Whether molecule is taken by lysosome only then targeted to mitochondria or directly taken up by mitochondria through different route? Please help.
Firstly, I think you may be confusing lysosomes with endosomes? If your molecule were to enter the lysosome it would probably just be degraded there.
You would probably have to conjugate your molecule to a mitochondrial-targeting signal, such as a lipophilic cation, which may enable it to directly enter into the mitochondrial matrix (and no endosomal involvement). But thats all I can speculate. Maybe this paper would be helpful:
'Mitochondria-Targeted Small Molecule Therapeutics and Probes'
Robin A.J. Smith, Richard C. Hartley, and Michael P. Murphy. Antioxidants & Redox Signaling. December 15, 2011, 15(12): 3021-3038. doi:10.1089/ars.2011.3969.
http://online.liebertpub.com/doi/full/10.1089/ars.2011.3969
Hi Kamal,
what kind of molecule are you trying to target to mitochondria? For example if you want to target expression of exogenous proteins, you can use a mitochondrial targeting sequence in your vector. That's how various fluorescent probes for example, are made mitochondria-specific. Or is your question about getting a molecule from the media into the cell and then to the mitochondria specifically? This would depend on a variety of factors (such as charge), but getting into the cell would definitely be your first hurdle. Or are you talking in an animal, not culture?
Hii Cummins,
Thanks for the reply.
I want to target exogenous peptide to the mitochondria. Do it need targeting moiety if i attach cell penetrating peptide alone with exogenous peptide? I also want to know whether it would be first taken up by lysosome and then released to mitochondria or directly enter into mitochondria without entering to lysosome. If i make it mitochondria specific then its route would be through lysosome or directly taken up by mitochondria? I am doing invito analysis i.e. cell culture studies.
Firstly, I think you may be confusing lysosomes with endosomes? If your molecule were to enter the lysosome it would probably just be degraded there.
You would probably have to conjugate your molecule to a mitochondrial-targeting signal, such as a lipophilic cation, which may enable it to directly enter into the mitochondrial matrix (and no endosomal involvement). But thats all I can speculate. Maybe this paper would be helpful:
'Mitochondria-Targeted Small Molecule Therapeutics and Probes'
Robin A.J. Smith, Richard C. Hartley, and Michael P. Murphy. Antioxidants & Redox Signaling. December 15, 2011, 15(12): 3021-3038. doi:10.1089/ars.2011.3969.
http://online.liebertpub.com/doi/full/10.1089/ars.2011.3969
If you are targeting proteins through genetic methods (transient and permanent), you could use specific signal peptide sequences that specifically translocate proteins to specific mitochondrial locations. These signal sequences will be cleaved off once it translocates to its location.
other method is described in this article freely available.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3231562/
Lypohilic cations (triphenyl phosphonium; TPP) are useful only to target chemical compounds.
Hope this would help your studies.
You will get perfect information regading targetting polypeptides/proteins into mitochondria in several Molecular Biology books. I find best and easily understandableinformation in Molecular Cell Biology by Lodish et al. (published by W.H. Freeman and Company, NY). Using specific peptide target sequences, you can target polypeptides even to a specific site in mitochondria (you can target protein of your choice to intermembrane space, within inner mitochondrial membrane or into stroma).
You can also find information on overall sorting and targetting of polypeptides in to various organelles amd membranes, in this book.
Regarding targetting mitochondrion as a whole into cells will be tough job, bust must be possible. I am sure someone will address this query of yours.
Hello Kamal!
I would suggest you to fuse your exogenous peptide to MLS of EndoG. It is known that under natural conditions MLS-EndoG targets directly mitochondria and it is also cleaved off after penetration. As far as I still remember the MLS is the 1-48 amino acids of EndoG propeptide. Check diverse EndoG publications. I hope I could be helpful. Good luck!
Photodynamic therapy using an agent that localizes in mitochondria, followed by eposure to light, will cause very specific damage to mitochondria. This generally leads to an apoptotic outcome as cytochrome c is released.
Dear Kamal,
you can simply apply a MitoTracker to your cells. And it will stain the mitochondira in vivo. They are amazing fast runners in the cell! You should have a frame rate from at least 2s in a time series to see how the move, and where they go (e.g. nuclei is usually easily detectable by the shape, a round area without mitochondira).
@Robert: Microtracker is for tracking the mitochondria as it is a targeting dye. but what sequence should i choose to target peptide to mitochondria?
you can select target peptide sequence of any of the proteins/polypeptides that are translated in cytosol and then targetted into specific loaction in mitochondria. For instance, you can use target peptide sequences of Matric Hsc70 to target polypeptide into matrix, you can use target peptide sequence of cytochrome b2 to target your polypeptide to intermembrane space, you can use targetpeptide sequences of ADP/ATP antiporter for positioning your polypeptide in innermembrane etc.
We used target peptide sequence of SSU (smaller subunit) of Rubisco to taget a bacterial gene for choline oxidase into chloroplasts in plants like chickpea, Indian mustard, sorghum etc.
You can make your own synthetic target peptide sequence, based on the known target peptide sequences that help in targetting polypeptide of your choice into site of your wish. We infact designed our target peptide sequence and got this target peptide sequence synthesized through Entelechon, Regensberg (DE). That saved our time.
You can look into publications from Prof. Walter Neupert and his team.
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