Research Talk 2005 YO

58
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

description

Research Talk 2005 Yulia Ovechkina

Transcript of Research Talk 2005 YO

Page 1: Research Talk 2005 YO

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

Page 2: Research Talk 2005 YO

Microtubules are polymers composed of tubulin dimers

Tubulin heterodimer

Protofilament

Microtubule- end + end

αααα ββββ

Page 3: Research Talk 2005 YO

Microtubules play a fundamental role in various cellular

functions

intracellular transport

cell motility

mitosis

cell shape and polarity

----

+++

----

+++

----

+++

----

+++

Page 4: Research Talk 2005 YO

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.

Page 5: Research Talk 2005 YO

C

N

N

O

N C

O

OCH3H

NCH2CH2CH2CH3H

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

yeast tubulin but not brain tubulin.

•causes microtubule depolymerization in fungal

and yeast cells.

•binds ß-tubulin subunit of fungal and yeast

microtubules but has low affinity for mammalian

tubulin.

Page 6: Research Talk 2005 YO

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

Page 7: Research Talk 2005 YO

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?

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Morphology of chromatin and microtubules in control

Aspergillus nidulans germlings

MT

DAPI

PHASE

Interphase Mitosis

10 µµµµM

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Time in Min

% of germlings in mitosis

10

5

15

20

25

30

35

40

30 60 90 120

45

0

Spongistatin 1 causes a 3 fold elevation of the mitotic

index, whereas benomyl causes a 7 fold elevation of the

mitotic index

spongistatin 1 [25 µµµµg/ml]

benomyl [2.4 µµµµg/ml]

solvent control

Page 10: Research Talk 2005 YO

solvent control, 90 min Benomyl, 30 min

Spongistatin, 30 min Spongistatin, 60 min Spongistatin, 90 min

10 µµµµM

Spongistatin 1 mechanism of action may involve a

novel microtubule-severing activity

Page 11: Research Talk 2005 YO

30 60 90 1200

20

40

60

80

100

30 60 90 1200

20

40

60

80

100

30 60 90 1200

20

40

60

80

100

solvent control Benomyl Spongistatin 1

normal mts fragmented mts no mts

While Benomyl quickly depolymerizes all microtubules,

Spongistatin 1 triggers rapid fragmentation of

microtubules

% of germlings

% of germlings

% of germlings

Time in Min Time in Min Time in Min

Page 12: Research Talk 2005 YO

Benomyl Spongistatin Control

MT

DAPI

Spongistatin 1 does not prevent mitotic spindle formation;

however, the spindles are shorter than in control germlings

10 µµµµM

Page 13: Research Talk 2005 YO

Spongistatin 1 causes a two fold elevation of the spindle

mitotic index

30 60 90 120

1

2

3

4

5

6

7

8

0

9

Time in Min after Adding Spongistatin 1

% of germlings with spindles

Spongistatin 1 (25 µµµµg/ml)

solvent control

Page 14: Research Talk 2005 YO

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.

Page 15: Research Talk 2005 YO

Mechanism and Regulation of Microtubule Depolymerizing Activity

of a kinesin related protein, MCAK

Part 1b

Page 16: Research Talk 2005 YO

Growth (polymerization) phase

Shrinkage

(depolymerization) phase

Catastrophe Rescue

GTP–tubulinGDP–tubulin

GTPGDP

Reproduced from Kinoshita et al.,

Trends in Cell Biology 2002

Microtubules are dynamic polymers

αααα ββββ

ββββ

αααα

GTP - bound tubulin

GDP - bound tubulin

Polymerization state

Depolymerization state

Page 17: Research Talk 2005 YO

In vivomicrotubule dynamics are regulated by a balance between

MT stabilizing proteins and MT destabilizing proteins.

XMAP215/TOG

CLIP-170; CLASPs

APC; EB-1

Tau, MAP2, MAP4

MCAK

Op18/Stathmin

Microtubules are much more dynamic in vivo than in vitro

Reproduced from Wittmann et al.,

Nat Cell Biol 2001

XMAP215

MCAK

Page 18: Research Talk 2005 YO

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).

