Control of Power Generation in Actin-Myosin Gels Yamamoto Sho.

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Presentation transcript:

Control of Power Generation in Actin-Myosin Gels Yamamoto Sho

Way of Control over Power Generation in Actin-Myosin gels ３、 exchange used dialysis membrane ４、 Photolysis of caged １、 Temperature adjustment in the sample generation ２、 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 membrane3. Jan Scrimgeour,*ab Jae Kyu Cho,c Victor Breedveldc and Jennifer Curtisab,Soft Matter,2011

Problem Prepolymer 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

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 ofin 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

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.