Alex Palazzo is an Assistant Professor in the Department of Biochemistry at The University of Toronto.
I've been busy preparing for a lab meeting - I'm presenting tomorrow. These last few months have been weird - since I've submitted my paper, about 1+1/2 months ago, I've been testing various theories, and toying with some ideas. Recently I knocked down two related proteins using siRNAs. Unexpectedly this treatment resulted in a striking morphological change. The knockdown cells don't appear to be dying, and their doubling rate is only slightly altered as compared to control cells but they now have all these micro-blebs.
Bellow the fold are two pictures, the first being the knockdown cells, the second being the control cells.
siRNA treated cells:
Notice the little micro-blebs appearing around the whole periphery of the knockdown cells. These blabs are smaller then most blebs and vary a bit between cells (notice that the cell at the very bottom of the micrograph has extremely small blebs). In addition, it looks like the cells are well spread and so cell attachment does not seem to be compromised.
Control cells (the nuclei are a bit out of focus because I wanted to examine the cell periphery):
So do you have any idea about what might be happening with the cells? Do these cells phenocopy any condition that you know of?
Comments
It's hard to say without knowing anything about the proteins you've knocked down. Those "blebs" look a bit like some things I've seen when my protein of interest activated the Unfolded Protein Response in Saccharomyces. The cells were fine, and they made protein, but it was sort of "stuck" in these microvesicles just below the cell membrane. In my case, though, the morphology of the membrane wasn't really altered, there were just these punctate regions immediately to the interior.
Posted by: Jason | July 16, 2007 12:24 PM
Well I'll just say that the two proteins are RNA binding factors.
Posted by: apalazzo | July 16, 2007 12:31 PM
Looks like your nuclei have changed shape too.
Posted by: Acme Scientist | July 16, 2007 9:23 PM
Yeah, a good friend of mine wrote to me that he thought the nuclear shape looks lobular and that the cytoplasm looks a little denser. It turned out that mRNA export in the knockdown cells were just fine ... another negative result.
Posted by: apalazzo | July 18, 2007 12:01 PM
off-target effect?
mis-regulation of mRNA transport (e.g. of actin or something cytoskeletal that is in the wrong place and/or being translated too much or too little?)
it kind of reminds me of nocodazole treatment.
Posted by: Joolya | July 19, 2007 3:27 PM
They don't look like blebs to me (you obviously wondered about apoptosis, hence "they don't seem to be dying") -- they look like vesicles clustering at the pm and to some extent at the nucleus (ER?). Some kind of knock-on effect causing increased secretion of something, or messing up receptor recycling?
Yeah, I got nothing, I'm just guessing here.
Posted by: Bill | July 26, 2007 12:28 AM
i think it looks like a giant whale's penis
Posted by: mick | November 5, 2007 5:47 PM