How do cells position their division plane?

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1 How do cells position their division plane? Nicolas Minc Group Leader Institut Jacques Monod Contact:

2 Cells divide at specific location C. Elegans zygote Sea urchin Embryos 10 mic Fission yeast cells

3 Division plane positioning Xenopus cells Drosophila epithelium C. Elegans embryo Zebrafish embryo Sea cucumber embryo (Synapta maculata)

4 How do cells position their division plane????

5 Outline 1- A historical view on the question 2- The steps of cell divison 3- Microtubule forces 4- Some classical case studies 5- Predicting division position and orientation

6 Outline 1- A historical view on the question 2- The steps of cell divison 3- Microtubule forces 4- Some classical case studies 5- Predicting division position and orientation

7 Old rules for division positioning Sachs rules for plant cells: Daughter cells of same size and division plane perpendicular to existing wall Sachs, 1878; Besson and Dumais, PNAS 2011

8 Old rules for division positioning Sea cucumber Synapata Hertwig s rules for animal cells Division at cell center of mass and perpendicular to longest axis Oscar Hertwig s constrained frog egg Hertwig, 1884; Minc and Piel, Trends Cell Biol, 2012

9 Old rules for division positioning Sea cucumber Synapata Oscar Hertwig s constrained frog egg What has changed in our understanding of this basic process?

10 Ouline 1- A historical view on the question 2- The steps of cell divison 3- Microtubule forces 4- Some classical case studies 5- Predicting division position and orientation

11 The Different steps of cell division Animal cells Plant cells Metaphase Cytokinesis Anaphase

12 The mitotic spindle Microtubules DNA In animal cells mitotic spindle organises both karyokinesis and cytokinesis Astral Microtubules play a key role in mediating position and orientation of spindle

13 Ouline 1- A historical view on the question 2- The steps of cell divison 3- Microtubule forces 4- Some classical case studies 5- Predicting division position and orientation

14 Microtubules are dynamic Tubulin-GFP in fission yeast

15 Microtubules are polarized

16 Microtubules can generate forces Minus-end motors: Dynein, kinesin-14 Depolymerizing factors: kinesin-13, kinesin-8

17 Dominant modes of force generation

18 Dominant modes of force generation MT pushing and the problem of cell size fpush ~ 1/L^2 Sufficient for small cells (fission yeast..~5mic) Not sufficient for large cells (eggs ~ mic)

Pulling with a limiting numbers of sites works in centering

19 Dominant modes of force generation MT pulling at the cortex Pulling may be unstable in centering fpull~ Constant Pulling with a limiting numbers of sites works in centering fpull~ L^2 Grill et al. Dev Cell 2005, Wuhr et al. Curr Biol 2009

20 Dominant modes of force generation MT pulling in the cytoplasm fpull~ L Cytoplasmic forces: Dynein-mediated vesicle friction? Wuhr et al. Curr Biol 2009, Kimura et al. 2011

21 Mechanics of spindle positioning Microtubules / DNA / Centrosomes (C. elegans zygote, P. Gonczy Lab) Microtubules generate forces to position the centrosomes, nuclei or spindle Forces are biased by different cues : Polarity components, cell adhesion, geometry

22 Ouline 1- A historical view on the question 2- The steps of cell divison 3- Microtubule forces 4- Some classical case studies 5- Predicting division position and orientation

23 Asymetric divisions?

24 Asymmetric first division in C. elegans embryo Hyman Lab Youtube Channel

25 Direct evidence for asymmetric force generation Grill et al. Nature 2001, Science 2003

Asymmetric first division in C. elegans embryo Asymmetry in polarity factors lead to asymmetry in pulling forces exerted by astral microtubules.

26 Asymmetric first division in C. elegans embryo Asymmetry in polarity factors lead to asymmetry in pulling forces exerted by astral microtubules. Microtubule pulling forces are exerted by minus end directed motors (Dynein) that pull on microtubules -> position spindle Morin and Bellaiche, Dev cell 2011

27 Other Asymmetric classical divisions S. Cerevisiae Drosophila Neuroblast

28 Symetric divisions?

29 A-The fission yeast cell WT Chang et al. JCS, 1997; Padte et. al. Curr Biol 2006; Celton-Morizur et. al. JCS 2006

30 The fission yeast cell WT Mid1 mutant Mid1-GFP Mid1 links Nuclear positioning to cytokinesis Chang et al. JCS, 1997; Padte et. al. Curr Biol 2006; Celton-Morizur et. al. JCS 2006

31 The fission yeast cell Centrifuge nucleus Without MTs How is the nucleus position in the center?? Tran et. al., JCB 2001 Daga et al. PNAS 2005, Curr Biol 2006

32 B. Cell shape and cell division positioning Observation of cleavage planes in early embryogenesis The two poles of the division figure come to lie in the direction of the greatest protoplasmic mass. (Hertwig, 1884 ) How does it work?

33 Model system: Sea Urchin embryos - Round and apolarized - Large cells/ large organelles - Cells obtained in large batch Hoechst staining (DNA) Time in minutes after fertilization

34 b. Cell shape and cell division 100mm Minc et al, Cell, 2011

35 b. Cell shape and cell division How does the cell sense its shape? How does the cell define its center/ its geometry?

