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Fanni Gergely
Cancer Research UK
Cambridge Research Institute
Tuesday
Case Study I. CDK5RAP2: Study of the centrosome and microtubule
cytoskeleton in DT40 cells
•generation of knockouts
•detecting truncated proteins
•finding phenotype(s) : time-lapse light microscopy
•proliferation and clonogenic assays
•microtubule cytoskeleton assays (depolymerisation, regrowth,
microtubule spin-down)
•centrosome isolation and microtubule assembly assays in vitro
How to use DT40 for cell biology?
The centrosome
microtubule organising centre
microtubules
γ-tubulin
ring complex
pericentriolar matrix (PCM)
mother/daughter
centrioles
Joo-Hee Sir
Centrosome duplicates once and only
once per cell cycle
procentrioles
S
G1
G2
Centriole
disengagement
M
Bipolar spindle formation
Centrosome
separation
Centrosome
maturation
Centrosomes have multiple roles
centrosomes provide structural support for cytoskeletal
elements (mitotic spindle, cilia, flagella)
centrosomes are signalling platforms (Chk1, Mcph1)
centrosomes have cell-type/tissue specific functions
centrosomes may be instructive in stem cell division (age of
centrioles)
Our questions
how does the centrosome organise microtubules during interphase
and mitosis?
how does the centrosome assemble? are there subdomains within
the centrosome?
>200 proteins
centriole
microtubule
assemby?
anhcoring?
microtubule
nucleation?
Advantages of DT40 cells for our studies:
centrosomal proteins are very stable; siRNA does not offer a
good option
centrosome proteins are large and modular; DT40 offers a
system to study specific domains
the majority of centrosomal proteins are not essential;
knockouts survive
little is known about the regulation of centrosome proteins;
manipulate regulatory sites in situ in DT40
centrosomal protein complexes have not been explored;
purify protein complexes from DT40 cells in which proteins
are tagged in situ
Centrosome dysfunction is linked with human diseases
Bardet-Biedl-syndrome
Polycystic kidney disease
Lissencephaly
Primordial Dwarfism
Autosomal Primary Recessive Microcephaly
brain is 20-30% of normal size
sloping forehead
all MCPH proteins are spindle pole- or
centrosome-associated (AspM, CenpJ, CDK5RAP2,
MCPH1, Stil)
MCPH proteins are highly conserved; do they define a special
centrosomal complex or a pathway?
CDK5RAP2
in fission yeast Mto1 and Pcp1 are required for interphase
cytoplasmic and mitotic microtubule organisation, respectively
(Sawin et al 2004, Sawin and Tran, 2006 and Fong et al, 2010)
in Drosophila, centrosomin (CNN) is required for proper centrosome
function (Megraw at et al, 1999, Zhang and Megraw, 2007, Lucas and
Raff, 2007, Dobbealere et al, 2008)
CNN has 2 human orthologues (Myomegalin and CDK5RAP2)
CDK5RAP2 is mutated in primary microcephaly (Bond et al, 2005)
Targeting strategy to disrupt the
cdk5rap2 gene
cdk5rap2 locus
89kb
3.8kb
5.2kb
2
1
3
4
5
6
39 40
7 8
Targeting constructs
cnn1
cnn2
Neo(D)/
Puro(E)
cnn1-/- targeted locus
1
2
6
7 8
44
Neo(D)/
Puro(E)
A
B
41
42
cnn2-/- targeted locus
1
38
39 40
A
1
A
2
6
F
7 8
G
44
Neo(C)/
Puro(D)
STOP
STOP
C D/E
cnn1lox targeted locus
43
43 44
Neo(C)/
Puro(D)
STOP
STOP
41
B
C/D
cnn1loxcnn2-/- targeted locus
44
1
2
6
39 40
41
43 44
Neo(C)/
Puro(D)
STOP
STOP
Finding your truncated product
Antibody
wt
cnn1
cnn1lox
cnn2
CDK5RAP2 merged
cnn1loxcnn2
CDK5RAP2 merged
wt
wt cnn1-/- cnn1lox wt cnn2+/- cnn2-/210 kDa
110kDa
55kDa
*
*
CDK5RAP2
ΔCNN2
wb:
CDK5RAP2
wb:
α-tubulin
cnn2-/interphase
mitosis
Disrupted alleles of cdk5rap2 gene
Antibody
wt
cnn1
cnn1lox
cnn2
cnn1loxcnn2
?
