GLIM

Golgi protein Localization by Imaging centers of Mass 

Golgi protein Localization by Imaging centers of Mass (GLIM) is a quantitative super-resolution method used to map protein distribution along the Golgi's cis-to-trans axis. GLIM can achieve a practical accuracy of < 30 nm under conventional wide-field or spinning disk confocal microscopy. It provides a numerical Localization Quotient (LQ) to define sub-Golgi positions, making it more quantitative than qualitative methods like immuno-EM or standard super-resolution techniques.

Steps

• Acquisition: 

Cells are treated with nocodazole to disperse the Golgi into individual disk-shaped ministacks. Samples are triply labeled with a cis-marker (GM130), a trans-marker (GalT-mCherry), and the test protein.

• Analysis: 

The sub-pixel center of fluorescence mass is calculated to represent the position of each Golgi protein. To correct for chromatic shift aberrations of the microscope, multi-color fluorescent beads are imaged under identical conditions to calibrate the microscope and subsequently to correct the centers of fluorescence mass of Golgi ministacks. The distance between two proteins is defined as the distance between their respective centers of fluorescence mass. The LQ is calculated as the ratio of the distance from the test protein to GM130 to that from GalT-mCherry to GM130.

Selection Criteria of analyzable ministacks

1. Signal-to-Noise Ratio (SNR) criterion: The total intensity of the Golgi ministack must be > 30*SD in each channel.

2. Axial angle or distance criterion: The projected distance, d1, between GM130 and GalT-mCherry must be > 70 nm to exclude vertically oriented stacks.

3. Collinearity criterion: /tan(a)/ or /tan(b)/ ≤ 0.3. This ensures centers align along a one-dimensional Golgi axis.

Download

Protocol: 

• GLIM Protocol in PDF.

Macros:

1. GLIM_A_Bead particle analysis.ijm

2. GLIM_B_Bead output 3 channels.ijm

3. GLIM_1_Golgi particle analysis.ijm

4. GLIM_2_Golgi ROI inspection.ijm

5. GLIM_3_Golgi output 3 channel data.ijm

Training images:

• Zipped folder containing training images for GLIM.

References

1. Tie HC, Mahajan D, Chen B, Cheng L, VanDongen AM, Lu L. (2016) A novel imaging method for quantitative Golgi localization reveals differential intra-Golgi trafficking of secretory cargoes. Mol. Biol. Cell. 27:848-861.

2. Tie HC, Chen B, Sun X, Cheng L and Lu L. (2017) Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass. J. Vis. Exp. (126), e55996.