BIOPHYSICS OF THE CELL SURFACE

In this group of Projects our aim is to understand how cells dynamically organize their cell surface and perform all the functions associated with the surface of the cell. Examples include remodeling of the plasma membrane, cell - cell interactions, lateral redistribution of proteins in the membrane and membrane repair. We study these physical phenomena like filopodia dynamics, cell surface repair by annexins, membrane curvature sensing by proteins, lipid order in plasma membranes and resulting protein segregation into distinct lipid phases. To explore these phenomena we use confocal imaging, super resolution (STORM), optical trapping, plasmonic heating and membrane manipulation tools.

Specific questions include:

What kind of dynamics do filopodia exhibit?

The cell surface contains mechanically active antennae-like structures called filopodia. Our lab has contributed significantly to the understanding of the biophysical properties of these structures. We have found that they have the ability to rotate, pull and form helical buckles – all at the same time. These properties have been measured quantitatively using optical trapping in conjunction with 3D confocal imaging (Lejinse et al. PNAS, 2015).

Funding: Villum Foundation (2012-2015)

How do cells cope with plasma membrane ruptures?

Currently, we are exploring the mechanism of plasma membrane repair in living cells. Cells are punctured using thermoplasmonics which allows nanoscopic holes to be formed in the cell membrane. Several annexins are involved in the cell repair and we are doing parallel imaging and puncturing to reveal the mechanism of the cell surface repair machinery. In parallel we study the biophysical properties (curvature affinity and mechanical effects of protein binding) of the same proteins in models systems like GUVs and plasma membrane vesicles.

Collaborators: Robert Daniels (FDA), Jesper Nylandsted (Kræftens Bekæmpelse), Adam Cohen Simonsen (SDU), Himanshu Khandelia (SDU)

Funding: Novonordisk synergy grant (2019-2022).

How do proteins affect membrane mechanics?

Proteins which sense and generate membrane curvature are often found to mechanically stiffen membranes. We have studied the mechanical effects of proteins bound within nanotubes (Ramesh et al. Sci. Rep., 2013 & Barooji et al. Sci. Rep., 2016). The mechanical effect is typically correlated with the ability of the protein to oligomerize.

Do proteins sense lipid order and membrane curvature?

Both peripheral and integral proteins are found to sense the lipid ordering in plasma membranes. Influenza virus protein Neuraminidase was found to sort exclusively into lipid disordered domains in isolated plasma membranes (Moreno et al. ACS Nano, 2019).

Membrane curvature also constitutes another mechanism of lateral organization of proteins. We have shown that F-BAR (Ramesh et al. Sci. Rep., 2013), I-BAR (Barooji et al. Sci. Rep., 2016), Annexin V (Moreno et al. ACS Nano, 2019) and GPCR (Rosholm et al. Nat. Chem. Biol., 2017) effectively sense membrane curvatures and the sensing correlates with the molecular shape of the protein.

Collaborators: Robert Daniels (FDA), Jesper Nylandsted (Kræftens Bekæmpelse), Dimitrios Stamou (Nanoscience Center, KU), Szabolcs Semsey (SNIRP BIOME)

Funding: Villum Foundation (2012-2015) and Sapere Aude DFF (2015-2019).

Can the plasma membrane of living cell be isolated with all its content intact?

During recent years we have focused on development of a method for isolation of the plasma membrane from living cells. This method is highly attractive since it allows us to investigate the plasma membrane proteins in their natural membrane environment, and in absence of the cellular skeleton and other organelles. The isolated membrane forms a spherical vesicle containing the proteins from the mother cell and also internal cytosolic proteins. Importantly, these proteins preserve their correct orientation in the membrane which is in striking contrast to reconstitution of proteins in membranes. This work was published in ACS Nano in 2019 (Moreno et al.) where we showed the feasibility of using these vesicles for optical manipulations and for studying lipid phase behaviors.

Collaborators: Robert Daniels (Food and Drug Administration, USA), Jesper Nylandsted (Kræftens Bekæmpelse), Szabolcs Semsey (SNIRP BIOME).

Development of optical tools for manipulation of the cell surface

Optical trapping of nanostructures

Our group has a long history of quantitative characterization of the optical trapping of nanostructures and micro particles. Trapping potentials and heating effects have been extensively characterized and published in numerous high impact journals (Chemical Reviews, ACS Nano, Nano Letters, Nanoscale, Scientific Reports).

