A single-molecule view on protein aggregation

FOM - 13.0411
Voortgangsverslag 2012
FOM-programma nr. 127
'A single-molecule view on protein aggregation'
Stichting voor Fundamenteel Onderzoek der Materie
www.fom.nl
mei 2013
Inhoudsopgave
Woord vooraf van de programmaleider ..................................................................................................... 2
Hoogtepunt uit het FOM Jaarboek 2012...................................................................................................... 4
Fact sheet per 1 januari 2013 ......................................................................................................................... 5
Historisch kwantitatief overzicht van input en output ............................................................................. 7
Promoties ......................................................................................................................................................... 7
Personele bezetting in 2012 ........................................................................................................................... 8
Output 2012 ................................................................................................................................................... 10
Werkgroep FOM-L-11 .................................................................................................................................. 10
Werkgroep FOM-L-23 .................................................................................................................................. 11
Werkgroep FOM-L-34 .................................................................................................................................. 12
Werkgroep FOM-T-15 .................................................................................................................................. 12
Werkgroep FOM-V-21 ................................................................................................................................. 13
Werkgroep Biophysics ................................................................................................................................. 13
Bijlage bij de outputgegevens ..................................................................................................................... 14
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Woord vooraf van de programmaleider
Introduction: The FOM programme 'A Single Molecule View of Protein Aggregation' (SMPA) aims
to aims to apply an array of innovative single-molecule techniques, augmented byselected ensemble and computational biophysics approaches, to yield an unprecedented molecular and dynamic
view on protein aggregation.
Added value: There is clear added value in the complementary approaches that are represented in
this programme. We have not yet fully achieved all potential synergy, and continue to work on it.
That said, the junior scientists know to find each other, and there is quite some interaction at this
level between the groups. The move of the Heutink group to Tübingen (see below) raises some
logistical challenges; the move of programme leader to Amsterdam should help further nucleate
interactions geographically.
Personnel changes: The last open PhD position was filled in in the course of 2012. We are now at full
strength. A major change was the announcement of the move of the group of Peter Heutink from
the VUmc to the DZNE in Tübingen, Germany, effected over the period November 2012 – February 2013. Prof. Heutink retains an appointment at the VUmc. The group continues to actively collaborate in the programme.
Progress: Most of the projects have made progress commensurate with the duration of the
appointments of the respective oios and postdocs, and the first manuscripts are in preparation.
Summaries of individual project progress are given below:
- Tans: Single molecule force spectroscopy studies of alpha-synuclein: This project has developed, albeit
with delays due to technical issues, the optical tweezers setup proposed. Force spectroscopy
studies have focused on the aggregation of a model system, Maltose Binding Protein, and the
effect of the chaperone Hsp70 on this aggregation. Force spectroscopy on alpha-synuclein has
not been performed yet; Woodside in Canada has performed the experiments originally proposed here, although these results are not yet published. Tans is in discussion with Woodside
about constructs and fall-back experiments. This project is behind schedule on the specific
measurements on alpha-synuclein.
- Bolhuis: Multiscale modeling of alpha-synuclein aggregation: An extensive all-atom explicit and
implicit solvent molecular dynamics (MD) study of α-synuclein monomer, and dimers has been
performed. The monomer collapses to elongated conformations while maintaining a surprisingly high (50- 75%,) of α-helical content, which might be a consequence of poor sampling.
Sampling was enhanced using the replica exchange molecular dynamics (REMD) method and
combined with the Well-Tempered Metadynamics biasing technique. Preliminary data shows
that α-synuclein conformations slowly adapt much lower helical content (23%). A publication
on these results is in preparation.
- Subramaniam: Quantitative measurement and super-resolution visualization of oligomeric aggregatemembrane interactions: We have established a supported lipid bilayer (SLB) platform, and have
systematically studied the effect of monomeric alpha-synuclein on SLBs. We observe that alphasynuclein forms micrometer sized clusters in SLBs causing membrane damage and strongly
decreases lipid fluidity starting from protein to lipid ratios 1:10. Using a mutant lacking 71-82
residues, we show that this amino acid stretch seems to be involved in membrane damage and
fluidity. A manuscript is in preparation.
