Group meeting 13th Dec. 2015 Kenji Nishizawa

Molecular crowding in cells (Callaway, E. 2008 Nature) (Goodsell 2009) Escherichia coli This illustration shows a cross-section of a small portion of an Escherichia coli cell. The cell wall, with two concentric membranes studded with transmembrane proteins, is shown in green. A large flagellar motor crosses the entire wall, turning the flagellum that extends upwards from the surface. The cytoplasmic area is colored blue and purple. The large purple molecules are ribosomes and the small, L-shaped maroon molecules are tRNA, and the white strands are mRNA. Enzymes are shown in blue. The nucleoid region is shown in yellow and orange, with the long DNA circle shown in yellow, wrapped around HU protein (bacterial nucleosomes). In the center of the nucleoid region shown here, you might find a replication fork, with DNA polymerase (in red-orange) replicating new DNA.  © David S. Goodsell 1999.  【cytoplasm】 highly concentrated ~300 mg/ml (ϕ ~30vol%) Mechanical property in cells (viscoelasiticity) 2

Crowding glass（colloids） glass transition glass super cooled 0.494 0.545 ~0.58 ~0.64 φ Colloidal suspension Concentration φ dependency of η Molecular crowding cause the rapidly increase η of in colloids. 粘性率 η/η０ What is in a living cell? φ 濃度 (Hunter et al. 2012 Reports on Progress in Physics)

Studying the molecular crowding effect Contents in my study Colliodal suspension （model systems） Viscosity of in vitro model systems My study Cell extrcts （multi） Viscosity in living cells living cells （ non-equilibrium ） Studying the molecular crowding effect on viscosity in a living cell

Measurements （Passive MR） QPD Displacement of a particle Fast Fourier Transform power spectral density Fluctuation-dissipation theorem Kramers-Kronig relations + Diameter １μm response function Generalized Stokes relation Complex shear moduli　 viscosity

Cell extract：concentration dependency of η Vogel-Fulcher-Tammann式 　　　細胞抽出液(大腸菌)

η rapidly increased as the protein concentration becomes higher Cell extract：concentration dependency of η prokaryotic cell：E. coli eukaryotic cell：xenopus eggs eukaryotic cell（commercial）HeLa cells 　　　細胞抽出液(大腸菌) 　　　細胞抽出液(カエルの卵） 　　　細胞抽出液(HeLa) conc. in a living cell η rapidly increased as the protein concentration becomes higher

Viscosity in living cells Colliodal suspension （ model systems ） Viscosity of in vitro model systems Is it difeerrent　from　 Viscosity in living cells? Cell extrcts 細胞抽出液 （equilibrium） Viscosity in living cells Living cells （ non-equilibrium）

Change concentration in a living cell Osmotic pressure Concentration of mavromoleules↑ New mediam drainage （３７℃） (Zhou et al. 2009 PNAS)

Result: Added glucose [m2/Hz]

Result: Added glucose

Mechanical properties in living cells（Viscoelastic properties） Cytoskeleton Athermal forrce Molecular crowding

Mechanical properties in living cells（Viscoelastic properties） Cytoskeleton Athermal forrce Molecular crowding

(C) Mechanical properties in living cells（Viscoelastic properties） Destruction s Cytoskeleton Athermal forrce Molecular crowding

Complex shear moduli G in a living cell （real part） （imaginary part）

Destruction of actin network Cytochalasin B Cytochalasins are known to bind to the barbed, fast growing plus ends of microfilaments, which then blocks both the assembly and disassembly of individual actin monomers from the bound end. Once bound, cytochalasins essentially cap the end of the new actin filament. Latrunculin B It binds actin monomers near the nucleotide binding cleft with 1:1 stoichiometry and prevents them from polymerizing. Administered in vivo, this effect results in disruption of the actin filaments of the cytoskeleton. This property has been used to great effect in the discovery of cadherin distribution regulation.

Change concentration in a living cell Change mediam which was added chemical New mediam drainage : Cells are dispersed on this area.

Effects of cytochalasin B May 9th G’ G’’ G’ (cytochalasin B) G’’ (cytochalasin B)

Effects of cytochalasin B April 27th G’ G’’ G’ (cytochalasin B) G’’ (cytochalasin B)

Effects of cytochalasin B April 4th G’ G’’ G’ (cytochalasin B) G’’ (cytochalasin B)

Effects of cytochalasin B March 24th G’ G’’ G’ (cytochalasin B) G’’ (cytochalasin B)

Effects of latrunculin B Quadrant photodiode

Effects of latrunculin B Quadrant photodiode

Effects of latrunculin B Quadrant photodiode

Effects of latrunculin B Quadrant photodiode

Calibration for MR in living cells

Displacement response Calibration in MR QPD Refractive index water n0 40% gly n1 cell ncell lens nl air 1 [v] Displacement response [m]

Displacement response C Calibration in MR QPD sample ns particle np [v] Displacement response C [m] Trap stiffness k

Relationship between k and C In glycerol

Displacement response in cells PID Quadrant photodiode sin wave Feedback loop x,y-Piezo stage

C cell C g k g Calibration in MR From the Relationship between k and C In glycerol Glycerol 40 w% In cells ×A calibration factor C cell C g 1) 2) k g trap stiffness 3) 2) ×A

Alignment 40% glycelole n1 Laser Quadrant photodiode Refractive index water n0 40% gly n1 cell ncell lens nl air 1 Quadrant photodiode Laser

Alignment (pattern 1) Water n0 Laser Quadrant photodiode Refractive index Water n0 water n0 40% gly n1 cell ncell lens nl air 1 Quadrant photodiode Laser

Alignment (pattern 2) Water n0 Laser Quadrant photodiode Refractive index Water n0 water n0 40% gly n1 cell ncell lens nl air 1 Quadrant photodiode Laser

Alignment (pattern 3) 40% glycelole n1 Laser Quadrant photodiode Refractive index 40% glycelole n1 water n0 40% gly n1 cell ncell lens nl air 1 Quadrant photodiode Laser

Alignment (pattern 1) Refractive index water n0 40% gly n1 cell ncell lens nl air 1

Alignment (pattern 1) Refractive index water n0 40% gly n1 cell ncell lens nl air 1 In cells cell water L a 細胞内部測定の際も で変えていないので

Alignment (pattern 2) 同様にして、

Alignment (pattern 2) cell water water n0 40% gly n1 cell ncell lens nl

Alignment (pattern 2) cell water

Alignment (pattern 3) cell water water n0 40% gly n1 cell ncell lens nl Calibはpattern 2と同じになるはず

C cell C g k g Calibration in MR Glycerol In cells 40 w% calibration factor C cell C g 1) 2) k g trap stiffness 3) 2) ×A