1. Alpha-Actinin is Required for Tightly Regulated Remodeling of the Actin Cortical Network during Cytokinesis Svetlana Mukhina Speaker: Jerry Qiu, Kunkka Liao

2. Introduction Cytokinesis Contractile Ring Hypothesis Actinin

3. Contractile Ring

4. Actinin

5. Dynamics of α-actinin during cell division of mammalian cells

6. Quantification of α-actinin-GFP intensity at the cleavage furrow

7. FRAP analysis of α-actinin-GFP during cytokinesis

8. Overexpression of α-actinin causes an increase in equatorial actin filaments, delayed cytokinesis and cytokinesis failure

9. Frequency of multinucleation in cells transfected with GFP, α-actinin-GFP, or non-transfected neighboring cells

10. A control NRK cell, or an NRK cell overexpressing α-actinin-GFP at a high level, was monitored by time-lapse phase and/or fluorescence optics

11. Rate of FRAP for GFP-actin

12. FRAP analysis of GFP-actin in cells overexpressing α-actinin- CherryFP during cytokinesis

13. NRK cells transiently transfected with α-actinin-GFP or control ontransfected NRK cells were stained with antibodies against myosin II and Hoechst 33258

14. Quantification of myosin II fluorescence intensity in whole cells and at both the equatorial and polar regions of cells overexpressing α-actinin-GFP compared with control cells

15. Overexpression of α-actinin-GFP inhibits actin turnover and causes the accumulation of actin filaments along the equator, without affecting myosin II organization.

16. Depletion of α-actinin causes global decrease in cortical actin filaments, accelerated cytokinesis and ectopic furrowing

17. CALI: Chromophore assisted laser inactivation. It has been used successfully to inactivate α-actinin in interphase cells

18. One side of the equatorial region of a dividing NRK cell expressing α-actinin-GFP or GFP only was irradiated by a laser beam and monitored by time-lapse phasecontrast optics

19. Ratio of furrow ingression between the laser-irradiated side and the nonirradiated side of the equator in cells expressing α-actinin-GFP, cells expressing GFP alone, and nontransfected cells with or without laser irradiation

20. This suggests that weakening of the cortical actin network by local inactivation of α-actinin may facilitate ingression.

21. Next, they used the RNA interference technique (RNAi) to deplete α-actinin globally from the cell. Cells microinjected with either of two different siRNAs directed against α- actinin(siRNA-1 and 2) showed ~70% reduction of α-actinin level, while those injected with a scrambled siRNA showed normal α-actinin distribution and level.

22. Global depletion of α-actinin caused severe defects in the integrity of the cortical actin network during cell division. In about 40% of cells depleted of α- actinin, equatorial cortex suddenly and quickly collapsed. The average duration of cytokinesis in cells depleted of α-actinin that showed rapid furrow ingression was around 400 seconds, as compared to 700–800 seconds in control noninjected cells and cells microinjected with scrambled siRNA.

23. Average duration of cytokinesis in control cells or cells depleted of α-actinin

24. Surprisingly, they occasionally observed precocious cortical ingression before anaphase onsetin about 10% of cells depleted of α-actinin,although chromosome congression andseparation was normal Moreover, in some α-actinin depleted cells, furrow ingression occurred both along and outside the equator after chromosome separation, producing anuclear cell fragments.Such accelerated cytokinesis and ectopic furrowing were not observed in cells microinjected with scrambled siRNA. These results suggest that α-actinin is required for the regulation of furrow ingression along the equator and for the prevention of ectopic furrowing during cell division.

26. Quantification of actin filaments fluorescence intensity at both the equatorial and the polar regions of cells depleted of α-actinin compared with control cells

27. In contrast, depletion of α-actinin had no effect on either the organization or total amount of myosin II,suggesting that cross- linking of actin filaments by α-actinin did not directly regulate myosin II.organizationtotal amount

28. NRK cells microinjected with or without siRNA against α-actinin diluted in fluorescent dextran were fixed and stained with antibodies against myosin II

29. Quantification of myosin II fluorescence intensity at both the equatorial and polar regions of cells depleted of α-actinin compared with control cells

30. As myosin II is essential for cytokinesis, they examined if accelerated cytokinesis and/or ectopic furrowing induced by depletion of α-actinin require myosin II activity. Blebbistatin, a known inhibitor for myosin II,was applied to cells microinjected with siRNA against α-actinin. While the control had no effects on accelerated cytokinesis and ectopic furrowing in cells depleted of α-actinin,no furrows were detected in cells depleted of α-actinin and treated with blebbistatin. Thus, as for normal furrows,myosin II activity is required for accelerated cytokinesis and ectopic furrowing.

31. FRAP analysis of α-actinin-GFP during cytokinesis of cells treated with blebbistatin

32. Discussion Functions of α-actinin along and outside the equator Comparison of the function of α-actinin in cytokinesis between fission yeast and mammalian cells Comparison of the function of α-actinin in cytokinesis between fission yeast and mammalian cells Regulation of the cortical actin network by α-actinin during cytokinesis Regulation of the cortical actin network by α-actinin during cytokinesis A hypothesis model of cytokinesis

33. Functions of α-actinin along and outside the equator A. Inward forces are present not only along the equator, but on the entire cortex from the early stage of mitosis. B. Furrow ingression requires remodeling rather than accumulation of actin filaments. C. Against the simplest version of the contractile ring hypothesis.

34. Functions of α-actinin along and outside the equator D. Cross-linking of actin filaments by α-actinin is required to resist global inward forces by increasing cortical rigidity in outside the equator or before the onset of cytokinesis. E. α-Actinin at the equator regulates furrow ingression speed after cytokinesis initiates. F. Inhibition of actin disassembly and actomyosin-based cortical contraction is most likely responsible for the delay in ingression.

35. Functions of α-actinin along and outside the equator G. Disruption of cell-cell or cell-substrate contacts may not associate with precocious ingression caused by the depletion of α-actinin. H. The role of α-actinin at the furrow is remodeling actin network. BACK

36. α-Actinin in cytokinesis between fission yeast and mammalian cells Ain1p ---- the α-actinin-like protein in yeast

37. α-Actinin in cytokinesis between fission yeast and mammalian cells Ain1pα - Actinin SpeciesYeastMammal Spectrin-like DomainsTwoFour Ring Formation++ Actin FormatBundlesNetwork Molecular LengthShortLong Cross-link PackedTightlyLoosely Contraction RingEasily foundHardly detected FunctionProper formation actin ringRemodeling of actin filaments

38. α-Actinin in cytokinesis between fission yeast and mammalian cells Although the cytokinetic components are well conserved between fission yeast and mammalian cells, the regulation of furrow ingression likely depends on the organization of the contractile ring. BACK

39. Regulation of the cortical actin network by α-actinin and myosin II during cytokinesis α-Actinin Function A. Stiffen the actin network. The stiffer network is easily to deform under a slow force by cytokinesis. B. Suppress local fluctuations shape changes in motor activities ingression during the cytokinesis. C. Increase in viscoelasticity of the isotropic actin network before furrow initiation.

40. Myosin II Function A. The forces rupture the association between actin and α-actinin. Cause “strain softening”. B. Increasing forces promote contraction and release of α-actinin from the equator, causing a decrease in cortical viscoelasticity and rearrangement of the cross-linked actin network, leads to change the cell shape. Regulation of the cortical actin network by α-actinin and myosin II during cytokinesis BACK

41. Hypothesis Model Step 1Activating Furrow Ingression Step 2Releasing Actin Network Step 3Cell Separating