Page 19: Research Talk 2005 YO

DAPI MTsMCAK

10µµµµmDAPI MTsEGFP-MCAK

MCAK localizes to kinetochores and centrosomes during

mitosis

Page 20: Research Talk 2005 YO

MCAK depolymerizes MTs in vivo when is overexpressed

in cells

EGFP-MCAK MTs 10µm

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A dominant negative hypir MCAK mutant localizes to the

same subcellular structures as endogenous MCAK but

does not depolymerize microtubules

Page 22: Research Talk 2005 YO

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)

Page 23: Research Talk 2005 YO

How does MCAK depolymerize microtubules?

•MCAK depolymerizes MTs from both ends.

•MCAK is a processive depolymerase.

•MCAK binding induces a conformational

change in the tubulin dimer at the MT ends

which leads to destabilization of MT lattice.

αααα ββββ

ββββ

αααα

GTP - bound tubulin favors

polymerization state.

GDP - bound tubulin favors

depolymerization state

ATP

ADP + Pi

ATP

ADP + Pi

Page 24: Research Talk 2005 YO

The neck + motor of MCAK is the minimal sufficient

structure for full depolymerizing activity

αααα αααα ααααββββ ββββ ββββ

MCAK

αααα ββββMCAK

MOTORN-term C-termNECK

MOTORNECK

Page 25: Research Talk 2005 YO

What is the role of the neck domain in the microtubule

depolymerization activity of MCAK?

αααα αααα ααααββββ ββββ ββββ

MCAK

αααα ββββMCAK

Page 26: Research Talk 2005 YO

MOTORN-term C-termNECK

The neck of MCAK is positively charged

A182-D218 neck domain is predicted to be a highly charged hydrophilic helix :

182 ARRKSCIVKEMEKMKNKREEKRAQNSEIRIKRAQEYD 218

-HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH---

A182 D246ARRKSCIVKEMEKMKNKREEKRAQNSEIRIKRAQEYDSSFPNWEFARMIKEFRVTIECHPLTLTD

+++ +- -+ + + + - - ++ - + ++ -

- - + + - + - -

-

D218

Page 27: Research Talk 2005 YO

Two sides of a highly charged hydrophilic helix found in

the hamster MCAK neck

Side with the

most

NEGATIVELY-

charged residues

Side with the

most

POSITIVELY-

charged residues

A182

Q215

Page 28: Research Talk 2005 YO

The microtubule exterior is negatively charged

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

RED is - charge

BLUE is + charge

Electrostatic forces play important role in kinesin-MT interactions

Page 29: Research Talk 2005 YO

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.

αααα ααααββββ ββββ αααα ββββMCAK

αααα ββββMC

AK

αααα ααααββββ ββββ

Page 30: Research Talk 2005 YO

To test the model we generated MCAK mutants with deletions and

alanine substitutions of highly conserved positively charged amino

acids in the neck domain

MOTORN-term C-termNECK

A182 E201 D218 E232 D246ARRKSCIVKEMEKMKNKREEKRAQNSEIRIKRAQEYDSSFPNWEFARMIKEFRVTIECHPLTLTD

A182 D246

A182

A182

A182

E232

D218

E201

D218E201

Deletions in the neck domain are indicated by a flanking amino acid number.

EGFP-

A182 D246

ARRKSCIVKEMEKMKNKREEKRAQNSEIRIKRAQEYDSSFPNWEFARMIKEFRVTIECHPLTLTD

+++ + + ++ ++ + ++

+ + +

Arrows indicate alanine substitutions of positively charged amino acids

Page 31: Research Talk 2005 YO

In vivo depolymerization assay is a fast and simple way

to test for defects in the MT depolymerizing activity

EGFP-MCAK MTs 10µm

Mean GFP fluorescence intensity Mean MT fluorescence intensity

Page 32: Research Talk 2005 YO

Deletion of the neck domain inhibits the MT

depolymerizing activity of MCAK

0

500

1000

1500

2000

2500

3000

EGFP

control

WT MCAK ∆ A182-

D246

MCAK

∆ A182-

E232

MCAK

∆ A182-

D218

MCAK

∆ A182-

E201

MCAK

∆ E201-

D218

MCAK

0

500

1000

1500

2000

2500

3000

EGFP

Control

∆ A182-D218MCAK

Control

EGFP fluorescence MT fluorescence

Mean Fluorescence Intensity

Page 33: Research Talk 2005 YO

0

500

1000

1500

2000

2500

3000

EGFP control WT MCAK R210A; K212A;

R213A MCAK

R183A; R184A;

K185A MCAK

K198A; R199A;

