Neurons relay information via specialized presynaptic compartments for neurotransmission. Unlike conventional organelles, the specialized apparatus characterizing the neuronal presynapse must form de novo. How the components for presynaptic neurotransmission are transported and assembled is poorly understood. Our results show that the rare late endosomal signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P ₂ ] directs the axonal cotransport of synaptic vesicle and active zone proteins in precursor vesicles in human neurons. Precursor vesicles are distinct from conventional secretory organelles, endosomes, and degradative lysosomes and are transported by coincident detection of PI(3,5)P ₂ and active ARL8 via kinesin KIF1A to the presynaptic compartment. Our findings identify a crucial mechanism that mediates the delivery of synaptic vesicle and active zone proteins to developing synapses.

Live-cell imaging is a powerful technique to study the dynamics and mechanics of various biological molecules like proteins, organelles, DNA, and RNA. With the rapid evolution of optical microscopy, our understanding of how these molecules are implicated in the cells’ most critical physiological roles deepens. In this review, we focus on how spatiotemporal nanoscale live-cell imaging at the single molecule level allows for profound contributions towards new discoveries in life science. This review will start by summarizing how single-molecule tracking has been used to analyze membrane dynamics, receptor–ligand interactions, protein–protein interactions, inner- and extra-cellular transport, gene expression/transcription, and whole organelle tracking. We then move on to how current authors are trying to improve single-molecule tracking and overcome current limitations by offering new ways of labeling proteins of interest, multi-channel/color detection, improvements in time-lapse imaging, and new methods and programs to analyze the colocalization and movement of targets. We later discuss how single-molecule tracking can be a beneficial tool used for medical diagnosis. Finally, we wrap up with the limitations and future perspectives of single-molecule tracking and total internal reflection microscopy.

In p53-deficient cancers, targeting cholesterol metabolism has emerged as a promising therapeutic approach, given that p53 loss dysregulates sterol regulatory element-binding protein 2 (SREBP-2) pathways, thereby enhancing cholesterol biosynthesis. While cholesterol synthesis inhibitors such as statins have shown initial success, their efficacy is often compromised by the development of acquired resistance. Consequently, new strategies are being explored to disrupt cholesterol homeostasis more comprehensively by inhibiting its synthesis and intracellular transport. In this study, we investigate a previously underexplored function of PI5P4Ks, which catalyzes the conversion of PI(5)P to PI(4,5)P ₂ at intracellular membranes. Our findings reveal that PI5P4Ks play a key role in facilitating lysosomal cholesterol transport, regulating lysosome positioning, and sustaining growth signaling via the mTOR pathway. While PI5P4Ks have previously been implicated in mTOR signaling and tumor proliferation in p53-deficient contexts, this work elucidates an upstream mechanism that unifies these earlier observations.