Fluorescent foci quantitation for high-throughput analysis
Main Article Content
Keywords
kinetochore, fluorescence, yeast, cerevisiae, ImageJ
Abstract
A number of cellular proteins localize to discrete foci within cells, for example DNA repair proteins, microtubule organizing centers, P bodies or kinetochores. It is often possible to measure the fluorescence emission from tagged proteins within these foci as a surrogate for the concentration of that specific protein. We wished to develop tools that would allow quantitation of fluorescence foci intensities in high-throughput studies. As proof of principle we have examined the kinetochore, a large multi-subunit complex that is critical for the accurate segregation of chromosomes during cell division. Kinetochore perturbations lead to aneuploidy, which is a hallmark of cancer cells. Hence, understanding kinetochore homeostasis and regulation are important for a global understanding of cell division and genome integrity. The 16 budding yeast kinetochores colocalize within the nucleus to form a single focus. Here we have created a set of freely-available tools to allow high-throughput quantitation of kinetochore foci fluorescence. We use this ‘FociQuant’ tool to compare methods of kinetochore quantitation and we show proof of principle that FociQuant can be used to identify changes in kinetochore protein levels in a mutant that affects kinetochore function. This analysis can be applied to any protein that forms discrete foci in cells.
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References
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33. London N, Biggins S (2014) Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint. Genes Dev 28: 140-152.
34. Musacchio A, Salmon ED (2007) The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 8: 379-393.
35. Warren CD, Brady DM, Johnston RC, Hanna JS, Hardwick KG, et al. (2002) Distinct chromosome segregation roles for spindle checkpoint proteins. Mol Biol Cell 13: 3029-3041.
36. Doncic A, Ben-Jacob E, Einav S, Barkai N (2009) Reverse engineering of the spindle assembly checkpoint. PLoS One 4: e6495.
37. Pearson CG, Yeh E, Gardner M, Odde D, Salmon ED, et al. (2004) Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase. Curr Biol 14: 1962-1967.
38. Miranda JJ, De Wulf P, Sorger PK, Harrison SC (2005) The yeast DASH complex forms closed rings on microtubules. Nat Struct Mol Biol 12: 138-143.
39. Westermann S, Avila-Sakar A, Wang HW, Niederstrasser H, Wong J, et al. (2005) Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex. Mol Cell 17: 277-290.
40. Aravamudhan P, Felzer-Kim I, Joglekar AP (2013) The budding yeast point centromere associates with two Cse4 molecules during mitosis. Curr Biol 23: 770-774.
41. Aravamudhan P, Felzer-Kim I, Gurunathan K, Joglekar AP (2014) Assembling the protein architecture of the budding yeast kinetochore-microtubule attachment using FRET. Curr Biol 24: 1437-1446.
2. Tkach JM, Yimit A, Lee AY, Riffle M, Costanzo M, et al. (2012) Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress. Nat Cell Biol 14: 966-976.
3. Chong YT, Koh JL, Friesen H, Duffy K, Cox MJ, et al. (2015) Yeast Proteome Dynamics from Single Cell Imaging and Automated Analysis. Cell 161: 1413-1424.
4. Lisby M, Rothstein R, Mortensen UH (2001) Rad52 forms DNA repair and recombination centers during S phase. Proc Natl Acad Sci U S A 98: 8276-8282.
5. Alvaro D, Lisby M, Rothstein R (2007) Genome-wide analysis of Rad52 foci reveals diverse mechanisms impacting recombination. PLoS Genet 3: e228.
6. Biggins S (2013) The composition, functions, and regulation of the budding yeast kinetochore. Genetics 194: 817-846.
7. Cheeseman IM (2014) The kinetochore. Cold Spring Harb Perspect Biol 6: a015826.
8. Westermann S, Drubin DG, Barnes G (2007) Structures and functions of yeast kinetochore complexes. Annu Rev Biochem 76: 563-591.
9. Crasta K, Ganem NJ, Dagher R, Lantermann AB, Ivanova EV, et al. (2012) DNA breaks and chromosome pulverization from errors in mitosis. Nature 482: 53-58.
10. Schliekelman M, Cowley DO, O'Quinn R, Oliver TG, Lu L, et al. (2009) Impaired Bub1 function in vivo compromises tension-dependent checkpoint function leading to aneuploidy and tumorigenesis. Cancer Res 69: 45-54.
11. Sheltzer JM, Blank HM, Pfau SJ, Tange Y, George BM, et al. (2011) Aneuploidy drives genomic instability in yeast. Science 333: 1026-1030.
12. Stirling PC, Bloom MS, Solanki-Patil T, Smith S, Sipahimalani P, et al. (2011) The complete spectrum of yeast chromosome instability genes identifies candidate CIN cancer genes and functional roles for ASTRA complex components. PLoS Genet 7: e1002057.
13. Sun W, Yao L, Jiang B, Guo L, Wang Q (2014) Spindle and kinetochore-associated protein 1 is overexpressed in gastric cancer and modulates cell growth. Mol Cell Biochem 391: 167-174.
