Hi guys,
I am working on live-cell imaging of primary cultured neurons. I want to identify the formation of synapses in my live-imaging. I doubt the transfection of synaptic markers as they may go to every where, even though using specific promoters. Is there any way to offer direct evidence? Most importantly, I want to know where the new formed synapses are.
Any ideas will be appreciated!
Best,
Yu
Hi Yu,
Are you interested in pre- or post-synaptic growth? Our lab uses CaMKII driven synaptophysin-tdTomato to assess synaptogenesis. We take before and after treatment images and look for changes within the same cell over time. This way we can control for over- or under-expressing cells. We also use primary neurons. I would have a little more faith in transfection efforts, I would say ~95% of the reporter does in fact localize to presynaptic terminals and we have increased synaptogenesis when adding TSA (positive control) and mitochondrial mobilization when adding Veratridine (again expected and supporting accurate presynaptic locale).
Bear in mind cultured cells are not in their native 3D tissue signaling environment. There is protein mistrafficking that will occur naturally when the system is changed (as in growing a brain cell in a plate rather than a brain). A well designed transfected reporter should reliably display protein expression just as good (if not better) than a primary antibody. That being said, the Arnold Lab in USC (http://dornsife.usc.edu/arnold) has created custom intrabodies for intracellular tagging of proteins and they have one for PSD95. Its avaibale on addGene, Ive used it and generated some great images with it but the signal to noise is rather low (too low for automated segmentation).
If you have any other questions feel free to ask!
Best,
Kyle
Article Recombinant Probes for Visualizing Endogenous Synaptic Prote...
Hi Yu,
Are you interested in pre- or post-synaptic growth? Our lab uses CaMKII driven synaptophysin-tdTomato to assess synaptogenesis. We take before and after treatment images and look for changes within the same cell over time. This way we can control for over- or under-expressing cells. We also use primary neurons. I would have a little more faith in transfection efforts, I would say ~95% of the reporter does in fact localize to presynaptic terminals and we have increased synaptogenesis when adding TSA (positive control) and mitochondrial mobilization when adding Veratridine (again expected and supporting accurate presynaptic locale).
Bear in mind cultured cells are not in their native 3D tissue signaling environment. There is protein mistrafficking that will occur naturally when the system is changed (as in growing a brain cell in a plate rather than a brain). A well designed transfected reporter should reliably display protein expression just as good (if not better) than a primary antibody. That being said, the Arnold Lab in USC (http://dornsife.usc.edu/arnold) has created custom intrabodies for intracellular tagging of proteins and they have one for PSD95. Its avaibale on addGene, Ive used it and generated some great images with it but the signal to noise is rather low (too low for automated segmentation).
If you have any other questions feel free to ask!
Best,
Kyle
Article Recombinant Probes for Visualizing Endogenous Synaptic Prote...
One more point, if the mistargeting you see is always surrounding the nucleus its actually the reporter being translated in the rough ER. This is appropriate as its part of the expression cycle, but it needs to be excluded. Exclusion can be done easily by expressing a cytosolic or nuclear-tagged reporter alongside your synaptic marker.
Hi Kyle,
Thank you for your detailed explanation and the advise about mistargeting and proteins on ER. The FingRs for PSD95, which you recommended, seems interesting to me. I will read the paper first. Maybe we can have better discussion after I understand it.
Anyway, special thanks to your tips about the 'mistargeting' proteins in ER, that is something I did not realize this possibility previously. @Kyle
Yu
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