Project details

or


Nuclear Envelope deformation in Ageing

Keywords:
Ageing, Nuclear Envelope, Nuclear Pore Complex

Researchers:
dr. L.M. Veenhoff

Nature of the research:
Ageing Nucleus: The nuclear envelope in which Nuclear Pore Complexes (NPC) are embedded is an important barrier and scaffold structure in eukaryotic cells. Past research has yielded valuable insights into the mechanisms that regulate transport of soluble molecules such as transcription factors and mRNA. In contrast, much less is known about the mechanisms responsible for transport of membrane proteins from the outer membrane of the nuclear envelope, where they are synthesized, to the inner membrane. What is clear, is that the membrane proteins at the inner membrane fulfil essential roles in normal cell homeostasis. For instance, they interact with chromatin and bring telomeres and silenced genes to the nuclear periphery. Also clear is that upon ageing the nuclear envelope deforms in diverse organisms, including yeast and human, and mutations in nuclear periphery or inner membrane-resident proteins can induce accelerated ageing phenotypes. The NPCs are long-lived and thus suspect to the accumulation of damage and consequent malfunction.

Fields of study:
cell biology molecular imaging mass spectrometry

Background / introduction
Genomes are non-randomly arranged, and this spatial organization of the chromosomes within the nucleus is thought to play a critical role in the regulation of gene expression (9). The proteins of the inner membrane play a large role in establishing the spatial organization by tethering specific regions to the periphery. Initial studies in isolated rat liver nuclear envelopes, identified a couple of dozen proteins that reside specifically in the inner membrane(10). More recent proteomic studies performed in different tissues identified more putative inner membrane proteins, suggesting that nuclear envelope composition is tissue-specific (11, 12). There is much to uncover at the specific cellular localisation of the inner membrane even in young healthy cells. But, what happens to this critical barrier, the nuclear envelope, with ageing is even less understood. To date, our knowledge of the function of the proteins of the inner membrane is restricted to only a handful of proteins, notably EMERIN, LBR, LEM2, MAN1, SUN1, LAP2-beta. Mutations or misregulation of genes encoding these membrane proteins of the inner membrane show aberrant nuclear morphology and give rise to a variety of diseases, jointly called laminopathies including lipodystrophies, progeria syndromes and neuromuscular disorders (13, 14), all of which are strongly age-related. Interestingly, mutations in Lamin-A, which is normally a soluble protein, result in permanent Lamin-A farnesylation and thereby constitutive anchoring to the inner nuclear membrane which causes Progeria, a premature ageing syndrome(15). Also upon normal physiological ageing the levels of the the farnesylated form of Lamin increase. Nuclear shape changes are associated with ageing of diverse organisms such as yeast (Fig. 2), worms, and humans (16, 17), but the underlying molecular mechanisms are not well understood.
Scr1/Heh1 and Heh2 exhibit a domain organization similar to their higher eukaryotic LEM2 and MAN1 homologues, with two transmembrane segments, an N-terminal LEM domain and a MAN1-C-terminal Homology Domain (5). The LEM (Lamin, Emerin, Man1)-domains are characteristic of inner membrane proteins and can interact with the nuclear lamina and/or chromatin-binding factors, thereby providing anchoring sites for chromatin at the nuclear periphery in order to modulate higher order chromatin structure (19-21). Telomeres and rDNA repeats are the main silent chromatin domains at the nuclear periphery in baker’s yeast. Src1 is indeed associated with subtelomeric regions (22-24) and it is involved in regulation of expression of genes located in these regions. Scr1 also associates with the rDNA region that, alike the telomere region, is highly repetitive and susceptible to recombination. Both telomere anchoring and rDNA stability have strong connections to ageing. A link to nuclear envelope deformation and Scr1/Heh1 and heh2 function is apparent from our observations that overexpression of Scr1/Heh1, Heh2 and simple inner membrane proteins with the NLS-L-TM domain composition cause gross deformations of the nuclear envelope (Fig 4A, unpublished).