14. Ryan SD, Britigan EM, Zasadil LM, Witte K, Audhya A, et al. (2012) Up-regulation of the mitotic checkpoint component Mad1 causes chromosomal instability and resistance to microtubule poisons. Proc Natl Acad Sci U S A 109: E2205-2214.
15. Sotillo R, Hernando E, Diaz-Rodriguez E, Teruya-Feldstein J, Cordon-Cardo C, et al. (2007) Mad2 overexpression promotes aneuploidy and tumorigenesis in mice. Cancer Cell 11: 9-23.
16. Tomonaga T, Matsushita K, Ishibashi M, Nezu M, Shimada H, et al. (2005) Centromere protein H is up-regulated in primary human colorectal cancer and its overexpression induces aneuploidy. Cancer Res 65: 4683-4689.
17. Joglekar AP, Salmon ED, Bloom KS (2008) Counting kinetochore protein numbers in budding yeast using genetically encoded fluorescent proteins. Methods Cell Biol 85: 127-151.
18. Joglekar AP, Bouck DC, Molk JN, Bloom KS, Salmon ED (2006) Molecular architecture of a kinetochore-microtubule attachment site. Nat Cell Biol 8: 581-585.
19. Lawrimore J, Bloom KS, Salmon ED (2011) Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome. J Cell Biol 195: 573-582.
20. Cieslinski K, Ries J (2014) The yeast kinetochore - structural insights from optical microscopy. Curr Opin Chem Biol 20: 1-8.
21. Zou H, Rothstein R (1997) Holliday junctions accumulate in replication mutants via a RecA homolog-independent mechanism. Cell 90: 87-96.
22. Thomas BJ, Rothstein R (1989) The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptionally regulated gene. Genetics 123: 725-738.
23. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, et al. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14: 115-132.
24. Sherman F (2002) Getting started with yeast. Methods Enzymol 350: 3-41.
25. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, et al. (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285: 901-906.
26. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9: 671-675.
27. Sluder G, Wolf DE (2013) Digital microscopy. San Diego: Academic Press. 672 p.
28. Cheeseman IM, Anderson S, Jwa M, Green EM, Kang J, et al. (2002) Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p. Cell 111: 163-172.
29. Wisniewski J, Hajj B, Chen J, Mizuguchi G, Xiao H, et al. (2014) Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres. Elife 3: e02203.
30. Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, et al. (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7: R100.
31. Herbert AD, Carr AM, Hoffmann E (2014) FindFoci: a focus detection algorithm with automated parameter training that closely matches human assignments, reduces human inconsistencies and increases speed of analysis. PLoS One 9: e114749.
32. Wu JQ, McCormick CD, Pollard TD (2008) Chapter 9: Counting proteins in living cells by quantitative fluorescence microscopy with internal standards. Methods Cell Biol 89: 253-273.
33. London N, Biggins S (2014) Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint. Genes Dev 28: 140-152.
34. Musacchio A, Salmon ED (2007) The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 8: 379-393.
35. Warren CD, Brady DM, Johnston RC, Hanna JS, Hardwick KG, et al. (2002) Distinct chromosome segregation roles for spindle checkpoint proteins. Mol Biol Cell 13: 3029-3041.
36. Doncic A, Ben-Jacob E, Einav S, Barkai N (2009) Reverse engineering of the spindle assembly checkpoint. PLoS One 4: e6495.
37. Pearson CG, Yeh E, Gardner M, Odde D, Salmon ED, et al. (2004) Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase. Curr Biol 14: 1962-1967.
38. Miranda JJ, De Wulf P, Sorger PK, Harrison SC (2005) The yeast DASH complex forms closed rings on microtubules. Nat Struct Mol Biol 12: 138-143.
39. Westermann S, Avila-Sakar A, Wang HW, Niederstrasser H, Wong J, et al. (2005) Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex. Mol Cell 17: 277-290.
40. Aravamudhan P, Felzer-Kim I, Joglekar AP (2013) The budding yeast point centromere associates with two Cse4 molecules during mitosis. Curr Biol 23: 770-774.
41. Aravamudhan P, Felzer-Kim I, Gurunathan K, Joglekar AP (2014) Assembling the protein architecture of the budding yeast kinetochore-microtubule attachment using FRET. Curr Biol 24: 1437-1446.
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Published
Jul 19, 2015
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Ledesma-Fernández E, Thorpe PH. Fluorescent foci quantitation for high-throughput analysis. J Biol Methods [Internet]. 2015 Jul. 19 [cited 2024 Apr. 19];2(2):e22. Available from: https://jbmethods.org/index.php/jbm/article/view/62
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Author Biographies
Elena Ledesma-Fernández, The Francis Crick Institute
Ph.D. Student at the Francis Crick InstitutePeter H. Thorpe, The Francis Crick Institute
Group Leader at the Francis Crick Institute
