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.

Appendix

Dielectric Models

When exposed to an AC electric field, cells experience mechanical forces, pressures and torques based on the electrostatic interactions of the induced cell dipole with the applied field. The cell dipole (μ) is proportional to the applied field (E ₀) and the complex dielectric polarizability of the cell: χ* (μ ∝ E ₀ · χ*). Among a variety of AC electrokinetic phenomena, dielectrophoresis (DEP) and multi-cell rotation (MCR) are most critical for electrofusion. Thus, cell alignment by positive DEP is required to produce stable cell chains prior to fusion, whereas the MCR effect may lead to destabilization or even to disruption of cell chains.

Exerted by an inhomogeneous field, the dielectrophoretic force (F _DEP) is proportional to the cell volume V (∝r ³, with r = cell radius), the field gradient ∇E ₀ and the real part of polarizability χ* (Jones 1995):

$$ F_{DEP} = 2\pi r^{3} \varepsilon_{\text{e}} \nabla E_{0}^{2} Re\chi^{ * } $$

(1)

In addition to the DEP force, MCR can occur in a linear AC field if cell chains are misaligned with respect to the field, E ₀ (Holzapfel et al. 1982). In case of two adjacent cells, the following relation holds for the frequency-dependent MCR speed, Ω_MCR:

$$ \Upomega_{\text{MCR}} = \frac{{3\varepsilon_{e} }}{8\eta }E_{0}^{2} \sin 2\theta \frac{{{\text{Im}}^{ 2} \chi^{ * } }}{{4 - \text{Re} \chi^{ * } }} $$

(2)

where θ is the angle between the field E ₀ and the line connecting the centers of the two cells and η is the viscosity of the suspending medium.

In a rotating field, the cell rotation, Ω_ROT, is determined by the imaginary part of χ*:

$$ \Upomega_{ROT} = - \frac{{\varepsilon_{\text{e}} E_{0}^{2} }}{2\eta }{\text{Im }}\chi^{*} $$

(3)

As evident from Eqs. 1–3, DEP, MCR and ROT are interrelated phenomena linked to each other through the complex polarizability (χ*), which in turn depends on the dielectric cell structure. Using an approach described elsewhere (Jones 1995), we derived mathematical expressions for the χ* of single- and double-shelled dielectric particles.

In the present study, the ROT and DEP responses of isolated protoplasts of P. pastoris could be approximated quite well with the single-shell model (SSM). Given that the protoplast radii (r ≈2 μm) are much larger than the thickness of the plasma membrane (~10 nm), a simplified expression of the complex polarizability (χ*) was used:

$$ \chi^{*} = \frac{{r\;C_{m}^{*} \left( {\varepsilon_{i}^{*} - \varepsilon_{e}^{*} } \right) - \varepsilon_{i}^{*} \varepsilon_{e}^{*} }}{{r\;C_{m}^{*} \left( {\varepsilon_{i}^{*} + 2\varepsilon_{e}^{*} } \right) + 2\varepsilon_{i}^{*} \varepsilon_{e}^{*} }} $$

(4)

where the complex permittivity (ε*) is defined as ε* = ε − jσ/ω, with ε as the real permittivity (F/m) and σ as the conductivity (S/m) of the medium (subscript “e”) and cytosol (subscript “i”); j = (−1)^1/2; and ω = 2πf is the radian field frequency. The complex area-specific membrane capacitance is given by C ^*_m  = C _m − j G _m/ω, where C _m (F/m²) and G _m (S/m²) are the real membrane capacitance and conductance per unit area, respectively.

The ROT spectra of single-shelled cells can also be presented as a superposition of two Lorentzian peaks:

$$ \Upomega \left( f \right) = 2A_{1} \frac{{\left( {f/f_{{{\text{c}}1}} } \right)}}{{1 + (f/f_{{{\text{c}}1}} )^{2} }} + 2A_{2} \frac{{\left( {f/f_{{{\text{c}}2}} } \right)}}{{1 + (f/f_{{{\text{c}}2}} )^{2} }} $$

(5)

where A ₁ and A ₂ are the amplitudes of the anti- and cofield peaks centered at f _c1 and f _c2, respectively. Equation 5 was used for to determine the f _c1 and f _c2 values from the ROT spectra, as shown in Fig. 2a.

Provided that σ_i ≫ σ_e ≫ σ_m, the SSM yields the following relationship between the f _c1, radius r, σ_e and membrane properties:

$$ f_{c1} \cdot r = \frac{{\sigma_{e} }}{{\pi \cdot C_{m} }} + \frac{{r \cdot G_{m} }}{{2\pi \cdot C_{m} }} $$

(6)

Equation 6 was used in this study to determine the C _m and G _m values from the f _c1 data obtained by the CRF technique.

The double-shell model (DSM), applied here to walled P. pastoris cells, yields the following expression for complex polarizability (χ*):

$$ \chi^{*} = \frac{{C_{m}^{*} r\left( {\varepsilon_{w}^{*} \left( {\left( {a^{3} + 2} \right)\varepsilon_{i}^{*} + 2\left( {a^{3} - 1} \right)\varepsilon_{w}^{*} } \right) - \varepsilon_{e}^{*} \left( {\left( {a^{3} - 1} \right)\varepsilon_{i}^{*} + \left( {2a^{3} + 1} \right)\varepsilon_{w}^{*} } \right)} \right) - a\varepsilon_{i}^{*} \varepsilon_{w}^{*} \left( {\left( {2a^{3} + 1} \right)\varepsilon_{e}^{*} - 2\left( {a^{3} - 1} \right)\varepsilon_{w}^{*} } \right)}}{{C_{m}^{*} r\left( {2\varepsilon_{e}^{*} \left( {\left( {a^{3} - 1} \right)\varepsilon_{i}^{*} + \left( {2a^{3} + 1} \right)\varepsilon_{w}^{*} } \right) + \varepsilon_{w}^{*} \left( {\left( {a^{3} + 2} \right)\varepsilon_{i}^{*} + 2\left( {a^{3} - 1} \right)\varepsilon_{w}^{*} } \right)} \right) + 2a\varepsilon_{i}^{*} \varepsilon_{w}^{*} \left( {\left( {2a^{3} + 1} \right)\varepsilon_{e}^{*} + \left( {a^{3} - 1} \right)\varepsilon_{w}^{*} } \right)}} $$

(7)

where a = r/(r − d), with r and d denoting the cell radius and the thickness of the cell wall, respectively. Subscript “w” refers to the cell wall. The meaning of other symbols is the same as in Eqs. 1–6.