Bloom Lab Summary

Resolving the Structure of Chromatin at the Centromere
in Saccharomyces Cerevisiae
Julian Haase
Bloom Lab

• Cohesin enriched approximately 3-fold in a 50kb region flanking the centromere
(Weber et al., PLOS, 2006)
How is successful chromosome segregation achieved?
• Faithful segregation of chromosomes to daughter cells is essential; failure
leads to aneuploidy, which can lead to cancer and diseases such as Down’s
syndrome (trisomy 21) and Edward’s syndrome (trisomy 18).
• The centromere is a chromosomal locus that is required for mitosis and acts as
the site of kinetochore formation. The histone H3 variant CENPA (Cse4) is
incorporated here.
• The kinetochore is large multi-protein complex consisting of over 70 proteins
that are recruited to the centromere. This then serves to mechanically link the
chromosomes to microtubules, through microtubule binding components
such as Ndc80.
• Once sister chromatids are properly attached to opposite poles via
microtubules, tension is generated across the spindle. This tension fulfills
checkpoints that allow segregation to continue.
• Tension is achieved by holding sister chromatids together prior to anaphase.
• The cohesin complex holds sisters together.

Are centromere proximal lacO arrays bound together during metaphase?
How do we reconcile the 3-fold enrichment of cohesin at centromeres
with separated centromere proximal lacO arrays?
Does this model accurately portray live cell imaging of
centromeres and centromere proximal DNA?
Outer Spots – Spindle pole bodies
Inner spots – CEN3 proximal lacO arrays
(1.1kb from Cen3)
Pearson et al., Journal Cell Biol., 2001
1 um

What is the path of DNA at the centromere?
Can we visualize cohesin enrichment at the centromere?
How do we resolve the organization of cohesin at the centromere?
• Deconvolution
• Model Convolution
What is the spatial confinement of pericentric chromatin?
What is the significance of kinetochore anisotropy?
Are there any mutants that regulate kinetochore anisotropy?

Chromosome Conformation Capture (3C)
A method to detect the interaction frequency between two points in the genome. This
can be used to infer the spatial arrangement and physical structure of a chromatin fiber.
1) Crosslink
2) Digest
3) Ligate
4) Reverse Crosslinks
5) PCR
1) Crosslink
2) Digest
3) Ligate
4) Reverse Crosslinks
5) PCR
Cen3
Cen3
15kb 23kb
50kb
50kb
Decker et al., Science, 2002

What is the conformation of chromatin near centromeres?
Yeh and Haase et al., Current Biology, 2008
WT ChrIII 15kb
(2.41)
WT ChrIII 23kb
(1.64)
WT ChrIII 50kb
(0.25)
mcd1-1 ChrIII 15kb
(1.54)
nuf2-60 ChrIII 15kb
(2.25)
galCen3 ChrIII 15kb
(1.18)
WT ChrXI 12.3kb
(2.49)
Uncrosslinked
(1.15)
0
0.5
1
1.5
2
2.5
0 0.5 1 1.5 2 2.5 3
Pericentricvsarmexperimentalratio
Pericentric vs arm control ratio
Chromosomal Interaction Frequency

Intra-strand cohesin
Inter-strand cohesin
C-loop
Kinetochore attachment
Proposed path of centromeric DNA: the C-loop
• Accounts for cohesin
enrichment at pericentric
DNA
• Predicts centromere
proximal lacO separation
seen in live cells
• Predicts the increase in
chromosomal looping at
pericentric DNA seen by 3C

What is the structure and function of the cohesin complex?
Intra-strand cohesin
Inter-strand cohesin
• Four protein complex
• Holds sister chromatids together
•Cleaved at anaphase onset

Can we detect cohesin enrichment at pericentric chromatin by fluorescence?
End on view
Smc3 Spc29
Ndc80Smc3
Side on view
Spc29Smc3

What are some limitations of light microscopy?
Verdaasdonk et al., Journal of Cellular Physiology, 2014
Airy discs
and rings
The blurring of
light by a
microscope,
the point
spread
function (PSF),
can be
approximated
by a Gaussian
distribution
Abbe
diffraction
limit
Spots within
the Abbe
limit appear
as a single
diffraction
limited spot.

