3D Imaging of Lipid-Guided Vesicle Trafficking Along the Cytoskeleton.

Abstract

Labeling lipids in vivo is a crucial step in exploring detailed molecular mechanisms coordinating phospholipid signaling and membrane trafficking. Using a lipid-binding domain fused with various fluorescent proteins (FPs) [such as single-colored FP, photoactivated-FP and FPs based on Förster resonance energy transfer (FRET)], lipid biosensors are used to monitor the spatiotemporal distribution, concentration, and dynamic characteristics of targeted lipids. Membrane trafficking is accompanied by rearrangement of membrane lipids and the cytoskeleton. 3D live-cell super-resolution (SR) imaging microscopy detects directional vesicle trafficking events in relation to lipids and the cytoskeleton near the plasma membrane (PM) in 3D space. 3D single-molecule co-tracking (smCo-tracking) provides time-lapse images of 3D colocalization between targeted lipids and vesicles. A dwell time pattern indicates the movement of vesicles towards exocytic sites at the PM. 3D single particle tracking (SPT) could track the overlapping trajectory of vesicles and lipids and display the distance to the cytoskeleton. Lipid biosensors provide a noninvasive and natural labeling for exploring the spatiotemporal dynamics and homeostasis of lipid in vivo. 3D live-cell SR imaging microscopy enables rapid, targeted access, deep imaging within samples at high spatial resolutions for real-time visualization of dynamic biological processes in vivo. 3D smCo-tracking analysis offers simultaneous tracking of different molecules to determine lipid-guided vesicle trafficking in a spatiotemporal organized manner. The 3D SPT technique could provide an accurate estimation of trajectories and spatiotemporal colocalization of vesicles and targeted lipids in 3D space, providing a high-precision visualization and depth track of directional vesicle trafficking. Directional vesicle trafficking is a complex and tightly organized process. Identifying specific lipids and other related regulators will contribute to the exploration of lipid vesicle–cytoskeleton interactions that drive directional delivery of cargos under various stress stimuli, particularly pathogen attacks. For 3D live-cell SR imaging microscopy, how to best balance the imaging speed, spatial-temporal resolution, and volumetric SR imaging capability remains a challenging task. Given that lipid derivatives cannot be genetically encoded, the brighter, more photostable and appropriate fluorophores visualizing the dynamic changes of targeted lipids in concentrations remain to be developed in plant cells. This work is supported by the National Natural Science Foundation of China ( 32030010 , 31900162 , 31770197 , 32000182 ), the Program of Introducing Talents of Discipline to Universities (111 project, B13007 ), and Young Elite Scientists Sponsorship Program by CAST ( 2019QNRC001 ). No interests are declared.