression which recapitulate nutrient-sensing signaling. Furthermore, Opto-OGT can be customized to be localized at specific subcellular sites. By targeting OGT to the plasma membrane, we demonstrate downregulation of site-specific AKT phosphorylation and signaling outputs in response to insulin stimulation. Thus, Opto-OGT is a powerful tool to define the role of O-GlcNAcylation in cell signaling and physiology.

cell biology↗