An essential role of profilin is to facilitate formin-mediated actin assembly

1. An essential role of profilin is to facilitate formin-mediated actin assembly Andrew Bestul Kovar Lab SuperCytoGroup March 18, 2013

2. Cells store actin monomers bound to profilin Actin monomers bound to Profilin (1) Profilin inhibits nucleation (2) Profilin-actin will add to the fast growing ‘barbed’ end Actin monomer ‘pointed’ end ‘barbed’ end PPPPP Ezezika et al., JBC 2009 Poly-L-Proline

3. Cells store actin monomers bound to profilin Actin monomers bound to Profilin (1) Profilin inhibits nucleation (2) Profilin-actin will add to the fast growing ‘barbed’ end Actin monomer ‘pointed’ end ‘barbed’ end PPPPP Current Model: Profilin has role in all actin processes by maintaining a pool of available actin monomers and profilins are interchangeable. Ezezika et al., JBC 2009 Poly-L-Proline

4. Non-fission yeast profilins fail to complement a fission yeast profilin-ts (cdc3-124) strain Profilin (cdc3)-ts strain after 72 hours at the restrictive temperature, 36ºC. Profilins expressed on plasmid under the control of the nmt81 promoter. SpPRF = S. pombe ScPFY = S. cerevisiae AtPRF1 = A. thaliana CePFN-1 = C. elegans MmPRF1 = M. musculus SjPRF = S. japonicus HsPROI = H. sapiens

5. Why do the non-complementing profilins fail to complement the cdc3-124 strain?

6. Why do the non-complementing profilins fail to complement the cdc3-124 strain? Goal: Find the function of profilin that allows complementation and makes profilin essential for cell viability.

7. Strategy • Mutate residues on a budding yeast profilin (ScPFY) towards the fission yeast profilin (SpPRF). • Find ScPFY mutants that can now complement the profilin-ts strain and see what function of profilin has been fixed.

8. Creating a directed random mutagenesis library ScPFY is 52% identical and 72% similar to SpPRF. 16/39 amino acid differences were targeted. = Single nucleotide degeneracy = SpPRF specific residue Residue involved with actin-binding Residue involved with PLP-binding

9. Creating a directed random mutagenesis library ScPFY is 52% identical and 72% similar to SpPRF. 16/39 amino acid differences were targeted. Green = PLP-binding residues Pink = Actin-binding residues Red = Mutated Actin-binding residues N Blue = Other Mutated residues C

10. 9-mutant budding yeast profilin (9-Mut) complements the fission yeast profilin-ts strain DAPI/Calcofluor Profilin-ts complementation assay after 72 hours at 36ºC Empty Vector SpPRF ScPFY ScPFY(9-Mut) = >2 nuclei = 2 nuclei = 1 nucleus

11. Biochemical analysis of 9-mutant budding yeast profilin Biochemical Property ScPFY(9-Mut) Muscle Actin Binding Nucleotide Exchange Fission Yeast Actin Binding PLP/Cdc12(FH1) Binding PIP2 Binding

12. Biochemical analysis of 9-mutant budding yeast profilin Biochemical Property ScPFY(9-Mut) Muscle Actin Binding ScPFY Nucleotide Exchange ScPFY Fission Yeast Actin Binding ScPFY PLP/Cdc12(FH1) Binding ScPFY PIP2 Binding SpPRF

13. Fission yeast has three distinct actin structures Arp2/3 Complex Formin For3 Formin Cdc12

14. ScPFY(9-Mut) is mostly fixed for the minor actin patches in cdc3-124 strain • Profilins integrated at Ura locus. • LifeAct-mCherry marker, 32°C for 2 hrs. • 120 sec. movies WT cdc3-124 SpPRF ScPFY 9-Mut

15. Patch Tracking Example LifeAct-mCherry

16. The cdc3-124 strain has minor actin patch defects Individual Patch Tracking LifeAct-mCherry

17. ScPFY(9-Mut) profilin is wild-type for actin patches in cdc3-124 strain Individual Patch Tracking

18. ScPFY(9-Mut) profilin is wild-type for endocytosis in cdc3-124 strain

19. The effect of cdc3-124 strain on the contractile ring Rlc1-GFP WT cdc3-124 • Rlc1-GFP (myosin marker) • After 2 hours at 32ºC • 120 min. movies

