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Research Talk 2005 Yulia Ovechkina

Research Talk 2005 Yulia Ovechkina

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  • 1. Research Talk Part 1: Microtubule destabilizing activities of an antimitotic agent, Spongistatin 1, and a kinesin related protein, MCAK. Research Talk Part 2: Role of actin-polymerizing proteins, WASP and HS1, in B cell surface receptor activation and internalization. Yulia Ovechkina, Ph.D. University of Washington
  • 2. Microtubules are polymers composed of tubulin dimers α β Tubulin heterodimer Protofilament - end Microtubule + end
  • 3. Microtubules play a fundamental role in various cellular functions mitosis cell motility cell shape and polarity ---- ---- ---- ---- intracellular transport +++ +++ +++ +++
  • 4. Tubulin is the target for an increasing number of anticancer and antifungal drugs •Antimicrotubule drugs disrupt cellular microtubules and prevent formation of a functional spindle, resulting in the accumulation of cultured cells in the G2/M phase of the cell cycle through specific inhibition of mitosis.
  • 5. Benomyl is a antimicrotubule, antifungal agent which is widely used worldwide on a large variety of crops •inhibits in vitro assembly of purified fungal and O H yeast tubulin but not brain tubulin. C NCH2 CH2 CH2 CH3 O N H •causes microtubule depolymerization in fungal N C OCH3 and yeast cells. N •binds ß-tubulin subunit of fungal and yeast microtubules but has low affinity for mammalian tubulin.
  • 6. Spongistatin 1 isolated from the marine sponge Hyrtios erecta is a potent antimitotic, antimicrotubule agent in mammalian cells •inhibits tubulin polymerization in vitro •causes microtubule depolymerization in vivo •exhibits antimitotic activity by disrupting normal mitotic spindle assembly, cell division and inducing apoptosis
  • 7. In addition to its activity in mammalian cells, spongistatin 1 has a broad-spectrum antifungal activity •What is a mechanism of spongistatin 1 antifungal activity? •Is Spongistatin 1 antifungal activity due to its antimicrotubule activity?
  • 8. Morphology of chromatin and microtubules in control Aspergillus nidulans germlings Interphase Mitosis MT DAPI PHASE 10 µM
  • 9. Spongistatin 1 causes a 3 fold elevation of the mitotic index, whereas benomyl causes a 7 fold elevation of the mitotic index 45 spongistatin 1 [25 µg/ml] 40 % of germlings in mitosis benomyl [2.4 µg/ml] 35 solvent control 30 25 20 15 10 5 0 30 60 90 120 Time in Min
  • 10. Spongistatin 1 mechanism of action may involve a novel microtubule-severing activity solvent control, 90 min Benomyl, 30 min Spongistatin, 30 min Spongistatin, 60 min Spongistatin, 90 min 10 µM
  • 11. While Benomyl quickly depolymerizes all microtubules, Spongistatin 1 triggers rapid fragmentation of microtubules solvent control Benomyl Spongistatin 1 100 100 100 80 80 80 % of germlings % of germlings % of germlings 60 60 60 40 40 40 20 20 20 0 30 60 90 120 0 30 60 90 120 0 30 60 90 120 Time in Min Time in Min Time in Min normal mts fragmented mts no mts
  • 12. Spongistatin 1 does not prevent mitotic spindle formation; however, the spindles are shorter than in control germlings Benomyl Spongistatin Control MT DAPI 10 µM
  • 13. Spongistatin 1 causes a two fold elevation of the spindle mitotic index 9 8 % of germlings with spindles 7 6 5 Spongistatin 1 (25 µg/ml) 4 solvent control 3 2 1 0 30 60 90 120 Time in Min after Adding Spongistatin 1
  • 14. Conclusions 1. Spongistatin 1 acts as an antimicrotubule, antimitotic agent in A. nidulans. 2. Spongistatin 1 mechanism of action may involve a novel microtubule-severing activity.
  • 15. Part 1b Mechanism and Regulation of Microtubule Depolymerizing Activity of a kinesin related protein, MCAK
  • 16. Microtubules are dynamic polymers Growth (polymerization) phase Polymerization state α β GTP - bound tubulin GDP Catastrophe Rescue GTP Depolymerization state β Shrinkage α (depolymerization) phase GTP–tubulin GDP - bound tubulin GDP–tubulin Reproduced from Kinoshita et al., Trends in Cell Biology 2002
  • 17. Microtubules are much more dynamic in vivo than in vitro MCAK XMAP215 Reproduced from Wittmann et al., Nat Cell Biol 2001 In vivo microtubule dynamics are regulated by a balance between MT stabilizing proteins and MT destabilizing proteins. XMAP215/TOG MCAK CLIP-170; CLASPs Op18/Stathmin APC; EB-1 Tau, MAP2, MAP4
  • 18. Mitotic Centromere Associated Kinesin (MCAK) is a protein of particular interest 1. MCAK is one of two major microtubule-destabilizing proteins in cells. 2. MCAK may be an important contributor to tumorgenesis: • MCAK is overexpressed in cancer cells; • Depletion of MCAK from kinetochores results in chromosome segregation defects, which in turn leads to aneuploidy (abnormal number of chromosomes).