Thermoplasmonics for manipulation of membranes and cells

The ability of nanostructures to heat upon irradiation using biologically harmless near infrared light, has opened up an array of new possibilities to manipulate soft matter and living cells. We exploit the local heating generated in optically trapped nanostructures for nanopuncturing of cells, local melting of membranes (Andersen et al. Soft Matter, 2014) and fusion of membranes (Rørvig-Lund et al. Nano Lett., 2015) and cells (Bahadori et al. Nano Res., 2017 & Bahadori et al., ROPP, 2018) to mention a few examples, see also (Jauffred et al. Chem. Rev., 2019).

Optical extraction of nanotubes from plasma membrane vesicles

High membrane curvatures are generated by optical extraction of nanotubes from cells and vesicles. This has allowed us to investigate the membrane curvature affinity of G-protein coupled receptor (Rosholm et al. Nat. Chem. Biol., 2017), BAR domain proteins (Ramesh et al. Sci. Rep., 2013, Barooji et al. Sci. Rep., 2016), several annexins and the virus protein Neuraminidase (Moreno et al. ACS Nano, 2019).

The membrane nanotubes also allow investigation of protein and lipid mobility by combing the optical trapping with fluorescence recovery after photobleaching (FRAP) experiments.

Collaborators: Robert Daniels (FDA), Jesper Nylandsted (Kræftens Bekæmpelse), Dimitrios Stamou (Nanosceince Center, KU)

Stem cell decision making

Stem cells are un-differentiated cells with the potential to differentiate into any specialized cell of an organism.

There does not yet exist a complete understanding of how biological, chemical and physical factors act in concerto to determine the destiny of a stem cell. The novel StemPhys center joins forces of stem cell biologists from DanStem and theoretical and experimental physics from the Niels Bohr Institute with the goal of significantly progressing our quantitative understanding of stem cell commitment.

StemPhys combines unique stem cell lines with expertise in modelling, bio-imaging, and mechanical manipulation of living matter. One goal is to control the differentiation of stem cells and produce long-living functional cells.

Our research on pancreatic progenitors, with the capacity to generate beta cells producing insulin, could produce new prospects for stem-cell based treatment of diabetes, and our work on liver progenitors could enhance drug development by providing hepatocytes for drug screening. Long-term goals include the development of methods to possibly reverse differentiation of stem cells.

StemPhys is a Danish National Research Foundation Center of Excellence (2015-2021).

Read more at StemPhys.nbi.ku.dk >>

Laser activated nanoparticles for tumor elimination

Irradiated metallic nanoparticles absorb part of the incoming laser light and the absorbed energy will be released in the surroundings as heat. In LANTERN we utilize this effect to develop a novel nanoparticle based cancer therapy.

Such plasmonic nanoparticles are extremely efficient light-to-heat converters and if a metallic nanoparticle is resonant, or close to resonant with the incoming light, the temperature elevation can easily reach hundreds of degrees Celsius.

Within the project we both develop a laser activated assay for targeted drug delivery and a laser activated photo-thermal treatment of cancer, both of these strategies being based on nanoparticles and being usable in concerto. The effects of the therapy are evaluated by PET imaging. The project is a collaboration between physicists at the NBI and molecular imaging and cancer experts at Panum / Rigshospitalet.

Supported by a Synergy Grant from the Novo Nordisk Foundation (2015-2018).

Previous projects:

Force mapping during cancer cell division and invasion

The cells of living tissues are constantly exposed to forces, they experience tension, compression, pressure and shear stress. Also, cells can themselves grow and divide, change shape and actively move, hence exerting forces on their microenvironment.

In this project, we focus on the mechanics of cancer cell invasion, both in terms of how cancer cells differ from non-cancerous cells, and the mechanical interaction between cancer cells and their microenvironment. Cancer cells are deadly once they cross the endothelial barrier of the blood stream, invade into local and distant tissue, and proliferate in vital organs.

Thus, we will focus on mapping the mechanical forces and dynamics associated with these processes to better understand the mechanisms and uncover potential avenues of inhibition. This project is a collaboration between physicists from NBI and cancer cell biologists from BRIC, KU.

Supported by a Research Project 2 Grant from the Danish Research Councils (2015-2018).