Immunofluorescence-Based Study of Cargo Transport Mediated by Cytoskeleton-Associated Molecular Complexes

The cytoskeleton plays a fundamental role in orchestrating the intracellular cargo transport, providing a dynamic scaffold that ensures the appropriate sorting, movement, and distribution of membrane-bound compartments. Through the coordinated activity of microtubules and actin filaments, cargo vesicles are driven along defined routes to ensure efficient intracellular trafficking. Among the molecular machineries that interact with the microtubule network, the intraflagellar transport (IFT) system has emerged as a key regulator of vesicular dynamics. Initially identified for its essential role in ciliogenesis, IFT is now recognized as a broader trafficking coordinator, mediating intracellular transport in different cellular processes. In T lymphocytes, IFT20, in collaboration with other IFT components, orchestrates the selective targeting of recycling vesicles within the endosomal system, promoting the accurate delivery of key receptors and signaling molecules. This chapter provides a detailed immunofluorescence-based protocol to study the role of the IFT system in vesicular trafficking, with a focus on its involvement in receptor recycling to the immune synapse and the retrograde transport of the mannose-6-phosphate receptor to the trans-Golgi network.