Tuesday, December 12, 2017

So, this Cactus walks into a nucleus...

Recently, on this blog, I've been musing about our foundational assumptions and how they color our thinking (for example, see here and here). In my last post, I briefly described a situation where this has affected my lab. In one of our computational studies, our model explained the experimental data better when two straightforward assumptions were made (see below). The problem was, one of those assumptions implied that Cactus can be found in the nucleus, even though it has always been conceived as a strictly cytoplasmic protein. This baseline conception colored some people's reaction so strongly as to reject our assumption, even though it was plausible.

The question I therefore raised at the end of the last post was whether our two assumptions subsequently needed to be validated experimentally? I am itching to get to that question, but before we address it, we should walk through what those two assumptions were and what experimental data are explained by the model. However, if you don't wish to read those details, here is a brief summary: The first assumption was not only straightforward, but it also provided an obvious way to match known data. The second assumption was more plausible than its denial, and resulted in a model that answered a problem with our understanding of the Dorsal gradient. Now, on to the details:
At the start of interphase, when the nuclei reform, molecules in the cytoplasm are "accidentally" captured by the nuclei. This includes Cactus (Cact) and Dorsal (dl)/Cact complex. But if dl/Cact complex is in the nucleus, that implies some of the fluorescence we measure stems from dl/Cact complex, which is transcriptionally inactive.

First assumption: After mitosis, when the nuclear envelope reforms, the contents of the nucleus (accidentally) reflect the contents of the surrounding cytoplasm. In other words, when the nuclear envelope "encloses" the nucleus, whatever protein-sized molecules happen to be in the cytoplasm at that time can get enclosed as well.

The reason why we made this assumption is that, as soon as interphase begins, there is fluorescence in the nuclei. We saw that in live embryos expressing a Dorsal-GFP tag (see Reeves et al., 2012). There are further details to this, but suffice it to say that a simple way to solve this problem is if nuclei at the start of interphase do not begin empty.

Second assumption: If our first assumption was correct, and the nuclei do not begin interphase empty, then a chain of important implications ensues. The first implication is that both Cactus and Dorsal/Cactus complex could reside in the nucleus. That in turn implied that it is possible that some of the fluorescence we measure, either in live Dorsal-GFP embryos, or in fixed embryos fluorescently immunostained against Dorsal, originated from Dorsal/Cactus complex. And this implied that our fluorescent measurements were not of the active Dorsal gradient, but of the total Dorsal gradient (free Dorsal + Dorsal/Cactus complex). At this point, the only way to infer the active Dorsal gradient is to use our computational model.

The reason why we made this assumption was that it seemed more obvious than its denial. However, this second assumption resulted in a large boon for our model: it now was able to correctly predict the expression of genes that depend on Dorsal signaling. Without going into too much detail, we have known since 2009 that the Dorsal gradient is too narrow to express genes like sog and dpp, which have borders roughly 50% of the way around the embryo (see here for more explanation). However, our model predicted the active Dorsal gradient (as opposed to the measured, total Dorsal gradient) may indeed be broad enough to place the sog and dpp gene expression borders.

Now that we have the details of our assumptions and implications in hand, in the next post I will discuss why I no longer think experimental validation of our assumptions is necessary.

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