Journal: Journal of Virology

Article Title: Visualization and tracking of tubule-derived, fluorescent-labeled NS1 as a marker of bluetongue virus in living cells

doi: 10.1128/jvi.00896-25

Figure Lengend Snippet: Subcellular localization and aggresome formation of NS1. ( a and b ) BSR cells were transfected with the lifeact-mCherry, sec61b-mCherry, and LAMP1-mCherry expression plasmids for 12 h, infected with BTV1-NS1-552eGFP (MOI = 0.1), fixed, and then infected with BTV1-NS1-552eGFP (MOI = 0.1) or BTV1-WT (MOI = 0.1) and probed with antibodies against β-tubulin and γ-tubulin to label cell microtubules and the MTOC (red). Hoechst 33342 staining (blue). Bar, 20 µm. ( c ) BSR cells were infected with BTV1-NS1-552eGFP (MOI = 0.1) or BTV1-WT (MOI = 0.1), fixed, and probed with antibodies against vimentin (red). The nuclei were stained with Hoechst 33342 (blue). Bar, 20 µm.

Article Snippet: Lifeact-mCherry (catalog no. 193300), sec61b-mCherry (catalog no. 90994), and LAMP1-mCherry (catalog no. 45147) were purchased from Addgene (Watertown, MA, USA).

Techniques: Transfection, Expressing, Infection, Staining

Journal: bioRxiv

Article Title: Temporally extended and reversible neuronal silencing with Aion

doi: 10.1101/2022.02.25.481932

Figure Lengend Snippet: (A) Left: Engineering strategy yielding Aion and Phobos CADN , two new blue-shifted, step-function ACRs with temporally extended conducting states. Mutation of the D156 residue to either C or N (yielding Aion and Phobos CADN , respectively) greatly slows down the closing kinetics of the ACRs compared to its parental construct Phobos CA . The generated variants are highlighted by a black frame. Right: Schematic of Aion indicating point mutations (triangles) at the relative position within the respective transmembrane helix. (B) Representative photocurrent traces of the slow-cycling ACRs Phobos CA , Aion, Phobos CADN , and Gt ACR1 CA activated by a short 20 ms light pulse at indicated wavelengths and irradiance of 3.35 mW/mm 2 (460nm) and 3.14 mW/mm 2 (505 nm). (C) Left: quantification of peak photocurrent ( I p ) and photocurrent 30 s post illumination ( I 30s ) for each ACR. Right: photocurrent ratio at 30 s compared to the peak. (D) Normalized photocurrent after light shutoff. (E) Closing time constants (τ off ) for each ACR. For (C) and (E), black lines correspond to mean values ± SEM, and circles are single measurement data points (n PhobosCA = 5 -6 cells, nAion = 5 - 10 cells, n PhobosCADN = 8 - 9 cells, n Gt ACR1CA = 6 cells). Kruskal- Wallis test with Dunn’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For HEK-cell expression, mutations were introduced in previously described ACR constructs of Gt ACR1 ( ) (Addgene #85464) and Phobos CA ( ) (Addgene #98169) using the QuikChange II kit (Agilent Technologies, Santa Clara, CA).

Techniques: Mutagenesis, Residue, Construct, Generated

Journal: bioRxiv

Article Title: Temporally extended and reversible neuronal silencing with Aion

doi: 10.1101/2022.02.25.481932

Figure Lengend Snippet: ( A ) Representative current traces of kinetically modified Phobos CA variants and Gt ACR1 CA . ACR variants were illuminated with 500 ms (row 1-4) or 20 ms (last row) actinic light at indicated wavelengths, while termination of photocurrents was accelerated with orange or red light for 10 s as indicated. ( B ) and ( C ) temporally expanded photocurrent traces as shown in (A) showing different modes of inactivation (B) and accelerated closing kinetics of Phobos CA variants (C). ( D ) Quantification of the residual photocurrent after orange or red-light application. While Gt ACR1 CA showed a 30 % photocurrent reduction when red light was applied 2 s post initial activation, application after 30 s caused partial re-opening of the channel. Phobos CA variants could be fully closed by orange light. (E) Quantification of the accelerated channel closing of Phobos CA variants with orange light.

Article Snippet: For HEK-cell expression, mutations were introduced in previously described ACR constructs of Gt ACR1 ( ) (Addgene #85464) and Phobos CA ( ) (Addgene #98169) using the QuikChange II kit (Agilent Technologies, Santa Clara, CA).