- Huber: Probing lipid-triggered amyloid nucleation by spin-label EPR: Fibril structures were investigated with EPR to provide a reference for the ultimately sought structures of oligomeric species.
The vacancy on this project was the last to be filled in, and the PhD student on the project has
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focused on learning the aggregation and fibrillization protocols and the relevant advanced EPR
methods.
- Briels/Den Otter: Simulations of alpha-synuclein aggregate formation: A generic Brownian dynamics simulation programme has been developed for patchy particles, accounting for translational and rotational motion. This programme has been tested and validated, and is an
important step towards the coarse-grained model for alpha-synuclein. A manuscript is in preparation.
- Aartsma/Canters: Optical characterization of alpha-synuclein in the cell with single molecule sensitivity and Oxidative stress and disturbance of metal homeostasis as triggers of alpha-synuclein misfolding
and aggregation in vivo: Despite investing a significant amount of time in optimizing fluorescence
imaging conditions to observe single, labeled alpha-synuclein in live cells, the autofluorescence
of the cell appears to be major challenge. As a fallback option, the focus is now on larger aggregates, and on investigating the cellular uptake of alpha-synuclein in different aggregation
states, and on the fate of these aggregates once they are incorporated. We have obtained high
quality movies in real time over periods of hours. Improved imaging resolution using STORM
techniques are underway. In vitro experiments on oligomeric species include characterizing
these intermediates by a variety of different techniques: single-molecule (dual color) FCS and
other optical measurements, mass spectrometry, and electron microscopy. Dual-color FCS facilities are in place now, and software for data analysis has become available. The difficulty is that
all the experiments have to be done in situ, i.e. in freshly prepared gel, because the aggregates
are metastable and appear to dissociate in solution.
- Heutink: Alpha-synuclein misfolding and aggregation in vivo in relation to genetic factors: The original proposal to use the specific interaction of a PDZ binding motif with the corresponding PDZ
domain for in vivo and real-time monitoring of alpha-synuclein aggregation failed spectacularly. The fallback option is to use GFP-labeled synuclein, and to compare with Myc labeled
synuclein to rule out effects of GFP fusion on aggregation. Further, the prion-like transmission
of alpha synuclein between cells has been investigated, including development of a high content screening assay. We have been able to show that wild type protein can transmit between
the different cell populations and familial mutations affect the rate at which the protein is
transmitted suggesting transmission could be relevant to disease pathogenesis. Deciphering the
pathways that are involved in alpha-synuclein transmission may represent new therapeutic targets to block the spread of protein misfolding.
Network activities: Full consortium meetings are held at ~8 month intervals (January 2012, September 2012, July 2013). Several smaller group meetings have been held – the Twente and Leiden
teams meet on about a 6-8 week interval. This frequency is likely to increase with the impending
move of the programme leader to AMOLF. An application for a Lorentz Center workshop on the
physics of protein aggregation is in preparation.
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Hoogtepunt uit het FOM Jaarboek 2012
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Fact sheet per 1 januari 2013
FOM - 10.1721/2
datum: 01-01-2013
APPROVED FOM PROGRAMME
Number
127.
Title (code)
A single-molecule view on protein aggregation (SMPA)
Executive organisational unit
BUW
Programme management
Prof.dr. V. Subramaniam
Duration
2011-2015
Cost estimate
M€ 2.5
Concise programme description
a. Objectives
This programme aims to apply an array of innovative single-molecule techniques, augmented by
selected ensemble and computational biophysics approaches, to yield an unprecedented molecular
and dynamic view on protein aggregation. We will bridge molecular and cellular perspectives
with well-controlled in vitro experiments complemented by innovative single molecule and superresolution methods to follow aggregation interactions within cells.
b. Background, relevance and implementation
Understanding protein nucleation and aggregation is one of the major challenges in contemporary
biophysics and, in the context of disease, one with great medical relevance. The ambition of this
programme is to unravel the physical mechanisms that underlie the dynamics of nucleation and
formation of early aggregate species. At the core of the programme is the physics of protein
folding, conformational dynamics and protein-protein and protein-membrane interactions.
