Tetrahymena thermophila mutants: Fat, Mouthless and Balloon

Thanks to the wonderful ASSETT program, I had the opportunity to procure various temperature sensitive mutants of Tetrahymena thermophila. These grow normally at room temperature, but at 37℃ /98.6℉ they display defects due to mutations in genes involved in cell division (hence called temperature sensitive mutants).

My first step in reproducing these phenotypes was to find a way to make a 37℃ /98.6℉ incubator in my home lab. Following is a description of my low-tech hack. The culture tubes were placed in a water bath (a plastic bowl) which was further placed under an inverted glass jar. The glass jar was heated using a light bulb that was further controlled using a digital thermostat. I repurposed the thermostat from a heating mat that I use regularly to germinate seeds in winter (see heating mat).

The cultures were first grown for a couple of days in NEFF medium and then were incubated overnight at 37℃ /98.6℉ (temperature shift). I would love to hear of other incubator-design hacks from the community. One which is portable would make it even cooler.

Following videos show the division mutants. First let us look at wild type Tetrahymena thermophila under a foldscope so as to get a comparative base.

Now let us look at the mutant called Fat. Here the cell becomes round and “fat”, and shows an abnormal swimming pattern. Note that in this video, the cells have been slowed down with a detaining solution. Also video starts after 10s or so.

This mutant is called Mouthless as it doesnt have a mouth. In this instance the population was low, perhaps as I had it at the higher temperature so long that most cells just died. Mutant spherical cells are seen.

This is by far the most spectacular of the mutants and is called Balloon. I think the Balloon mutant is telling us something important — which is the range of ciliate shapes shouldnt come as a surprise. Here a single mutant shows a variety of shapes. Perhaps ciliate shape diversity could involve polymorphisms in such genes? A corollary to that would be that several ciliates might not be as different from each other as their shapes suggest.

Genetic mutants played a big role in understanding the biology of an organism, they still do. Thousands of mutants in model organisms changed the way we understood the biology of various life forms. However, in the past most mutants were isolated either naturally or by exposing cells to mutagens and selecting mutants. Now with genome sequences, we can alter specific genes and look for phenotypes. However, mutants still provide deep insights, so it is important to keep looking carefully and you might be on to something interesting if you find a mutant.

2 Comments Add yours

  1. Manu Prakash says:

    Lake: I love love love this post. It pokes at some very deep Questions – which you elude in your collorary.

    I have been thinking of “shape” in ciliates – like many other folks in the past. I have come to some conclusions that shape
    Is not solely encoded in the genomeof ge ciliates – and your corollary comes very close to that same idea. The cortical skeleton is crucial for setting up the shape – mis-regulation of the same carries an error in construction – and that would propagate to all progeny. Another way to say that is the “drawing board” a mother is working with is itself morphed – and once that happens, new shape will emerge. In another way, if the mother was using construction rules to build a cell geometry, but somehow the graph paper she was drawing the daughters on had a “transform” – say inclined lines instead of perpendicular line – the shape that emerges would be different.

    Let’s chat so I can explain this better. Beautiful post / also love your sub titles. I have such a huge backlog of posts – but you have just fired me up for getting my posts done.

    Fantastic post!

    Cheers
    Manu

    Cheers
    Manu

  2. laksiyer says:

    Dear @Manu. I know exactly what you mean. I am reminded of a series of experiments that one of my teachers (Karl Aufderheide) introduced me to in grad school of a famous paramecium researcher called Tracy Sonneborn (I will try and dig out those papers). In that Sonneborn surgically reversed a small section of the cilia of Paramecium causing that section to be in antiphase with the rest of the cilia in the organism. Descendants of these paramecia also maintained the same antiphase section. Karl used to always say that this is evidence of non-DNA based inheritance. In the old days I was much harsher on such views, but after what you say and what I have seen, this might be so for ciliates, or perhaps certain sub-cellular structures that might be templating descendant structures?

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