Visualizing intra-Golgi localization and transport by side-averaging Golgi ministacks

Area of Science:

• Cell Biology
• Molecular Biology
• Microscopy

Background:

• The mammalian Golgi apparatus, composed of flattened membrane sacs called cisternae, plays a crucial role in protein modification and transport.
• Understanding the molecular organization and intra-Golgi transport of cargos remains a significant challenge.
• Resolving Golgi proteins at the cisternal level using light microscopy is difficult.

Purpose of the Study:

• To develop and apply a novel microscopy approach for visualizing Golgi cisternal organization and intra-Golgi transport.
• To achieve unprecedented spatial resolution of Golgi protein distribution and cargo movement.

Main Methods:

• Development of a side-averaging approach for analyzing microscopy images.
• Acquisition of side-view images of nocodazole-induced Golgi ministacks using Airyscan microscopy.
• Image transformation, alignment, intensity normalization, and averaging of >30 Golgi proteins.

Main Results:

• Uncovered the organization of pre-Golgi, cis, medial, trans, and trans-Golgi network membranes with high spatial resolution.
• Observed the progressive transition of a synchronized cargo wave from the cis to the trans-Golgi.
• Confirmed that constitutive cargos exit at the trans-Golgi, while secretory targeting to the trans-Golgi network is signal-dependent.

Conclusions:

• The developed side-averaging method provides high-resolution insights into Golgi organization and intra-Golgi transport.
• The study elucidates the spatial organization of Golgi compartments and the dynamics of cargo trafficking.
• Findings reinforce the distinct mechanisms of cargo exit from the Golgi and targeting to the trans-Golgi network.