Hello, I've seen similar "crystals" in HEK cells when expressing certain (but not most) mVenus-fusion proteins. mVenus is similar to mYFP. I now wonder if this effect depends on my protein being cleaved, releasing mVenus. Will look into this.
Elena ---- Original message ---- >Date: Sat, 16 Feb 2013 16:14:58 -0600 >From: CCP4 bulletin board <[email protected]> (on behalf of Artem >Evdokimov <[email protected]>) >Subject: Re: [ccp4bb] Sighting of Protein Crystals in Vivo?! >To: [email protected] > > Note: the crystals in my paper are not regular GFP, > they're special GFP from a marine Copepod. I've seen > GFP crystals (eGFP) before in other cells, though. > Bacillus thuringiensis (an organism I work with > every day) makes beautiful crystals of parasporal > insecticidal toxins when it sporulates. Hundreds of > these proteins are known, and many of them (about > 60% or so) produce geometrically perfect shapes > (cubes, rectangles, etc.) The internet is rife with > images of these if you care to see them. We are very > interested in FEL studies of those crystals but > we've not contacted the FEL folks yet (if any of > them are reading, please feel free to comment back > to me directly). This is probably the most > abundant/diverse source of biologically crystallized > protein material (at least as far as I know). > Artem > > On Sat, Feb 16, 2013 at 4:04 PM, A. Radu Aricescu > <[email protected]> wrote: > > Hi Artem, Jacob, > > I've personally never seen such crystals despite > using for many years various stand-alone GFP > variants (including eGFP) as transfection controls > in HEKs and other mammalian cell lines, but now I > believe you and owe Jacob an apology for being > skeptical when I first saw his pics :-)) > > But, I can't stop wondering what is the secret? > Must be something else than just overexpression... > How often do you see such crystals occurring (what > % of GFP-expressing cells), and how stable are > they? Could this be related to the expression > system, transfection procedure, broader cell > culture conditions... Artem finds GFP crystals in > HeLa cells (have you also tried HEKs?), while > Jacob's experience seems to be the opposite > (admittedly using a different GFP flavour)... > > It just feels like this "in cell" crystallization > might become a very powerful tool if one could > harness it! > > Best wishes, > radu > > ------------------------------------------ > A. Radu Aricescu, PhD > University Research Lecturer > > University of Oxford > Wellcome Trust Centre for Human Genetics > Division of Structural Biology > Roosevelt Drive, Oxford OX3 7BN > United Kingdom > Phone: +44-1865-287564 > Fax: +44-1865-287547 > > ---- Original message ---- > >Date: Sat, 16 Feb 2013 15:38:12 -0600 > >From: CCP4 bulletin board <[email protected]> > (on behalf of Artem Evdokimov > <[email protected]>) > >Subject: Re: [ccp4bb] Sighting of Protein > Crystals in Vivo?! > >To: [email protected] > > > > In addition to the above mentioned references > you > > can also see: > > Charcot-Leyden crystals > > (http://www.ncbi.nlm.nih.gov/pubmed/6508005) > > and my own ( :) ) figure 1 > > > http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618374/figure/f1/ > > Cheers, > > Artem > > > > On Fri, Feb 15, 2013 at 11:46 PM, Zhijie Li > > <[email protected]> wrote: > > > > Hi Jacob, > > > > Interesting topic. > > > > This reminds me the posters I saw on ACA > 2010, on > > the femto-second infrared laser based > instrument . > > That instrument utilizes the nonlinear > optical > > properties of crystals of chiral > molecules to > > detect very small crystalline materials > from > > amorphous background: the crystals will > double the > > frequency of the laser, turning the > infrared light > > to visible light. I cannot recall the > exact name > > of the technology now, unfortunately. > > > > Your case of observing in vivo GFP > crystals is a > > little special in that the crystals are > > fluorescent. I guess if we scan cells > > over-expressing proteins with the above > mentioned > > instrument, we might find that many > proteins will > > do the same in cells. > > > > Naturally occurring in vivo crystals are > not very > > rare. If we do not restrict the topic to > proteins, > > then it is well known that many viruses > readily > > crystallize in the host cell's nuclei and > the > > resulting crystals or crystalline arrays > can be > > observed under EM. And if we do not > restrict the > > cells to mammalian cells, then there come > the > > famous BT crystals. > > > > In addition, I just did some internet > search and > > here are some interesting results: > > > > 1) Viral protein crystals can form in > HEK cells > > infected by adenovirus > > > (http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002894) > > 2) Bacterial infection can cause the > infected > > epithelial cells to form pathological > > crystal-containing inclusion bodies in the > cytosol > > > (http://www.ncbi.nlm.nih.gov/pubmed/8940763). > > 3) Crystalline inclusion bodies are > found in > > rabbit embryos > > > (http://dev.biologists.org/content/44/1/31.full.pdf) > > and epididymis of the nine-banded > > > > armadillo(http://www.sciencedirect.com/science/article/pii/S0022532073800073). > > Actually if google "crystalline inclusion > body", > > there will be tons of literatures. > > 4) IgG crystallized in the ER when over > expressed > > from a highly optimized CHO expression > system > > > (http://www.jbc.org/content/286/22/19917.abstract). > > This is particularly interesting as we > know that > > whole IgGs are not so prone to > crystallize, > > although the author do state that > "Crystallizing > > propensity was due to the intrinsic > > physicochemical properties of the model > IgG". > > > > > > Given the prevalence of in vivo > crystallization, > > especially considering their correlation > with > > inclusion bodies, I think it is reasonable > to > > suspect that there are some cases that the > > inclusion bodies formed during over > expression of > > transgenic proteins in E. coli are > crystalline. I > > expect that we will be enlightened on this > issue > > by somebody on the BB soon. > > > > Zhijie > > > > > > From: Jacob Keller > > Sent: Friday, February 15, 2013 2:44 PM > > To: [email protected] > > Subject: [ccp4bb] Sighting of Protein > Crystals in > > Vivo?! > > Dear Crystallographers, > > I was looking at some live, control HEK > cells > > expressing just eGFP, and to my great > surprise, > > saw littered across the dish what appeared > to be > > small fluorescent needles (see > attached--sorry > > about the size, but it's only ~1MB total.) > Can > > these possibly be fortuitous protein > > crystals? They were too small to mount I > think, > > and for what it's worth, > parallel-transfected HeLa > > cells did not have these things. But, some > needles > > could be seen in the DIC images as well, > and the > > needles were only fluorescent with GFP > filter > > sets, and not CFP, YFP, or texas red > filters. I > > thought of whale myoglobin crystallizing > on the > > decks of ships, but never thought I would > see > > this.... > > Jacob > > > > -- > > > ******************************************* > > Jacob Pearson Keller, PhD > > Postdoctoral Associate > > HHMI Janelia Farms Research Campus > > email: [email protected] > > > *******************************************