How do we overcome the blurring of light?
Verdaasdonk et al., Journal of Cellular Physiology, 2014
Deconvolution
restores light blurred
by the point spread
function to the
original point source
without loss of data.
Smc3-GFP
Original Deconvolved
Smc3-GFP before
and after the
application of
nonlinear iterative
deconvolution

Can we get a clearer picture of the organization of cohesin using deconvolution?
Smc3 GFP side on view
deconvolvedoriginal
Smc3 GFP end on view
deconvolvedoriginal

How is cohesin organized in the mitotic spindle?
Smc3
Ndc80
Spc29
Smc3
Spc29 Smc3
By generating surface renders from deconvolved images stepping through the spindle, we expect cohesin is confined
to a hollow barrel shaped region encompassing the spindle.

Populate geometric shape
with fluorophores
Convolve with
experimental PSF
Analyze and compare experimental
and modelled imagesExperimental PSF
What is model convolution?
Model convolution provides subpixel accuracy of the position of fluorescently labelled proteins.
Stephens et al., MBoC, 2013
• Takes the opposite approach of deconvolution. It generates an understanding of the
possible fluorophore distributions that give rise to an experimental image.
• This can be used to gain insight to the number of molecules,
the distribution of molecules, dynamics, and more.

Can model convolution be used to predict the structure of spindle components?
Winey et al., 1995;
Gardner et al., 2005

Can model convolution be used to predict the structure of spindle components?

550nm
500 nm
What is the structural organization of cohesin in the mitotic spindle?
• Cohesin is enriched 3-fold along pericentric chromatin
• Imaging tells us cohesin is organized along the spindle axis
• Using deconvolution, model convolution and surface rendering we conclude
cohesin is arrayed as a hollow cylinder encompassing the spindle during metaphase.

Can we determine localization with sub pixel accuracy using large population data sets?
Using large population data sets (n>200), we can generate positional density
maps which show the frequency with which something can be found at a given
location.
Haase and Mishra et al., Current Biology, 2013

How do we validate heatmaps as a method we trust?
Use heatmaps to measure known values from in vivo measurements of kinetochore components.
• Average discrepancy between heatmap
and SHREC values is 5.3 nm.
• Heatmaps faithfully reproduce
measurements from high localization
accuracy techniques.
Joglekar et al., Current Biology, 2009
Haase et al., Current Biology, 2012
• SHREC - Single molecule High Resolution
Colocalization: two dimensional (XY)
measurement with high localization
accuracy (10nm).
10 nm
• Compare SHREC measurements of intra-
kinetochore distances to heatmap
measurements to validate.
Heatmap
values
SHREC
values

How do chromatin heatmaps compare to cohesin localization?
LacO 1.1kb from Cen3 WT
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Y= 285.1nm ± 68.9nm
X= 354.5nm ± 74.1nm
n= 240
lacO 1.1kb from Cen3 LacO 1.8kb from Cen15 Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Y= 257.3nm ± 76.8nm
X= 405.2nm ± 136.4nm
n= 228
lacO 6.8kb from Cen15 LacO 3.8kb from Cen3 Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Y= 326.2nm ± 110nm
X= 420.6nm ± 175.3nm
n= 208
lacO 8.8kb from Cen3
Centromere proximal chromatin fills a volumetric space similar to that predicted by cohesin visualization
-Width of cohesin barrel encompasses the spread in the Y dimension of chromatin heatmaps
-Both cohesin and chromatin show decreased localization at the spindle axis
Stephens et al., JCB, 2011

Do inner and outer kinetochore components have the same degree of anisotropy?
Ndc80 Metaphase
Ndc80 Anaphase
C-Ndc80
575.56
N-Cse4
659.95
400
450
500
550
600
650
700
750
800
Spot “Height” in Metaphase
C-Ndc80
1.09
N-Cse4
1.23
1
1.1
1.2
1.3
Spot Anisotropy in Anaphase
(spot “height”/spot “width”)
Broad Cse4 localization pattern similar to that observed by Wisniewski, et al., eLife, 2014
Haase et al., Current Biology, 2012
Cse4 Metaphase
Cse4 Anaphase

