Hi,

That´s a very interesting possibility, but we have not been able to actually identify such intermediates probably due to a couple of reasons: 1) The nucleoporin we have mostly used as a handle for NPC purification is the nuclear basket component Mlp1, and the nuclear basket is probably one of the last NPC parts that gets assembled during its biogenesis; 2) Although we have been able to collect many thousands of particles for cryo-EM, it might still not be enough for the classification to identify intermediates that would represent only a small fraction and that could potentially not be easy to identify and align. Still, something to think about for the future and that could potentially be feasible using the right conditions.

Thanks for the answer.

Hi,

I am sorry to say that there is no magic recipe for that, it is completely case-dependent and it usually requires to invest time and effort on the optimization of the affinity purification conditions. We will try to show some general guidelines for the selection of proper conditions that could help maximize the recovery of the tagged protein and its interactome using as an example the nuclear pore complex.

The work of several groups (including ours) indicate that the size (diameter) of the purified NPCs is smaller than the one of an in-situ NPC in an actively growing cell. It is however quite similar to the size of an in situ NPC in cells where active transport and nuclear envelope tension have been reduced. Thus the NPCs could undergo a dilation-constriction cycle that seems to be related to the nuclear envelope tension.

Hi,

The use of cross-linkers in protein complex characterization is a broad topic that would require a detailed discussion depending on the specific system and downstream application desired. But just to give you a general overview, there are two steps in which we have used cross-linkers for characterizing native protein complexes: 1) To stabilize the complex at the time of cell breakage and solubilization, in which case we have usually followed the method published by Subbotin & Chait (DOI: 10.1074/mcp.M114.041095) where a small concentration of crosslinker was used to stabilize the complex before isolation; 2) To characterize the connectivity and proximity of the different components of the complex once it is already isolated using cross-linking and mass spectrometry (doi: 10.1074/mcp.M114.041673; doi: 10.1038/nmeth.3617; DOI: 10.1016/j.tibs.2015.10.008). In both cases the advantages are that the use of the cross-linkers could help stabilize the complex and make it less prone to disassembly, specially in the case of labile components, and also provide a wealth of data about the arrangement of the components when combined with MS. The dangers could be decrease of the purification yields, difficulties processing the sample and potential artifacts mainly when the cross-linking regime is not properly titrated and defined (over cross-linking). This is a key step that requires to spend some time defining the conditions (time, temperature, concentration, quenching, etc) that would be optimal for your system. The references above could be a good starting point to help identify conditions that could be applied to your system. Another pitfall is that successful and reliable mass spectrometry requires expertise that could be difficult to find or expensive.

I hope this helps. Best.

Yes, most definitely. We have successfully used it to isolate NPCs that have been then used to generate cryo-electron tomography and single particle cryo-EM maps of the baker’s yeast NPC.

Hi,

In our hands, the Dynabeads™ M-270 Epoxy are the ones that perform the best. They should be coupled to a suitable antibody/nanobody that could capture your protein of interest or the tag used to label it.

Hi,

Our method was not designed with industrial applications in mind, thus I cannot indicate one, but we think it should be possible to adapt it for biotechnological and biomedical applications.

Hi,

The main advantages respect to previous methods used to isolate Saccharomyces cerevisiae NPCs are that our method is much faster, provides a much higher yield of NPCs and could be applied to virtually any yeast genetic background.

Hi,

We flash-freeze them in liquid nitrogen and then store them at -80C. The storage buffer is 20mM Hepes-KOH pH 7.4, 20mM sodium chloride, 50mM potassium acetate, 2mM magnesium chloride, 0.1% Tween-20, 10% glycerol, 1mM DTT. The glycerol acts as a cryo-preservative and under these conditions the nuclear pore complexes maintain their native state after isolation for several weeks.