Sumana Raychaudhuri

In neurons, many membrane proteins that are synthesized in the cell body must be efficiently delivered to axons to regulate neuronal connectivity. Transcytosis is an atypical transport mode in which membrane proteins internalized from soma membranes are transported to axons in an anterograde fashion. Here, we characterized the trafficking dynamics and mechanism of transcytosis of the receptor TrkA from the soma in response to nerve growth factor (NFG) signaling at the axon in mouse sympathetic neurons. Live imaging and electron microscopy of compartmentalized cultures revealed that soma surface–derived TrkA proteins underwent dynamic transport in axons, with changes in speed, direction, and the vesicular organelles that carried them as they moved from proximal to distal axon compartments. In mice, soma surface–labeled TrkA proteins were observed in sympathetic nerve terminals, demonstrating that transcytosis occurs in vivo. Transcytosed TrkA proteins were enriched at presynaptic varicosities, bouton-like structures that store and release neurotransmitters. Disrupting its transcytosis by introducing a point mutation into TrkA reduced the number and size of presynaptic sites and decreased synaptic transmission in vivo and in culture. These findings provide mechanistic insight into an atypical mode of receptor trafficking and demonstrate its physiological relevance in sympathetic neuron connectivity in mice.

This protocol describes freezing neurons under high pressure after stimulation and imaging the neurons under electron microscope afterwards. At first, hippocampal neurons were cultured form embryonic tissues, treated with drug for 7 days, frozen in high pressure freezer. After freeze substitution and embedding in Epoxy based resins, thin sections were cut, stained and imaged at 100000x magnification under electron microscope. Images were segmened blinded using FiJi and custom macro and data were analyzed in Matlab.

Inflammation-associated fibroblasts (IAFs) are associated with progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial cells is unknown. Here, we developed an in vitro model whereby human colon fibroblasts are induced by specific cytokines and recapitulate key features of IAFs in vivo. When cocultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid expansion and barrier disruption due to swelling and rupture of individual epithelial cells. Colonoids cocultured with IAFs also show increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated by a paracrine pathway involving prostaglandin E ₂ and its receptor EP4, leading to protein kinase A -dependent activation of the cystic fibrosis transmembrane conductance regulator. EP4-specific chemical inhibitors effectively prevented IAF-induced colonoid swelling and restored normal proliferation and genome stability. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a therapeutic avenue to mitigate inflammation-associated epithelial injury.

Oxysterol-binding protein (OSBP)-related proteins (ORPs) are lipid-binding proteins that are conserved from yeast to humans. They are implicated in many cellular processes including signaling, vesicular trafficking, lipid metabolism, and nonvesicular sterol transport. All ORPs contain an OSBP-related domain (ORD) that has a hydrophobic pocket that binds a single sterol. ORDs also contain additional membrane-binding surfaces, some of which bind phosphoinositides and may regulate sterol binding. Studies in yeast suggest that ORPs function as sterol transporters, perhaps in regions where organelle membranes are closely apposed. Yeast ORPs also participate in vesicular trafficking, although their role is unclear. In mammalian cells, some ORPs function as sterol sensors that regulate the assembly of protein complexes in response to changes in cholesterol levels. This review will summarize recent advances in our understanding of how ORPs bind lipids and membranes and how they function in diverse cellular processes.

Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein–related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.