Protein-tagging in DT40 cells
Uses of in situ tagging
replaces need for chicken-specific antibody
reports on how complete gene disruption is
allows detection of truncated proteins after gene disruption
(western and immunofluorescence)
Another truncated product
tag-wt
tag-cnn1lox
protein G
(tagCDK5RAP2) merged
w
t
ta
gcn wt
n
ta 1 lox
gcn
n1
lo
x
protein G
(tagCDK5RAP2) merged
tag-wt
tag-CDK5RAP2/
tag-ΔCNN1
210kDa
GsTAP
(protein G+strep bp)
protein G
protein G
(tag-ΔCNN1) merged (tag-ΔCNN1) merged
wb:
protein G
tag-cnn1lox
72kDa
Bürckstümmer et al, 2006 Nat Methods
cell lines
protein
product
wt
CDK5RAP2
tag-wt
lox
c1
cnn1-/-
tag-CDK5RAP2
tag-cnn1lox
cnn1lox
cnn2-/-
tag- CNN1
cnn1lox cnn2-/-
wb:
α-tubulin
55kDa
CNN1
interphase
Immunoblot
Subcellular
Localisation
protein
detectable
interphase
mitotic
centrosome centrosome
?
?
(35%)
CNN2
CNN1 CNN2
?
(16%)
?
?
?
yes
no
? not known
mitosis
So far...
ΔCNN1 and ΔCNN2 truncated products are expressed in
knockouts
ΔCNN1 and ΔCNN2 truncated products are absent from
interphase centrosomes but are present at low levels in
mitotic centrosomes
Are the knockout lines as ʻfitʼ as wild-type DT40?
(assaying proliferative potential of cells)
DIC microscopy
late
passages
Late passages
(3-4
weeks in culture)
film cells for 22-26 hours
(CO2 chamber is a must)
cellcell
numbers
numbers
1.0E+08
wt
1.0E+07
het
1.0E+06
del
1.0E+05
0
24
48
72
time (hrs)
time (hours)
Arno Alpi
determine
# of cell divisions
# of cell deaths
length of cell division
etc.
Are the knockout lines as ʻfitʼ as wild-type DT40?
(assaying proliferative potential of cells)
DIC microscopy
late
passages
Late passages
(3-4
weeks in culture)
film cells for 22-26 hours
(CO2 chamber is a must)
cellcell
numbers
numbers
1.0E+08
wt
1.0E+07
het
1.0E+06
del
1.0E+05
0
24
48
72
time (hrs)
time (hours)
determine
# of cell divisions
# of cell deaths
length of cell division
etc.
Arno Alpi
26 hours
wt
cnn1
2 cell
divisions
42%
17%
death
1%
11%
Assaying proliferative potential of cells
% cells with colony forming ability
100
Single cell
Colony
7 or 10 days
80
60
40
20
0
CNN2+/+
CNN2+/-
CNN2-/-
CNN1-/-
CNN1-/CNN2+/-
CNN1-/CNN2-/-
BUT WHY?
find out whether the centrosome and the microtubule
cytoskeleton are intact
Microtubule depolymerisation and
regrowth
microtubules in DT40 are cold-stable
depol
use 2 μg/ml nocodazole for depolymerisation
for regrowth wash cells with DMSO+medium
B
cnn1-/-
wt
cnn1-/-
wt
α-tubulin
merged
0 min
5 min
volume of α-tubulin
associated with centrosome (µm3)
regrowth
9
8
7
6
5
4
3
2
1
0
p<0.27 (N.S)
wt
wt
cnn1
cnn1-/--/-
5 min
Microtubule assembly on
centrosomes in vitro
synchronise
harvest
lyse (0.25% Triton)
pellet
centrosomes onto
coverslip
Buffer only
centrin
α-tubulin
wt
add tubulin, GTP
and taxol to
centrosomes and
incubate at 30C
End of Part I.
*
centrosomes detach from spindle poles in cnn1-/- cells
DIC imaging shows that cnn1-/- cells divide less and die more often
Clonogenic potential of cnn1-/- cells is reduced
centrosomes can assemble microtubules in cnn1-/- cells in vivo
many unanswered questions....are centrosomes intact otherwise in cells? how
does centrosome detachment arise? how dynamic is centrosome detachment?
we need microscopy...
Seeing is believing...microscopy in
DT40 cells
Thursday
Case Study II. CDK5RAP2: the use of microscopy in DT40 cells
•zooming in: electron microscopy
•finding phenotype(s) in mitotic cells: confocal microscopy (fixed
samples) and spinning disk confocal microscopy (live)
•tagging proteins in mutant backgrounds
•identifying and correlating mitotic spindle and centrosome
phenotypes
Centrosomes detach from the mitotic
spindle poles in cnn1-/- cells
centrin
TACC3
merged
wt
Centrin
(centrioles)
*
cnn1-/-
TACC3 (PCM
+spindle)
*
*
cnn2-/-
partially detached
centrosome
% prometaphase/
metaphase cells
70
detached
centrosome
60
50
40
30
20
10
0
wt
cnn1-/-
cnn1lox
cnn2-/- cnn1lox
cnn2-/-
Detective work starts here...
