1. Group meeting
13th Dec. 2015
Kenji Nishizawa

2. Molecular crowding in cells
(Callaway, E 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

3. 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 Reports on Progress in Physics)

4. 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

5. 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

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

7. 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

8. 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）

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

10. Result: Added glucose
[m2/Hz]

11. Result: Added glucose

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

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

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

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

16. 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.

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

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

19. Effects of cytochalasin B April 27thG’
G’’
G’ (cytochalasin B)
G’’ (cytochalasin B)

20. Effects of cytochalasin B April 4thG’
G’’
G’ (cytochalasin B)
G’’ (cytochalasin B)

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

22. Effects of latrunculin B
Quadrant photodiode

23. Effects of latrunculin B
Quadrant photodiode

24. Effects of latrunculin B
Quadrant photodiode

25. Effects of latrunculin B
Quadrant photodiode

26. Calibration for MR in living cells

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

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

29. Relationship between
k and C In glycerol

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

31. 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

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

33. 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

34. 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

35. 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

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

37. 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

38. Alignment (pattern 2)
同様にして、

39. Alignment (pattern 2) cell water water n0 40% gly n1 cell ncell lensnl

40. Alignment (pattern 2)
cell
water

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

42. 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