36 Interphase microtubules sense shape and orient division DNA Microtubules + DMSO + Nocodazole. Division axis is set early in interphase/early prophase. Centering and orientation is MT dependent and actin independent. MT asters probe cellular space and orient the nucleus by exerting pulling forces

37 Theoretical model of division axis definition Key hypothesis: Each MT pulls with a force that scales with its length. Hamaguchi, Hiramoto, 1980 Wühr, Tan, Parker, Detrich, Mitchison, 2010

38 Torque (A.U.) Theoretical model of division axis definition Probability density (A.U.) Key hypothesis: Each MT pulls with a force that scales with its length. => Compute global torque exerted by the two MT asters on the nucleus Division axis angle, a Division axis angle, a See Théry M, Jiménez-Dalmaroni A, Racine V, Bornens M, Jülicher F., Nature 2007

39 Experiments Theory Theoretical model of division axis definition All parameters fixed, only cell shape varies

40 Ouline 1- A historical view on the question 2- The steps of cell divison 3- Microtubule forces 4- Some classical case studies 5- Predicting division position and orientation

41 Predicting spindle orientation in an adult tissue Section drawing from pigeon testis (M. Guyer, 1900)

42 Predicting spindle orientation in an adult tissue Section drawing from pigeon testis (M. Guyer, 1900)

43 Predicting spindle orientation in an adult tissue Section drawing from pigeon testis (M. Guyer, 1900) Model Suggest that cell geometry may be a major cue for division positioning in these cells

44 Predicting cleavage plane orientation Carvhalo et. al. Nat Cell Biol 2013 (with C-P Heisenberg)

45 Predicting cleavage plane orientation Carvhalo et. al. Nat Cell Biol 2013 (with C-P Heisenberg)

46 Predicting division orientation in tissues Drosophila thorax epithelium Bosveld et al., Submitted, (with Y. Bellaiche)

(amphibians, fishes..) - Set by various signals (cell shapes, maternal cues, yolk.

47 Continuing projects : Predicting embryonic cleavage patterns Sea Urchins Frogs - Cleavage patterns are stereotyped and invariant among groups of species (amphibians, fishes..) - Set by various signals (cell shapes, maternal cues, yolk..) - Yet they are labile, suggesting they may rely more on self-organization designs than determinism

48 Novel model framework to predict cleavage patterns 1- Model to predict division position in 3D ; can account for surface cues, yolk.. Anaëlle

49 Novel model framework to predict cleavage patterns 1- Model to predict division position in 3D ; can account for surface cues, yolk.. Anaëlle 2- Model to predict blastomere shapes and Rearrangements (Surface evolver)

50 Novel model framework to predict cleavage patterns F Polarity cap, Yolk gradient Surface Evolver 3D division model Surface Evolver Shape Division : volumes and initial cell-cell contacts Shapes And so on Complete in silico modeling of embryos development!

51 Predicting cleavage patterns Zebrafish Kimmel et al. 1995

52 WT Zebrafish Blastodisc Yolk A B Blastodisc Yolk Predicting cleavage patterns Model Zebrafish Hypothesis: Microtubule length-dependent forces

53 A WT Zebrafish Blastodisc Yolk B Predicting cleavage patterns Model Blastodisc Zebrafish Yolk Top view

54 A WT Zebrafish Blastodisc Yolk B Predicting cleavage patterns Model Blastodisc Zebrafish Yolk C Experiment (Olivier et al., 2010) Top view F Model Top view Experiment (Olivier et al., 2010) C Top view D Side view 1 F G Model Top view Side view 1 Switch of orientation E Side view 2 H Side view 2 D Side view 1 Side view 1 G D Side view 1 Side view 1 G Side view 1 L Experiment (Urven et al., 2006) Switch of orientation Switch of orientation

55 Predicting cleavage patterns A WT xenopus B Hypothesis: Microtubules pull with length dependent forces, and are less stable in yolk Yolk granules MTs Xenopus C Model

56 Predicting cleavage patterns D h H AVCR = H/h Experiment (*) Model * Yokota et al.,

57 Predicting cleavage patterns Macromeres Micromeres Sea urchin

58 Take Home - Division positioning and orientation relies on forces and torques exerted by cytoskeletal elements onto nuclei or spindles - These forces are influenced by different cues: Internal polarity, cell geometry, adhesion.. - These cues may act in additive manner in tissues to generate the wide diversity of division patterns. - Integration between biological and quantitative approach at the heart of these questions

59 Acknowledgments Morphogenetic puzzle team Collaborators Arezki Boudaoud, ENS Lyon Etienne Couturier, MsC, Paris Carl Philipp Heisenberg, IST, Vienna Yohanns Bellaiche, Curie, Paris Alex Mc Dougal, Villefranche David Burgess, Boston College Fred Chang, Columbia University Past members: Francois Rousset, Henry De Belly, Marguerite Lapierre Yonatan Zegman, Alexis Campetelli, Rima Seddiki OPEN POSITIONS FOR MASTERS Paris IJM Armin Pombe 100m 10m 10cm 10μm

Pulling forces in Cell Division

Pulling forces in Cell Division Frank Jülicher Max Planck Institute for the Physics of Complex Systems Dresden, Germany Max Planck Institute for the Physics of Complex Systems A. Zumdieck A. J.-Dalmaroni