Why do centrosomes fall off in cnn1-/- cells?
NuMA
dynein/dynactin
abnormal centrosome structure?
abnormal spindle pole?
Transmission Electron Microsopy in DT40
harvest cells
fix in 1% warm
glutareldehyde
post-fix in
osmium tetroxide
dehydrate
pellet in increasing 30-100% EtOH
washes
cut sections
(50-100nm)
embed using
Epoxy kit
Transmission electron microsopy
Joo-Hee Sir
Joo-Hee Sir
Serial sections in electron microsopy
+/+
-/-
Alexis Barr
-/-
1nnc
tw
cnn1
1nnc
cnn1
-/-
wt
Why do centrosomes fall off in cnn1-/- cells?
NuMA
dynein/dynactin
abnormal centrosome structure?
NO!
abnormal spindle pole?
Fixing cells for fluorescent microsopy
seed cells onto poly-lysine coated coverslips (allow cells to settle for 15
minutes); avoid centrifugation if possible
What do I want to see?
Find the right fixation conditions for your antigen/antibody...
cytoskeleton: 100% -20C methanol; 3% formaldehyde/paraformaldehyde
(extraction 0.1-0.5% Tween or Triton); 0.5-1% glutaraldehyde/1-3%
paraformaldehyde mix (quench afterwards!)
chromatin-binding/nuclear protein: 1-4%paraformaldehyde (extraction
0.1-0.8% Triton)
make sure that your antibody is specific!!!!!
block cells in 5-10%BSA or in 5-10%FCS for 5-30 minutes prior to primary
antibody
mount in antifade medium
Laser-scanning confocal microscopy
A point light source for illumination (laser)
A point light focus within the specimen
A pinhole at the image detecting plane
These three points must be aligned accurately to
each other in the light path of image formation to
obtain confocal image. Confocal effects result in
supression of out-of-focal-plane and stray light in
the final image. Confocal allows optical sectioning.
The architecture of cnn1-/- spindles
5μm
5μm
Why do centrosomes fall off in cnn1-/- cells?
NuMA
dynein/dynactin
abnormal centrosome structure?
NO!
abnormal spindle pole?
NO!
NuMA
dynein/
dynactin (p150)
Abnormal attachment?
Centrosome purification
Purified centrosomes
3.
% sucrose:
Fractions:
70
2
50
3
70
40
4
5
6
7
WCE
2.
WCE
1.
2
50
3
4
40
5
6
7
19kDa
centrin 1
55kDa
60% sucrose
70%, 50%,
40% sucrose
collect fractions
41kDa
γ-tubulin
62kDa
Plk1
210kDa
protein G
(tagAKAP450)
111kDa
p150glued
tagAKAP-cnn1lox
DT40
tagAKAP-wt
DT40
pellet centrosomes
from each fraction
Fraction 3
Fraction 4
γ-tub
421.8
288.7
Plk1
471.4
803.1
AKAP450
5.6
48.1
p150
0
0
% of protein in cnn1lox compared to
wt centrosomes
Immunofluorescence confirms lack of tagged
AKAP450 in mitotic cnn1-/- centrosomes
akap450 locus
1
48
49
50
51
52
GsTAP Neo
targeting construct
STOP
tag-akap locus
1
48
49
50
51 52
GsTAP Neo
STOP
Protein G
(AKAP450)
γ-tubulin
Protein G
(AKAP450)
merged
wt
tagAKAPcnn1lox
tagAKAPwt
tagAKAPcnn2-/-
γ-tubulin
in situ tag replaces need for chicken-specific antibody
merged
Our final model
CDK5RAP2 is required for efficient cell proliferation
CDK5RAP2 links centrosome with spindle poles
CDK5RAP2 recruits dynactin and AKAP450 into the
centrosome
NuMA, Dynein
spindle pole
AKAP450
dynactin
(p150)
CDK5RAP2
centrosome
Centrosome detachment from spindle poles does not prevent bipolar spindle
formation
Centrosome within the spindle pole contributes to maintaining spindle length
and proper chromosome alignment
Fixed cells are not ideal to study a
dynamic process...
Spinning disc confocal microscope
*
Rotating disc has thousands of pinholes
field of view is scanned in single exposure, thus
allowing the capture of confocal images at high speed
(great for z-stacks)
multiple points are collected simultaneously, sample
receives less laser light
spinning disk confocal microscopy is well suited to
high speed 3D imaging of living systems
disadvantages: a huge amount of light is lost through
the Nipkow disc; cannot image low fluorescent intensity;
background can be high due to pinhole crosstalk
Time-lapse microscopy on DT40 cells
Leibowitz media
ibidi dish