Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies

Abstract

The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2-4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.

Label-free purification and characterization of optogenetically engineered cells using optically-induced dielectrophoresis

“…The results on the membrane capacitances of the engineered cells may vary with the measurement techniques and conditions. [53][54][55] However, our results on the membrane capacitances of the HEK293 cells and HEK293-ChR2 cells by stable transfection are very similar to those by electrorotation as reported in the literature 53 owing to the same cell type and protein type, the same isotonic solution, and a similar measuring principle (red dotted rectangle in Fig. 6d), which fully demonstrates that the ODEP-based approach for determining the membrane capacitance of optogenetically engineered cells is effective and reliable.…”

Expansion Microscopy for Cell Biology Analysis in Fungi

“…By contrast, in U. maydis sporidia cell wall digestion was successful at any culture age due to its yeast-like growth. The importance of the culture conditions in cell wall digestion for protoplast generation has been reported previously in filamentous fungi (Reilly and Doering, 2010;Li et al, 2017) and yeasts (Terpitz et al, 2012).…”

The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination

“…To facilitate the generation of reliable electrophysiological data, we carried out patch-clamp experiments in mammalian cells expressing CarO::YFP. We chose this experimental model because patch-clamp experiments of fungal protoplasts are complex 32 33 34 and high-quality F. fujikuroi protoplasts could only be obtained in a narrow time-window. In contrast, the use of a stable CarO::YFP-expressing mammal cell line allowed us to record abundant and reproducible data.…”

“…Data analysis was performed using Clampfit 10.3 (Molecular Devices Co.) and Origin 8.5 (OriginLab Corporation, Northampton, USA). Patch-Clamp experiments were performed using a perfusion-chamber described in detail before 32 . Pipettes (GB150F-8P, Scientific-Instruments, Hofheim, Germany) with a tip opening diameter of 1.5 μm exhibited a resistance of 3–5 MΩ in standard bath solution.…”