1. 1. Resolving the Structure of Chromatin at the Centromere in Saccharomyces Cerevisiae Julian Haase Bloom Lab
2. 2. 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 Downs syndrome (trisomy 21) and Edwards 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.
3. 3. 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
4. 4. 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?
5. 5. 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
6. 6. 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
7. 7. 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
8. 8. 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?
9. 9. 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
10. 10. Can we detect cohesin enrichment at pericentric chromatin by fluorescence? Yeh and Haase et al., Current Biology, 2008 End on view Smc3 Spc29 Ndc80Smc3 Side on view Spc29Smc3
11. 11. 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?
12. 12. 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.
13. 13. 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
14. 14. 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
15. 15. 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.
16. 16. 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?
17. 17. 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.
18. 18. Can model convolution be used to predict the structure of spindle components? Winey et al., 1995; Gardner et al., 2005 Stephens et al., MBoC, 2013
19. 19. Stephens et al., MBoC, 2013 Can model convolution be used to predict the structure of spindle components?
20. 20. Stephens et al., MBoC, 2013 Can model convolution be used to predict the structure of spindle components?
21. 21. 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. Yeh and Haase et al., Current Biology, 2008
22. 22. 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?
23. 23. 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
24. 24. 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
25. 25. 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
26. 26. 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?
27. 27. 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
28. 28. 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 Cse4Spc29 Cse4 Cse4Spc29 Cse4
29. 29. 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
30. 30. 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?
31. 31. 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
32. 32. 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 Haase and Mishra et al., Current Biology, 2013 Absence of Pat1 decreases Cse4 footprint to that of Ndc80 Cse4 Metaphase Cse4 pat1D Metaphase Cse4 Anaphase Cse4 pat1D Anaphase
33. 33. Can deconvolution confirm the Pat1 dependent nature of kinetochore anisotropy? Ndc80 GFP deconvolved Metaphase Anaphase Ndc80Spc29 Ndc80 deconvolvedoriginal deconvolvedoriginal Ndc80Spc29 Ndc80 Cse4Spc29 Cse4 Cse4Spc29 Cse4 Ndc80 pat1DSpc29 Ndc80 pat1D Ndc80 pat1DSpc29 Ndc80 pat1D Cse4 pat1DSpc29 Cse4 pat1D Cse4 pat1DSpc29 Cse4 pat1D
34. 34. Does loss of Pat1 alter the localization mapping of the inner kinetochore? Y= 94.6nm 94.3nm X= 219.3nm 76.0nm n=1032 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 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
35. 35. 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 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 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
36. 36. 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
37. 37. Does Cse4 association to CEN change in the absence of Pat1? Haase and Mishra et al., Current Biology, 2013 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.
38. 38. 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 Haase and Mishra et al., Current Biology, 2013 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
39. 39. 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
40. 40. The Structure of Chromatin at the Centromere in Saccharomyces Cerevisiae Julian Haase Bloom Lab University of North Carolina at Chapel Hill
41. 41. 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
42. 42. Put extra slides after this point
43. 43. 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.
44. 44. 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. > Yeh and Haase et al., Current Biology, 2008
45. 45. 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 Yeh and Haase et al., Current Biology, 2008
46. 46. Degree of looping Yeh and Haase et al., Current Biology, 2008
47. 47. 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
48. 48. 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 Yeh and Haase et al., Current Biology, 2008
49. 49. 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 Yeh and Haase et al., Current Biology, 2008
50. 50. Images of cohesin + kinetochores Yeh and Haase et al., Current Biology, 2008 11.10.11 #12611.10.11 #44
51. 51. WT cohesin time series Frap Scope, unbinned t0m t3m t6m t9m t12m t15m t18m t0m t5m t10m t15m t20m
52. 52. 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
53. 53. Confocal WT End On view Smc3 GFP Smc3 GFP deconvolved Smc3 GFP Smc3 GFP deconvolved
54. 54. Confocal WT Side On view Smc3 GFP Smc3 GFP deconvolved Smc3 GFP Smc3 GFP deconvolved
55. 55. Frap Scope cohesin Smc3GFP, Spc29RFP, Ndc80mCherry Surface Render 11.10.11 #12611.10.11 #44
56. 56. 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
57. 57. 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
58. 58. Simulations of clustering Andrew Stephens Cory W. Quammen & Russell M. Taylor II UNC Computer Science
59. 59. Model convolution of mitotic spindle structures Stephens et al., MBoC, 2013
60. 60. 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 Integrated Intensity: 35753 Anaphase: Ndc80 GFP Width: 556nm (3.47% decrease from metaphase) Max Intensity: 820 (9.59% increase from metaphase) Integrated Intensity:37299 (4.32% increase from metaphase) Decrease in width represents decrease in radius of spherical structure, not just a 2D decrease!
61. 61. Metaphase: WT Cse4 GFP Width: 655nm Max intensity: 352 Integrated Intensity: 16741 Metaphase: mre11D Cse4 GFP Width: 541nm Max intensity: Integrated Intensity: Wildtype vs mre11D spot shapes, perpendicular to spindle axis 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) Anaphase: mre11D Cse4 GFP Width: 537nm Max intensity: Integrated Intensity:
62. 62. 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 Haase and Mishra et al., Current Biology, 2013
63. 63. 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 Haase and Mishra et al., Current Biology, 2013
64. 64. 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 Haase and Mishra et al., Current Biology, 2013
65. 65. 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 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
66. 66. Heatmap Validation Haase and Mishra et al., Current Biology, 2013
67. 67. Haase and Mishra et al., Current Biology, 2013 Heatmap Validation
68. 68. 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
69. 69. 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)
70. 70. SIM: H2B-GFP Spc29-RFP Metaphase SIM: Smc3-GFP Spc29-RFP Metaphase