Protein misfolding and aggregation lies at the heart of a range of devastating diseases, including
neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The underlying physics
is poorly understood, and has broader significance, including in the assembly of food proteins to
provide texture, supramolecular assembly of proteins to form functional complexes, and
biologically templated formation of novel materials.
Ensemble biophysics approaches are not well suited to capture the dynamics and structural
changes associated with individual folding and binding transitions, which are critical to a
mechanistic understanding of the earliest steps in protein aggregation. The transient nature,
inherent heterogeneity, and low numbers of early stage aggregates necessitate single molecule
spectroscopy approaches and other innovative methods that can detect distributions of structures
in ensembles. We will investigate the physical mechanisms underlying the dynamics of nucleation
and formation of early aggregate species of human α-synuclein, focusing on three key questions:
1. How do multiple α-synucleins aggregate?
2. How do early aggregates perturb phospholipid membranes?
3. What are the cellular and genetic triggers of aggregation?
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The programme consortium will use state of the art experimental and computational biophysics
approaches, complemented by methods from protein chemistry and molecular biology and
genetics of neurodegenerative disease to unravel the knotty problem of protein aggregation.
Funding
salarispeil cao per 01-07-2012
bedragen in k€
< 2012
2013
2014
2015
2016
2017
> 2018
Totaal
FOM-basisexploitatie
1.177
570
528
217
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2.492
FOM-basisinvesteringen
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Doelsubsidies NWO
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Doelsubsidies derden
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1.177
570
528
217
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2.492
Totaal
Source documents and progress control
a) Original programme proposal: FOM-10.1238
b) Ex ante evaluation:
FOM-10.1422
c) Decision Executive Board:
FOM-10.1720
Remarks
The final evaluation of this programme will consist of a self-evaluation initiated by the programme
leader and is foreseen for 2016.
EK
Subgebied: 100% FL
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par. HOZB
Historisch kwantitatief overzicht van input en output
personeelsaantallen (in gerealiseerde fte)
WP/V
WP/T
oio
NWP
0,8
2,3
0,3
Input
2011
2012
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1,8
1,3
2011
-
overige wetenschappelijke publicaties
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2012
-
3
Output
proefschriften
5,5
totaal op activiteitenniveau *
(in k€ )
258
overige producten van
wetenschappelijke activiteit
2
vakpublicaties
7
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* Bedragen na afsluiten boekjaar.
Promoties
2011
Geen.
2012
Geen.
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528
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Personele bezetting in 2012
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Output 2012
Werkgroep FOM-L-11
Werkgroepleider
Prof.dr. E.J.J. Groenen
Affiliatie
Binnen de werkgroep actieve
U(H)D's
FOM-programma
Universiteit Leiden
Titel van het project + nummer
Dr. M. Huber
A single-molecule view on protein aggregation
Probing lipid-triggered amyloid nucleation by spin-label EPR
10SMPA04
FOM medewerker(s) op het project
Naam
Soort Personeel
P. Kumar
oio
Datum in dienst
15 jun 2012
Datum uit dienst
14 jun 2016
1. Academische publicaties
a. Publicaties in gerefereede tijdschriften
1. M. Drescher, M. Huber, and V. Subramaniam, Hunting the Chameleon: Structural
Conformations of the Intrinsically Disordered Protein Alpha-Synuclein, Chembiochem, 13,
761-768, 2012
2. M. Robotta, C. Hintze, S. Schildknecht, N. Zijlstra, C. Jungst, C. Karreman, M. Huber, M. Leist,
V. Subramaniam, and M. Drescher, Locally Resolved Membrane Binding Affinity of the NTerminus of alpha-Synuclein, Biochemistry, 51, 3960-3962, 2012
3. Maryam Hashemi Shabestari, Ine M.J. Segers-Nolten, Mireille M.A.E. Claessens,
