research talk 2005 yo
DESCRIPTION
Research Talk 2005 Yulia OvechkinaTRANSCRIPT
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
Microtubules are polymers composed of tubulin dimers
Tubulin heterodimer
Protofilament
Microtubule- end + end
αααα ββββ
Microtubules play a fundamental role in various cellular
functions
intracellular transport
cell motility
mitosis
cell shape and polarity
----
+++
----
+++
----
+++
----
+++
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.
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.
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
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?
Morphology of chromatin and microtubules in control
Aspergillus nidulans germlings
MT
DAPI
PHASE
Interphase Mitosis
10 µµµµM
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
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
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
Benomyl Spongistatin Control
MT
DAPI
Spongistatin 1 does not prevent mitotic spindle formation;
however, the spindles are shorter than in control germlings
10 µµµµM
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
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.
Mechanism and Regulation of Microtubule Depolymerizing Activity
of a kinesin related protein, MCAK
Part 1b
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
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
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).
DAPI MTsMCAK
10µµµµmDAPI MTsEGFP-MCAK
MCAK localizes to kinetochores and centrosomes during
mitosis
MCAK depolymerizes MTs in vivo when is overexpressed
in cells
EGFP-MCAK MTs 10µm
A dominant negative hypir MCAK mutant localizes to the
same subcellular structures as endogenous MCAK but
does not depolymerize microtubules
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)
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
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
What is the role of the neck domain in the microtubule
depolymerization activity of MCAK?
αααα αααα ααααββββ ββββ ββββ
MCAK
αααα ββββMCAK
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
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
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
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
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
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
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
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
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
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
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.
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%
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
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.
Part II
Role of actin cytoskeleton in BCR activation and
signal propagation
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
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
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.
Upon stimulation, B cell surface receptor (BCR) clusters
and undergoes internalization
BCR
BCR
actin
- ααααIgM
+ ααααIgM
actin
BCR
ActinααααIgM
HS-1 is recruited to BCR signalosome in activated B
splenocytes
BCR HS1
HS1BCR
- ααααIgM
+ ααααIgM
Phosphorylated PLCγγγγ2 colocalize with BCR cap in
activated B splenocytes
pPLCγ2BCR
BCR pPLCγ2
- ααααIgM
+ ααααIgM
HS1 deficient B splenocytes exhibit impaired BCR
clustering
BCR
BCR
- ααααIgM
+ ααααIgM
actin
actin
BCR
ActinααααIgM
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
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
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
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
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
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
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.
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
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
Alexa 488 transferrin based internalization assay in a
human B cell line, BL2
BCR
Transferrin
Merge
No stimulation 1 min stimulation 5 min stimulation
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