PhD Thesis – Actin-based Force Generation in the Vascular Endothelium

Prof. Dr. Dietmar Manstein

The research group “Structure and Function of Molecular Motors” at Hannover Medical School (MHH) is recruiting two PhD students to pursue a project aiming at the characterization of actin-based force generating complexes in the human vascular endothelium.


Research Topic


The diversity of tropomyosin isoforms contributes critically to the functional specificity of a wide range of actin-dependent processes in nonmuscle cells. It is now well recognized that different tropomyosin isoforms selectively recruit different myosin motors to specific cellular actin filaments, in processes that gear the mechanical activity and organisation of actin-based contractile structures. While other researchers have described the Tpm-mediated effects in terms of a local gatekeeper function, our results provide evidence for a more elaborate form of regulation, where the tropomyosin isoforms present in the complex defines key parameters of the myosin motors once they are bound to the actin filament. While there is no significant difference between filaments formed from b- and g-actin, the combination of tropomyosin and myosin isoforms defines the duty-cycle, thermodynamic coupling, maximal velocity, and strain-sensitivity of product release steps. Accurate modelling of this more intricate scenario requires a paradigm shift with regard to experimental design and data interpretation. In the context of the proposed project, we will examine the composition, localization dynamics and function of actin-based force-generating complexes in vascular endothelial cells. Our goal is to identify the major contractile complexes present in vascular endothelial cells, to elucidate their function-defining structural features, to deduce first principles that allow an accurate modelling of the impact of disease mutations and allosteric effector molecules on their chemo-mechanical properties, thermal stability, and protein folding stability and dynamics.

 

The project includes the following tasks:

  •  Identify prototypic force-generating complexes that drive cytoplasmic cargo transport, membrane remodelling, vesicle formation, and discrete steps in regulated endo- and exocytosis, cell migration, and cytokinesis in VECs. Special focus will be given to the force-generating role of the different cytoskeletal isoforms of actin, myosin, and Tpm that support cell migration, cytokinesis, membrane remodelling, vesicle formation, cytoplasmic cargo transport, and discrete steps in regulated endo- and exocytosis.
  • Determine the dynamic behaviour and changes in relative abundance of distinct actin-based force generating complexes in VECs, as a consequence of external trigger events.
  • Reconstitute prototypic complexes from the purified proteins, and characterize their kinetic and force generating properties.
  • Probe selected design features of individual components of actin-based force generating complexes using in vitro and cell-based model systems.
  •  Determine high-resolution structures of reconstituted actin-based force generating complexes and their components.
  •  Model the consequences brought about by changes in isoform composition, binding small allosteric effector molecules, and disease mutations.
  • Test the model’s validity with the help of reconstituted complexes containing genetically modified components.
  • Determine the allosteric code that controls the functional behaviour of actin-based force generating complexes

 

The project is funded by the DFG and will be performed in the context of internal and external collaborations. Structural studies are performed in part at the Helmholtz Synchrotron Facility PETRA III and the Centre of Structural Systems Biology on the DESY campus in Hamburg-Bahrenfeld.

 

Please send your application to:
Schille.Joanna@mh-hannover.de

with the following documents:
1. Short 1-page CV
2. Copies of relevant certificates