Health

Health

chaired by Loes Segerink & Jeroen Cornelissen

Abstracts

Disordered and ordered subensembles of the unstructured protein alpha-synuclein in cells, Amin Fakhree (NBP)

Alpha-synuclein (αS), an intrinsically disordered protein, is thought to be the major player in synucleinopathies such as Parkinson’s disease, though the exact function(s) of αS is not known. In vitro experiments show that αS is disordered in buffer, nevertheless, in the presence of lipid membranes αS can adopt an alpha-helical structure. Furthermore, biomolecular and cellular imaging techniques showed that αS is associated with membrane related processes inside the cell. Based on these in vitro and in vivo reports, one would expect to be able to identify alpha-helical structure of αS in cells. In contrast, NMR and EPR studies seem to suggest that αS remains disordered inside the cell.

To identify conformationally different subensembles of αS, we microinjected SH-SY5Y cells with small amounts of FRET pair labeled αS. We imaged the cells using ultrasensitive microscopy, and determined the FRET index of the microinjected αS. We observed a significant difference between FRET index of the cytosolic αS and αS bound to small vesicles. This clearly shows that there are at least two structurally distinct subensembles of αS inside cells: 1) the disordered form in the cytosol, 2) the membrane associated form. Our data indicates that the disordered nature of monomeric αS is not fully preserved in cells. The sensitivity and ability to image and laterally resolve conformational differences, makes our method very well suited to single out function-dependent conformational subensembles.

Pentafluorophenyl-based single-chain nanoparticles: A versatile platform towards protein mimicry, Jan-Willem Paats (BNT)

Mimicking the structure and activity of proteins is a highly desired goal for functional polymeric materials. Through precise positioning of monomers (i.e. amino acids), proteins adopt a specific 3 dimensional morphology, which in turn leads to the remarkable variety in properties. Even though perfect control over monomer sequence remains elusive at present, strides have been made to obtain bio-inspired macromolecules with protein-resembling structures, such as single-chain polymer nanoparticles (SCNPs). SCNPs are well-defined soft objects, formed by intramolecularly crosslinked linear polymers, which resemble the size and approximate the structure of proteins. By introducing reactive groups into the precursor backbone, SCNPs may be used post-formation as a template to introduce different functionalities towards mimicking proteins and biomaterials. 

In this work, we present the formation of pentafluorophenyl-based single chain nanoparticles (PFP-SCNPs). Subsequently, PFP-SCNPs are modified post-formation to introduce a variety of functional groups. Through sequential one-pot functionalization, water-soluble particles are obtained with pendant peptides (glutathione and a 6xHis tag), with retention of biological function. Furthermore, PFP-SCNPs are equipped with fluorescent dyes for cell studies, as well as alkyne handles for facile radiolabeling. Cell viability studies reveal no toxicity in hCMEC/D3 cells. The facile post-formation modification provides excellent control over the particle’s characteristics, such as polarity, hydrophilicity and functional groups. Combined with the positive biocompatibility results, PFP-SCNPs are therefore a promising candidate towards a variety of protein-like applications, ranging from targeted therapy to homogeneous catalysis.

Engineering vascularized tissues, Jeroen Rouwkema (BME)

An optimal engineered tissues will need to contain a vascular network; either to supply the cells in the tissue with nutrients after implantation, or to ensure a physiological tissue response when the tissue is used as a screening platform. Especially when the tissue is engineered for implantation purposes, this network needs to be properly organized, including macrovascular structures but also microvascular capillaries, to accommodate a functional connection with the vasculature of the patient. Within our group, we explore the use of chemical and mechanical cues to control and optimize vascular organization within engineered tissues. We combine this with bio-fabrication technology, such as 3D bio-printing and the use of tissue building blocks, to engineer a spatially controlled starting situation. With this approach, our aim is to control tissue remodeling and maturation, resulting in a vascular network that resembles a vascular tree. 

tba, Srirang Manohar (BMPI)

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