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 <CCP4BB@JISCMAIL.AC.UK> (on behalf of Artem >Evdokimov <artem.evdoki...@gmail.com>) >Subject: Re: [ccp4bb] Sighting of Protein Crystals in Vivo?! >To: CCP4BB@JISCMAIL.AC.UK > > 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 > <zhijie...@utoronto.ca> 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: CCP4BB@JISCMAIL.AC.UK > 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: j-kell...@northwestern.edu > *******************************************
