Preparation of Retinal Samples for Volume Electron Microscopy.

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level precision. Within the retina, various types of neurons establish synaptic connections in the inner and outer plexiform layers. While conventional EM techniques have yielded valuable insights into retinal subcellular organelles, their limitation lies in providing 2D image data, which can hinder accurate measurements. For instance, quantifying the size of three distinct synaptic vesicle pools, crucial for synaptic transmission, is challenging in 2D. Volume EM offers a solution by providing large-scale, high-resolution 3D data. It is worth noting that sample preparation is a critical step in Volume EM, significantly impacting image clarity and contrast. In this context, we outline a sample preparation protocol for the 3D reconstruction of photoreceptor axon terminals in the retina. This protocol includes three key steps: retina dissection and fixation, sample embedding processes, and selection of the area of interest.

Cumulative effects of weakly repressive regulatory regions in the 3' UTR maintain PD-1 expression homeostasis in mammals.

PD-1 has become a common target for cancer treatment. However, the molecular regulation of PD-1 expression homeostasis remains unclear. Here we report the PD-1 3' UTR can dramatically repress gene expression via promoting mRNA decay. Deletion of the PD-1 3' UTR inhibits T cell activity and promotes T-ALL cell proliferation. Interestingly, the robust repression is attributable to cumulative effects of many weak regulatory regions, which we show together are better able to maintain PD-1 expression homeostasis. We further identify several RNA binding proteins (RBPs) that modulate PD-1 expression via the 3' UTR, including IGF2BP2, RBM38, SRSF7, and SRSF4. Moreover, despite rapid evolution, PD-1 3' UTRs are functionally conserved and strongly repress gene expression through many common RBP binding sites. These findings reveal a previously unrecognized mechanism of maintaining PD-1 expression homeostasis and might represent a general model for how small regulatory effects play big roles in regulation of gene expression and biology.