Nuclear import of DNA: genetic modification of plants

Nuclear import of DNA: genetic modification of plants

gene delivery by Agrobacterium tumefaciens

T. Tzfira & V. Citovsky. 2001. Trends in Cell Biol. 12: 121-129

VirE2 binds ssDNA in vitro

forms helical complex with “telephone cord” morphology Scanning Transmission Electron Microscopy

V. Citovsky et al., J. Mol. Biol. 271: 718 (1997)

VirE2 binds ssDNA in vitro

forms helical complex with “solenoid” morphology Transmission Electron Microscopy in negative stain

A. Abu-Arish et al., J. Biol. Chem. 279: 25359-363 (2004).

3D structure of VirE2-ssDNA complex by electron microscopy

iterative procedure

raw images raw images

2D reference projections 2D reference projections initial

3D model

multi-reference multi-reference

alignment alignment assign azimuthal assign azimuthal

angles to angles to raw images raw images

apply in-plane transformations to

raw images

back-project search for helical symmetry

parameters and calculate residuals

select parameters for minimal residual and randomize slightly to

avoid local traps

impose helical symmetry repeat the cycle until convergence

to a stable 3D structure

require stable symmetry parameters and uniform reference distribution

symmetrized 3D volume

asymmetric 3D volume

E. Egelman. 2000. A robust algorithm for the reconstruction of helical filaments using single-particle methods. Ultramicroscopy 85: 225-234.

3D structure of VirE2-ssDNA complex by electron microscopy

E. Egelman. 2000. A robust algorithm for the reconstruction of helical filaments using single-particle methods. Ultramicroscopy 85: 225-234.

3D structure of VirE2-ssDNA complex by electron microscopy

outer dia. 15.7 nm; rise 5.1 nm 4.3 VirE2/turn; ~ 19 bases/VirE2 putative ssDNA site along inner dia.

corroboration by tetrameric rings

hypothesis: entry into the nucleus starts with delivery via the cytoskeleton.

difficult to test due to radial organization.

approach: map a tricky problem onto a tractable one

arrival to destination vs. path in random medium methods: tracer – Agrobacterium T-complex

control nuclear targeting by mutation

particle tracking – fluorescence microscopy statistical analysis

follow the tracer through a random network then identify direction along single filament

Use of T-complex to probe intracellular dynamics

Xenopus egg extract – cytoskeletal reconstitution microtubules & actin

long, semi-flexible protein filaments

associating with molecular motors: myosin, kinesin, dynein

http://en.wikipedia.org/wiki/Image:Xenopus_laevis.jpg

assay: embed VirE2-fl.ssDNA complex in random arrays of microtubules and filamentous actin

does nuclear localization signal invoke active transport?

Structural polarity of microtubules:

conventional kinesin moves “minus” to “plus”

cytoplasmic dynein moves “plus” to “minus”

RΔt=

[

]

RdR/2Dtexp[^−R2/4Dt]

〈x²t〉=

∑

x=0

∞

x² Px , t 

〈x²t〉=2d Dt^γ, γ 1,=1,1 excursions:

probability distribution:

mean square displacement:

(Gaussian)

tracer: T-complex of Agrobacterium tumefaciens

gene transfer to plants ssDNA w/ VirD2, VirE2

VirE2 VirE2 VirE2 VirE2 VirE2

VirD2

5’ 3’

VirD2

B Guralnick, G Thomsen, V Citovsky Plant Cell 1996

VirE2 effective only in plants

reversal of 2 a.a. “restores” NLS wtVirE2 = plVirE2

mutVirE2 = anVirE2

tracer: T-complex of Agrobacterium tumefaciens import to reconstituted nuclei

wtVirE2 = plVirE2 mutVirE2 = anVirE2

standard motility assay: transport along microtubules

h. scale 13 µm, time interval 0.25 sec

anVirE2 plVirE2

assay: embed VirE2-fl.ssDNA complex in random arrays of microtubules and filamentous actin

does the NLS invoke active transport?

statistical assay by probability distribution histogram.

result: microtubules involved in active movement. nocodazole.

result: F-actin responsible for sub-diffusion.

Cytochalasin restores conventional diffusion.

〈 x²〉~t^3/4

sensitivity to vanadate: dynein is the relevant motor.

recall directionality.

result: dynein is the relevant motor. focus on MSD.

summary: mean square displacements.

9 mut-an Cytochalasin D .93 1 active

8 wt-pl Cytochalasin D

+ Nocodazole .97 1 passive

Brownian

7 wt-pl Cytochalasin D .97 1 passive

Brownian

6 mut-an mAb70.1 .77 3/4 passive

constrained

5 mut-an Vanadate .76 3/4 passive

constrained

4 mut-an AMP-PNP .98 1 active

3 mut-an Nocodazole .75 3/4 passive

constrained

2 mut-an untreated .99 1 active

1 wt-pl untreated .75 3/4 passive

constrained expt VirE2 Extract Measured γ ^Interpret γ ^movement

〈x²〉~t^γ

model: directed walk on random lattice

on arriving at a junction, direction of a turn is pre-determined

“random walk” vs. “random velocity field”

why ordinary diffusion scaling?

assume:

in-plane microtubules lie along x,y

mesh size l, velocity v, step duration τ

stacked in z. random walk.

RVF:

same scaling as the classical random walk.

τ=l v D_z=l²

2τ ≈ lv 2

〈x²















^

^

l



〈r²









〈 r

 t  〉≈  ^{l }

 ^t

model: random velocity field, scaling argument

back to biology:

statistical motility assay shows that nuclear targeting leads to active centripetal delivery on microtubules.

H Salman, A Abu-Arish, S Oliel, A Loyter, J Klafter, R Granek, M Elbaum.

Biophys. J. 89: 2134-2145 (2005).