Cell and Developmental Biology University of Michigan

Research

Cellular Biophysics of macromolecular protein machines with in vivo and in vitro fluorescence microscopy techniques and biophysical analysis. Current focus is on the molecular mechanisms of kinetochore function and regulation during cell division.

Figure 1 Nanoscale architecture of the kinetochore. Determined using live-cell fluorescence microscopy methods.

Emergent molecular mechanism of kinetochore force generation and signaling

Background: The lab studies how a eukaryotic cell accurately segregates its genome during cell division. Accurate chromosome segregation during mitosis ensures that each daughter cell inherits one and only one copy of each chromosome in the mother cell. This seemingly simple goal requires highly sophisticated protein machinery that executes mechanical functions that generate motile forces to move chromosomes and biochemical functions that establish cell cycle timing. The kinetochore, a highly complex macromolecular machine (Fig. 1), is at the center of both mechanical and biochemical processes of chromosome segregation.

Research areas & techniques: The kinetochore and the centromere, the unique chromosomal locus that nucleates the kinetochore, constitute the two main research areas in the lab. We use an interdisciplinary approach.

Figure 2 Dimensions of the Dam1 ring. Does the ring exist in vivo?

The main technical expertise of the lab is in developing high-resolution fluorescence microscopy methods that can quantify molecular properties such as their nanoscale localization and dynamics (super-resolution microscopy), their distribution (FRET), and their orientation (Fluorescence Polarization microscopy) in live cells. We use these tools to study how kinetochore proteins are organized at the nanoscale (e.g. the Dam1 complex shown in figure 2) and how they move to achieve their function. We also work with recombinant proteins in vitro in single molecule assays using spectroscopic methods and TIRF microscopy.

Projects:

1. How does the kinetochore generate force to move chromosomes

2. How does the kinetochore generate a biochemical signal for the spindle assembly checkpoint?

3. How does the centromere (chromosomal foundation of the kinetochore) act as a "tensiometer" for the kinetochore?

Disease relevance: Impairment or misregulation of the kinetochore leads to errors in chromosome segregation, generating aneuploid cells. These cells possess abnormal chromosome numbers, and they are a major cause of tumorigenesis, and a hallmark of cancerous tumors. Therefore, understanding the molecular mechanisms underlying kinetochore function and regulation is a highly active area in cancer cell biology. Developmental defects arising from chromosome missegregation are also linked to defects in kinetochore and centromere (the chromosome locus that nucleates the kinetochore) function. A long term goal of the lab is to design artificial kinetochores for genetic engineering.

Lab Members

• Kaushik Gurunathan
  Research Fellow
• Isabella Felzer Kim
  Research Lab Technician
• Pavithra Aravamudhan
  Graduate Student
• Alan Goldfarb
  Undergradate Student
• Ted Chen
  Undergradate Student

Publications

Representative Publications

1. Johnson, K., Joglekar A. P., Hori, T., Fukagawa, T., Salmon E. D. (2010) Vertebrate kinetochore protein architecture: protein copy number J Cell Biol. 189(6):937-43.
2. Joglekar A. P., Bloom K., Salmon E. D. (2010) Mechanisms of force generation with end-on kinetochore-microtubule attachments Curr. Op. Cell Biol 22(1):57-67.
3. Bloom K., and Joglekar A. P. (2010) Toward building a chromosome segregation machine Nature 463:446-56.
4. Joglekar A. P. and DeLuca J. G. (2009) Chromosome segregation: Ndc80 can carry the load Curr. Biol. 19(10):R404-7.
5. Joglekar A. P., Bloom K., Salmon E. D. (2009) In vivo protein architecture of the eukaryotic kinetochore with nanometer accuracy Curr. Biol. 19(8):694-99.
6. Wan X., OQuinn R.P., Pierce H. L., Joglekar A. P., DeLuca J. G., Desai  A., Yen T. J., Salmon E. D. (2009) Protein architecture of the human kinetochore microtubule attachment site Cell 137(4):672-84.
7. Ribeiro S., Gatlin J. C., Dong Y., Joglekar A. P., Cameron L., Hudson D. F., McEwen B. F., Salmon E. D., Earnshaw W. C., Vagnarelli P. (2009) Condensin regulates the stiffness of vertebrate centromeres Mol. Biol. Cell 20(9):2371-80.
8. Gardner M. K., Bouck D. C., Paliulis L. V., Meehl J. B., OToole E. T., Haase J., Soubry A., Joglekar A. P., Winey M., Salmon E. D., Bloom K., and Odde D. J. (2008) Kinesin-5 motors mediate chromosome congression by promoting disassembly of longer microtubules Cell 135(5): 894-906.
9. Joglekar A. P., Bouck D., Finley K., Liu X., Wan Y., Berman J., He X., Salmon E.D., Bloom K. (2008) Molecular architecture of kinetochore-microtubule attachment sites is conserved in point and regional centromeres J. Cell Biol. 181(4): 587-94.
10. Bouck D., Joglekar A. P., Bloom K. (2008) Design Features of a Mitotic Spindle: Balancing tension and compression at a single microtubule kinetochore interface in budding yeast Ann. Rev. Genet. 42:335-59.
11. Joglekar A. P., Salmon E. D., Bloom K. (2008) Counting kinetochore proteins in budding yeast using genetically encoded fluorescent proteins  Methods Cell Biol. 85:127-51.
12. Yeh E., Haase J., Paliulis L. V., Joglekar A. P., Bond L., Bouck D., Salmon E. D., Bloom K. S. (2008) Pericentric chromatin is organized into an intramolecular loop in mitosis Curr. Biol. 18(2):81-90.
13. Gardner M. K., Haase J., Mythreye K., Molk J. N., Anderson M., Joglekar A. P., O'Toole E. T., Winey M., Salmon E. D., Odde D. J., Bloom K. (2008) The microtubule-based motor Kar3 and plus end-binding protein Bim1 provide structural support for the anaphase spindle  J. Cell Biol. 180(1):91-100.
14. Kudryashov S. I., Joglekar A. P., Mourou G., Herbstman J. F., Hunt A. J. (2007) Nanochannels fabricated by high-intensity femtosecond laser pulses on dielectric surfaces Appl. Phys. Lett. 91:141111.
15. Joglekar A. P., Bouck D., Molk J., Bloom K., Salmon E. D. (2006) Molecular architecture of a kinetochore-microtubule attachment site Nature Cell Biol. 8:581-85.
16. Joglekar A. P., Liu, H. H., Meyhofer E., Mourou G., Hunt, A. J. (2004) Optics at critical intensity: Applications to nanomorphing PNAS USA 101(16): 5856-61.
17. Joglekar A. P., Liu H. H., Meyhofer E., Mourou G., Hunt, A. J. (2003) A Study of the deterministic character of optical damage by femtosecond laser pulses and applications to nanomachining Appl. Phys. B - Lasers 77(1): 25-30.
18. Joglekar A. P., Hunt, A. J. (2002). A simple, mechanistic model for directional instability during mitotic chromosome movement Biophys. J. 83(1): 42-58.
19. Kunte, K., Joglekar, A. P., Ghate, U., Pramod, P. (1999) Patterns of butterfly, bird and tree diversity in the Western Ghats Current Science 77 (4): 577-86.