Bart D. van Rooijen, Vinod Subramaniam, Martina Huber, Elucidating the Alpha-Synuclein
Fibril Fold by Pulsed EPR, Biophysical Journal, 102, 454a, 2012
2. Voordrachten, posters, prijzen en nevenactiviteiten
b. Overige voordrachten en posters op (internationale) conferenties en andere
(wetenschappelijke) bijeenkomsten
1. M. Huber, Amyloid protein aggregation by EPR, GDCh Fachgruppe Magnetische Resonanz
34th Annual Discussion Meeting, 17 sep 2012, 22 sep 2012, Halle, Germany
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Werkgroep FOM-L-23
Werkgroepleider
Prof.dr. T.J. Aartsma
Affiliatie
Universiteit Leiden
FOM-programma
A single-molecule view on protein aggregation
Titel van het project +
nummer
Optical characterization of alpha-synuclein in the cell with single
molecule sensitivity 10SMPA06
FOM medewerker(s) op het project
Naam
Soort Personeel
M.M. Apetri
postdoc
Datum in dienst
01 okt 2011
Datum uit dienst
30 sep 2014
2. Voordrachten, posters, prijzen en nevenactiviteiten
b. Overige voordrachten en posters op (internationale) conferenties en andere
(wetenschappelijke) bijeenkomsten
1. Mihaela Apetri, Optical Characterization of ?-Synuclein Aggregation in Living Cells with
Single Molecule Sensitivity, Dutch Meeting on Molecular and Cellular Biophysics, 01 okt
2012, 02 okt 2012, Veldhoven, Nederland
Werkgroepleider
Prof.dr. T.J. Aartsma
Affiliatie
FOM-programma
Universiteit Leiden
A single-molecule view on protein aggregation
Titel van het project +
nummer
Oxidative stress disturbance of metal homeostasis as triggers of alphasynuclein misfolding and aggregation in vivo 10SMPA08-2
FOM medewerker(s) op het project
Naam
Soort Personeel
M. Mucibabic
oio
Datum in dienst
01 jun 2012
Datum uit dienst
30 sep 2015
2. Voordrachten, posters, prijzen en nevenactiviteiten
b. Overige voordrachten en posters op (internationale) conferenties en andere
(wetenschappelijke) bijeenkomsten
1. Marija Mucibabic, Determination of Early Events in ?-Synuclein Aggregation by using
Single-Molecule Fluoresence, Dutch meeting on Cellular and Molecular Biophysics, 01 okt
2012, 02 okt 2012, Veldhoven, Nederland
2. Marija Mucibabic, Determination of Early Events in ?-Synuclein Aggregation by using
Single-Molecule Fluoresence, Dutch meeting on Protein research, Nucleic acids and Lipids
& Biomembranes, 10 dec 2012, 11 dec 2012, Veldhoven, Nederland
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Werkgroep FOM-L-34
Werkgroepleider
Prof.dr. G.W. Canters
Affiliatie
Universiteit Leiden
FOM-programma
A single-molecule view on protein aggregation
Titel van het project +
nummer
Oxidative stress and disturbance of metal homeostasis as triggers of
alpha-synuclein misfolding and aggregation in vivo 10SMPA08
FOM medewerker(s) op het project
Naam
Soort Personeel
M. Mucibabic
oio
Datum in dienst
01 okt 2011
Datum uit dienst
31 mei 2012
Geen output in 2012.
Werkgroep FOM-T-15
Werkgroepleider
Prof.dr. V. Subramaniam
Affiliatie
Binnen de werkgroep
actieve U(H)D's
FOM-programma
Universiteit Twente
Titel van het project +
nummer
Quantitative measurement and super-resolution visualization of
oligomeric aggregate-membrane interactions 10SMPA03
Dr. M.M.A.E. Claessens
A single-molecule view on protein aggregation
FOM medewerker(s) op het project
Naam
Soort Personeel
A.S. Iyer
oio
Datum in dienst
04 sep 2011
Datum uit dienst
03 sep 2015
N. Schilderink
01 sep 2011
31 aug 2016
TP/T
2. Voordrachten, posters, prijzen en nevenactiviteiten
b. Overige voordrachten en posters op (internationale) conferenties en andere
(wetenschappelijke) bijeenkomsten
1. Iyer, A.S., Stockl, M.T., Claessens, M.M.A.E. & Subramaniam, V., Probing alpha-synucleinmembrane interactions on supported lipid bilayers., Annual Dutch meeting on Molecular
and Cellular Biophysics, 01 okt 2012, 02 okt 2012, Veldhoven, Nederland
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Werkgroep FOM-V-21
Werkgroepleider
Prof.dr. P. Heutink
Affiliatie
Vrije Universiteit Amsterdam
FOM-programma
A single-molecule view on protein aggregation
Titel van het project +
nummer
Alpha-synuclein misfolding and aggregation in vivo in relation to
genetic factors 10SMPA07
FOM medewerker(s) op het project
Naam
Soort Personeel
S. Jain
postdoc
Datum in dienst
01 jun 2011
Datum uit dienst
31 mei 2013
Geen output in 2012.
