Contract no. NMP4-CT-2004-516989
Project acronym: ACTIVE BIOMICS
Project title: Active Biomimetic Systems
Instrument: STREP
Thematic Priority: NMP-3
Final publishable activity report
Period covered: from May 1, 2005 to October 30, 2008
Date of preparation: January 2009
Start date of project: May 1, 2005
Duration: 42 months
Project coordinator name: Prof. Reinhard Lipowsky
Project office: Dr Angelo Valleriani
Project coordinator organization name:
Max Planck Institute of Colloids and Interfaces
Coordinator email: active.BIOMICS@mpikg-golm.mpg.de
Coordinator fax: +49 (0)331 5679602
1. Project partners
This STREP involves the following active partners:
Max Planck Institute of Colloids and Interfaces, Potsdam, Germany (MPI)
FOM Institute for Atomic and Molecular Physics, Amsterdam, Netherlands (AMOLF)
Curie Institute, Paris, France (CURIE)
European Molecular Biology Laboratory, Heidelberg, Germany (EMBL)
Fritz Lipmann Institute, Jena, Germany (FLI)
Enzymology and structural biochemistry Lab., CNRS, Gif-sur-Yvette, France (LEBS)
Politecnico of Milano, Milano, Italy (PMILA)
University of Leipzig, Leipzig, Germany (ULEIP)
Institute of Chemistry of the Chinese Academy of Sciences, Beijing, China (ICCAS)
National Center for Nanoscience and Technology, Beijing, China (NCNST)
1
2. Project execution
2.1 Project structure
The project consisted of six scientific workpackages (WP) aimed at studying in detail
the different aspects of active biomimetic systems as mentioned below.
WP1 “Force generation by actin filaments”
Understanding the molecular mechanisms of site-directed growth and forcegeneration by actin filaments (LEBS, MPI, ULEIP). The focus of this WP was on:
Site-directed growth of filament bundles; Motor properties of formin; Actin network
in vesicles and liposomes; Coordinated actin turnover.
WP2 “Force generation by microtubules”
Theoretical and experimental studies of force generation by single and bundled
microtubules (AMOLF, EMBL, PMILA, MPI). The focus of this WP was on:
Single microtubules, effects of regulatory proteins; Microtubule bundles, experiments;
Microtubule bundles, theory and modeling.
WP3 “Force generation by stepping motors”
Experimental and Theoretical study of force generation by single motors (Curie,
EMBL, IMB, MPI, PMILA). The focus of this WP was on: Regulation of force
generation; Molecular mechanics of stepping motors; Membrane tubes pulled out by
kinesin motors.
WP4 “Transport by stepping motors”
Experimental and theoretical studies of transport by stepping motors on immobilized
and growing filaments (MPI, EMBL, IMB, ULEIP, ICCAS, NCNST). The focus of
this WP was on: Motor traffic on immobilized filaments, experiment and theory;
Processivity of myosin minifilaments; Motor traffic on growing filaments, theory and
experiment; Transport of kinesin-supported capsules along microtubules.
WP5 “Self-Organization in actin-based systems”
Self-organization of actin filaments based on crosslinking by myosin motors in order
to generate active biomimetic polymer networks (ULEIP,CURIE, LEBS, MPI). The
focus of this WP was on: Architecture of crosslinked actin networks; Active
actin/myosin bundles; Stability/instability and oscillatory behavior of active networks
WP6 “Self-Organization in microtubule-based systems”
Active assemblies built up from microtubules and kinesin motors using purified
proteins, such as natural molecular motors and artificial, 'designed' motors (EMBL,
AMOLF, FLI, MPI). The focus of this WP was on: Self-organized assemblies and
pre-structured environments, experiments; Regulation of self-organized assemblies,
experiments; Numerical simulations and theory
2
2.2 Project achievements and perspectives
We have performed experiments to describe the force generation of thick bundles of
actin filaments and their site-directed nucleation by surface anchored molecules. This
is the basis for fully controlled physical measurements of forces produced by
polymerization of actin against an obstacle and of the mechanical properties of the
resulting polymer arrays. In addition, measurements at the mesoscopic scale provided
further insight into the molecular mechanisms underlying motility.
We have explored different methods to construct bundles and meshworks of actin
filaments on quasi 2-dimensional biomimetic actin networks on pillared surfaces and
in arrays of laser traps. Crosslinking between filaments plays an important role in the
behavior of these systems. In the future, it will be possible to discover the presence of
cooperative phenomena due to crosslinking. We also constructed bundles of
microtubules and used optical tweezers in order to measure the generated forces. The
study of theses processes will help understanding several motility processes in cells,
such as filaments interacting with mitotic chromosomes.
In order to elucidate the transport properties within cells, we have studied in great
detail the molecular motor kinesin. On the one hand, the kinesin/microtubule
interaction was studied using molecular modelling and is shown to depend on the
state of the nucleotide binding pocket of the molecular motor. On the other hand, the
molecular structure of kinesin's motor head was also addressed from the
experimental side. By using recombinant methods, it was possible to modify this
structure and to elucidate the interplay between the nucleotide binding pocket and the
microtubule binding domain.
The motility of kinesin covers many length scales, from single mechanical steps with
a step size of a few nanometers to transport of cargo particles over centimeters or even
meters. We have shown that the kinesin motor is governed by a network of several
motor cycles that represent different chemomechanical pathways and act as a
gearbox for the motor.
We have also studied experimentally the behavior of kinesin in crowded environments
and, in particular observed its ability to bypass obstacles on the microtubule. This is
an important property that may greatly contribute to our understanding of traffic
inside cells. On the other hand, we have found out that kinesins, when immobilized on
a structured surface, pull microtubules over this surface with a gliding velocity that
can be controlled by the motor density.
We have developed models that represent a rather general molecular mechanism for
the bi-directional transport of cargo as frequently observed in eukaryotic cells. In
fact, in vivo, teams of kinesin motors are involved in the transport of cargo particles
over long distances up to centimeters. In many cases, the kinesin team competes with
a team of dynein motors that try to pull the cargo particle in the opposite direction.
When motors are attached to a membrane, a situation that may occur frequently
within cells, kinesin motors are able to pull membrane tubes. We have studied, both
theoretically and experimentally, membrane tubes created by teams of kinesin
3
motors out of large unilammellar vesicles. For some parameter values, the tube is
found to undergo oscillations in its length.
Both actin filaments and myosin motors are essential parts of the mechanisms
underlying muscular contraction. We have built several biomimetic systems by which
one can study the interplay between actin filaments and myosin motors at the
nanometer scale. This turns out to be a rather interesting possibility in order to
construct synthetic nano-muscles.
4