Group meeting 2016/5/13 Katsuhiro Umeda

Contents Introduction Active and passive MR with feedback Fucci My progress

Introduction Cytoskeletons Motor proteins generating the force Essential Cell Biology (© Garland Science 2010) http://www.seibutsushi.net/blog/2008/12/636.html 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

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

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

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.

G1 S G2 M Fucci Real-time visualization of cell-cycle progression 0.5- 1 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 http://www.amalgaam.co.jp/products/advanced/fucci.html

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)

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

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

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

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

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