PhD Thesis proposal form
Discipline
Biology
Doctoral School
Gènes, Génomes, Cellules
Thesis subject title: Regulatory networks of microtubule posttranslational modifications
 Laboratory name and web site: Institut Curie, Department of Signalling, Neurobiology and Cancer
Research team Janke: “The role of tubulin polymodifications in microtubule functions"
Web: http://perso.curie.fr/Carsten.Janke/
 PhD supervisor (contact person):
 Name: Carsten JANKE
 Position: Senior group leader Institut Curie (Research Director CNRS)
 email: Carsten.Janke@curie.fr
 Phone number: +33 1 69863127
 Thesis proposal:
Microtubules are key components of the eukaryotic cytoskeleton involved in a multitude of essential
cellular functions. In the past decades, thorough studies have elucidated the biophysical properties of
microtubules, their interactions with multiple microtubule-associated proteins and molecular motors,
as well as many specialized cellular functions of the microtubule network. While many of the basic
functions and properties of microtubules have been studied in great detail, microtubules have
generally been considered as homogeneous macromolecular assemblies. This is contrasted by the
obvious existence of microtubule identities in cells, which allow microtubules to acquire specific
properties for functional specialisation. Our group investigates an emerging regulatory mechanism
that could play a key role in generating distinct microtubule identities, the posttranslational
modifications of tubulin1.
We are interested in the specific role of the modifications polyglutamylation and polyglycylation in
the cell cycle control, neuronal differentiation and function as well as in ciliary functions. Previous
work has demonstrated that polyglutamylation is enriched on neuronal microtubules2. Very high
levels of polyglutamylation are present on axonemal microtubules in cilia and flagella3-5, and also on
centrioles, the core components of centrosomes and basal bodies6. Interphase microtubules of most
other mammalian cell types carry low levels of polyglutamylation, whereas microtubules of the
mitotic spindle and the midbody show elevated polyglutamylation levels7, indicating a possible role
of this modification in cell cycle control. Polyglycylation is completely absent from cytosolic
microtubules and labels exclusively the axonemes of most cilia and flagella8,9, which could point to a
very specific role of this modification in ciliary assembly and functions.
The research on these modifications was for many years hampered by the lack of solid functional
studies, which could not been carried out since the modifying enzymes had remained unknown. Our
group has identified most of the enzymes involved in the generation and removal of
polyglutamylation and polyglycylation10-14. Using these enzymes, our group is now using systematic
functional studies to understand the roles of polyglutamylation and polyglycylation in microtubule
functions. To understand the complexity of the modifications from a molecular to an organism level,
we have developed a systems research approach ranging from biochemistry to cell and mouse
biology, and we are also investigating links between tubulin modifications and diseases, such as
cancer, neurodegeneration and ciliopathies.
Despite the fact that we are now able to directly address the role of tubulin polymodifications in a
number of microtubule functions, we are still missing insight into the regulatory events that localize,
activate or inactive the modifying enzymes. When TTLL proteins are expressed in different cell lines
and primary cell culture, the overexpressed proteins rarely show strong localizations within the cells.
Some enzymes accumulate at the centrosomes, whereas some localize specifically into cilia15. Only
in rare cases, TTLLs localize to cytoplasmic MTs, indicating that those enzymes contain a MT
binding motif. The general conclusion from these observations is that most TTLL proteins alone
cannot efficiently localize to specific microtubules. It is therefore perceivable that binding partners
could exist that guide TTLLs to specific microtubule localizations in cells in order to specifically
modify these microtubules.
The present PhD project will focus on the mechanisms that regulate the modifications of
microtubules in different physiological contexts. The goal is to identify binding partners for several
TTLL enzymes to study the mechanisms that specify the functions of different TTLLs within cells. A
first insight into how interacting proteins could act on TTLLs has been found for the TTLL1
complex. The biochemical purification of this enzyme revealed four binding partners. One of the
subunits, PGs1, can autonomously localize to the pericentriolar space when overexpressed in cells.
As none of the other proteins of this complex localized specifically within the cells, this suggested
that PGs1 is the localization subunit of the TTLL1 complex10,16.
All TTLLs are expressed at very low levels, even in cells with high levels of tubulin modifications,
such as neurons or ciliated cells. We have developed specific antibodies for each of the TTLLs, and
have never been able to detect an endogenous protein with them, neither in immunohistochemistry
nor in immuno blots. In contrast, when the enzymes are biochemically enriched, some antibodies
detected the endogenous proteins10. Following these observations, we will isolate TTLL binding
partners by purifying endogenous protein complexes from native sources, instead of overexpressing
the TTLL enzymes for pull-down experiments. The rationale is that binding partners might be
equally low expressed and might not associate efficiently to the overexpressed TTLLs. Biochemical
purification methods could be partially deduced from our experience with the purification of the
TTLL1 complex16. A final step in the purification could be an affinity purification using our specific
antibodies for TTLLs. The purified proteins will be analysed by mass spectrometry, and the function
of each novel component will be tested by localization, binding and functional assays in cultured
cells.
Depending on the identity of the interacting proteins, follow-up functional studies will be designed.
For instance, localization subunits of TTLL enzymes could be used to study the dynamic subcellular
distribution of the modifying enzymes and their protein complexes during the cell cycle, neuronal
differentiation or ciliogenesis. It is equally possible that some of the identified interacting proteins
will link the modifying enzymes to signalling pathways. In this case, the role of this pathway in the
activity and cellular functions of the proteins will be studied. Finally, we also expect that we could
identify new substrates of the modifying enzymes, which could also give rise to functional studies on
their role in cells.