20. ScPFY(9-Mut) rescues contractile ring assembly in cdc3-124 strain • Profilins integrated at Ura locus in a profilin-ts strain • Rlc1-GFP marker, 32°C for 2 hrs. • 120 min. movies WT cdc3-124 SpPRF ScPFY 9-Mut

21. Fission yeast has three distinct actin structures Arp2/3 Complex Formin For3 Formin Cdc12

22. Correlation between cdc3-124 strain complementation and utilization by fission yeast formin Cdc12 In presence of 2.5 µM actin and 50 nM Cdc12

23. ScPFY(9-Mut) is utilized better by Cdc12 in spontaneous actin assembly assay than ScPFY

24. ScPFY(9-Mut) is utilized better than wt ScPFY by Cdc12 in TIRF microscopy A+F+2 µM SpPRF 1.5 µM Actin + 5 nM Cdc12 Red = Control Green = Formin-associated Orange = Not determinable Control = 11.6 sub/s Formin = 0.4 sub/s Control = 10.3 sub/s Formin = 14.7 sub/s A+F+2 µM ScPFY A+F+2 µM 9Mut Control = 11.6 sub/s Formin = 4.3 sub/s Formin = 9.2 sub/s

25. Conclusions • The ScPFY(9-Mut) is biochemically similar to the wild-type ScPFY except in PIP2 binding. • The ScPFY(9-Mut) is utilized better than wild-type ScPFY by fission yeast formin Cdc12 in vitro. • The ScPFY(9-Mut) also rescues contractile ring assembly, meaning that it is being utilized better by Cdc12 in vivo. • Proper utilization by formin is an essential role of profilin in the cell.

26. Conclusions • This improvement in utilization could be due to a profilin-formin FH2 interaction that increases the dissociation rate of profilin for subsequent rounds of elongation.

27. Implications for a formin-profilin interaction Actin Bni1 FH2 ScPFY

28. Implications for a formin-profilin interaction

29. Implications for a formin-profilin interaction Steric hinderance as a model of formin specification of profilin isoforms predicts the stair stepping model of formin translocation

30. Future Directions • Is there an interaction between formin FH2 domain and profilin at the barbed end? • Use a photo-activatible crosslinker on the formin and then incubate formin, actin and profilin together, activate the crosslinker and see whether you can pull-down FH2 and profilin together. (Would need to cleave off FH1 domain after activation) • Do directed mutational analysis on Cdc12 FH2 and see if mutating sites at this area of Cdc12 to “bulkier” subunits allows it to utilize various profilins. • Perform surface plasmon resonance on FH1-profilin-actin complex to elucidate whether an interaction at this level has been rescued in the ScPFY(9-Mut). • Determine crystal structures of mutant profilins to see whether the loops on profilin have shifted. • What effect do profilin isoforms have on For3 and actin cables? • TIRF analysis of For3 with SpPRF, ScPFY and ScPFY(9-Mut). • Track labeled Myo52 in profilin integration strains to see whether the actin cables are useable tracks or are somehow deficient.

31. Acknowledgments Kovar Lab Cristian Suarez Dennis Zimmermann Jen Sees Jenna Christensen Jon Winkelman Tom Burke Yujie Li Thesis Committee Bob Keenan Mohan Gupta Michael Glotzer Jon Staley Former Kovar Lab Agnieszka Grzegorzewska Erin Neidt MCB Training Grant U of C Cytoskeleton SuperGroup

32. For3 utilization of profilin-actin seems less selective than Cdc12

33. Implications for a formin-profilin interaction Inside budding yeast cell Inside fission yeast cell Inside fission yeast cell

34. Implications for a formin-profilin interaction Cdc12 Cdc12 HsPROI AtPRF1

35. Implications for a formin-profilin interaction Cdc3-124 strain complementation assay Ch5: M69I, D83Q Ch9: D83Q, A84K Ch11: D83Q, V105T Ch13: A84K, V105T

36. Implications for a formin-profilin interaction mDia1 DAAM1 FMNL3 HsPROI SpPRF