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Yeast: Microtubules assembly

Temperature control of microtubules assembly

S. pombe is a very powerful organism to study microtubules assembly. The easy manipulation of fission yeast, the short dividing time and the existence of temperature-sensitive mutant strain allows researcher to dissect the role of microtubules in various aspects of S. pombe biology. Temperature is a controlling factor of microtubule assembly.
What if you could polymerize and depolymerize microtubules in a precise and ultra-fast fashion or combine microtubules studies with any of your temperature-sensitive mutant fission yeast ? With our CherryTemp temperature controller you can switch from 5-45C in seconds, it is published, easy to use and fits easily with any microscope settings.

Microtubules and cellular processes

Temperature-control-microtubule-fission-yeast (3)Microtubules are polymeric and very dynamic structures, which are part of the cytoskeleton of every cells. They play a crucial role in various cellular processes. During cell division they are important for chromosomes segregation in mitosis. They also ensure intra-cellular transport, nucleus and organelles movement. They participate in cell polarity and cellular migration. Microtubules, along with intermediate filament and actin filament can be seen as the bone and muscle of a cell, they really define cell architecture and shape. The role of microtubules in S.pombe cell division, growth and polarity has been very well documented (S.Martin, 2009; Piel and Tran, 2009).

 

Microtubules structure

Temperature-control-microtubule-fission-yeastA microtubule is a hollow cylinder and a polarized non-covalent polymer, with a plus and a minus end. A microtubule is composed of 13 protofilaments, each of them composed of alfa and beta-tubulin heterodimers. There are three phases in microtubule assembly: a nucleation, an elongation and an equilibration phases. The nucleation phase consists in the formation of the alfa and beta-tubulin dimers. This phase is called nucleation as alfa and beta dimers are the “nucleus” chore components of microtubules. In S.pombe, microtubule nucleation occurs in perinuclear microtubules organizing center called spindle pole body. The elongation phase is a growing phase, during which tubulin dimers are added to the microtubule plus end allowing microtubules to elongate away from the MTOC. Finally when the concentration of polymerized actin is equivalent to that of cytoplasmic free actin, the microtubule has reached an equilibration phase.

 

Microtubules depolymerization is controlled by temperature

temperature-control-microtubule-fission-yeastMicrotubules are very dynamic structures, with a constant addition and removal of tubulin dimers at the microtubule plus end tip. When tubulin dimers depolymerize and microtubule start to shrink this is refer to as “catastrophe”. The switch from a catastrophe phase to a growing phase is called “rescue”phase.

A number of drugs can interfere with microtubule polymerization, among them colchicine has been widely used in laboratory. Interestingly, once microtubules are formed, their stability becomes temperature dependent (Lodish, 2000). Subjecting the cells to low temperature (6C) is enough to obtain microtubule depolymerization, along with re localization of microtubule-associated protein to the perinuclear domain. This cold-shock is reversible and with heat microtubules can polymerize again.

Interestingly, fishes swimming in cold water, posess tubulin which is cold resistant and can polymerize at very low temperature (-1.8C).

 

Microtubule assembly during yeast cell division

temperature-control-microtubule-fission-yeast2During cell division, spindle microtubules are attached on one side to the spindle pole bodies and on the other side to chromosomes to pull them apart and into the future two daughter cells. During the cell cycle, if spindle microtubules are defective, the spindle checkpoint blocks mitosis progression. Taking advantage of a series of cold and heat-sensitive S.pombe beta-tubulin mutant along with temperature-shift experiments Paul Nurse’s team (Castagnetti et al., 2010) showed that while microtubules depolymerization triggers spindle checkpoint, it could quickly be bypassed with heat, and notably at high temperature. Indeed, after microtubules depolymerization, cytokinesis was delayed of 2 hours at 20 C, 45 min at 25C and no significant delays were observed at 32 C (Castagnetti et al., 2010). Following on these findings, they made the very interesting observation that without spindle microtubules, S.pombe can still undergo unusual nuclear fission.

 

Studying microtubules with temperature-shift experiment

temperature-control-microtubule-fission-yeast (2)Microtubules are dynamic, they grow, shrink, undergo catastrophe in a very short time. Microtubule half-life is short, it ranges from 1 to 10 min. Doing cold shock to induce microtubules depolymerization followed by heat-shock to revert it and combining it with live cell imaging or video time-lapse can become very tricky. With CherryTemp you can do just that, switch temperature (5 to 45 C) in seconds, program cycle of polymerization and depolymerization. In a very nice study, Phong T Tran’s team (Fu et al., 2009) used CherryTemp to perform temperature-shift experiments and look at the specific role of microtubule associated protein and motor protein in spindle elongation. CherryTemp is very easy and very efficient temperature controller device to perform ultra-fast activation, inactivation of temperature-sensitive proteins and dissect mechanisms that otherwise would be out of reach.

Reference

C.Fu, J J. Ward, I.Loiodice, G.Velve-Casquillas, FJ. Nedelec, and PT. Tran, Phospho-Regulated Interaction between Kinesin-6 Klp9p and Microtubule Bundler Ase1p Promotes Spindle Elongation, Developmental cell, 2009
S. Castagnetti, S. Oliferenko, P. Nurse Fission Yeast Cells Undergo Nuclear Division in the Absence of Spindle Microtubules, PLoS Biology, 2010
S. Martin Microtubule-dependent cell morphogenesis in the fission yeast, trends in Cell Biology, 2009
H.Lodish, Molecular Cell biology, WH Freeman edition.
M.Piel and PT. Tran Cell Shape and Cell Division in Fission Yeast , Current Biology 2009

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


How does CherryTemp™ work?

CherryTemp reaches such performance thanks to electronically controlled Peltier element and advanced microfluidic technology.

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