Systems-level imaging reveals assembly and synaptic delivery of a multi-organelle ensemble during T cell killing

Cytotoxic T lymphocytes (CTLs) undergo a dynamic intracellular polarisation of their secretory compartments as they recognise and destroy cancerous cells. How multiple organelles are coordinated in space and time during this process is unknown. To enable high-dimensional systems-level imaging of multiple organelles and their relationships at high spatiotemporal resolution we use multispectral super-resolution microscopy and volumetric live-cell imaging of CTLs interacting with their targets. Organelle polarisation is initiated by perinuclear endoplasmic reticulum (ER) contacting the immune synapse, while calcium signalling promotes multi-organelle clustering around the centrosome at the rear of the cell. This creates a multi-organelle ensemble that then translocates around the nucleus to the synapse. We track organelle interactions during dynamic polarisation revealing a shift from ER-dominated to Golgi-dominated contacts as organelles reach the synapse. These findings show that CTL polarity is built through staged, calcium-driven formation of a multi-organelle assembly. More broadly, this work establishes a framework for systems-level imaging of the organelle network in cells as they deliver effector functions.