How does MitoTracker Bind?
J. S. John
stained chick embryo cells using Mitotracker. Our professor said he
never found out from Invitrogen how it really works but they told him
it binds to the lipid. Anyone know more precisely how it does?
This spec sheet says some stuff but I can't understand it.
http://probes.invitrogen.com/media/pis/mp07510.pdf
"The cell-permeant MitoTracker® probes contain a mildly thiol-reactive
chloromethyl moiety
for labeling mitochondria (Figure 1).
Figure 1. The intracellular reactions of our fixable
mitochondrion-selective dye, MitoTracker® Orange CM-H2TMRos. When
this cell-permeant probe enters an actively respiring cell, it is
oxidized to MitoTracker® Orange CMTMRos and sequestered
in the mitochondria, where it reacts with thiols on proteins and
peptides to form an aldehyde-fixable conjugate."
The above quote was from that sheet.
So after the stain enters the mitochondria, what happens?
Thanks
Nathan McCorkle
>
> We're going to be doing fluorescent microscopy in my lab class and we
> stained chick embryo cells using Mitotracker. Our professor said he
> never found out from Invitrogen how it really works but they told him
> it binds to the lipid. Anyone know more precisely how it does?
>
> This spec sheet says some stuff but I can't understand it.
>
> http://probes.invitrogen.com/media/pis/mp07510.pdf
>
> "The cell-permeant MitoTracker® probes contain a mildly thiol-reactive
> chloromethyl moiety
> for labeling mitochondria (Figure 1).
> Figure 1. The intracellular reactions of our fixable
> mitochondrion-selective dye, MitoTracker® Orange CM-H2TMRos. When
> this cell-permeant probe enters an actively respiring cell, it is
so first it gets into the cell
> oxidized to MitoTracker® Orange CMTMRos and sequestered
> in the mitochondria, where it reacts with thiols on proteins and
> peptides to form an aldehyde-fixable conjugate."
once in the mito, it binds sulfur amino acids, which either you fix by
adding some reagent that contains aldehyde, or which it does on its on
with aldehydes in the mito.
>
> The above quote was from that sheet.
>
> So after the stain enters the mitochondria, what happens?
>
basically it metabolizes so that it reacts with light differently than
non-activated-by-metabolism stain molecules... maybe to differentiate
bacteria from eukaryotes, as well as living vs non-living.... or maybe
some cell types dont have the metabolic pathway to activate the stain.
> Thanks
>
I think :P
--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Nathan McCorkle
protein pump that it mentions:
http://en.wikipedia.org/wiki/Rhodamine#Rhodamine_123
basically the dye gets metabolised, then sequestered into the mito,
then sulfur groups abstract the Cl on the bottom of the molecule,
binding it to big proteins. The PDF says that there are other types of
dyes, but they suffer from washing out during cell fixing steps, which
this dye doesn't suffer from. It does however not work well in dead
cells, which other dyes can.
J. S. John
> basically the dye gets metabolised, then sequestered into the mito,
> then sulfur groups abstract the Cl on the bottom of the molecule,
> binding it to big proteins. The PDF says that there are other types of
So the compound binds to the membrane proteins.
"where it reacts with thiols on proteins and peptides to form an
aldehyde-fixable conjugate." [1]
So this would likely form the peptide bind with a sulfur containing
AAs like cysteine or methionine?
> dyes, but they suffer from washing out during cell fixing steps, which
> this dye doesn't suffer from. It does however not work well in dead
> cells, which other dyes can.
>
Any idea why this would happen?
Nathan McCorkle
>
> On Wed, Oct 21, 2009 at 6:16 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
>
>> basically the dye gets metabolised, then sequestered into the mito,
>> then sulfur groups abstract the Cl on the bottom of the molecule,
>> binding it to big proteins. The PDF says that there are other types of
>
> So the compound binds to the membrane proteins.
bound, but potentially, the data sheet says the dye gets sequestered
into the mito, so it could then interact with any protein inside,
including membrane ones.
> "where it reacts with thiols on proteins and peptides to form an
> aldehyde-fixable conjugate." [1]
>
> So this would likely form the peptide bind with a sulfur containing
> AAs like cysteine or methionine?
>
>> dyes, but they suffer from washing out during cell fixing steps, which
>> this dye doesn't suffer from. It does however not work well in dead
>> cells, which other dyes can.
>>
> Any idea why this would happen?
to get pulled out by ethanol or whatever solvent is used in the
fixation process.
J. S. John
> No I think they are just intra-mito proteins, not necessarily membrane
> bound, but potentially, the data sheet says the dye gets sequestered
> into the mito, so it could then interact with any protein inside,
> including membrane ones.
What makes Mito tracker specific to the mitochondria membrane?
Wouldn't it bind to the cell membrane proteins also?
Nathan McCorkle
metabolized and then it stays there... Not sure, I can look more at
the last link in this list in a few hours, but gotta go to a thesis
defense on 3d protein visualisation now
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WBK-4P6M8P4-4&_user=47004&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1061331114&_rerunOrigin=google&_acct=C000005018&_version=1&_urlVersion=0&_userid=47004&md5=4a0d94df2e60a2bc218dd3cde5180f17
http://molpharm.aspetjournals.org/cgi/content/abstract/45/6/1145
http://bbc.mcw.edu/Computation/models/text/Mitochondrial%20Inner%20Membrane%20Electrophysiology%20Assessed%20by%20Rhodamine-123%20Transport%20and%20Fluorescence.pdf
J. S. John
>
> Take a look at these, I think once it gets into the mito it gets
> metabolized and then it stays there... Not sure, I can look more at
> the last link in this list in a few hours, but gotta go to a thesis
> defense on 3d protein visualisation now
>
>
I'm reading this one right now. Not a quick reader though.