  • 19. MCAK localizes to kinetochores and centrosomes during mitosis MCAK DAPI MTs µ 10µm EGFP-MCAK DAPI MTs
  • 20. MCAK depolymerizes MTs in vivo when is overexpressed in cells 10µm EGFP-MCAK MTs
  • 21. A dominant negative hypir MCAK mutant localizes to the same subcellular structures as endogenous MCAK but does not depolymerize microtubules
  • 22. Inhibition of endogenous MCAK by a dominant negative MCAK mutant results in results in chromosome segregation defects metaphase anaphase Microtubules EGFP-MCAKmut Chromosomes (in blue)
  • 23. How does MCAK depolymerize microtubules? ADP + Pi ATP •MCAK depolymerizes MTs from both ends. •MCAK is a processive depolymerase. •MCAK binding induces a conformational ATP change in the tubulin dimer at the MT ends ADP + Pi which leads to destabilization of MT lattice. β α β α GTP - bound tubulin favors GDP - bound tubulin favors polymerization state. depolymerization state
  • 24. The neck + motor of MCAK is the minimal sufficient structure for full depolymerizing activity N-term NECK MOTOR C-term NECK MOTOR K MCAK MCA α β α β α β α β
  • 25. What is the role of the neck domain in the microtubule depolymerization activity of MCAK? K MCAK MCA α β α β α β α β
  • 26. The neck of MCAK is positively charged N-term NECK MOTOR C-term A182 D218 D246 ARRKSCIVKEMEKMKNKREEKRAQNSEIRIKRAQEYDSSFPNWEFARMIKEFRVTIECHPLTLTD +++ +- -+ + + + - - ++ - + ++ - - - - + + - + - - A182-D218 neck domain is predicted to be a highly charged hydrophilic helix : 182 ARRKSCIVKEMEKMKNKREEKRAQNSEIRIKRAQEYD 218 -HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH---
  • 27. Two sides of a highly charged hydrophilic helix found in the hamster MCAK neck A182 Side with the Side with the most most POSITIVELY- NEGATIVELY- charged residues charged residues Q215
  • 28. The microtubule exterior is negatively charged RED is - charge BLUE is + charge The electrostatic map of microtubule exterior was obtained with the computational evaluation of electrostatic potentials by N. A. Baker, D. Sept, S. Joseph, M. J. Holst, and J. A. McCammon, Proc. Natl. Acad. Sci. USA, 2001 Electrostatic forces play important role in kinesin-MT interactions
  • 29. Proposed MCAK neck function The positively-charged neck of MCAK acts as an electrostatic tether to anchor MCAK to the negatively-charged microtubules in order to increase the processivity of MT depolymerization. K MCA K β MCA α α β α β α β α β α β
  • 30. To test the model we generated MCAK mutants with deletions and alanine substitutions of highly conserved positively charged amino acids in the neck domain N-term NECK MOTOR C-term EGFP- A182 A182 E201 D218 E232 D246 D246 ARRKSCIVKEMEKMKNKREEKRAQNSEIRIKRAQEYDSSFPNWEFARMIKEFRVTIECHPLTLTD ARRKSCIVKEMEKMKNKREEKRAQNSEIRIKRAQEYDSSFPNWEFARMIKEFRVTIECHPLTLTD +++ + + ++ ++ + ++ + + A182 + D246 A182 E232 Arrows indicate alanine substitutions of positively charged amino acids A182 D218 A182 E201 E201 D218 Deletions in the neck domain are indicated by a flanking amino acid number.
