MyoGrad internal PhD project announcement 2015
Project leader Berlin
Philipp Selenko
E-Mail / Phone
Name of Institute/Research Unit
selenko@fmp-berlin.de
Institute for Molecular Pharmacology
Berlin/InCell NMR
Project leader Paris
Catherine Coirault
E-Mail / Phone
Name of Institute/Research Unit
c.coirault@institut-myologie.org
Center of research in Myology/
INSERMU974
Title of project:
Nuclear response to mechanical stress: Role of emerin phosphorylation
and implications for nuclear envelopathies
Summary of project:
Background - Nuclear envelopathies, commonly known as laminopathies, are a rapidly
expanding group of human hereditary diseases that are caused by gene mutations of nuclear envelope
proteins. One of the most frequent and best studied example is Emery-Dreifuss muscular dystrophy
(EDMD), a skeletal myopathy that leads to progressive muscule weaknening, joint contractures and
cardiac malefunctions. Mutations in the EMD gene, encoding the emerin protein, cause the X-linked
form of EDMD, whereas mutations in the LMNA gene, encoding A-type lamins are responsible for the
autosomal form of the disease.
The molecular mechanisms underlying EDMD remain elusive. Both emerin and A-type lamins
contribute to nuclear architecture and function in physically connecting the cell nucleus to the
cytoskeleton, which enables forces to be transmitted from the extracellular matrix and the cytoskeleton
to the nuclear interior 1. Mutations in the LMNA gene that cause LMNA-related congenital muscular
dystrophy result in defective mechano-response behaviors in human myoblasts that are characterized by
an impairement in sensing matrix stiffness and the inability to withstand mechanical stretching of the
extracellular matrix 2. Emerin is anchored at the inner nuclear membrane and interacts with a large
number of proteins: structural components of the nuclear envelope (SUN1, SUN2, nesprins) and of the
nucleoskeleton (lamins, actin), DNA binding proteins (barrier-to-autointegration factor (BAF),
transcription factors) and signaling molecules (-catenin). Recently, phosphorylation of emerin tyrosines
was proposed to occur in response to force and to mediate the nuclear response to tension 3. Thus, emerin
might act as a signaling-dependent switch that triggers the expression of mechanosensitive genes.
Hypothesis/Aims
The Myograd PhD project will adress
i)
ii)
iii)
to what extent emerin is phosphorylated in response to cell stretching conditions,
how phosphorylation influences the structure of emerin and its in vitro and
cellular binding behavior
how emerin missense mutations seen in EDMD deregulate phosphorylation and
the mechanosensitive response to forces.
Methodology
Experiments will be performed on primary and immortalized muscle cells from EDMD patients (P22L
and DK37 emerin missense mutations, available in group of Catherine Coirault) and control individuals.
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To determine whether emerin missense mutations induce defects in mechanosensing, mechanosensibility patterns will be assessed with cell culture experiments under specific stretching conditions (3D
cultures). Tyrosine phosphorylation of emerin will initially be investigated by Western-blotting with
phospho-specific antibodies 3 and mass spectrometry (in collaborations with Dr. J. Armengaud, CEA
Marcoule), and under different conditions of kinase/phosphatase inhibition. We will use additional
siRNA silencing strategies or drugs to modulate the dynamics of the cytoskeletons, including myosin,
actin, microtubule and members of cell-matrix adhesion complexes. The interplay between emerin phosphorylation and these regulators will be analyzed by the Paris-team, under defined condition of mechanical stress.
Next, possible structural alterations of emerin in response to tyrosine phosphorylation-, triggered by
different mechanical forces-, will be studied by in-cell Nuclear Magnetic Resonance (NMR) spectroscopy, which enables the simultaneous deduction of emerin’s phosphorylation status and its overall structural properties (using a functional, truncated nucleoplasmic version of emerin, aa1-187). Besides wildtype emerin, we will employ this in-cell NMR approach to also study several EDMD-related mutants of
the protein (i.e., S54F, del95-99, Q133H, P183H and P183T). Preparation of these emerin samples will
be carried out in collaboration with the team of Dr. S. Zinn-Justin, CEA Gif-sur-Yvette, who will spend
a sabbatical year with the group of Dr. Selenko in Berlin. Impact of emerin tyrosine phosphorylation on
its binding behavior to BAF, lamin and actin will be analyzed by complementary biophysical methods
including Isothermal calorimetry (ITC) (Dr. S. Zinn-Justin).