Werkgroep Biophysics
Werkgroepleider
S.J. Tans
Affiliatie
FOM-programma
FOM-instituut AMOLF
Titel van het project +
nummer
Single molecule force spectroscopy studies of alpha-synuclein
10SMPA01
A single-molecule view on protein aggregation
FOM medewerker(s) op het project
Naam
Soort Personeel
S.V. Bezrukavnikov
oio
Datum in dienst
01 apr 2011
Datum uit dienst
31 mrt 2015
V. Sunderlikova
01 jun 2012
31 mei 2013
TP/V
2. Voordrachten, posters, prijzen en nevenactiviteiten
b. Overige voordrachten en posters op (internationale) conferenties en andere
(wetenschappelijke) bijeenkomsten
1. S. Bezrukavnikov, A. Mashaghi, S.J. Tans, The effect of DnaK chaperone system on a protein
folding pathway, Single Molecule Approaches to Biology GRC, 15 jul 2012, 20 jul 2012,
Vermont, USA
2. S. Bezrukavnikov, S.J. Tans, Single molecule force spectroscopy studies: protein folding,
SMPA meeting, 18 sep 2012, 20 sep 2012, Utrecht, the Netherlands
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Bijlage bij de outputgegevens
Dit voortgangsverslag met de outputgegevens is tot stand gekomen aan de hand van de input van
de onderzoekers van zowel de FOM-instituten als de universitaire werkgroepen. Dit verslag bevat
een dwarsdoorsnede van de geleverde input. De programmaleider heeft de output akkoord
bevonden en een woord vooraf geschreven over de voortgang van het programma. Bij alle gegevens staat een datum of een periode. Omdat er enige tijd zit tussen de totstandkoming en publicatie van dit overzicht, geeft dit dus geen actueel beeld. Doel is dan ook de voortgang en bereikte
resultaten te laten zien uit het peiljaar. Voor de volledigheid staat hieronder de originele vragenlijst voor de onderzoekers vermeld, plus het relevante deel van de e-mail met instructies. AMOLF
en DIFFER hebben een vragenlijst op maat gekregen die op details afwijkt van deze versie.
Geachte professor,
Hierbij ontvangt u een formulier voor het opgeven van de output van het jaar 2012. In het formulier staan de titel van het project, het projectnummer en de FOM-medewerker(s) al vermeld. U
kunt eventueel betrokken U(H)D's ook opgeven in het formulier. We verzoeken u deze informatie
terug te zenden vóór 1 februari 2013. Lees deze e-mail in zijn geheel aandachtig door voor de tips
bij het invullen en sla regelmatig uw bestand op!
Achtergrond outputverzameling
Ieder jaar verzamelt FOM de output van alle projecten die bij FOM lopen. De informatie die we
daaruit verkrijgen gebruiken we om te voldoen aan de verplichting aan NWO om te rapporteren
over het totaal aantal publicaties, proefschriften, octrooien, etc. Daarnaast gebruiken we de informatie om tabellen in het Jaarboek van FOM te kunnen weergeven. Ook wordt aan de werkgemeenschapscommissies een jaarlijks outputverslag gestuurd waarin in detail over alle lopende
projecten binnen de FOM-programma's wordt gerapporteerd. Op de website komen rond de
zomer de voortgangsverslagen die jaarlijks in de werkgemeenschapscommissies besproken worden, beschikbaar als download bij de fact sheets van de FOM-programma's.
Welke output opgeven?
Wij verzoeken u alleen output op te geven die toegeschreven kan worden aan het betreffende project en alleen uit het peiljaar. Dus het verzoek is om alleen de output van de FOM-medewerkers
die op het formulier staan op te geven.
Alvast mijn hartelijke dank voor uw medewerking,
Met vriendelijke groet,
Gabby Zegers
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