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 <[log in to unmask]> (on behalf of Artem Evdokimov <[log in to unmask]>)
>Subject: Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!
>To: [log in to unmask]
>
> 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
> <[log in to unmask]> 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 <[log in to unmask]>
> (on behalf of Artem Evdokimov
> <[log in to unmask]>)
> >Subject: Re: [ccp4bb] Sighting of Protein
> Crystals in Vivo?!
> >To: [log in to unmask]
> >
> > 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
> > <[log in to unmask]> 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: [log in to unmask]
> > 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: [log in to unmask]
> >
> *******************************************
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