Conclusion.
The goal of this PhD project is to determine the role of intracellular emerin phosphorylation in response to mechanical stress, especially with regard to emerin-related mutations in muscular dystrophies.
To this end, cell- and molelcular-biology methods will be employed in combination with advanced biophysical approaches, including high-resolution in-cell NMR spectroscopy. The respective PhD candidate
will be exposed to a highly interdisciplinary research environment and be trained in multiple cuttingedge methodologies. The joint acquisition of this complementary set of skills will embellish the candidate with unique advantages for his/her future career in science.
Benefit of Paris-Berlin cooperation.
The French PI (Dr. C. Coirault) is part of the team of Dr. G. Bonne located at the Center of
Research in Myology (Paris) and has developed an experimental cell culture approach to follow cell
architecture and molecular signaling during application of a mechanical force on fibroblasts and
myoblasts at different stages of differentiation. Furthermore Dr. Gisèle Bonne has discovered that
mutations in lamin A caused Emery-Dreifuss Muscular Dystrophy 4, and since then, her team has
established different primary and immortalized cell lines from EDMD patients and control individuals.
The German PI (Dr. P. Selenko) at the Leibniz Institute of Molecular Pharmacology (FMP
Berlin) has pioneered in-cell NMR approaches for structural and functional studies in native cellular
environemnts. The Berlin in-cell NMR group is only European research team with this expertise. The
Selenko team has access to excellent NMR infrastructure and is well equipped for all cell biological
aspects of the project. The Berlin group is part of the European Instruct Initiative and has, over the past
couple of years, successfully completed several similar projects in collaborative efforts
(https://www.structuralbiology.eu/nmr/).
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Project outline (please precise whether the doctorate will be in Paris or Berlin in the table below)
1 Half year
2 Half year
3 Half year
4 Half year
5 Half year
6 Half year
Berlin –recombinant
emerin production and
in vitro phosphorylation
assays (in
collab. with
SZJ)
Paris – cell
culture assays, analysis of mechanosensitivity, emerin
phosphorylation status, mass
spectrometry (collab.
with JA)
Paris – kinase/phosphatase
Inhibitors,
role of
phosphorylation,
emerin mutants
Berlin – incell phosphorylations, structural analysis
Berlin –
comparative
analysis mutant emerin
Paris –gene
transfection
and silencing, emerin
interaction
network,
phosphorylation & interactions
Further information
PhD students currently in the group in Paris:
Name of PhD stud
Name of PI
Year of enrolment
Funding source
Martina Fischer
C Coirault/P Knauss
2013
UPMC-Myograd
Christine Technau
G Bonne/S Wissler
2012
DFG-Myograd
Maria Chatzifrangkeskou
G Bonne/P Knauss
2013
DFG-Myograd
Choline Macquart
G Bonne, A Muchir
/M Gotthardt
2014
UPMC-Myograd
Margot Saunier
V Allamand
2014
UPMC
Year of enrolment
Funding source
PhD students currently in the group in Berlin:
Name of PhD stud
Name of PI
None (all PostDoc’s)
Three recent publications of the PI in Paris (Name of PI in bold):
1.
Bertrand AT, Ziaei S, Ehret C, Duchemin H, Mamchaoui K, Bigot A, Mayer M, Quijano-Roy S,
Desguerre I, Lainé J, Ben Yaou R, Bonne G, Coirault C. Cellular microenvironments reveal defective mechanosensing responses and elevated YAP signaling in LMNA-mutated muscle precursors. J Cell Sci 2014 ;127(Pt 13):2873-84
2.
