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Auto-reverse nuclear migration in bipolar vertebrate cells on micropatterned surfaces B. Szabó, Zs. Környei,A. Czirók, G. Csúcs, J. Zách, D. Selmeczi,

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Presentation on theme: "Auto-reverse nuclear migration in bipolar vertebrate cells on micropatterned surfaces B. Szabó, Zs. Környei,A. Czirók, G. Csúcs, J. Zách, D. Selmeczi,"— Presentation transcript:

1 Auto-reverse nuclear migration in bipolar vertebrate cells on micropatterned surfaces B. Szabó, Zs. Környei,A. Czirók, G. Csúcs, J. Zách, D. Selmeczi, T. Vicsek Motivation Methods - micropatterning - video-microscopy - inhibitors, immuno- cytochemistry - digital image processing Results Model

2 Nuclear positioning/migration versus bipolarity Bipolar cells are abundant in nature Fission yeast (J.Cell.Sci., 1998, 701) Where ? Yeasts (meiotic prophase, fast oscillations) In roots hairs (oscillations) Epithelium (positioning) Nucleokinesis (locomotion) Ventricular zone (developing vertebrate brain) Arabidopsis thaliana root hairs (Mol. Biol. Of the Cell)

3 Nuclear positioning/migration versus bipolarity Positioning of nuclei in the epithelium (wing, fruitfly) Ventricular zone (developing vertebrate brain) G1MG2SG1 Lumen of neural tube Newly formed chick neuronal tube (Nature, , 83)

4 METHODS: Nuclear motility assay Micropatterning: Alternating adhesive/nonadhesive strips are prepared by microcontact printing. “Stamps” are made of PDMS obtained from a silicon “negative” produced by photolitogaphy (design, photoresist layer on metal film, chemical etching using the patterned metal film) Stamping: PDMS stamp Inking with the protein solution Short drying Backfill with passivating PLL-PEG Stamping – transfer of the protein structures

5 METHODS: Long-term videomicroscopy

6 Inhibitors: Drugs targeting cytoskeletal proteins were used (vinblastine, taxol --> MT dynamics; vanadate, AMP-PNP, ML-7 --> mol. motors; cytochalasin --> F-actin) Immunocytochemistry: combining selective staining with videomicroscopy (comparing spatial information with velocity data) Digital image processing: recording data from video sequences, calculating velocities of nuclei, and determining such features of the oscillatory motion as distribution of periods, effect of drugs on average velocity, etc METHODS (continued)

7 Results A typical experiment: Adhesive: fibronectin Strip width: 20 micron Cell: C6 glioma cell line Duration: 36 hours A single cell:

8 Auto-reverse motion in bipolar cells C6 – yes, average period 5 h Primary mouse fibroblast: - yes, average period 1.8 h Primary mouse astroglia: migration, no oscillations 3T3 nuclei migrate, no reverse U87 no nuclear motility C6 on plain on stripe fibroblast on stripe

9 Time dependence of the position of a nucleus and the two corresponding edges of the cell Time dependence of the velocity of the above nucleus

10 Distribution of the maximum velocities in an ensemble of C6 cells Peaks are at +/- 50 micron/hour Distribution of the duration of the periods in an ensemble of the above cells. Peak is at 5 hours

11 Effect of drugs Inhibition of microtubule dynamics: e.g., adding after one day 20 nM Vinblastine blocks auto-reverse migration Drugs targeting actin and molecular motors did not have effect

12 Results from immunocytochemistry The microtubules run parallel to the main axis (even over the nucleus, where their number density is significantly smaller) Staining for microtubules

13 Centrosome position is correlated with the direction of nuclear motion Staining for  tubulin reveals that the centrosome is never located in the front part of the nucleus no migration centrosome

14 Forces involved in nuclear migration From data on the viscosity of the cell plasma and the velocity/size of the nucleus, using Stoke’s law, we estimate the force needed to be in the range pN The polimerization of one microtubule generates appr. 1pN, thus a few dozen can push/pull the nucleus Microtubule polymerization dynamics is a source of force (From Molecular Biology of the Cell )

15 Experimental evidence for microtubules pushing an aster in a microfabricated chamber (PNAS, 94, )

16 MODEL Based on our following observations: a) Array of microtubules is highly organized b) Centrosome is on the trailing side c) Estimated force is high enough ( dynein can play some role close to the cortex) Reversal of direction is due to the repositioning of the centrosome (pushed by microtubules) at the edge of the cell Only microtubules playing the most important role in the migration of the nucleus are shown (there are many more)

17 CONCLUSIONS - First direct observation of oscillatory nuclear migration in vertebrate cells - Proposition/demonstration of a nuclear motility assay for in vitro study - Determination of the main characteristics of auto-reverse nuclear migration - Evidence for the major role of MT dynamics and the position of the centrosome - Presentation of a corresponding model Acknowledgements: Hung. Natl. Sci Funds: OTKA and NKFP

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