In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi

Abstract

Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.

Keywords: Nanofabrication and nanopatterning; Optics and photonics.

Fig. 1. In vivo laser nanosurgery of the cell wall of the filamentous fungus Phycomyces blakesleeanus using fs laser pulses and patch-clamp recording of the released fungal protoplast membrane current activity after the surgical procedure.

a Bright-field and b TPEF image of the plasmolyzed and labeled hypha before surgery. A 20×2 spot-wise pattern was positioned on the cytoplasm-free section of the cell wall. The average laser power in the sample plane for imaging was 1.1 mW (dwell time 2.5 µs) at 730 nm. c TPEF image of the same hypha after surgery. The surgical incision is indicated by the arrow. The laser power at the sample plane for the surgery was 6.1 mW (dwell time 1 s) at 730 nm. The color intensity bars for the TPEF signal are as follows: violet/blue, lowest TPEF signal; and dark red, highest TPEF signal. The color intensity bar is linear and covers the entire range of the data. Scale bar: 10 μm. d Bright-field image of the same hypha with the protoplast released through the surgical incision after laser cutting. e Bright-field image of the patch-clamp pipette in contact with the membrane of the protoplast released through the surgical incision. Scale bar: 20 μm. All images were taken with a Zeiss 40× 1.3 oil objective. f Top: Representative single-channel current recordings obtained from the released protoplast at V_h of +20 mV and 0 mV. o: open channel current level; c: closed channel current level. The calibration bar is on the right side. Bottom: current-voltage (IV) dependency of the recording shown above. On the abscissa, the reversal potentials of the main ions in the bath and pipette solutions are shown, indicating that the current is carried mainly by glutamate. The obtained conductance (g) is given above the linear fit through the measured points. Recorded in SolB. g Representative current recorded from the entire protoplast membrane in the whole-cell configuration. The cells were recorded in SolA with a low-chloride pipette solution. The voltage stimulation protocol used to obtain the recordings is shown in the inset. The calibration bar is at the bottom

Fig. 2. Laser nanosurgery procedure main steps and turning points for successful removal of the Phycomyces blakesleeanus hyphae cell wall.

The osmolarity of the extracellular solution at each step of the procedure is indicated on the blue gradient strip. A detailed description of the procedure can be found in the Materials and Methods section. The distribution of the average laser power used for nanosurgery of P. blakesleeanus cell walls is shown in the upper graph, and the distributions of the sizes of the protoplasts released in the two consecutive steps (bottom) are shown as histograms with the upper bin boundary on the abscissa

Fig. 3. SEM images of the Phycomyces blakesleeanus cell wall after the femtosecond laser nanosurgery.

a TPEF image of the fs laser-generated incision in the hyphal cell wall. The average laser power in the sample plane was 1.0 mW for imaging (dwell time 2.5 µs) and 7.5 mW for surgery (dwell time 1 s). Scale bar: 5 µm. b SEM image of the same laser-generated incision shown in a. Scale bar: 2 µm. c TPEF image of the fs laser-made incision. The average laser power in the sample plane was 1.0 mW for imaging (dwell time 2.5 µs) and 7.6 mW for surgery (dwell time 1 s). Scale bar: 5 µm. All TPEF images were acquired with a Zeiss 40× 1.3 oil objective. The color intensity bars for the TPEF signal are as follows: violet/blue, lowest TPEF signal; and dark red, highest TPEF signal. The color intensity bar is linear and covers the entire range of the data. d SEM image of the entire laser-generated incision, same as that shown in c. Scale bar: 2 µm. e Hyphal cell wall thickness (d) of the incision shown in d. Scale bar: 0.5 µm. f Released protoplast through a laser incision made with 6.7 mW average power at 730 nm. Scale bar: 1 µm

Fig. 4. Parameters affecting the probability of protoplast release.

Probability of protoplast release from the nanosurgery-produced incision, superimposed on the corresponding histogram bin of parameter values. a Distance of the laser incision from the hyphal protoplast during nanosurgery: distribution and dependence of the probability of protoplast release. b Hyphal width at the site of nanosurgery: distribution and dependence of the probability of protoplast release. c Length of surgical incision: distribution and dependence of the probability of protoplast release. d In a separate series of experiments, the effect of increased calcium concentration on the probability of protoplast release was measured as a function of incision length in the nanosurgical phase of the procedure, without subsequent steps of deplasmolysis. High [Ca²⁺]: 30 mM, as used in the surgical protocol. The hyperosmotic solution used was 600-617 mOsm throughout the protoplast release period. Standard [Ca²⁺]: 3 mM and 1 mM (pooled data). The hyperosmotic solution used was 555-560 mOsm throughout the protoplast release time. For each 3 µm incision size bin in the histogram, the corresponding number of protoplasts obtained divided by the number of surgical sections performed is shown above the probability curve. In all graphs, the abscissa represents the upper limit of the bin. For a–c: n_tot(incisions) = 203 (a); 209 (b); 208 (c). n_tot(protoplast) = 71(a), 81(b), 112 (c). For d: n_tot(incisions) = 272 (Standard Ca²⁺); 148 (High Ca²⁺). n_tot(protoplast) = 43 (Standard Ca²⁺); 55 (High Ca²⁺)

Fig. 5. Nanosurgery applied to different sites on the hyphae.

TPEF images of the hyphae with a laser surgical incision at different locations: a apex, b middle, c “neck” and d lateral branch of the labeled hypha. The white arrows indicate a laser-made incision in the hyphal cell wall. All images were taken with a Zeiss 40× 1.3 oil objective. The color intensity bars for the TPEF signal are as follows: violet/blue, the lowest TPEF signal; and dark red, the highest TPEF signal. The color intensity bar is linear and covers the entire range of the data. Scale bar: 10 μm. e Probability of the protoplast release at each site (apex, middle, neck and lateral branch), with the corresponding number of incisions obtained. f Osmotic conditions during the protoplast release at the different sites are shown as fractions of the total number of protoplasts released at each site in each of the solutions used: “postsurgery”= 620 mOsm; “bath chamber” = 595 mOsm

Fig. 6. Summary of the properties of the obtained currents.

a Major types of currents present in the protoplast membrane categorized according to ion permeability. Ion permeability was determined from IV plots under asymmetric conditions, as shown in the bottom panel of Fig. 1g. b Conductance ranges determined for major protoplast types of ion currents, with representative current recordings shown on the right. Unselective anionic current recording, inside-out, V_h = +80 mV, bath solution with high glutamate content (solB), g = 15 pS; Organic acid-carried current, outside-out, V_h = +30 mV, bath solution with nitrate (solA), g = 40 pS, activity modes indicated above current recording. Cl⁻ -selective current, outside-out, V_h = +80 mV, solB, g = 12 pS. o: open channel current level; c: closed channel current level. The calibration bar for each recording is presented on the right side

Fig. 7. Schematic drawing of the NLSM setup for nanosurgery and cell wall imaging.

Ti:Sa fs laser for cell surgery and TPEF imaging, VNDF motorized variable neutral density filter, Sh shutter, GSM galvanometer-scanning mirrors, L1 and L2 - beam expander, MDM main dichroic mirror (cutoff 700 nm), Obj. microscopic objective 40 × 1.0 physiological, Sam. sample, Con. aspheric condenser lens, TL tube lens, BS/M beam splitter or mirror toggle, Cam. camera, F VIS filter 400–700 nm for CFW fluorescence, L3, L4 focusing lens, TPEF PMT photomultiplier tube for TPEF signal, PD photodiode, AD/DA acquisition card