Research question / problem definition
Your work will be to investigate what triggers nuclear envelope deformation and to dissect how chromatin organisation at the periphery changes with ageing
Workplan
In Baker's yeast you will perform a genetic screen to identify genes that are essential for nuclear envelope deformation. The nuclear envelope deformation will be induced artificially by overexpression of an inner membrane protein. The deformations are monitorred by fluorescence microscopy. The data analysis part will be challenging but will allow you to formulate and test possible hypothesis of what genes affect NE deformation and how this may relate to ageing.
Time and preference dependent we will set up chromatin IP experiments to map chromatin at the periphery during ageing.
References
1. J. M. Cronshaw, A. N. Krutchinsky, W. Zhang, B. T. Chait, M. J. Matunis, J. Cell Biol. 158, 915 (2002).
2. M. P. Rout et al., J. Cell Biol. 148, 635 (2000).
3. F. Alber et al., Nature 450, 695 (2007).
4. L. T. Burns, S. R. Wente, Curr. Opin. Cell Biol. 24, 341 (2012).
5. M. C. King, C. P. Lusk, G. Blobel, Nature 442, 1003 (2006).
6. A. C. Meinema et al., Science 333, 90 (2011).
7. A. C. Meinema, B. Poolman, L. M. Veenhoff, Traffic. (2013).
8. A. C. Meinema, B. Poolman, L. M. Veenhoff, Nucleus. 3, 322 (2012).
9. P. Fraser, W. Bickmore, Nature 447, 413 (2007).
10. E. C. Schirmer, L. Florens, T. Guan, J. R. Yates, III, L. Gerace, Science 301, 1380 (2003).
11. N. Korfali et al., Nucleus. 3, 552 (2012).
12. J. S. Gomez-Cavazos, M. W. Hetzer, Curr. Opin. Cell Biol. 24, 775 (2012).
13. I. Mendez-Lopez, H. J. Worman, Chromosoma 121, 153 (2012).
14. H. J. Worman, C. Ostlund, Y. Wang, Cold Spring Harb. Perspect. Biol. 2, a000760 (2010).
15. R. D. Goldman et al., Proc. Natl. Acad. Sci. U. S. A 101, 8963 (2004).
16. E. Haithcock et al., Proc. Natl. Acad. Sci. U. S. A 102, 16690 (2005).
17. M. Webster, K. L. Witkin, O. Cohen-Fix, J. Cell Sci. 122, 1477 (2009).
18. S. S. Lee, V. Avalos, I, D. H. Huberts, L. P. Lee, M. Heinemann, Proc. Natl. Acad. Sci. U. S. A 109, 4916 (2012).
19. Y. Gruenbaum, A. Margalit, R. D. Goldman, D. K. Shumaker, K. L. Wilson, Nat. Rev. Mol. Cell Biol. 6, 21 (2005).
20. N. Wagner, G. Krohne, Int. Rev. Cytol. 261, 1 (2007).
21. P. Meister, A. Taddei, Curr. Opin. Genet. Dev. (2013).
22. S. E. Grund et al., J. Cell Biol. 182, 897 (2008).
23. S. Rodriguez-Navarro, J. C. Igual, J. E. Perez-Ortin, Yeast 19, 43 (2002).
24. K. Mekhail, J. Seebacher, S. P. Gygi, D. Moazed, Nature 456, 667 (2008).
25. M. A. D'Angelo, M. Raices, S. H. Panowski, M. W. Hetzer, Cell 136, 284 (2009).
26. J. N. Savas, B. H. Toyama, T. Xu, J. R. Yates, III, M. W. Hetzer, Science 335, 942 (2012).
27. V. Menendez-Benito et al., Proc. Natl. Acad. Sci. U. S. A 110, 175 (2013).
28. G. van den Bogaart, A. C. Meinema, V. Krasnikov, L. M. Veenhoff, B. Poolman, Nat. Cell Biol. 11, 350 (2009).
29. F. Alber et al., Nature 450, 683 (2007).
30. S. Gauci, L. M. Veenhoff, A. J. Heck, J. Krijgsveld, J. Proteome. Res. 8, 3451 (2009).
31. S. B. van, S. Henikoff, Nat. Biotechnol. 18, 424 (2000).
32. J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, H. Ewers, Nat. Methods 9, 582 (2012).
33. E. Wiederhold, L. M. Veenhoff, B. Poolman, D. J. Slotboom, Mol. Cell Proteomics. 9, 431 (2010).
34. E. M. Green, Y. Jiang, R. Joyner, K. Weis, Mol. Biol. Cell 23, 1367 (2012).
35. L. M. Veenhoff, E. H. Heuberger, B. Poolman, EMBO J. 20, 3056 (2001).
36. D. M. Simpson, R. J. Beynon, Anal. Bioanal. Chem. 404, 977 (2012).
37. S. Mohammed, A. Heck, Jr., Curr. Opin. Biotechnol. 22, 9 (2011).
38. S. Lemeer, A. J. Heck, Curr. Opin. Chem. Biol. 13, 414 (2009).
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