  • 31. In vivo depolymerization assay is a fast and simple way to test for defects in the MT depolymerizing activity Mean GFP fluorescence intensity Mean MT fluorescence intensity 10µm EGFP-MCAK MTs
  • 32. Deletion of the neck domain inhibits the MT Mean Fluorescence Intensity depolymerizing activity of MCAK 3000 3000 EGFP MCAK ∆ A182-D218 Control Control 2500 2500 2000 2000 1500 1500 1000 1000 500 500 0 0 EGFP WT MCAK ∆ A182- ∆ A182- ∆ A182- ∆ A182- ∆ E201- control D246 E232 D218 E201 D218 MCAK MCAK MCAK MCAK MCAK EGFP fluorescence MT fluorescence
  • 33. Removal of the positively charged amino acids from the neck inhibits the MCAK’s depolymerization activity Mean Fluorescence Intensity 3000 3000 EGFP MCAK 3-4 substitutions 7-10 subs Control Control 2500 2500 2000 2000 1500 1500 1000 1000 500 500 0 0 EGFP control WT MCAK R210A; K212A; R183A; R184A; K198A; R199A; R183A; R184A; R183A; R184A; R213A MCAK K185A MCAK K202A; R203A K185A; K198A; K185A; K198A; MCAK R199A; K202A; R199A; K202A; R203A MCAK R203A;R210A; K212A; R213A MCAK EGFP fluorescence MT fluorescence
  • 34. Neutralization of positive charges in the MCAK’s neck also inhibited MT depolymerizing activity in vitro A182 I253 S583 A182-S583 neck motor No A182- A182 I253 S583 Motor A182- Ala- D218- I253- S583 S583 A182-Ala Ala-neck motor Control S583 S583 -S583 D218 I253 S583 s p s p s ps p s p D218-S583 motor Tubulin I253 S583 I253-S583 motor 92 ±4 10 ±4 13 ±5 5±1 S P The numbers are percentages of depolymerized tubulin after subtraction of no-motor control.
  • 35. Aurora B kinase phosphorylates MCAK in vitro at three positions: Ser 92, Ser 106/Ser108/Ser112, and Ser 186 N-term NECK MOTOR C-term S92 S106 S186 S108 S112
  • 36. Aurora B, a serine/threonine kinase, is a key regulators of the mitotic cell division process •Aurora B is expressed and active at the highest level during mitosis phase of the cell cycle. •Aurora B kinase regulates cell division and its checkpoints, errors of which can lead to aneuploidy or genetic instability. •Aurora B is overexpressed in many human cancers, and elevated expression has been correlated with chromosomal instability.
  • 37. Phosphorylation inhibits MCAK’s MT depolymerizing activity in vitro IgG beads - + - + AurB AurB beads + - + - MCAK + + - - AurB s p s p s p s p 52 ±6 90 ± 4 MCAK S P Tubulin PO4 AurB 52% 90% S P 1 2 3 4 5 6 7 8 PO4 The numbers are percentages of depolymerized tubulin after subtraction of no-motor control.
  • 38. Point mutants data also suggest that phosphorylation decreases the MT depolymerizing activity of MCAK Fluorescence Intensity EGFP WT S92E S92A S92E S92A MCAK S186E S186A S106E S106A S108E S108A S112E S112A S186E S186A EGFP fluorescence MT fluorescence
  • 39. Conclusions •Removal of positive charges from the neck domain either by deletions or alanine substitutions inhibits MT depolymerizing activity of MCAK in vitro and in vivo. •The neck of MCAK may function as electrostatic tether to confer processivity to the motor domain by anchoring it to the MT ends. •MCAK is phosphorylated by Aurora B kinase in vitro. •Phosphorylation inhibits the MT depolymerizing activity of MCAK in vitro and in vivo.
  • 40. Part II Role of actin cytoskeleton in BCR activation and signal propagation
  • 41. Nucleation of filamentous actin mostly depends on activation of the Arp2/3 complex Activated Arp2/3 complex binds to the side of an existing actin filament and nucleates assembly of a new actin filament. The resulting branch structure is Y-shaped.
  • 42. Two major protein families can activate Arp2/3 mediated actin polymerization: WASP and HS1/cortactin WASP and HS1/cortactin may simultaneously interact with Arp2/3 complex to synergistically promote actin assembly. Adapted from Weaver et al., Current Biology 2002
  • 43. Our current hypothesis: WASP and HS1 provide a link between activation of BCR and actin cytoskeleton remodeling •Actin polymerization is involved in recruiting signaling molecules into membrane lipid raft microdomains which serve as signaling platforms. •Force of actin polymerization helps to merge lipid raft microdomains together leading to accumulation of signaling proteins and amplification of initial signal from the surface receptors. •Actin polymerization is critical for a cell surface receptor down-regulation by endocytosis which usually terminates signaling from the receptor.