Michelet P, Carreira S, Demoule A, Amour J, Langeron O, Riou B, Coirault C. Effects of Acute
Respiratory and Metabolic Acidosis on Diaphragm Muscle Obtained from Rats. Anesthesiology
2015, in press
Cowling BS, Chevremont T, Prokic I, Kretz C, Ferry A, Coirault C, Koutsopoulos O, Laugel V,
Nomero NB, Laporte J. Reduced dynamin 2 expression rescues X-linked Centronuclear Myopathy.
J Clin Invest 2014 3;124(3):1350-63
3.
.
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Three recent publications of the PI in Berlin (Name of PI in bold):
Freedberg DI, Selenko P. Live cell NMR. Annu Rev Biophys. 2014;43:171-92.
1.
2.
3.
Kosten J, Binolfi A, Stuiver M, Verzini S, Theillet FX, Bekei B, van Rossum M, Selenko P. Efficient modification of alpha-synuclein serine 129 by protein kinase CK1 requires phosphorylation of
tyrosine 125 as a priming event. ACS Chem Neurosci. 2014 Dec 17;5(12):1203-8.
Theillet FX, Binolfi A, Frembgen-Kesner T, Hingorani K, Sarkar M, Kyne C, Li C, Crowley PB,
Gierasch L, Pielak GJ, Elcock AH, Gershenson A, Selenko P Physicochemical properties of cells
and their effects on intrinsically disordered proteins (IDPs). Chem Rev. 2014 Jul 9;114(13):6661714.
Successful PhDs since 2005 that were supervised by the PI in Paris:
Name of PhD stud
Guellich Aziz
Year PhD diploma
2007
Publications by the PhD candidate relative to the PhD project
(Names of PhD student and PI in bold)
1.
2.
3.
4.
Guellich A, Damy T, Lecarpentier Y, Conti M, Claes V, Samuel JL, Quillard J, Hébert JL, Pineau
T, Coirault C. Role of oxidative stress in cardiac dysfunction of PPARalpha-/- mice. Am J Physiol
Heart Circ Physiol. 2007 Jul;293(1):H93-H102.
Guellich A, Damy T, Conti M, Claes V, Samuel JL, Pineau T, Lecarpentier Y, Coirault C. Tempol
prevents cardiac oxidative damage and left ventricular dysfunction in the PPAR-α KO mouse. Am
J Physiol Heart Circ Physiol. 2013 Jun 1;304(11):H1505-12
Salvi N, Guellich A, Michelet P, Demoule A, Le Guen M, Renou L, Bonne G, Riou B, Langeron
O, Coirault C. Upregulation of PPARbeta/delta is associated with structural and functional changes
in the type I diabetes rat diaphragm. PLoS One. 2010 Jul 8;5(7):e11494
Coirault C, Guellich A, Barbry T, Samuel JL, Riou B, Lecarpentier Y. Oxidative stress of myosin
contributes to skeletal muscle dysfunction in rats with chronic heart failure. Am J Physiol Heart
Circ Physiol. 2007 Feb;292(2):H1009-17.
Successful PhDs since 2005 that were supervised by the PI Berlin:
Name of PhD stud
Rossukon Thongwichian
Year PhD diploma
2012
Publications by the PhD candidate relative to the PhD project
(Names of PhD student and PI in bold)
1.
2.
3.
4.
Thongwichian R, Selenko P, In-cell NMR in Xenopus laevis oocytes.
