I want to carry out calcium signaling experiments in MSC cultured in 3D scaffolds.
Due to equipment constrains, I only can exite the fluorocrome probe above 450nm. So, I am considering to use Fluo3/FuraRed. However, I am concern about how to place the calcium indicator in the proper compartment of the nucleus. Then, I also consider to use calcium-binding fluorescent indicators like cameleons, pericams, or aequorins fused to a Nuclear Localization Signal (NLS).
I could be very wrong, but is there a gradient of calcium
between the nucleus and the rest of the cell?
(I was under impression that nuclear pores allow
ions to pass freely)
Both, most synthetic calcium indicators and calcium ions can indeed enter the nucleus through the pores. In my experiments I saw that Oregon Green BAPTA-1, AM gives a bright nuclear staining pattern in neurons. The calcium signals in nucleus were similar to those in the other cytosolic areas.
All the chemical Ca2+-sensitive dyes (Fura/Fluo/OGB) localizes also in the nucleus, where the signal is usually brigher, as underlined by Samira. As already pointed out, in most of the cell types, there are few differences in calcium dynamics in the cytosol vs nucleus, since Ca2+ freely permeates through nuclear pores. Moreover, keep in mind that most of the Ca-sensitive dyes increases the diffusion of Ca2+ cations (i.e. the diffusion of free Ca2+ is slower than the diffusion of Ca2+ bound to the dye).
IMHO, genetically encoded calcium sensors are always the best choice when investigating specific subcellular compartments. In addition, ratiometric probes (cameleon-like) are preferable than single-wavelenght probes (GCaMP-like), when feasible. If you plan to make a new costruct from scratch (by adding a NLS signal to an already available Ca2+ probe), go with last generation cameleons (Nature Methods 11, 175–182 (2014) doi:10.1038/nmeth.2773) if you want a ratiometric FRET based sensor, or with last generation GCaMP6 (Nature 499, 295–300 (18 July 2013) doi:10.1038/nature12354) if you want a single-wavelenght indicator. Also aequorin can be fine, but this heavily depends on the kind of experiments you need to carry out.
I agree with Diego.
In my old lab we had good nuclear targeting of cameleon by fusing the probe with histone 2b (H2B).
I, too, am a fan of the genetically encodable tools, especially if your interests are specific to Ca2+ levels in the nucleus as these tools can minimize fluorescence transients that arise outside of your structure of interest. In addition to what has been said, if you plan to use genetic tools, think about how you plan to transduce the construct into your cells of interest.
Given the description you gave of your experimental setup, I'd recommend that you use an NLS fusion sensor packaged into a lentivirus. This method of gene delivery ensures high penetrance as well as relatively fast expression of the construct (around two days post infection).
If you decide to go that route, check out the U Penn and UNC vector cores. Chances are, they have a packaged virus available in their respective catalogues that will fit your needs.
Thank you all for your commentaries and suggestions.
As I would like to work with primary cells, I would go for small fluorescent probes such as Fluo4.
Why it is better the engineered and targeted protein Ca indicators than the small dyes when both can be compartmentalised and ROI selection can be done?
...following up on this:
The Looger lab just published their H2B-GCaMP6 fusions (AAV plasmids) on addgene.
https://www.addgene.org/browse/article/17925/
We just finished our own with classic NLS sequence instead of H2B.
Did anyone ever compare NLS and H2B concerning nuclear localization efficiency etc?
Hi Tobias et al-
Any update on how nuclear localized GCaMPs work? Seems super handy for eliminating neuropil signal (for example in endoscopy where it can't be resolved and just adds to background), but also potentially bad for nuclear calcium related events to have a ton of GCaMP in the nucleus.
Or other ways to limit GCaMP to the soma?
Thanks in advance!
Jen
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