I sent an email to my professor about what I learned so far and he replied:
" The odd thing is that mitotracker Green-FM (which is the one we
used) seems to have somewhat different properties from the others,
since it appears to stain mitochondria on dead cells --such as cheek
smears, that are dried down before staining. It may be that some
oxidative activity survives the treatment, but it seems unlikely.
Note also that in the cheek cells, it survives despite the lack of
fixation. It also seems to have a significant background membrane
staining, sugesting that there is a lipid component to the reaction.
In past years, we have been unable to get clear mitotracker staining
on the chicken cells. This year, we modified the procedure, following
the ideas suggested by the Invitrogen information, and we added the
mitotracker solution to the living cultures prior to aldehyde
fixation. As you saw, there was very little stain in the fixed cells,
and there were major problems with cell attachment and survival. There
were also many odd circular patches with punctate fluorescence all
over the slides. This stain clearly had nothing to do with the cells.
My suspicion is that it represents some form of micelle that
developed in the culture medium and then bound to the glass.
Interestingly, when we looked at the chicken cells before fixation,
but after mitotracker exposure, we did see convincing mitochondrial
stain, but all of the cells were floating. Bummer.
As you can see, there seems to be more to the Mitotracker Green-FM
than is in the Invitrogen description. In fact, if you compare the
literature in the "manual" with the "handbook", you will find some
inconsistencies between the two sources."
According to him, Mitotracker green FM is not like the other ones. I
wonder what's going on.
Dakota
gives an error, or the paper the person you forwarded linked.
Here is the invitrogen info packet on the MitoTracker stains
http://probes.invitrogen.com/media/pis/mp07510.pdf
"For the MitoTracker® Green FM probes, use a slightly lower
concentration (20–200 nM). At higher
concentrations, these probes tend to stain other cellular structures. "
and from here, potentially useful
http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Probes-for-Organelles/Probes-for-Mitochondria.html#head2
"MitoTracker Green FM Dye
Mitochondria in cells stained with nanomolar concentrations of
MitoTracker Green FM dye (M7514, ) exhibit bright green,
fluorescein-like fluorescence (, , ). The MitoTracker Green FM probe
has the added advantage that it is essentially nonfluorescent in
aqueous solutions and only becomes fluorescent once it accumulates in
the lipid environment of mitochondria. Hence, background fluorescence
is negligible, enabling researchers to clearly visualize mitochondria
in live cells immediately following addition of the stain, without a
wash step.
Unlike MitoTracker Orange CMTMRos and MitoTracker Red CMXRos, the
MitoTracker Green FM probe appears to preferentially accumulate in
mitochondria regardless of mitochondrial membrane potential in certain
cell types, making it a possible tool for determining mitochondrial
mass. Furthermore, the MitoTracker Green FM dye is substantially more
photostable than the widely used rhodamine 123 fluorescent dye and
produces a brighter, more mitochondrion-selective signal at lower
concentrations. Because its emission maximum is blue-shifted
approximately 10 nm relative to the emission maximum of rhodamine 123,
the MitoTracker Green FM dye produces a fluorescent staining pattern
that should be better resolved from that of red-fluorescent probes in
double-labeling experiments. The mitochondrial proteins that are
selectively labeled by the MitoTracker Green FM reagent have been
separated by capillary electrophoresis."
The MitoGreen FM isn't retained after fixation, and seems to
necessitate using smaller concentrations to avoid non mitochondrial
specific lipid binding. It seems that the other MitoTrackers are
based on fluorescence of the oxidized state of the dye after it
couples with cysteine residues, or is oxidized in the
mitochondria...but this MitoGreen FM and the other "FM" trackers only
fluoresce when they accumulate in lipid deposits.
As the forwarded original poster suspected, there might be some
liposome/micelle formation going on which is picking up the stain and
not allowing any of it to reach the mitochondria.
Have they tried another mito tracker stain besides the Green FM? It
seems to be the most finicky tracker dye after reading the info
packet, and requires a very specific experimental protocol as listed
in invitrogens attached packet.
The Mitochondria has two membranes and a lot of internal surface area
so I can imagine that the largest, if not perhaps 2nd largest
concentration of lipids within the cell membrane lipid bilayer itself
would be the mitochondria or the endoplasmic reticulum. Just a guess
though
Just wrote all this thinking it was forwarded from a stranger outside
the list...so might have rambled on a bit more than needed after
realizing it seemed to be a question already posted by Nathan or
PhillyJ.
It'd be neat to see some pictures of the failed/successful stains though.
Jeswin
> Maybe it's just on my end but that paper you linked doesn't load and
> gives an error, or the paper the person you forwarded linked.
>
this question. I haven't thought about it since. Anyway, if you can
get the article, here is the pubmed link:
http://www.ncbi.nlm.nih.gov/pubmed/17372838
Graduating sucks, you lose access to journals :(