Techniques: Modification, Activation Assay

Journal: bioRxiv

Article Title: Temporally extended and reversible neuronal silencing with Aion

doi: 10.1101/2022.02.25.481932

Figure Lengend Snippet: ( A ) Maximum-intensity projection images of two-photon stacks showing expression of Aion (i) Phobos CADN (ii) and Gt ACR1 CA (iii) in CA1 pyramidal neurons of rat organotypic hippocampal slice cultures. Fluorescence intensity is shown as inverted gray values. Insets show magnified view of the apical dendrite. ( B ) Representative photocurrent traces of Aion (i), Phobos CADN (ii) and Gt ACR1 CA (iii), evoked by a 20 ms light pulse at the respective peak activation wavelength (460 nm for Aion and Phobos CADN , 525 nm for Gt ACR1 CA ) and equal irradiance of 10 mW/mm 2 . Channel closing was accelerated with red-shifted light (1 s, 10 mW/mm 2 , 595 nm for Aion and Phobos CADN , 660 nm for Gt ACR1 CA ) at 197 s after channel opening. Note that red light at 198 s elicited partial opening of Gt ACR1 CA instead of closing, indicated by an outward current after light stimulation. The black arrows at 30 and 120 s after channel opening indicate the time points at which stationary photocurrent amplitudes were quantified in C-E. ( C ) Quantification of peak photocurrent density for Aion, Phobos CADN and Gt ACR1 CA . ( D ) Quantification of photocurrent amplitude at 30 s and 120 s after channel opening with respect to peak current. ( E ) Photocurrent ratio at 120 s compared to 30 s. ( F ) Same as (B) but channel closing was accelerated 35 s after opening. ( G ) Residual current upon channel closing 35 s after opening. Photocurrent amplitude was measured in the 5 s before and after the closing light pulse. ( H ) Quantification of photocurrent amplitude upon channel closing 198 s after opening with respect to the peak photocurrent. For (C-E, G-H), mean values ± SEM are shown (black lines) together with single measurement data points (circles, n = 5 - 16 cells). Kruskal-Wallis test with Dunn’s multiple comparisons test, *p < 0.05, **p < 0.01.

Article Snippet: For HEK-cell expression, mutations were introduced in previously described ACR constructs of Gt ACR1 ( ) (Addgene #85464) and Phobos CA ( ) (Addgene #98169) using the QuikChange II kit (Agilent Technologies, Santa Clara, CA).

Techniques: Expressing, Fluorescence, Activation Assay

Journal: bioRxiv

Article Title: Temporally extended and reversible neuronal silencing with Aion

doi: 10.1101/2022.02.25.481932

Figure Lengend Snippet: ( A ) Left: Membrane voltage trace showing reversible suppression of depolarization-induced action potentials (APs) by photoswitching Aion between open and closed state with a blue (460 nm, 20 ms, 10 mW/mm 2 ) and an orange light pulse (595 nm, 1 s, 10 mW/mm 2 ), respectively. Right: Quantification of spike rate during current injection at indicated time intervals: before channel opening (0-5 s), after channel opening (5-45 s), and after channel closing (45-55 s) in Aion-expressing CA1 neurons (n = 10 cells). ( B ) Same as (A) but for neurons expressing Gt ACR1 CA (n = 5 cells). Channel was opened with a green (525 nm, 20 ms, 10 mW/mm 2 ) light pulse. Note that in Gt ACR1 CA -expressing cells AP firing could not be recovered immediately after illumination with red light (660 nm,1 s, 10 mW/mm 2 ). ( C ) Current ramps were injected into Aion-expressing neurons to induce APs before and after illumination with a short blue light pulse (460 nm, 1 s, irradiances from 0.001 to 10 mW/mm 2 ). For each ramp, the injected current at the time of the first AP was defined as the rheobase. Example membrane voltage traces are shown for the trial in which a light intensity of 10 mW/mm 2 was used. ( D ) Quantification of the rheobase shift and ( E ) the relative change in the number of current ramp-evoked APs over 47 s after light stimulation (460 nm, 1 s, 10 mW/mm 2 ) (n = 7 cells). ( F ) Number of APs evoked during the first current ramp after opening of Aion with 1 s blue light at indicated irradiances. Significant AP block was achieved at 0.1 mW/mm 2 (n = 7 cells). ( G-J ) Same experiment as shown in (C-F) except that CA1 neurons expressed Gt ACR1 CA (n = 6-7 cells) and 525 nm-light was used for channel opening. Note that Gt ACR1 CA silencing efficacy decayed already within the 1 st minute after light stimulation (I,J). For (A,B, D-F, and H-J) filled circles represent single measurement data points and black circles correspond to medians, Friedman test, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant.

Article Snippet: For HEK-cell expression, mutations were introduced in previously described ACR constructs of Gt ACR1 ( ) (Addgene #85464) and Phobos CA ( ) (Addgene #98169) using the QuikChange II kit (Agilent Technologies, Santa Clara, CA).

