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Thermosensitive mutations

Use of temperature-sensitive mutant in S. pombe fission yeast for live-cell imaging

Working with S. pombe is working with the most powerful tool in genetics. You can scan all aspect of cell division, microtubule assembly, cell polarity and do phenotype-genotype analyses in a science minute! Experiments go faster in fission yeast, it is the beauty of this model. So how about going even faster in your research?.

S.pombe use in biology

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S. pombe (Schizosaccharomyces pombe) is an African fission yeast (in fact, pombe means beer in Swahili) isolated in 1893 from millet beer. It is a unicellular eukaryote with caracteristic rode shape cells. S. pombe diameter is around 3 micrometers and depending on its cell cycle phase status, a cell can measure between 8 and 13 micrometers. With a generation time of 2 to 4 hours, and straightforward genetics, S.pombe was at the origin of groundbreaking work on cell cycle regulation, work at the origin of Sir Paul Nurse 2001 medicine Nobel prize (along with L. Hartwell and T.Hunt). In 2002, S. pombe full genome sequence was issued (Wood et al., 2002): it is composed of around 14 million base pair with 4824 genes. Yeast genetics studies have been at the forefront of genetic with regards to the use of temperature-sensitive mutant to dissect cell division and cellular pathways regulation.

 

Using temperature-sensitive mutant S. pombe

Temperature-sensitive mutant fission yeast is a commonly used genetics tool to study essential genes. It is a fast and effective technique to conditionally inactivate genes and question the role of any protein in a time-fashion manner. Thermo-sensitive mutant proteins function correctly at permissive temperature (25 C) but are unable to function at high (36 C), restrictive, temperature, on this basis, hypomorphic proteins (protein acting at sub-optimal level) can be obtained at intermediate temperature (Ben-Aroya et al., 2010). Temperature-sensitive mutations are point mutations which can for instance affect the correct folding of the protein. Some well-studied temperature-sensitive mutant fission yeasts affect cell cycle progression, mutant cells adopt a characteristic elongated shape when shifted to non-permissive temperature.

 

What is your favorite Temperature-sensitive mutant fission yeast ?
Temperature-sensitive-mutant-fission-yeast-live-cell-imaging-cell-division

Thermo-sensitive mutation is used to study cell cycle progression, spindle orientation, microtubule assembly, cell polarity, enviromental-sensing mechanisms, protein localization. For instance, in a very elegant study published in Developmental cell journal, Huang et al investigated the positionning of cell division plane, using live cell Imaging and temperature sensitive mutant, they found that Tea1p, Tea4p and Pomp1p, localized at the cell extremities, inhibit the assembly of cell division machinery at the cell ends, hence allowing the precise positioning of cell division plane. Bratman and Chang used temperature-sensitive Clasp protein, a stabilizing microtubule factor, to study the precise localization and interaction between Clasp and microtubule (Bratman and chang 2007).

 

Do temperature-shift experiments with S.pombe
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The availability of all these temperature-sensitive mutants, the possibility to easily perform live-cell imaging and track fluorescent-tagged genes, is opening a world of scientific experiments possibilities. With S.pombe, one can really watch experimental modification as they happen. If you want to shift from permissive to restrictive temperature in seconds and prevent temperature leakiness you can use our ultra-fast CherryTemp temperature controller. With CherryTemp you can also increment your sample temperature in a very precise step by step-fashion and look at the effect on your protein localization, or on your yeast behavior: e.g cell’s elongation when it is blocked in S phase. With CherryTemp, shifting from semi-restrictive to restrictive or going back to permissive temperature can be done in seconds; our system is both accurate and stable!

Reference

Scott V. Bratman and Fred Chang, Stabilization of Overlapping Microtubules by Fission Yeast CLASP, Dev Cell, 2007
Shay Ben-Aroya, Xuewen Pan , Jef D. Boeke and Philip Hieter ,Making temperature-sensitive mutants, Methods Enzymol. 2010
Wood et al., The genome sequence of Schizosaccharomyces pombe, Nature 2002
Yinyi Huang, Ting Gang Chew, Wanzhong Ge, and Mohan K. Balasubramanian Polarity Determinants Tea1p, Tea4p, and Pom1p Inhibit Division-Septum Assembly at Cell Ends in Fission Yeast, Dev Cell, 2007

S. Ben-Aroya, X. Pan, JD. Boeke, and P. Hieter, Making temperature-sensitive mutants, Methods Enzymol. 2010


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