Taken together, the PhD project in our lab aims to insert the newly discovered enzymes for tubulin
polyglutamylation and polyglycylation into the regulatory landscape of cells and organisms. The
project is based on established methods in our lab, and challenges new frontiers at the same time. We
have a solid network of national and international collaborators that will assist us in our project. The
Institut Curie is an internationally recognised centre of excellence, hosting under the same roof basic
research ranging from physics over cell biology, developmental biology to cancer research.
Moreover, the presence of clinicians at the institute gives researchers direct access to clinical
research.
References:
1. Janke, C. & Bulinski, J.C. Post-translational regulation of the microtubule cytoskeleton:
mechanisms and functions. Nat Rev Mol Cell Biol 12, 773-786 (2011).
2. Audebert, S. et al. Developmental regulation of polyglutamylated alpha- and beta-tubulin in
mouse brain neurons. J Cell Sci 107, 2313-2322 (1994).
3. Lechtreck, K.F. & Geimer, S. Distribution of polyglutamylated tubulin in the flagellar apparatus
of green flagellates. Cell Motil Cytoskeleton 47, 219-235 (2000).
4. Million, K. et al. Polyglutamylation and polyglycylation of alpha- and beta-tubulins during in
vitro ciliated cell differentiation of human respiratory epithelial cells. J Cell Sci 112, 4357-4366
(1999).
5. Schneider, A., Plessmann, U. & Weber, K. Subpellicular and flagellar microtubules of
Trypanosoma brucei are extensively glutamylated. J Cell Sci 110 ( Pt 4), 431-437 (1997).
6. Bobinnec, Y. et al. Glutamylation of centriole and cytoplasmic tubulin in proliferating nonneuronal cells. Cell Motil Cytoskeleton 39, 223-232 (1998).
7. Regnard, C., Desbruyeres, E., Denoulet, P. & Eddé, B. Tubulin polyglutamylase: isozymic
variants and regulation during the cell cycle in HeLa cells. J Cell Sci 112, 4281-4289 (1999).
8. Bré, M.H. et al. Axonemal tubulin polyglycylation probed with two monoclonal antibodies:
widespread evolutionary distribution, appearance during spermatozoan maturation and possible
function in motility. J Cell Sci 109, 727-738 (1996).
9. Plessmann, U. & Weber, K. Mammalian sperm tubulin: an exceptionally large number of
variants based on several posttranslational modifications. J Protein Chem 16, 385-390 (1997).
10. Janke, C. et al. Tubulin polyglutamylase enzymes are members of the TTL domain protein
family. Science 308, 1758-1762 (2005).
11. van Dijk, J. et al. A targeted multienzyme mechanism for selective microtubule
polyglutamylation. Mol Cell 26, 437-448 (2007).
12. Rogowski, K. et al. Evolutionary divergence of enzymatic mechanisms for posttranslational
polyglycylation. Cell 137, 1076-1087 (2009).
13. Wloga, D. et al. TTLL3 Is a tubulin glycine ligase that regulates the assembly of cilia. Dev Cell
16, 867-876 (2009).
14. Rogowski, K. et al. A family of protein-deglutamylating enzymes associated with
neurodegeneration. Cell 143, 564-578 (2010).
15. van Dijk, J. et al. Polyglutamylation Is a Post-translational Modification with a Broad Range of
Substrates. J Biol Chem 283, 3915-3922 (2008).
16. Regnard, C. et al. Characterisation of PGs1, a subunit of a protein complex co-purifying with
tubulin polyglutamylase. J Cell Sci 116, 4181-4190 (2003).
 Publications of the laboratory in the field:
Janke C, Bulinski JC. Post-translational regulation of the microtubule cytoskeleton: mechanisms and
functions. Nat Rev Mol Cell Biol 2011, 12: 773-786.
Rogowski K, van Dijk J, Magiera MM, Bosc C, Deloulme J-C, Bosson A, Peris L, Gold ND, Lacroix
B, Grau MB, Bec N, Larroque C, Desagher S, Holzer M, Andrieux A, Moutin M-J, Janke C. A
family of protein-deglutamylating enzymes associated with neurodegeneration. Cell 2010, 143:
564-578.
Lacroix B, van Dijk J, Gold ND, Guizetti J, Aldrian-Herrada G, Rogowski K, Gerlich DW, Janke C.
Tubulin polyglutamylation stimulates spastin-mediated microtubule severing. J Cell Biol 2010,
189: 945-954.
Rogowski K, Juge F, van Dijk J, Wloga D, Strub J-M, Levilliers N, Thomas D, Bre M-H, Van
Dorsselaer A, Gaertig J, Janke C. Evolutionary divergence of enzymatic mechanisms for
posttranslational polyglycylation. Cell 2009, 137: 1076-1087.
van Dijk J, Rogowski K, Miro J, Lacroix B, Eddé B, Janke C. A targeted multienzyme mechanism
for selective microtubule polyglutamylation. Mol Cell 2007, 26: 437-448.
 Specific requirements to apply, if any:
We are looking for creative, independent candidates with a strong background in biology and
biochemistry. The candidate should be familiar with basic techniques such as molecular cloning,
protein purification, protein analysis, cell culture, light microscopy. Knowledge in mouse biology
would be an advantage. The laboratory language is English.