MicrobeHunter.com Microscopy Forum

Ciliates exhibit what is known as nuclear dimorphism where usually two (or more) nuclei co-exist. One, the micronucleus (MIC) is transcriptionally silent and acts as a germline carrier i.e. carries all the information required to make a new organism. The macronucleus (MAC) on the other hand, develops from the MIC whereby a huge portion of the MIC DNA (mostly repetitive sequence and genetic parasites known as transposons or jumping genes) is excised and eliminated. The remaining pieces of DNA (microchromosomes) are amplified many thousands of times and carry the information necessary for the day-to-day functioning of the organism.

Occasionally, when conditions go bad for example food becomes scarce, ciliates conjugate and exchange newly replicated micronuclei. Upon exchange these fuse (i.e. a process analogous to fertilization) after which a number of divisions occur. Some of the division products end up as the new MIC whereas others mature into the new MAC. The developing MACs are known as MAC anlagen and in the process of MAC maturation the old MAC is destroyed. The destruction of the old MAC depends on a specific gene expression program and is thus termed Programmed Nuclear Death.

Here I captured a few shots of Paramecium (I believe) stained with acridine orange (stains DNA in green) in conditions of epifluorescent microscopy. You can see the normal appearance with clearly distinct MIC and MAC. But you can also observe exconjugant cells undergoing programmed nuclear death. One can observe precisely 4 MAC anlagen present per cell along with multiple smaller nuclear fragments (products from the death of the old MAC) that would eventually be resorbed.

Excellent work Wes! The images are not only instructive but also attractive. Can you give me any idea (order of magnitude: minutes, hours, days) of how long it takes from completion of conjugation to completion of the programmed nuclear death process - i.e. complete resorption of the fragments of the old MAC?
Tom W.

Very interesting Wes!

This is absolutely gorgeous! Such a clear record of the post-conjugal cell, after the old macronucleus disintegrates and before the final series of divisions. I think some of the smaller fragments might include recently divided micronuclei (the post-conjugal synkaryon, which undergoes a few mitoses before the cell itself splits)? Congratulations on achieving such great results.

Well done, Wes! Excellent images and interesting subject .

Kirby

Element 56 wrote: ↑

Wed Sep 02, 2020 1:17 pm

Well done, Wes! Excellent images and interesting subject .

Kirby

+1 !

Excellent work!

Going to have to read up on this! Thanks for posting!!

Thank you everyone for the interest and kind comments.

tgss wrote: ↑

Wed Sep 02, 2020 11:31 am

Can you give me any idea (order of magnitude: minutes, hours, days) of how long it takes from completion of conjugation to completion of the programmed nuclear death process - i.e. complete resorption of the fragments of the old MAC?

I wasn't sure so had to check the literature and it appears the parental macronucleus persists until the eighth cell cycle post conjugation. Under optimal laboratory conditions the doubling time of Paramecium tetraurelia and Paramecium caudatum is about 14 hours. Interestingly the initiation of macronuclear degeneration occurs around the fifth cell cycle following conjugation. So the short answer to your question is it takes days for complete MAC resorption. For the sake of completeness I also dug out the duration of the conjugation process which differs in Paramecium species, about 24 hours in Paramecium bursaria, about 15 hours in Paramecium caudatum, about 12 hours in Paramecium multimicronucleatum and about 6 h in Paramecium tetraurelia.

Bruce Taylor wrote: ↑

Wed Sep 02, 2020 1:16 pm

I think some of the smaller fragments might include recently divided micronuclei (the post-conjugal synkaryon, which undergoes a few mitoses before the cell itself splits)?

I absolutely agree here, however I am not sufficiently trained to tell you which ones would be the micronuclei (maybe one has to use immunofluorescence with MIC antibody markers or quantitative fluorocytometry to detect 2C DNA content but this is beyond the capabilities of my home "lab").

And I'd also like to take the opportunity to share with you a pair of conjugating Paramecia, this time in DIC. It would be interesting to explore chemical methods for the induction of conjugation and look at conjugating cells under the fluorescence scope to see if I can track the micronuclei.

Excellent work and images! Very inspirational.

Thank you hkv!

Wes wrote: ↑

Wed Sep 02, 2020 7:39 pm

tgss wrote: ↑

Wed Sep 02, 2020 11:31 am

Can you give me any idea (order of magnitude: minutes, hours, days) of how long it takes from completion of conjugation to completion of the programmed nuclear death process - i.e. complete resorption of the fragments of the old MAC?

I wasn't sure so had to check the literature and it appears the parental macronucleus persists until the eighth cell cycle post conjugation. Under optimal laboratory conditions the doubling time of Paramecium tetraurelia and Paramecium caudatum is about 14 hours. Interestingly the initiation of macronuclear degeneration occurs around the fifth cell cycle following conjugation. So the short answer to your question is it takes days for complete MAC resorption. For the sake of completeness I also dug out the duration of the conjugation process which differs in Paramecium species, about 24 hours in Paramecium bursaria, about 15 hours in Paramecium caudatum, about 12 hours in Paramecium multimicronucleatum and about 6 h in Paramecium tetraurelia.

Thank you Wes for the very complete answer. The duration of this process is much longer than I anticipated, extending as it does through several generations. One can't help wondering what the implications of this might be if it's persistence actually provides an evolutionary advantage. Thanks again for a very informative post and response.
Tom W.

Beautiful fluorescent images of Paramecium - bravo.
Cheers
RB

RobBerdan wrote: ↑

Wed Sep 02, 2020 10:40 pm

Beautiful fluorescent images of Paramecium - bravo.
Cheers
RB

Thank your Rob! I have to remember to follow your advice regarding fluorescence microscopy and shoot in raw format next time. I recall you posting at some point about how one could mimic deconvolution by shooting in raw and then altering certain settings (perhaps you can briefly refresh my memory which settings you alter).

Another beautiful image, Wes!
Kirby

Impeccable image.
Very impressive.
thanks

Very good work //KD

Thank you Kirby, Sabatini and KD.