Identification of electroporation sites in the complex lipid organization of the plasma membrane

Abstract

The plasma membrane of a biological cell is a complex assembly of lipids and membrane proteins, which tightly regulate transmembrane transport. When a cell is exposed to strong electric field, the membrane integrity becomes transiently disrupted by formation of transmembrane pores. This phenomenon termed electroporation is already utilized in many rapidly developing applications in medicine including gene therapy, cancer treatment, and treatment of cardiac arrythmias. However, the molecular mechanisms of electroporation are not yet sufficiently well understood; in particular, it is unclear where exactly pores form in the complex organization of the plasma membrane. In this study we combine coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis to identify how formation of pores depends on the local lipid organization. We show that pores do not form homogeneously across the membrane, but colocalize with domains that have specific features, the most important being high density of polyunsaturated lipids. We further show that knowing the lipid organization is sufficient to reliably predict poration sites with machine learning. Additionally, by analysing poration kinetics with Bayesian survival analysis we show that poration does not depend solely on local lipid arrangement, but also on membrane mechanical properties and the polarity of the electric field. Finally, we discuss how the combination of atomistic and coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis can guide the design of future experiments and help us to develop an accurate description of plasma membrane electroporation on the whole-cell level. Achieving this will allow us to shift the optimization of electroporation applications from blind trial-and-error approaches to mechanistic-driven design.

Molecular simulations reveal the free energy landscape and transition state of membrane electroporation

“…MD simulations confirmed the presence of hydrophobic and hydrophilic pores during pore formation, while the radii of stable open pores were in good agreement with experimental estimates [18]. The effect of lipid composition on pore formation has been investigated in detail by simulations [19–23]. To induce pores within accessible simulation times, pores have been formed under excessive non-equilibrium conditions by applying large transmembrane potentials of several volts, which would lead to membrane rupture after a pore has nucleated.…”

“…However, because good reaction coordinates (RCs) for driving pore formation were not available until recently, MD simulation did not lead to understanding of the free energy landscape of pore formation. Such understanding would be highly valuable to design electric pulses with desired effects on membranes [24], to rationalize the kinetics of pore opening and closing under conditions of different potentials or different lipid compositions [8, 25], or to translate results found for model membranes into complex biological membranes [23]. Here, we closed this gap and computed the free energy landscape of electroporation covering pore nucleation and expansion at experimentally relevant potentials.…”

Quantitative Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) Imaging of Individual Vesicles to Investigate the Relation between Fraction of Chemical Release and Vesicle Size

“…Since the only difference between the EP and LF samples is how they were transfected, we assume that the electroporation protocol affects how the vesicles transport constituents. Electroporation temporarily destabilizes the cellular membrane to create pores for small molecules to enter cells and could potentially influence the physical or structural properties of nanometer vesicle membranes [21] and alter membrane‐related function such as vesicle loading and diffusion dynamics [22] …”

“…Electroporation temporarily destabilizes the cellular membrane to create pores for small molecules to enter cells and could potentially influence the physical or structural properties of nanometer vesicle membranes [21] and alter membrane-related function such as vesicle loading and diffusion dynamics. [22] After stimulation, vesicle size decreases compared to Non stim in all groups (Figure 2A). This is expected since vesicle size decreases following release events to maintain osmotic equilibrium after the change in DA content.…”

Models of Electroporation and the Associated Transmembrane Molecular Transport Should Be Revisited

“…Filling the gaps in knowledge will require multiscale approaches including molecular modeling such as molecular dynamics simulations together with enhanced sampling methods to determine the free energy barriers for the formation of the different types of pores. In addition, experiments on model membrane systems of increasing complexity, including lipid bilayers with complex lipid mixtures, bilayers containing membrane proteins and/or cytoskeletal components, as well as cells genetically engineered to express selected cellular components (or knock out the expression) can be designed to provide the required information [82][83][84]86,87]. Further studies on the biological mechanisms which could help cells repair the membrane after electroporation are also needed [88].…”