Optimized fluorescent proteins for 4-color and photoconvertible live-cell imaging in Neurospora crassa

Abstract

Fungal cells are quite unique among life in their organization and structure, and yet implementation of many tools recently developed for fluorescence imaging in animal systems and yeast has been slow in filamentous fungi. Here we present analysis of properties of fluorescent proteins in Neurospora crassa as well as describing genetic tools for the expression of these proteins that may be useful beyond cell biology applications. The brightness and photostability of ten different fluorescent protein tags were compared in a well-controlled system; six different promoters are described for the assessment of the fluorescent proteins and varying levels of expression, as well as a customizable bidirectional promoter system. We present an array of fluorescent proteins suitable for use across the visible light spectrum to allow for 4-color imaging, in addition to a photoconvertible fluorescent protein that enables a change in the color of a small subset of proteins in the cell. These tools build on the rich history of cell biology research in filamentous fungi and provide new tools to help expand research capabilities.

Fig. 1.. NCU4502 promoter length assay using a luciferase reporter.

(A) Schematic of lchomologous recombination at the csr-1 locus were inserted into pRS426. Varying lengths of DNA up to 1 kb upstream of the translational start site (ATG) were inserted upstream of the firefly luciferase coding region into the vector using Gibson assembly. B) Western blot of luciferase expressed by varying lengths of NCU4502 promoter in liquid grown shaking culture under constant light at 25 °C.

Fig. 2.. In vivo properties of constitutively expressed fluorescent proteins in N. crassa.

(A) Schematic of fluorescent proteins expressing genetic constructs. 1 kb upstream and downstream targeting flanks for homologous recombination at the csr-1 locus were inserted into pRS426. Original or codon-optimized open reading frames of fluorescent protein were inserted downstream of the NCU04502-promoter-driven SON-1 coding region into the vector using Gibson assembly. Among the fluorescent proteins used, coding sequences for sGFP, mApple, mRuby3, mScarlet, mCherry, iRFP670 were used without modification. mNeonGreen, vsfGFP-9, mNeptune2.5, and mTagBFP2 were codon optimized by applying the Neurospora codon bias settings in SnapGene. (B) Confocal images showing mTagBFP2 or iRFP670 tagged SON-1, or miRFP670–2 tagged histone H1 in hyphal tips. (C) An example of brightness measurement. The maximum fluorescent intensities of 8×8 pixel ROIs (white squares) from nuclear membrane were used for quantification. (D) Fluorescent intensity measurements of RFPs and GFPs when tagging SON-1 in N. crassa. Each data point represents the mean of 10 measurements within 1 cell. For each fluorescent protein, n = 10 cells. (E) An example of photostability measurement. A 125 × 125 pixel ROI (yellow square) was created for each tip and the mean brightness of this region over the time course was used for quantification. (F) Bleaching profile of fluorescent proteins when tagging SON-1 in N. crassa. 95 % of corresponding lasers were used for bleaching. The percentage of excitation at a corresponding wavelength relative to maximum excitation are shown for each fluorescent protein tested (data from FPbase.org). Each curve represents the average of n ≥ 10 individual measurements. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.. Analysis of promoters for varying levels of constitutive expression.

Western blot analysis of luciferase to compare the relative strengths of 6 different constitutive promoters for use in overexpression or heterologous protein constructs. 30 μg of total protein from N. crassa lysates were run in each well. Tubulin was used as a loading control. The upper and lower images of the luciferase blots were contrasted differently in ImageJ to make lane 6 visible. Lanes 1–3 were highly saturated at high contrast and therefore omitted from the second row.

Fig. 4.. Fluorescence spectra, laser lines, and bandpass emission filters for 4 color imaging.

Plot of the spectral properties of fluorescent proteins for use in 4-color imaging in N. crassa. Numerical data for spectra was acquired from FPbase.org and was replotted to create this figure. Laser lines shown are specific to the instrument on which the images were acquired but are also common on many systems. Numerical data on band pass filters is from Chroma (https://www.chroma.com/spectra-viewer).

Fig. 5.. 4-color live cell imaging reveals 4-dimensions cellular structures of Neurospora crassa mycelium.

A) Schematic of BML and SON-1 tagging genetic construct using a bidirectional promoter. C. heterostrophus gpd-1 promoter was used to drive the expression of son-1 tagged by iRFP670. P. tritici-repentis toxA promoter was used to drive the expression of bml tagged by mTagBFP2. 1 kb upstream and downstream targeting flanks for homologous recombination at the csr-1 locus were inserted into pRS426. B) 3D rendered images of a hyphal tip from a culture grown on agar gel pads from the 4-color strain showing each channel or merged channel. C) Time lapse imaging of the living 4-color strain. The filled arrowheads (white and yellow) indicate nuclei moving along the cytoplasmic flow; the empty arrowhead indicates a nucleus moving against the cytoplasmic flow. Grey: SON-1^iRFP670, Red: WC-2^mApple, Yellow: CDC-11^mNeonGreen, Blue: BML^mTagBFP2. Scale bar = 10 μm. D) Single focal image of the nuclear envelope (SON-1 in red) and microtubules (BML in greys). Red arrows indicate dense microtubule networks associated with the nuclear envelope. E) Z-projection of an unhealthy region in N. crassa mycelium network with ring-like and tubular higher order septin structures (yellow). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6.. Tracking nuclear movement with a photoconvertible fluorescent protein mEos3.1.

A) Schematic of hH1^mEos3.1 tagging genetic construct. 1 kb upstream and downstream targeting flanks for homologous recombination at the csr-1 locus were inserted into pRS426. The whole coding sequence of histone H1 followed by codon-optimized mEos3.1 and V5 epitope tag was inserted downstream of the ccg-1 promoter into the vector using Gibson assembly. B) Time lapse imaging of histone H1 tagged with mEos3.1. The yellow square indicates the region chosen for photostimulation by 405 nm light to convert mEos3.1 from green to red. Images shown were acquired immediately before photoconversion, immediately after, and after 1.5 min of continuous imaging. The white arrow is indicating the direction of bulk cytoplasmic flow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)