Group meeting 2016/5/13 Katsuhiro Umeda.

Presentation on theme: "Group meeting 2016/5/13 Katsuhiro Umeda."— Presentation transcript:

1 Group meeting 2016/5/13 Katsuhiro Umeda

2 Contents Introduction Active and passive MR with feedback Fucci
My progress

3 Introduction Cytoskeletons Motor proteins generating the force
Essential Cell Biology (© Garland Science 2010) The dynamics of cellular constituents obviously do not obey statistics in thermodynamic equilibrium It is essential to quantify their viscoelasticity and activity away from equilibrium

4 Feedback Microrheology(MR)
Prove laser （830nm） PID Piezo stage AOD Drive laser （1064nm） Set point Measure Cell Feedback loop Camera Quadrant photodiode Displacement of the particle to the medium

5 Active MR : Radius of the particle The generalized Stokes relation
Response function Displacement External force The generalized Stokes relation Drive laser Prove laser : Radius of the particle Complex shear modulus

6 Passive MR and Active MR
Particle Response function FFT Fluctuation-dissipation theorem (Equilibrium only) Combining AMR and PMR and evaluating the difference between the results is a way to test for nonequilibrium behavior of a system.

7 G1 S G2 M Fucci Real-time visualization of cell-cycle progression
h 6-12 h 今回Fucciと呼ばれる細胞周期の進行をリアルタイムに観察できる蛍光プローブを用いて観察を行いました。 Fucciは細胞周期の特定の時期にのみ存在するGemininとCdt1というたんぱく質を利用しています。 Cdt1はDNA複製のライセンス化制御因子であり、G1期に増加し、その他の期では分解されています。Cdt1にmonomeric Kusabira-Orange2という蛍光たんぱく質を融合させて、G1期の核を赤色に蛍光発色させます。 一方、GemininはDNA複製のライセンス化阻害因子であり、期に増加し、G1期では存在しません。Gemininにmoromeric Azami-Green1という蛍光たんぱく質を融合させて、sG2M期の核を緑色に蛍光発色させます。 3-4 h 6-8 h

8 Methods and purposes Micro-rheology measurement of the same HeLa cell
After about 12 h G1 phase S or G2 phase Compare results of the same HeLa cell (Complex shear modulus and activity)

9 Photographs of a HeLa cell
S or G2 5μm 5μm 12 h Red Green 5μm 5μm

10 Result1 : Complex shear modulus
○ G’ △ G” Red : G1 Green : S or G2

11 Result2 : α”, ωC(ω)/2kBT (Activity)
Cnoneq(ω) ― ωC(ω)/2kBT (PMR) ● α” (AMR) α”, ωC(ω)/2kBT [m/N] Frequency [Hz] Frequency [Hz] 12 h G1 S or G2

12 The yesterday's experiment
G1 Death… This is the difficulty of the same HeLa cell measurement

13 Summary Measuring of a HeLa cell using by Active MR and Passive MR with feedback system Fuuci can easily determine G1 and/or S/G2/M phases of the cell cycle Although in lack of statistics, some tendency in over- all stiffness and their frequency dependence were observed