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Calcium ions in the Cell

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Calcium ions in the Cell

Postby someguy456345 » Sat Apr 24, 2010 3:05 pm

So I just had my final exam for Cell Biology and this was one of the questions on it I can't figure out, hoping someone may be able to shed some light/clear up the issues I have.

Calcimycin, a toxin produced by the bacterium Streptomyces chartreusensis, acts as a mobile ion-carrier that allows Ca2+ ions to freely cross eukaryotic cell membranes, and indictors like Fluo5N give off fluorescence when bound to these ions. When Fluo5N is introduced into the same kind of yeast cells we used in our discussion of the two-hybrid analysis, and these cells are then treated with calcimycin, what organelle would you expect to see fluoresce when the cells are examined by confocal microscopy?

A) Mitochondria
B) Lysosome
C) Proteasome
D) RER
E) None of the Above

Alot of things jump out at me right away. Firstly, I must mention we've heard nothing of the toxin nor the fluorophore in class, upon further research on the internet I've found that Calcimycin (aka 823187) does indeed bind and inhibit Mitochondria.

When looking at this question, it reminded me strongly of a previous year's exam question which substituted yeast for bacteria (no organelles) and I thought this might be the way the professor was trying to trick us. I would have thought that all organelles would fluoresce equally as the toxin would introduce Calcium into all parts of the cell.

I know that bacterial cell walls allow for generalized passage of ions (e.g. calcium or Fluo5N) but are fungal walls significantly different in their function. I would assume their walls would allow for generalized passage as well.

Also the mention of "the same kind of yeast cells used in our discussion of yeast 2-hybrid" is confusing as well. The only thing I can think of there is that the yeast are deficient in three amino acids (2 are plasmid markers, the third is a result of activation of the fish-bait gene activation). I don't think this is what he meant, moreso that the cells were susceptible to plasmid uptake (predominantly charged particles = similar to Fluo5N maybe)

In actuality, I picked mitochondria not because I knew it was inhibited, but because the three other organelles contain inner exoplasmic face vs a mitochondrial double membrane causing an inner cytosolic face (seemed like the only difference). However, in retrospect this may create difficulty for the Fluo5N to cross both membranes.

Thanks in Advance!
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Re: Calcium ions in the Cell

Postby dam13 » Thu Apr 29, 2010 10:38 pm

if its poisonous wouldnt u expect to find it in the lysosome? im not too cell savy but that would be my guess being that it processes food and foreign bodies, but i could also see how u could see it in the rough er bc that is where protein synth is taking place and i think ~75% of [ca+2] is stored. ha so i also would have some prbls w this question, cellbio always has so many things going on
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Postby JackBean » Fri Apr 30, 2010 5:50 am

dam13 has a point. THe question actually isn't about where the dye diffuses into, but where is calcium stored...
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby kolean » Sat May 01, 2010 3:06 am

Ca2+ is mostly stored in the ER (don't know about rER) and the mitochondria.
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Re: Calcium ions in the Cell

Postby nmremala » Sat May 01, 2010 7:52 am

There are TWO factors we have to consider : Calcimycin and the Calcium fluorescent dye

Calcimycin incorporates into the cell membranes : first into plasma membrane and then very slowly into other intracellular membranes i.e. the membranes of mitochondria, nucleus, ER, Lysosomes, Golgi etc. It has to diffuse through the aqueous cytoplasm to reach the latter membranes OR through the continuities of plasma membrane with these membranes. So, the main action of calcimycin will be through its incorporation in the plasma membrane. The result will be a lot of sudden influx of calcium ions into the cytoplasm and resultant dramatic rise of cytoplasmic calcium ion conc. This happens only when the extracellular Calcium ion conc. is much higher than the intracellular conc. This generally is the case. The intracellular Ca ion conc. are less than micromolar (most cells have 0.1 micromolar or less) and extracellular conc. are above millimolar; so the gradient is nearly 10,000 times.

The calcium fluorescent dyes fluoresce when they bind to calcium ions (they have different emission spectral characteristics in the bound versus unbound state). These dyes, being non-lipophilic in nature, donot cross the cellular membranes. These dyes are introduced into the cell through 1 of the 2 ways:

1. micro-injection of the conc. dye solution into cytoplasm. Generally injecting the dye solution into cell organelle is very difficult ( 1. You need have a very finely fabricated microsyringe, 2. You need to inject into sufficient number of organelle of the same type and 3. You need a microscope which can facilitate the visualisation of the organell during microinjection process, NO MICROSCOPE OPERATING WITH UV OR VISIBLE WAVELENGTHS OF LIGHT CAN GIVE SUFFICIENT MAGNIFICATION OF THE ORGANELLE INSIDE A LIVING CELL SO WE CAN DO MICROINJECTION)

OR

2. Incubation of the cells with the AM (acetoxy methyl) ester of the dye. The lipases of the cytoplasm cleave the ester bond to release the free dye into the cytoplasm. The possibility that a cell organell getting labelled with the dye is remote. So, only the cytoplasm gets labelled with the dye. Even if some amount of dye manages to get into the lumen of some cell organelle by some unknown mechanism, the amount will be very less and hence the signal from the lumen of the cell organelle will be comparatively very very less.

So, for all practical sense, it is the cytoplasm that can be labelled with the fluorescent dye.

Since cytoplasm/cytosol is not one of the options in the question, the answer must be none of these.
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