pHusion: A robust and versatile toolset for automated detection and analysis of exocytosis

Abstract

Exocytosis is a fundamental process used by all eukaryotic cells to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate the role exocytosis plays in neuronal morphogenesis, previously we developed computational tools with a graphical user interface (GUI) to enable the automatic detection and analysis of exocytic events (ADAE GUI) from fluorescence timelapse images. Though these tools have proven useful, we found that the code was brittle and not easily adapted to different experimental conditions. Here, we developed and validated a robust and versatile toolkit, we have named pHusion, for the analysis of exocytosis written in Image-Tank, a graphical programming language that combines image visualization and numerical methods. We tested this method using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types, and various exocytic markers to generate a flexible and intuitive package. Using pHusion, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are much longer lasting than those in immature murine cortical neurons, and that clustering of exocytic events occurs across cell types.

TRIM9 controls growth cone responses to netrin through DCC and UNC5C

“…Doxycycline (Sigma, D9891)-inducible adenoviral constructs were generated for Halo-TRIM9 and UNC5C-pHmScarlet using the adeno-X TM system 3 (Takara Bio, 631180), using the detailed protocol outlined by (O'Shaughnessy et al, 2024). The TRIM9-Halo construct was created using pcs2-Myc-TRIM9 plasmid (Winkle et al, 2014).…”