Is kinetochore anisotropy the result of light blurring?
• Broad non diffraction limited footprint of Cse4 remains distinct after deconvolution when compared to Ndc80
• Unlikely to be an imaging artifact
Metaphase Anaphase
Ndc80Spc29 Ndc80
deconvolvedoriginal
Ndc80Spc29 Ndc80
deconvolvedoriginal
Ndc80Spc29 Ndc80 Ndc80Spc29 Ndc80
Cse4Spc29 Cse4 Cse4Spc29 Cse4

Y= 94.6nm ± 94.3nm
X= 219.3nm ± 76.0nm
n=1032
Y= 116.2nm ± 102.95nm
X= 258.9nm ± 106.4nm
n=268
Y= 180.92nm ± 155.92nm
X= 286.85nm ± 120.9nm
n=1064
Do heatmaps give any insight into the anisotropy of kinetochores?
Cse4 Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
C terminal Ndc80 Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
N term Ndc80 Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1

What makes Pat1 a candidate for anisotropy regulation at the kinetochore?
Wang et al., , 1996
Pilkington et al., , 2008
Mishra et al., Genetics, 2013
• Structural component of the kinetochore, and has a conserved region which
mediates CEN association
• Associates with centromeres in an NDC10 dependent manner
• Loss of Pat1 delays sister chromatid separation, causes errors in segregation,
and leads to defects in structural integrity of chromatin near the centromere.
• Protein Associated with Topoisomerase II
• Involved in P-body assembly (non translating mRNAs and decapping factors)
More recently, Pat1 was found to have a role in chromosome segregation
independent of its function in P-body assembly and translation repression

Does Pat1 play a role in kinetochore anisotropy?
Ndc80 Metaphase Ndc80 pat1D Metaphase
Ndc80 Anaphase Ndc80 pat1D Anaphase
C-Ndc80
1.09
C-Ndc80
pat1D
1.09
N-Cse4
1.23
N-Cse4
pat1D
1.06
1
1.1
1.2
1.3
Spot Anisotropy in Anaphase
(spot “height”/spot “width”)
C-Ndc80
3,958.76
C-Ndc80
pat1D
3,321.30
N-Cse4
955.18
N-Cse4
pat1D
595.25
400
900
1400
1900
2400
2900
3400
3900
4400
Integrated Spot Intensity
C-Ndc80
575.56
C-Ndc80
pat1D
571.17
N-Cse4
659.95
N-Cse4
pat1D
569.87
400
450
500
550
600
650
700
750
800
Spot "Height" in Metaphase
Absence of Pat1 decreases Cse4 footprint to that of Ndc80
Cse4 Metaphase Cse4 pat1D Metaphase
Cse4 Anaphase Cse4 pat1D Anaphase

Can deconvolution confirm the Pat1 dependent nature of kinetochore anisotropy?
Ndc80 GFP
deconvolved
Metaphase Anaphase
Ndc80Spc29 Ndc80
deconvolvedoriginal deconvolvedoriginal
Ndc80Spc29 Ndc80
Ndc80
pat1DSpc29
Ndc80
pat1D
Ndc80
pat1DSpc29
Ndc80
pat1D
Cse4
pat1DSpc29
Cse4
pat1D
Cse4
pat1DSpc29
Cse4
pat1D

Does loss of Pat1 alter the localization mapping of the inner kinetochore?
Y= 94.6nm ± 94.3nm
X= 219.3nm ± 76.0nm
n=1032
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Y= 180.92nm ± 155.92nm
X= 286.85nm ± 120.9nm
n=1064
Cse4 Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Y= 108.8nm ± 102.6nm
X= 281.1nm ± 74.7nm
n= 200
Cse4 pat1KO Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1

