Super-resolution studies of the organisation of polarity regulators at the plasma membrane


Supervising PI

Joint supervision by Dr. Paul van Bergen en Henegouwen (Utrecht University) and U-Protein Express

ESR8 Sara Pompe

Project Description

Background

TheNanodisc orientation of the spindle determines the correct organisation of epithelial tissue. Different molecular complexes have recently been described with essential roles in spindle orientation. These molecules include different inositol lipids and the Gα-LGN-NuMA and EGFR/IQGAP1 protein complexes. The aim of this project is to determine how these protein:lipid complexes function, and how they are regulated spatiotemporally by both external and intracellular polarity signals and cues.

Objectives

  • Characterization of protein/lipid complexes involved in spindle orientation in mammalian epithelial cells.
  • Generation of molecular tools for high resolution imaging of spindle formation in 3D cell cultures of epithelial cells.
  • Functional analysis of identified regulatory components for asymmetric spindle orientation.

Summary of Results

In order to improve excisting medical approaches in their mode of action or to develop of a new therapeutically relevant antibody or nanobody(Oliveiraet al.,2013)in the field of molecular oncology it is essential to know very precisely about the structure of its target protein as well as its microenvironmental key players influencing the activation and translocation. Integral membrane proteins (MP), like the EGFR (Sigismund et al.,2017), embedded in lipid rafts are representing a major fraction of all targets in the field of molecular oncology due to their ease of accessibility at the cell surface and mostly overexpression. To study specifically the composition of EGF-induced clusters I have set up a procedure according to (Dörret al.,2016)for the extraction and purification of the epidermal growth factor receptor (EGFR) transmembrane protein including its native lipid environment viathe the nanodiscs forming copolymer styrene-maleic acid (SMA).The nanodiscs have been immune purified viaEGFR targeting biparatopic nanobodies (Fig 1.) for their proteomic analysis of novel key players involved in early stage of EGFR internalization. Subsequent mass spectrometry analysis of the EGFR containing nanodiscs showed the enrichment/recruitment of specific proteins in/to EGFR containing nanodiscs. In addition studies using immune fluorescence and super-resolution could confirm the membranous co-localization as wells as translocation of EGFR with the newly determined key players after EGF treatment indicating that they are playing a critical role in the cluster formation for the internalization of the receptor.

Figure 1: Extraction and purification of membrane proteins including their native lipid environment using the nanodiscs forming copolymer SMA.

Due to the invasive physical and chemical constraints of the current therapeutic strategies, it is still acentral challenge to deliver therapeutics over the blood brain barrier (BBB)into the brainand gain access to tumors with an efficient dose as well as no harmless impact towards healthy brain tissue (Azad et al.,2015), (Gabathuler, 2010).To address a targeted non-invasive delivery, I have been working on a new approachof nanobody-mediated endocytosis and directed transcytosis of the heparin-binding EGF-like growth factor (HB-EGF) receptor from the luminal to the abluminal side in polarized brain endothelial cells. This developed novel targeted strategy is very promising to circumvent the previous issues. HB-EGF is a very promising target for drug delivery into the brain since the receptor is constitutively present at the luminal side of polarized brain endothelial cells and has no natural endogenous ligands thus there is no competition with the transport of essential nutrients into the brain to ensure the homeostatic processes for an optimal neuronal function. The nanobodies been received by phage selection using a library that was derived from a serial immunization of 2 Llamas with the HB-EGF receptor (Fig. 2) (Pardon, E. et al.2014).

Figure 2: Serum evaluation of two Llamas SNL 166 and SNL 167 for their immune response against HB EGF (a) long and (b) short version after 28 days of injection with the ectodomains (1:1). Both Llamas show an increased immune response towards both versions of HB-EGF after 28 days compared to the non-immunized status (day 0). Thereby the immune response of SNL166 is higher compared to the one of SNL167.

If coupled to a drug this approach is very promising to be applied in vivoto enhance the delivery of the therapeutic across the BBB for an efficient tumor recurrence while leaving normal brain tissue unscathed. Furthermore the NBs can be engineered to be bispecific targeting on the one hand HB-EGF and on the other hand upregulated biomarkers presented by tumors (e.g. EGFRvIII a mutated form of EGFR in glioblastoma (Zadeh et al.,2013)). If this construct is then modified by coupling it to a fluorophore it would allow a precise localization of the tumor tissue viasuper-resolution imaging.

References

 

  1. Azad, T. D. et al.,Therapeutic strategies to improve drug delivery across the blood-brain barrier. Neurosurg Focus. 2015 March; 38(3): E9. doi:10.3171/2014.12.FOCUS14758
  2. Oliveira, S. et al.,Targeting tumors with nanobodies for cancer imaging and therapy. ELSEVIER, Journal of Controlled Release,Volume 172, Issue 3, (2013) 607–617.
  3. Sigismund, S., et al.,Emerging functions of the EGFR in cancer. Mol Oncol. 2018 Jan; 12(1): 3–20. Published online 2017 Nov 27. doi: 1002/1878-0261.12155
  4. Dörr, J. M. et al. The styrene–maleic acid copolymer: a versatile tool in membrane research. Eur Biophys J. 2016; 45: 3–21. Published online 2015 Dec 6. doi: 1007/s00249-015-1093-y
  5. Zadeh, G. et al.,EGFR and EGFRvIII in Glioblastoma Partners in Crime. Cell press previews. Vol. 24, Issue 4, P403-404, OCTOBER 14, 2013
  6. Gabathuler, R., Approaches to transport therapeutic drugs across the blood–brain barrier to treat brain diseases. Neurobiology of Disease. 37 (2010)48-57.
  7. Pardon, E. et al. A general protocol for the generation of Nanobodies for structural biology. Nat Protoc. 2014 March ; 9(3): 674–693. doi:10.1038/nprot.2014.039.