Techniques: Membrane, Injection, Expressing, Blocking Assay

Journal: bioRxiv

Article Title: Temporally extended and reversible neuronal silencing with Aion

doi: 10.1101/2022.02.25.481932

Figure Lengend Snippet: ( A ) Aion silencing capacity was evaluated over 10 minutes. Example membrane voltage trace of an Aion-expressing CA1 neuron showing reliable suppression of depolarization-induced APs (2 s current injections every 4.5 s) for 10 min by activating Aion with 2 short blue light pulses spaced 5 min apart (460 nm, 1 s, 10 mW/mm 2 ). (i, ii) Insets show magnified view of the membrane potential at the indicated time points. Voltage traces were median-filtered to calculate the change in membrane depolarization after light stimulation of Aion (as shown by the red arrows). ( B ) Quantification of Aion-mediated change in membrane depolarization over a time period of 5 min after light stimulation (460 nm, 1 s, 10 mW/mm 2 ) as shown in (A). Grey traces correspond to single neurons and black trace shows the median (n = 6 cells). ( C ) Time at which the 1 st current-evoked AP occurred after opening of Aion. Note that Aion reliably blocked all APs for 5 min after light stimulation. Grey circles represent single measurement data points and black line corresponds to median (n = 6 cells). ( D ) Gt ACR1 CA silencing capacity was evaluated over 5 minutes. Example membrane voltage trace of a Gt ACR1 CA -expressing CA1 neuron after activation of Gt ACR1 CA with a short green light pulse (525 nm, 1 s, 10 mW/mm 2 ). (i, ii) same as (A, i-ii) but for Gt ACR1 CA . ( E, F ) Same as (B, C) but for Gt ACR1 CA (n = 4 cells; 525 nm, 1 s, 10 mW/mm 2 ). Note that the change in membrane depolarization went back to baseline approx. 3.5 min after light stimulation (E) and neurons started firing within the first 200 s after light stimulation (F). ( G ) Left: Organotypic slices with CA1 cells expressing Aion were stimulated overnight (O/N) in a custom-made LED chamber inside the incubator (3 s 460-nm light pulse every 5 min, 0.3 mW/mm 2 ). Right: example membrane voltage trace of an Aion-expressing CA1 neuron recorded after 12 h of light stimulation in the incubator. Inset shows magnified view of the membrane potential during light stimulation. ( H ) Quantification of spike rate before and during the 5 minutes following light stimulation. ( I ) Resting membrane potential, membrane resistance, membrane capacitance, number of APs evoked by somatic current injection (300 pA, 500 ms) and amplitude of the 1 st AP in Aion-expressing cells after 12 h of light stimulation, compared to the following three control groups: Non-transfected CA1 pyramidal cells, cells expressing only the fluorescent protein citrine that were stimulated O/N under the same conditions as Aion-expressing cells and Aion-expressing cells without O/N stimulation. Black lines: mean values ± SEM, n Non-transfected = 6 cells, n citrine-only = 7 cells, n Aion = 7 cells, n Aion No light = 6 cells, one-way ANOVA, *p < 0.05, n.s. = not significant.

Article Snippet: For HEK-cell expression, mutations were introduced in previously described ACR constructs of Gt ACR1 ( ) (Addgene #85464) and Phobos CA ( ) (Addgene #98169) using the QuikChange II kit (Agilent Technologies, Santa Clara, CA).

Techniques: Membrane, Expressing, Activation Assay, Injection, Control, Transfection

Journal: bioRxiv

Article Title: Temporally extended and reversible neuronal silencing with Aion

doi: 10.1101/2022.02.25.481932

Figure Lengend Snippet: ( A ) Schematic model of nociceptive sensory neurons (C4da) and connected downstream neurons (A08n) in Drosophila larvae. ( B ) Nociceptive touch responses were strongly reduced after blue light in animals expressing Aion in A08n neurons, but not in controls (wt). The responses could be fully recovered after orange light exposure. ( C ) Larvae expressing Gt ACR1 in A08n neurons showed robustly reduced nociceptive responses under constant green light exposure. ( D ) Constitutive silencing by expression of Kir2.1 in A08n lead to similarly strong inhibition of larval nociception as with Aion. For (B-D) the behavioral response to a 50 mN mechanical stimulus is shown. ( E ) All-optical paradigm for inhibition of A08n neurons with Aion and activation of C4da neurons with Cs Chrimson. Repeated induction of nociceptive behavior with red light was strongly inhibited after blue light-induced Aion activation and largely reversed by orange light. ( F ) Activation of Aion expressed in A08n neurons with a single blue light pulse (5 s) resulted in reduction of nociceptive responses for at least 15 min and partially recovered only after 60 min. For (E-F) behavioral responses to Cs Chrimson activation in C4da neurons at the indicated time points are shown. Numbers of animals are indicated by white numbers in (B-F).

Article Snippet: For HEK-cell expression, mutations were introduced in previously described ACR constructs of Gt ACR1 ( ) (Addgene #85464) and Phobos CA ( ) (Addgene #98169) using the QuikChange II kit (Agilent Technologies, Santa Clara, CA).

Techniques: Expressing, Inhibition, Activation Assay