Can model convolution help us understand kinetochore anisotropy?
Y= 94.6nm ± 94.3nm
X= 219.3nm ± 76.0nm
n=1032
Y= 90.1nm ± 79.9nm
X= 219.1nm ± 65.3nm
n= 1000
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Ndc80 Simulated
Distance (nm)
Distance(nm)
0 130 260 390 520 650 780 910
650
520
390
260
130
0
-130
-260
-390
-520
-650 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1

Can model convolution provide insight into the broad localization density of Cse4?
Y= 94.6nm ± 94.3nm
X= 292.5nm ± 96.4nm
n=1000
Cse4 simulated
Distance (nm)
Distance(nm)
0 130 260 390 520 650 780 910
650
520
390
260
130
0
-130
-260
-390
-520
-650 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Y= 180.92nm ± 155.92nm
X= 286.85nm ± 120.9nm
n=1064
Cse4 Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Y= 190.2nm ± 141.3nm
X= 280.8nm ± 58.5nm
n= 1000
Cse4 1 centered 4 displaced
Distance (nm)
Distance(nm)
0 130 260 390 520 650 780 910
650
520
390
260
130
0
-130
-260
-390
-520
-650 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1

Does Cse4 association to CEN change in the absence of Pat1?
CEN association of Cse4 is reduced in pat1D strains by ~60%. Centromeric
levels of Cse4 were assayed by ChIP analysis of Cse4-Myc at CEN1, 3 and 5 and
non-CEN DNA in wild type and pat1D strains.

Is depletion of Cse4 at the centromere in pat1D strains is indicative of extra Cse4 molecules?
• 40% reduction in Cse4
fluorescence intensity
upon loss of Pat1
• 60% reduction of Cse4 at
CEN by ChIP upon loss of
Pat1
• Heatmaps show a change
in Cse4 footprint to one
similar to that of Ndc80
upon loss of Pat1
• Model convolution
cannot match WT Cse4
distribution without the
addition of extra
molecules
• Pat1 regulates
localization of an
accessory pool of Cse4

1x
Kinetochore -
Centromere
Attachment Site
16x
Kinetochore
Microtubules
~250nm diameter
Interpolar
Microtubules
Pericentric cohesin
barrel surrounding
spindle microtubules
~500nm diameter
Accessory molecules of
Cse4 distributed along
pericentric chromatin
Assembling the pieces
Using a diverse set of techniques (3C, deconvolution, model convolution, heatmaps) in conjunction with
widefield microscopy and ChIP, we reach the following conclusions:
C-loop

The Structure of Chromatin at the Centromere
in Saccharomyces Cerevisiae
Julian Haase
Bloom Lab
University of North Carolina at Chapel Hill

Acknowledgements
Bloom Lab
Current Members:
Kerry Bloom
Elaine Yeh
Josh Lawrimore
Former Members:
Ajit Joglekar
Jolien Verdaasdonk
Andrew Stephens
Rachel Haggerty
UNC Computer Science Department
Russ Taylor
Leandra Vicci
Cory Quammen
Basrai Lab
Munira Basrai
Prashant Mishra
UNC Physics Department
Michael Falvo
Salmon Lab
Ted Salmon
Aussie Suzuki

Put “extra” slides after this point

Fluorescence light distribution in an image
Point Spread Function (PSF) of Light
Light emitted from a point source is spread out.
150nM
30nM
Diffraction Limit – Image resolution is limited by the diffraction of light.

Chromatin conformation at Cen3
Cen3 15Kb
Crosslinked
Arm3 15Kb
Crosslinked
Cen3 15Kb
uncrosslinked
Arm3 15Kb
uncrosslinked
15kb (n=10)
112249 52518 9485 7367
Crosslinked 15kb
Cen3 vs. Arm3 Ratio
112249/52518 = 1.96
Uncrosslinked 15kb
Cen3 vs. Arm3 Ratio
9485/7367 = 1.25
Crosslinked Ratio vs. Uncrosslinked Ratio
((1.96/1.25)-1)*100 = 56%
From this, we can say that there is
increased physical proximity on
either side of Cen3 relative to a
region on the arm.
>