Methods in Molecular Biology 2012 895 33-41
Rose HM, Stuiver M, Thongwichian R, Theillet FX, Feller SM, Selenko P Quantitative NMR
analysis of Erk activity and inhibition by U0126 in a panel of patient-derived colorectal cancer cell
lines. Biochem. Biophys. Acta 2013 1834(7) 1396-401
Theillet FX, Rose HM, Liokatis S, Binolfi A, Thongwichian R, Stuiver M, Selenko P
Site-specific mapping and time-resolved monitoring of serine/threonine phosphorylation in vitro
and in mammalian cell extracts. Nature Protocols 2013 8(7) 1416-32
Theillet FX, Smet-Nocca C, Liokatis S, Thongwichian R, Kosten J, Yoon MK, Kriwacki R,
Landrieu I, Lippens G, Selenko P Cell signaling, post-translational protein modifications and
NMR spectroscopy. J. Biomol. NMR 2012 54(3) 217-36
Name of PhD stud
Beata Bekei
Year PhD diploma
2013
Publications by the PhD candidate relative to the PhD project
(Names of PhD student and PI in bold)
1.
2.
Bekei B, Rose HM, Herzig M, Dose A, Schwarzer D, Selenko P
In-cell NMR in mammalian cells: Part 1, Methods in Molecular Biology 2012 895 43-54
Bekei B, Rose HM, Herzig M, Selenko P
In-cell NMR in mammalian cells: Part 2.Methods in Molecular Biology 2012 895 55-66
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Bekei B, Rose HM, Herzig M, Stephanowitz H, Krause E, Selenko P
In-cell NMR in mammalian cells: Part 3. Methods in Molecular Biology 2012 895 67-83
Kosten J, Binolfi A, Stuiver M, Verzini S, Theillet FX, Bekei B, van Rossum M, Selenko P
Efficient modification of alpha-synuclein Serine 129 by protein kinase CK1 requires phosphorylation of Tyrosine 125 as a priming event. ACS Chemical Neuroscience 2014 5(12) 1203-8
3.
4.
Name of PhD stud
Silvia Verzini
Year PhD diploma
2013
Publications by the PhD candidate relative to the PhD project
(Names of PhD student and PI in bold)
Theillet FX, Binolfi A, Liokatis S, Verzini S, Selenko P Paramagnetic relaxation enhancement to
improve sensitivity of fast NMR methods: application to intrinsically disordered proteins. J. Biomol. NMR, 2011 51(4) 487-95
Kosten J, Binolfi A, Stuiver M, Verzini S, Theillet FX, Bekei B, van Rossum M, Selenko P
Efficient modification of alpha-synuclein Serine 129 by protein kinase CK1 requires phosphorylation of Tyrosine 125 as a priming event. ACS Chemical Neuroscience 2014 5(12) 1203-8
1.
2.
Name of PhD stud
Jonas Kosten
Year PhD diploma
2015
Publications by the PhD candidate relative to the PhD project
(Names of PhD student and PI in bold)
Kosten J, Binolfi A, Stuiver M, Verzini S, Theillet FX, Bekei B, van Rossum M, Selenko P
Efficient modification of alpha-synuclein Serine 129 by protein kinase CK1 requires phosphorylation of Tyrosine 125 as a priming event. ACS Chemical Neuroscience 2014 5(12) 1203-8
Theillet FX, Smet-Nocca C, Liokatis S, Thongwichian R, Kosten J, Yoon MK, Kriwacki R,
Landrieu I, Lippens G, Selenko P Cell signaling, post-translational protein modifications and
NMR spectroscopy. J. Biomol. NMR 2012 54(3) 217-36
1.
2.
References
1.
2.
3.
4.
Zwerger, M. et al. Myopathic lamin mutations impair nuclear stability in cells and
tissue and disrupt nucleo-cytoskeletal coupling. Human molecular genetics 22, 23352349 (2013).
Bertrand, A.T. et al. Cellular microenvironments reveal defective mechanosensing
responses and elevated YAP signaling in LMNA-mutated muscle precursors. Journal
of cell science 127, 2873-2884 (2014).
Guilluy, C. et al. Isolated nuclei adapt to force and reveal a mechanotransduction
pathway in the nucleus. Nature cell biology 16, 376-381 (2014).
Bonne, G. et al. Mutations in the gene encoding lamin A/C cause autosomal dominant
Emery-Dreifuss muscular dystrophy. Nature genetics 21, 285-288 (1999).
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