K202A; R203A

MCAK

R183A; R184A;

K185A; K198A;

R199A; K202A;

R203A MCAK

R183A; R184A;

K185A; K198A;

R199A; K202A;

R203A;R210A;

K212A; R213A

MCAK

0

500

1000

1500

2000

2500

3000

Removal of the positively charged amino acids from the

neck inhibits the MCAK’s depolymerization activity

MCAK

Control

3-4 substitutions 7-10 subsEGFP

Control

EGFP fluorescence MT fluorescence

Mean Fluorescence Intensity

Page 34: Research Talk 2005 YO

motorneck

A182 I253 S583

motorAla-neck

A182 I253 S583

motor

A182-S583

A182-Ala-S583

D218-S583

motor

I253 S583

I253-S583

Neutralization of positive charges in the MCAK’s neck

also inhibited MT depolymerizing activity in vitro

The numbers are percentages of

depolymerized tubulin after subtraction of

no-motor control.

s p s p s p s p s p

No

Motor

Control

A182-

S583

A182-

Ala-S583

D218-S583

I253-

S583

92 ±4 10 ±4 13 ±5 5±1

D218 I253 S583

Tubulin

S P

Page 35: Research Talk 2005 YO

MOTORN-term C-termNECK

S92 S106

S108

S112

S186

Aurora B kinase phosphorylates MCAK in vitro at three

positions: Ser 92, Ser 106/Ser108/Ser112, and Ser 186

Page 36: Research Talk 2005 YO

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.

Page 37: Research Talk 2005 YO

Phosphorylation inhibits MCAK’s MT depolymerizing

activity in vitro

s p s p s p s p

90 ±452 ±6

MCAK

AurB beads

IgG beads

+ -+ -

- -+ ++ +- -

1 2 3 4 5 6 7 8

The numbers are percentages of

depolymerized tubulin after subtraction

of no-motor control.

Tubulin

MCAK

AurB

AurB

AurB

S P

PO4

S P

PO4

52% 90%

Page 38: Research Talk 2005 YO

Point mutants data also suggest that phosphorylation

decreases the MT depolymerizing activity of MCAK

EGFP fluorescence

MT fluorescence

EGFP WT

MCAK

S92E

S186E

S92A

S186A

S92E

S106E

S108E

S112E

S186E

S92A

S106A

S108A

S112A

S186A

Fluorescence Intensity

EGFP WT

MCAK

S92E

S186E

S92A

S186A

S92E

S106E

S108E

S112E

S186E

S92A

S106A

S108A

S112A

S186A

Fluorescence Intensity

Page 39: Research Talk 2005 YO

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.

Page 40: Research Talk 2005 YO

Part II

Role of actin cytoskeleton in BCR activation and

signal propagation

Page 41: Research Talk 2005 YO

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.

Nucleation of filamentous actin mostly depends on

activation of the Arp2/3 complex

Page 42: Research Talk 2005 YO

Adapted from Weaver et al., Current Biology 2002

WASP and HS1/cortactin may simultaneously interact with Arp2/3

complex to synergistically promote actin assembly.

Two major protein families can activate Arp2/3 mediated

actin polymerization: WASP and HS1/cortactin

Page 43: Research Talk 2005 YO

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.

