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

Neuronal Cells

 

Chung-Chih Lin

Departmentof 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 0.3-1 micron,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, 0.3-1 micron, are in neurites and most of them are still.  Several

large dots of GFP-Rab3A, larger than 1 micron, 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.