  • 44. Upon stimulation, B cell surface receptor (BCR) clusters and undergoes internalization - αIgM BCR actin + αIgM BCR actin αIgM Actin BCR
  • 45. HS-1 is recruited to BCR signalosome in activated B splenocytes - αIgM BCR HS1 + αIgM BCR HS1
  • 46. γ Phosphorylated PLCγ2 colocalize with BCR cap in activated B splenocytes - αIgM BCR pPLCγ2 + αIgM BCR pPLCγ2
  • 47. HS1 deficient B splenocytes exhibit impaired BCR clustering BCR actin - αIgM actin + αIgM BCR αIgM Actin BCR
  • 48. BCR internalization in stimulated HS1 deficient B cells is similar to that in wild type B cells 700 600 500 Series1 HS1 KO 400 Series2 M FI WT 300 200 100 0 non 2 Abs 0 min 1 min 5 min 20 min
  • 49. Both HS1 deficient and wild type B cells have similar levels of calcium influx after stimulation of B cell receptor as determined by flow cytometric analysis 4000 Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A WT 3000 Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A 2000 1600 1000 1400 0 1200 0 200 400 600 Time Specimen_001_8 bl6 hbss 1.fcs 4000 1000 Wild type B cells + 10 ug/ml anti IgM Abs Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A HS1 KO B cells + 10 ug/ml anti IgM Abs HS1 KO 800 3000 0 200 400 600 2000 Time 1000 0 0 200 400 600 Time Specimen_001_9 hs hbss 1.fcs
  • 50. Total levels of polymerized actin are only modestly decreased in HS1 and WASp/HS1 deficient B cells Alexa-488 phalloidin staining 120 100 80 MFI 60 40 20 0 non- WT H S1 K O WA Sp s t a i ne d H S1 K O
  • 51. Simultaneous inhibition of both WASp and N-WASp proteins by Wiskostatin resulted in inhibition of BCR clustering and reduction of polymerized actin DAPI BCR Actin + 5uM Wiskostatin DAPI BCR Actin
  • 52. Simultaneous inhibition of both WASp and N-WASp proteins by Wiskostatin resulted in inhibition of BCR clustering in primary murine B cells + 5 uM Wiskostatin No stimulation
  • 53. Simultaneous inhibition of both WASp and N-WASp proteins resulted in a dose dependent inhibition of BCR- mediated calcium influx Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A 1000 800 10 ug/ml anti IgM Abs 600 5uM Wiskostatin + 10 ug/ml anti IgM Abs 400 + 5 uM Wiskostatin Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A 0 200 400 600 Time 900 800 700 10 ug/ml anti IgM Abs 600 0.5uM Wiskostatin + 10 ug/ml anti IgM Abs 500 400 + 0.5 uM Wiskostatin Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A 0 200 400 600 Time 900 800 700 10 ug/ml anti IgM Abs 600 0.1uM Wiskostatin + 10 ug/ml anti IgM Abs 500 400 + 0.1 uM Wiskostatin 0 100 200 300 400 500 Time
  • 54. Current approaches to study a link between actin cytoskeleton and BCR signaling •Depletion of B cell line of WASP and N-WASP by siRNA to assay defects of BCR signalosome and actin cap assembly. •Visualizing BCR cluster formation in HS-1 and WASP deficient primary B cells using live cell imaging using spinning disk a confocal microscope. •Visualizing protein-protein interactions between BCR signalosome components, WASP and HS-1 proteins by FRET technique. •Fluorescent microplate reader based adhesion assays in HS-1 and WASP deficient primary B cells with and without BCR engagement.
  • 55. Acknowledgements Berl Oakley Collaborators Katherine Jung Elizabeth Oakley Kathrin Jung George Pettit Natalie Prigozhina Cancer Research Institute Dept. of Molecular Genetics Arizona State University, AZ The Ohio State University, OH Leslie Wilson Linda Wordeman Cori Newton Mike Wagenbach University of California, CA Todd Maney Ayana Moore Dept. of Physiology and Biophysics Jason Swedlow University of Washington, WA Paul Andrews University of Dundee, UK Dept. of Immunology Children’s Hospital, Seattle WA Ron Milligan Carolyn Moores The Scripps Research Institute, CA
  • 56. Model of HS-1 involvement in BCR signaling Ag BCR DAG Lyn Syk Btk γ PLCγ2 IP3 Ca++ HS-1 Ag F-actin assembly and crosslinking BCR cluster assembly and maintainence which leads to signal amplification BCR Signaling amplification
  • 57. Alexa 488 transferrin based internalization assay in a human B cell line, BL2 No stimulation 1 min stimulation 5 min stimulation BCR Transferrin Merge
  • 58. The low level of free tubulin in cells transfected with MCAK is a result of a tubulin autoregulation mechanism 2500 Mean Tubulin Fluorescence 2000 1870 1645 1625 1500 1000 798 707 500 DMSO 0.01mM Noc 0.1mM Noc 0.01mM Noc 0.1mM Noc control for 15 min for 15 min for 12 hrs for 12 hrs 0 Non-treated cells, N=77 Cells treated with 1000 nM Cells treated with 100 nM Cells treated with 1000 nM Cells treated with 100 nM Nocodazole for 15 min, Nocodazole for 15 min, Nocodazole for 12 hr, Nocodazole for 12 hr, N=41 N=36 N=88 N=67