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Control of Power Generation in Actin-Myosin Gels
Yamamoto Sho
Way of Control over Power Generation in Actin-Myosin gels
3、 2Ca exchange used dialysis membrane
4、 Photolysis of caged
2Ca
1、 Temperature adjustment in the sample generation2、 ATP regeneration system used PC , CPK
1、 Temperature adjustment in the sample generation
・ All the preparations and implements were kept to be cold. ( about at 0 ℃ )
・ When observing,I put the samples on the THERMO PLATE which was set at 3 ℃
Result
At room temperature(24℃) On THERMO PLATE
Problem ・ I could not control the exact temperature of the “actin-myosin gels”.・ The probe particles swayed from one side to another.(Probably because of the watere current in the THERMO PLATE.)
Solution・ To cover a box to keep the inner temperature cold .
・ Not to use oil immersion lens.( The oil conduct heat of the objective lens to the samples.)
exchange used dialysis membrane2Ca 3.
Jan Scrimgeour,*ab Jae Kyu Cho,c Victor Breedveldc and Jennifer Curtisab,Soft Matter,2011
ProblemPrepolymer solution did not be polymerized.Polyethylene glycol diacrylate has MEHQ as inhibitor.
Solution
Distillation under reduced pressure Boiling point of MEHQ : 243 ℃ 760mmHg 126 ℃ 11mmHg
5.Cooling water 9.Vacuum pump
2Ca
In the cell , force generation on actin-myosin network is controlled by calcium ion concentration. After released by endoplasmic reticulum , calcium is combined with troponin , and tropomyosin is caused a structural change , and finally actin is made activated.
Function of in Muscular Tissue
Method・ F-actin and Tn-Tm complex are mixed to final concentrations of 1mg/ml each in the presence of 0.1M KCl , 0.1mM ATP , 10mM-TrisHCl(pH=8.0) and the mixture are heated at 45 ℃ for 15 min and then cool at a slow rate (about 0.3 deg/min) to room temperature.
To confirm F-actin is crosslinked with TN-TM , I should observe superprecipitation in the presence of Ca ion.
Hajime Honda7 and Sho Asakura,1988
(a) and (c) Video-recorded images of fluorescent actin filaments and (b) and (d) changes in position of the actin filaments brought about by their movement in 0.6 s at (a) and (b) pCa = 3, (c) and (d) pCa = 5.8 at 30℃.
Hajime Honda7 and Sho Asakura,1988
Hajime Honda7 and Sho Asakura,1988
The average verocities at pCa=3 of regulated actin(continuous line with filled circle)and unregulated actin(broken line with open triangles).The broken line with open circle shows the temperature-dependence of regulated actin-activated myosin ATPase activity.