3C product analysis
Condition
Cen3/Arm3
Crosslinked
DNA
Cen3/Arm3
Uncrosslinked
DNA
Percent increase following
crosslinking
xlinked-unxlinked
Unxlinked
Sample gel n P value
WT 15kb 1.96 ± .18 1.25 ± .15 56.46% 10 4.46E-08
WT 23kb 1.50 ± .05 1.21 ± .03 23.81% 5 1.02E-05
WT 50kb 0.68 ± .25 1.21 ± .30 -43.81% 5 2.82E-04
aF 15kb 1.90 ± .21 1.22 ± .07 56.02% 10 1.33E-08
aF 23kb 1.52 ± .03 1.21 ± .03 25.91% 5 6.37E-07
aF 50kb 1.13 ± .05 1.21 ± .04 -6.58% 5 .002
ndc10-1 15kb 1.21 ± .08 1.18 ± .09 2.03% 10 .55
ndc10-1, aF 15kb 1.21 ± .03 1.20 ± .02 1.56% 10 .76
mcd1-1 15kb 1.44 ± .14 1.18 ± .05 22.17% 10 3.89E-05
gal cen 15kb 1.23 ± .05 1.21 ± .06 1.76% 10 .67
P
xlinked
A
xlinked
P
unxlinked
A
unxlinked

Degree of looping

0
0.5
1
1.5
2
2.5
3
3.5
1 2 3 4 5
cen3vsarm3IntensityRatio
Actual Dilution of Cen3 vs Arm3
Intensity Ratios vs Actual Dilutions
Series1
3C artificial control

DIC
Smc3 is organized around the spindle axis
Confocal images, 100nm steps
Smc3 GFP Spc29 RFP
Smc3GFP
Spc29RFP
Smc3 localizes as two lobes of fluorescence along either
side of the spindle axis, when the spindle is viewed side
on. The lobes are inside the spindle pole bodies,
indicating the cohesin structure is shorter than the
spindle.
Find more/better images to
show here

DIC
This end-on view suggests cohesin is
organized in a cylindrical array.
Confocal images, 100nm steps
Smc3 localizes as a hollow circle when viewed
end on. Spindle pole bodies can be seen directly
in the center of this structure. This “doughnut”
shape, when considered along with the bi-lobed
distribution, suggests cohesin forms a cylinder
that wraps around the spindle.
Smc3 GFP Spc29 RFP
Find more/better images to
show here.
Smc3GFP
Spc29RFP

Images of cohesin + kinetochores
11.10.11 #12611.10.11 #44

WT cohesin time series
Frap Scope, unbinned
t0m t3m t6m t9m t12m t15m t18m
t0m t5m t10m t15m t20m

Side On End On
Width (nm) 417 485
St Dev (nm) 36 76
Side On End On
Original Deconvolved Original Deconvolved
Width (nm) 417 538* 485 559*
St Dev (nm) 36 60 76 30

Confocal WT End On view
Smc3 GFP
Smc3 GFP
deconvolved Smc3 GFP
Smc3 GFP
deconvolved

Confocal WT Side On view
Smc3 GFP
Smc3 GFP
deconvolved Smc3 GFP
Smc3 GFP
deconvolved

Frap Scope cohesin Smc3GFP, Spc29RFP, Ndc80mCherry Surface Render
11.10.11 #12611.10.11 #44

End On Decon Width
(inclusive)
pixels nm
7.75 502
10 648
8.5 551
8 518
8 518
9.5 616
Average Average
8.63 559
Side On Decon Width
(inclusive)
pixels nm
9 583
8 518
8 518
8.5 551
8 518
9 583
8 518
8 518
Average Average
8.31 538

Microscopy Assisted by Graphics and Interactive Convolution
(MAGIC)
How can we test if this proposed structure generates
the fluorescent pattern we see in vivo? With MAGIC!
Model Fluorescent
Image
Special thanks to Cory Quammen and Russ Taylor, members of the
Nanoscale Science Research Group, part of the Computer Science
Department at UNC-Chapel Hill
Magic
Image

Simulations of clustering
Andrew Stephens
Cory W. Quammen & Russell M.
Taylor II
UNC Computer Science

