What are the single dots in nucleus?

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Wed May 24 05:26:32 EST 2000


Hi ZhongLin,
Your images look quite interesting. As my lab is working on nuclear
architecture, we are quite familiar with dot-like structures in the
nucleus. These dots are usually considered "subcompartments" of the
nucleus that have a specific function (although the functions themselves
are mainly unclear...). Some of these "dots" are called coiled bodies,
gems, PML-bodies, and a search in Medline will give you quite a lot of
papers on these structures. 

In your particular case, more information would be necessary before
definitive statements can be made: 
1. is the dot only observed in transfected cells, or is it also present
in non-transfected cells and/or cells that have received the empty HA-vector?
2. Is there anything special in the regions of the coverslip with dotted
cells (higher than average cell density, cells with different morphology
or size?)
3. Do the dots also appear when you use a different method for
transfection (e.g. electroporation)?
4. Do the dots also appear in a different cell line?

If you see that the dots are specific for transfected cells, and also
appear in other cells and with other transfection methods, it is most
likely that, indeed, it is your protein. Your finding that not all cells
have the dot suggest that what you see are the cells that have actually
taken up the plasmid and make the protein, and the others are the
non-transfected background. If you transfect cells on the
plate/coverslip, it may well be that local differences in concentration
lead to regions of dot-free vs. dotted cells. This will particularly be
striking if you use transfection by calcium phosphate, because the
crystals become enriched in some regions due to vibrations either in the
incubator or when you move the plate.

We have observed differences between the localization of endogeneous
protein vs. transfected protein in some instances, so it would not be
too surprising to see the same with your protein. Of course this means
that we all have to be very cautious as to the interpretation of
localization studies on transfected cells...

Best wishes,
Frank


PS: You may think of using a GFP-construct of your protein to follow the
localization in live cells. This rules out potential artifacts inherent
in immunofluorescence experiments.





Zhonglin Chai wrote:
> 
> 
> Hello Everyone,
>     Time by time, when I immunofluorescent stained transient
> transfected HeLa cells I observed single dots in the nucleus of some
> cells. Those cells
> are found in limited areas of the coverslips. They seemed to me that
> they were infected/contaminated by some micro-organisms (?) which
> infected
> surrounding cells (if they can be detected by the Ab or conjugate I
> used). As shown in the following pictures, one cell got only one dot,
> if any, in its
> nucleus, but not all the cells (even though in the area where other
> cells got the dot). Mitotic cells also got a single dot in the mitotic
> chromosomes
> (metaphase), or one each on the separated chromosomes
> (telophase/anaphase). I don't believe this is the protein expressed by
> my transfection
> because I know the pattern is different. Do you have any idea what it
> could be?
>     The pictures below are examples showing HeLa cells transiently
> transfected with a HA-tagged protein (nucleolar protein) by
> electroperation,  fixed
> 
> with methanol, and stained with mouse McAb to HA epitope, and rabbit
> anti-mouse Ig-FITC conjugate (green). The nucleus are stained with a
> DNA
> dye (blue). A merged image of both green and blue is also shown (on
> the right).
> 
> [Image] [Image] [Image]
> 
> --
> ZhongLin Chai, PhD
> __________________________________________________________
> Department of Pathology and Immunology
> Monash University Medical School
> Alfred Hospital
> Commercial Rd, Prahran, VIC 3181, AUSTRALIA
> Telephone: (61 3) 9903 0698 (lab)
>            (61 3) 9903 0696 (office)
> Mobile:    0413 58 1940 or International: +61 413 58 1940
> Fax:       (61 3) 9903 0731
> email:     zhonglin.chai at med.monash.edu.au
> ___________________________________________________________
>






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