How to detect protein-cytoskeleton interaction? - (Jan/28/2009 )

Dear all,
I would like to ask you something about interaction of proteins with cytoskeleton. I do separation (by centrifugation) of (1%) triton x-100 soluble and insoluble fraction (cytoskeleton). A protein of my interest is in triton x-100 insoluble fraction, so I suppose that it interacts with cytoskeleton. But I would like to show this interaction (protein-cytoskeleton) clearly, for example, using a drug that would disrupt interaction of my protein with cytoskeleton, and then the protein would be released into supernatant and cytoskeleton would stay in pellet after re-centrifugation.
Do you have any experience with such a experiment - which drug(s) should I use?
How can I show in the best way, that my protein interacts with some cytoskeletal proteins?

Another general question regarding cytoskeleton. When one performs separation of cytoplasmic and nuclear fraction, the lamin B is typical nuclear protein. If I do separation of triton x-100 soluble and insoluble fraction the lamin B is mainly in triton x-100 insoluble fraction. Does it mean that lamin B protein is part of cytoskeleton?, i.e. cytoskeleton is not only in cytoplasma, but also in nucleus?

Thanks for your suggestions.
vik

victor.m on Jan 29 2009, 10:03 AM said:

By cytoskeleton- do you mean intermediate filaments in this case since you are describing insoluble fractions? I wouldn't necessarily assume your protein interacts with the "cytoskeleton" just because it is in the same fraction, unless you have other evidence otherwise. Assuming that it is- it will depend on which part of the cytoskeleton you are looking at - actin and Microtubules(MT) are dependent on ATP for assembly/disassembly are there are many specific drugs that affect their dynamics and interactions. Intermediate filaments don't require ATP for assembly are their dynamics are regulation through phosphorylation so you can consider kinase inhibitors or phosphatase inhibitors may disrupt your fraction. However these drugs may not do anything for your particular protein- have you seen any evidence for interaction (eg co-localisation with Immunofluoresence or from literature?)
As to your other point- lamin B is a nuclear intermediate filament protein, and as an intermediate filament protein you will find it in the insoluble fraction of your samples. It does contribute the structure of nucleus (its found in associated with the inner nuclear membrane) and there are many papers which explore its function and structure if you want to know more.



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