Model convolution of mitotic spindle structures

Wildtype spot shapes, perpendicular to spindle axis
Metaphase: Cse4 GFP
Width: 655nm
Max intensity: 352
Integrated Intensity: 16741
Anaphase: Cse4 GFP
Width: 642nm (1.98% decrease from metaphase)
Max Intensity: 387 (9.73% increase from metaphase)
Integrated Intensity:17496 (4.51% increase from metaphase)
Metaphase: Ndc80 GFP
Width: 576nm
Max intensity: 748
Anaphase: Ndc80 GFP
Decrease in “width” represents decrease in radius of spherical structure,
not just a 2D decrease!

Metaphase: WT Cse4 GFP
Width: 655nm
Max intensity: 352
Metaphase: mre11D Cse4 GFP
Width: 541nm
Max intensity:
Integrated Intensity:
Wildtype vs mre11D spot shapes, perpendicular to spindle axis
Anaphase: Cse4 GFP
Anaphase: mre11D Cse4 GFP
Width: 537nm
Max intensity:
Integrated Intensity:

C-Ndc80
575.56
C-Ndc80
pat1D
571.17
C-Ame1
666.44
C-Ame1
pat1D
671.53
N-Cse4
659.95
N-Cse4
pat1D
569.87
N-Cse4
xrn1D
583.18
400
450
500
550
600
650
700
750
800
1
Kinetochore Spot "Height" along Y axis in Metaphase
C-Ndc80
3,958.76
C-Ndc80
pat1D
3,321.30
C-Ame1
1,587.75
C-Ame1
pat1D
1,450.61
N-Cse4
955.18 N-Cse4
pat1D
595.25
N-Cse4
xrn1D
604.88
400
900
1400
1900
2400
2900
3400
3900
4400
Kinetochore Integrated Spot Intensity

C-Ndc80
60432
C-Ndc80
pat1D
59913
C-Ame1
48640
C-Ame1
pat1D
49998
N-Cse4
24355
N-Cse4
pat1D
32516
N-Cse4
xrn1D
33854
5000
15000
25000
35000
45000
55000
65000
75000
1
Whole Cell Fluorescence in Metaphase

Cse4 Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Y= 180.92nm
± 155.92nm
X= 286.85nm
± 120.9nm
n=1064
Cse4 pat1KO Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Y= 108.86nm
± 102.66nm
X= 281.08nm
± 74.74nm
n=200
Y= 102.28nm
± 95.33nm
X= 241.98nm
± 110.9nm
n=204
Cse4 Gal-Psh1 Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1

Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Y= 94.6nm
± 94.3nm
X= 219.3nm
± 76.0nm
n=1032
Y= 116.2nm
± 102.95nm
X= 258.9nm
± 106.4nm
n=268
C terminal Ndc80 Metaphase
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Ndc80 GFP at AA 410
Distance (nm)
Distance(nm)
0 130 259 389 518 648 778 907
648
518
389
259
130
0
-130
-259
-389
-518
-648 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Y= 118.2nm
± 100.6nm
X= 240.2nm
± 87.9nm
n=204
20.95nm 18.7nm
39.6nm

Heatmap Validation

Cse4 transcription is not affected in pat1D strains
Transcription of the CSE4 gene is not affected in
pat1∆ strains. Total RNA was extracted from
wild type and pat1D strains as determined by qRT-
PCR. Haase and Mishra et al., Current Biology, 2013

Future Directions
By what mechanisms are accessory molecules of Cse4 regulated?
-Pat1 prevents ubiquitination of Cse4?
-examine rates of ubiquitination in WT vs pat1D
-does increasing rate of ubiquitination in WT cells replicate Cse4 distribution in pat1D?
Do accessory Cse4 molecules serves as a “rapid response” to detachment events – mre11?
Super Resolution Imaging – Structured Illumination Microscopy (SIM)

SIM: H2B-GFP Spc29-RFP Metaphase
SIM: Smc3-GFP Spc29-RFP Metaphase

Bloom Lab Summary

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