Journal: bioRxiv

Article Title: A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions

doi: 10.1101/2024.02.15.580492

Figure Lengend Snippet: A) Schematic of the ER-mRuby3-GCaMP6-210. Calreticulin Signal Peptide and KDEL retention sequences are for ER targeting, GCaMP6-210 is the low affinity GCaMP. B) Airy scan images showing the expression of the ER-mRuby3-GCaMP6-150 in a hippocampal cell body (GCaMP6-210 - green, mRuby3 – red). Orange arrows mark examples of puncta that are thought to be the sensor undergoing degradation. Scale bar is 2 µm. C) Schematic of the ER-Halo-GCaMP6-150. Calreticulin Signal Peptide and KDEL retention sequences are for ER targeting, GCaMP6-150 is the low affinity GCaMP and HaloTag is the bacterial dehalogenase. D) Airy scan images showing the expression of the ER-Halo-GCaMP6-150 in a hippocampal cell body (GCaMP6-150 - green, HaloTag – red). HaloTag is loaded with JF-635. Scale bar is 2 µm.

Article Snippet: To construct the ER-GCaMP fused to mRuby3, mRuby3 was PCR amplified from the pKanCMV-mRuby3-18aa-actin ( ) (Addgene #74255).

Techniques: Expressing

Journal: eLife

Article Title: GABA B R silencing of nerve terminals

doi: 10.7554/eLife.83530

Figure Lengend Snippet: ( A ) Schematic of the experimental protocol. Neurons are stimulated with a brief action potential (AP) train (20 AP in 1 s) at three different [Ca 2+ ] e centered on the physiologic set point of 1.2 mM. Ionomycin, a Ca 2+ ionophore, is administered at the end of the experiment to convert fluorescence into absolute [Ca 2+ ] i . ( B ) An axon from a neuron expressing GCaMP6f localized to nerve terminals by synaptophysin (physin-GCaMP) at rest (top) and a difference image showing the response to 20 AP (bottom). The difference image is the mean peak (five frames) subtracted by the mean baseline (49 frames) in [Ca 2+ ] e 2 mM. For display, a representative subset of terminals was highlighted. Scale bar is 10 µm. ( C ) Traces of responses to 20 AP from the neuron shown in B, with [Ca 2+ ] e color-coded as in A. Expected Δ[Ca 2+ ] i at 0.8 and 1.2 mM is represented by dashed lines and calculated as a change in influx relative to 2.0 mM proportional to the ratios of [Ca 2+ ] e . Arrow bars show the difference between expected and measured Δ[Ca 2+ ] i . Traces are mean, with error (SEM) represented by gray lines in only pre-stimulus and peak frames for clarity (n=211 nerve terminals). ( D ) Summary of Δ[Ca 2+ ] i as a function of [Ca 2+ ] e (mean ± 95%CI, n=9 neurons). The x-axis intercept for measured changes in Δ[Ca 2+ ] i is 0.47 mM. *p<0.05, one-sample t -test compared to the expected value.

Article Snippet: The following published DNA constructs were used: VGLUT1-pHluorin (vGpH) , synaptophysin-GCaMP6f (physin-GCaMP) , cytosolic GCaMP6f , and GPI iGluSnFR3 v857(iGluSnFR) , which was a gift from Kasper Podgorski. mRuby3-synapsin1a (Addgene plasmid #187896) was generated by removing GFP from GFP-synapsin ( ) using restriction sites AgeI and BGIII, and substituting it in frame with mRuby obtained from pKanCMV-mRuby3-18aa-actin, which was a gift from Michael Lin (Addgene plasmid #74255).

Techniques: Fluorescence, Expressing

Journal: bioRxiv

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

doi: 10.1101/2022.09.12.507692

Figure Lengend Snippet: A) Schematic of fluorescent proteins expressing genetic construct. 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. B) Confocal images showing mTagBFP2 or iRFP670 tagged SON-1 in hyphal tips. C) 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. D) Bleaching profile of fluorescent proteins when tagging SON-1 in N. crassa . 95% of corresponding lasers were used for bleaching. Each curve represents the average of 3 individual measurements.

Article Snippet: Among the fluorescent proteins used, coding sequences for sGFP , mApple, mRuby3, mScarlet, mCherry, iRFP670 were used without modification, mApple-C1 was a gift from Michael Davidson (Addgene plasmid # 54631 ; http://n2t.net/addgene:54631 ; RRID:Addgene_54631) ( ). pKanCMV-mRuby3-18aa-Tubulin was a gift from Michael Lin (Addgene plasmid # 74256 ; http://n2t.net/addgene:74256 ; RRID:Addgene_74256) ( ). pmScarlet_C1 was a gift from Dorus Gadella and the CMV immediate-early promoter described herein was amplified from this plasmid, as well as the mScarlet encoding gene (Addgene plasmid # 85042 ; http://n2t.net/addgene:85042 ; RRID:Addgene_85042) (Bindels et al., 2017).

Techniques: Expressing, Construct, Homologous Recombination, Plasmid Preparation

Journal: bioRxiv

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

doi: 10.1101/2022.09.12.507692

Figure Lengend Snippet: 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 arrows (white and yellow) indicate nuclei moving along the cytoplasmic flow; the empty arrows indicate a nucleus moving against the cytoplasmic flow. Grey: SON-1 iRFP670 , Red: WC-2 mApple , Yellow: CDC-11 mNeonGreen , Blue: BML mTagBFP2 . Scale bar = 10μm. D) Single focal image of the nuclear envelope (SON-1 in red) and microtubules (BML in greys). Red arrows indicate microtubule patches associated with the nuclear envelope. E) Z-projection of an unhealthy region in N. crassa mycelium network with ring-like and tubular septin structures (yellow).

Article Snippet: Among the fluorescent proteins used, coding sequences for sGFP , mApple, mRuby3, mScarlet, mCherry, iRFP670 were used without modification, mApple-C1 was a gift from Michael Davidson (Addgene plasmid # 54631 ; http://n2t.net/addgene:54631 ; RRID:Addgene_54631) ( ). pKanCMV-mRuby3-18aa-Tubulin was a gift from Michael Lin (Addgene plasmid # 74256 ; http://n2t.net/addgene:74256 ; RRID:Addgene_74256) ( ). pmScarlet_C1 was a gift from Dorus Gadella and the CMV immediate-early promoter described herein was amplified from this plasmid, as well as the mScarlet encoding gene (Addgene plasmid # 85042 ; http://n2t.net/addgene:85042 ; RRID:Addgene_85042) (Bindels et al., 2017).

Techniques: Construct, Expressing, Homologous Recombination, Imaging