An Economic Method to Detect Dynamic Localization of GFP-Rab3A in Living Neuronal Cells

Chien-Chang Huang (黃建彰), Wei-Yu Chen (陳威宇) and Chung-Chih Lin (林崇智).  Department of Life Sciences, Chung-Shan Medical College, Taichung, Taiwan

 

Rab3A is one of small G proteins that regulate neurotransmitter release.  There are several lines of evidence indicating that Rab3A is involved in fusion of synaptic vesicles to plasma membrane.  But there is unclear where and when Rab3A plays a role in regulation of neurotransmission.  If Rab3A involves in membrane fusion, it must be there.  Therefore, dynamic localization of Rab3A will provide direct evidence to know location that Rab3A acts.

To visualize Rab3A in living cells, Rab3A fuses with a green fluorescent protein (GFP) and fusion protein is expressed in neuronal cells and fluorescence of GFP will indicate location of Rab3A protein.  Because Rab3A locates both in cytosol and membrane-bound compartments, lots of fluorescence background appears in our student-grade fluorescence microscope. When incident angle of excitation light is higher than critical angle, most of light is reflected and there is very intensive and thin exciting light near cell-glass interface, named evanescent wave, generated.  Because evanescent wave is concentrated about 30-300nm, there is no fluorophores, which is out of evanescent field, excited to generate fluorescent background.  Then background generated by out of evanescent field is eliminated and increase higher spatial resolution. This kind of technique is named evanescent wave fluorescence microscopy or total internal reflection fluorescence microscopy (TIRFM).  In prismless TIRFM, high NA objective and annular ring determine the path of excitation light to generate evanescent wave; we use these two parts to improve spatial resolution of our fluorescence microscope. 

Our improved setup can detect small dots of GFP-Rab3A, about 0.3-1mm, near perinuclear regions and neurites in neuronal cells.  Dynamic localization of GFP-Rab3A is recorded by conventional continuous microphotography, frame/8s.  The serial dynamic photos are scanned and the animation is made of scanned photos by GIF animator.  The angle and distance of trace and sizes of fluorescence dots are analyzed by NIH-image.  Small GFP-Rab3A dots, 0.3-1mm, are in neurites and most of them are still.  Several large dots of GFP-Rab3A, larger than 1mm, move among neurites and perinuclear region, and some of them are fused each other or break down into small vesicles. These results indicate that Rab3A is not only involved in vesicle docking but also in synaptic vesicle recycle.  From our results, slight modification of microscopy setup and uses of free softwares can reduce the cost to capture slow protein translocalization in living cells.