Thread: Membrane Fusion Anyone?

I wanted to know if anyone on this forum was interested in membrane fusion and more specifically the mechanisms involved in membrane fusion.

I have just completed my honours project at university, for which I was using molecular 'fishing' techniques with a view to determining their applications for studing the interactions of the proteins involved in fusion.

During the course of the project I have had some ideas of my own about this, although maybe too mechanistic.

Any opinions on this fascinating area of biology?!?

Cheers

G

Well, I don't have too much in-depth knowledge in the area, but it's potentially pertinent to the work I'm doing in a biomedical lab. We're trying to work out the genetic mechanisms of muscular dystrophy, mainly by looking at the effect of various genes on mouse models, mouse muscle cell lines, and human muscle cell lines.

One of our measures is the effect of certain genes on myotube formation, which involves multiple myoblast cells fusing together - and, of course, the fusion of their membranes. If regenerating muscle fibers are unable to fuse into mature myotubes, this could contribute to the overall muscle atrophy that is symptomatic of muscular dystrophy patients. The question then arises as to why exactly the myoblasts have difficulty fusing together; it's possible that genes involved in directing membrane fusion are negatively effected in the disease.

Unfortunately, we're not that far along at this point, but who knows - it may end up being an avenue of research one day. May I ask what the results of your project were?

Wow!

Sure, well obviously as I said in my initial post, it is all at what feels like a simplified level. But basically I purified recombinant his-tagged a-SNAP using an affinity column (Ni-nta).

I then looked at ways of showing that a-SNAP could form complexes with other proteins (ie SNAREs, NSF etc) while associated with nta beads. God that sounds simple.

Unfortunately it was only a 6 week project, with limited resources.

But the general outcome was that I was able to show that Syntaxin did form a complex with nta-bound a-SNAP, this was done using a method based on reading in this paper (last part of methods section):

The pre-vacuolar t-SNARE AtPEP12p forms a 20S complex
that dissociates in the presence of ATP
Diane C. Bassham and Natasha V. Raikhel
The Plant Journal (1999) 19(5), 599±603

I incubated the bound a-SNAP with cytosolic and membrane protein fractions from synamptosomes overnight, in various combinations (cytosol only, cytosol+membrane, membrane only) with 'a-SNAP only' as a control.

We only had an anti-body for syntaxin so thats why I only show syntaxin was able to bind. That was using Western Blot.

We had hoped to look at NSF binding using a non hydrolysable form of ATP (gamma-s?) but didn't have time

When I write it like that it seems amazing that it took 6 weeks!

Don't know if its of interest to you and I could probably have explained it more clearly, but thats the gist of it.

Cheers for your interest, any feedback would be appreciated :-D

Your work sounds interesting, it must be good to be doing research that has a potentially positive impact on something or someone.

I'm still unsure as to what I will do when I graduate, still looking for that job advert!

Thanks and regards

G