Nuclear import of DNA: genetic modification of plants

Transcription

1 Nuclear import of DNA: genetic modification of plants

2 gene delivery by Agrobacterium tumefaciens T. Tzfira & V. Citovsky Trends in Cell Biol. 12:

3 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)

4 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: (2004).

5 3D structure of VirE2-ssDNA complex by electron microscopy initial 3D model symmetrized 3D volume multi-reference alignment assign azimuthal angles to raw images raw images 2D reference projections repeat the cycle until convergence to a stable 3D structure apply in-plane transformations to raw images require stable symmetry parameters and uniform reference distribution back-project search for helical symmetry parameters and calculate residuals asymmetric 3D volume impose helical symmetry select parameters for minimal residual and randomize slightly to avoid local traps iterative procedure E. Egelman A robust algorithm for the reconstruction of helical filaments using single-particle methods. Ultramicroscopy 85:

6 3D structure of VirE2-ssDNA complex by electron microscopy E. Egelman A robust algorithm for the reconstruction of helical filaments using single-particle methods. Ultramicroscopy 85:

7 3D structure of VirE2-ssDNA complex by electron microscopy outer dia nm; rise 5.1 nm 4.3 VirE2/turn; ~ 19 bases/vire2 putative ssdna site along inner dia. corroboration by tetrameric rings

8 Use of T-complex to probe intracellular dynamics 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

9 Xenopus egg extract cytoskeletal reconstitution microtubules & actin long, semi-flexible protein filaments associating with molecular motors: myosin, kinesin, dynein

10 assay: embed VirE2-fl.ssDNA complex in random arrays of microtubules and filamentous actin does nuclear localization signal invoke active transport? excursions: [ 2 R Δt = x t Δt x t y t Δt y t 2 ] 1 2 probability distribution: RdR/ 2 Dt exp [ R / 4 Dt ] (Gaussian) 2 mean square displacement: x t = x 2 P x, t 2 x=0 2 γ x t =2d Dt, γ 1,=1, 1 Structural polarity of microtubules: conventional kinesin moves minus to plus cytoplasmic dynein moves plus to minus

11 tracer: T-complex of Agrobacterium tumefaciens gene transfer to plants ssdna w/ VirD2, VirE2 VirE2 effective only in plants reversal of 2 a.a. restores NLS 3 5 VirD2 wtvire2 = plvire2 mutvire2 = anvire2 VirD2 VirE2 VirE2 VirE2 VirE2 VirE2 B Guralnick, G Thomsen, V Citovsky Plant Cell 1996

12 tracer: T-complex of Agrobacterium tumefaciens import to reconstituted nuclei wtvire2 = plvire2 mutvire2 = anvire2

13 standard motility assay: transport along microtubules h. scale 13 µm, time interval 0.25 sec

14 assay: embed VirE2-fl.ssDNA complex in random arrays of microtubules and filamentous actin does the NLS invoke active transport? plvire2 anvire2

15 statistical assay by probability distribution histogram. result: microtubules involved in active movement. nocodazole.

16 result: F-actin responsible for sub-diffusion. Cytochalasin restores conventional diffusion. x 2 ~t 3 / 4

17 sensitivity to vanadate: dynein is the relevant motor. recall directionality.

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

19 summary: mean square displacements. x 2 ~t γ expt VirE2 Extract Measured γ Interpret γ movement 1 wt-pl untreated.75 3/4 passive constrained 2 mut-an untreated.99 1 active 3 mut-an Nocodazole.75 3/4 passive constrained 4 mut-an AMP-PNP.98 1 active 5 mut-an Vanadate.76 3/4 passive constrained 6 mut-an mab /4 passive constrained 7 wt-pl Cytochalasin D.97 1 passive Brownian 8 wt-pl Cytochalasin D + Nocodazole.97 1 passive Brownian 9 mut-an Cytochalasin D.93 1 active

20 model: directed walk on random lattice on arriving at a junction, direction of a turn is pre-determined random walk vs. random velocity field

21 model: random velocity field, scaling argument why ordinary diffusion scaling? assume: in-plane microtubules lie along x,y τ= mesh size l, velocity v, step duration τ RVF: v D z= stacked in z. random walk. x 2 t P yz 0, t l 2 v 2 t 2 l l P yz 0, t 2 2τ lv l l y 2 t Dz t r 2 t lv t same scaling as the classical random walk. l2 r t t 2 2

22 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: (2005).