Page 44: Research Talk 2005 YO

Upon stimulation, B cell surface receptor (BCR) clusters

and undergoes internalization

BCR

BCR

actin

- ααααIgM

+ ααααIgM

actin

BCR

ActinααααIgM

Page 45: Research Talk 2005 YO

HS-1 is recruited to BCR signalosome in activated B

splenocytes

BCR HS1

HS1BCR

- ααααIgM

+ ααααIgM

Page 46: Research Talk 2005 YO

Phosphorylated PLCγγγγ2 colocalize with BCR cap in

activated B splenocytes

pPLCγ2BCR

BCR pPLCγ2

- ααααIgM

+ ααααIgM

Page 47: Research Talk 2005 YO

HS1 deficient B splenocytes exhibit impaired BCR

clustering

BCR

BCR

- ααααIgM

+ ααααIgM

actin

actin

BCR

ActinααααIgM

Page 48: Research Talk 2005 YO

BCR internalization in stimulated HS1 deficient B cells is

similar to that in wild type B cells

0

100

200

300

400

500

600

700

non 2 Abs 0 min 1 min 5 min 20 min

MF

I

Series1

Series2

HS1 KO

WT

Page 49: Research Talk 2005 YO

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

0 200 400 600Time

800

1000

1200

1400

1600

Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A

Specimen_001_9 hs hbss 1.fcs

0 200 400 600Time

0

1000

2000

3000

4000

Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A

0 200 400 600Time

0

1000

2000

3000

4000

Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A

Specimen_001_8 bl6 hbss 1.fcs

Wild type B cells + 10 ug/ml anti IgM Abs

HS1 KO B cells + 10 ug/ml anti IgM Abs

WT

HS1 KO

Page 50: Research Talk 2005 YO

Alexa-488 phalloidin staining

0

20

40

60

80

100

120

non-

st ai ned

WT HS1 KO WASp

HS1 KO

MFI

Total levels of polymerized actin are only modestly

decreased in HS1 and WASp/HS1 deficient B cells

Page 51: Research Talk 2005 YO

BCR ActinDAPI

BCR ActinDAPI

Simultaneous inhibition of both WASp and N-WASp

proteins by Wiskostatin resulted in inhibition of BCR

clustering and reduction of polymerized actin

+ 5uM Wiskostatin

Page 52: Research Talk 2005 YO

No stimulation

+ 5 uMWiskostatin

Simultaneous inhibition of both WASp and N-WASp

proteins by Wiskostatin resulted in inhibition of BCR

clustering in primary murine B cells

Page 53: Research Talk 2005 YO

0 200 400 600Time

400

600

800

1000

Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A

10 ug/ml anti IgM Abs

5uM Wiskostatin + 10 ug/ml anti IgM Abs

0 200 400 600Time

400

500

600

700

800

900

Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A

0 100 200 300 400 500Time

400

500

600

700

800

900

Ratio: Indo-1 (violet)-A/Indo-1 (blue)-A

10 ug/ml anti IgM Abs

0.5uM Wiskostatin + 10 ug/ml anti IgM Abs

10 ug/ml anti IgM Abs

0.1uM Wiskostatin + 10 ug/ml anti IgM Abs

Simultaneous inhibition of both WASp and N-WASp

proteins resulted in a dose dependent inhibition of BCR-

mediated calcium influx

+ 5 uMWiskostatin

+ 0.5 uMWiskostatin

+ 0.1 uMWiskostatin

Page 54: Research Talk 2005 YO

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.

Page 55: Research Talk 2005 YO

Berl OakleyKatherine Jung

Elizabeth Oakley

Kathrin Jung

Natalie Prigozhina

Dept. of Molecular Genetics

The Ohio State University, OH

Linda WordemanMike Wagenbach

Todd Maney

Ayana Moore

Dept. of Physiology and Biophysics

University of Washington, WA

Dept. of Immunology

Children’s Hospital, Seattle WA

Acknowledgements

Collaborators

George PettitCancer Research Institute

Arizona State University, AZ

Leslie WilsonCori Newton

University of California, CA

Jason SwedlowPaul Andrews

University of Dundee, UK

Ron Milligan Carolyn Moores

The Scripps Research Institute, CA

Page 56: Research Talk 2005 YO

Model of HS-1 involvement in BCR signaling

Lyn Syk Btk PLCγγγγ2

DAG

Ca++

BCR

IP3

HS-1

F-actin assembly

and crosslinking

BCR cluster assembly and maintainence

which leads to signal amplification

Ag

Ag

BCR Signaling amplification

Page 57: Research Talk 2005 YO

Alexa 488 transferrin based internalization assay in a

human B cell line, BL2

BCR

Transferrin

Merge

No stimulation 1 min stimulation 5 min stimulation

Page 58: Research Talk 2005 YO

The low level of free tubulin in cells transfected with

MCAK is a result of a tubulin autoregulation mechanism

707798

16251645

1870

0

500

1000

1500

2000

2500

Non-treated cells, N=77 Cells treated with 1000 nM

Nocodazole for 15 min,

N=41

Cells treated with 100 nM

Nocodazole for 15 min,

N=36

Cells treated with 1000 nM

Nocodazole for 12 hr,

N=88

Cells treated with 100 nM

Nocodazole for 12 hr,

N=67

Mean T

ubulin F

luore

scence

DMSO

control

0.01mM Noc

for 15 min

0.1mM Noc

for 15 min

0.01mM Noc

for 12 hrs

0.1mM Noc

for 12 hrs