Journal: Science Advances

Article Title: α-Synuclein expression is required for somatodendritic dopamine release and immediate early gene induction

doi: 10.1126/sciadv.ady6978

Figure Lengend Snippet: ( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or α1D (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).

Article Snippet: Primary antibodies include anti-actin (mouse monoclonal, Sigma-Aldrich, #A5441, RRID: AB_476744; 1:1000), anti–glyceraldehyde-3-phosphate dehydrogenase (mouse monoclonal, Proteintech, #60004-1-Ig, RRID: AB_2107436; 1:1000), anti–pCREB (Ser 133 , rabbit monoclonal, Cell Signaling Technology, #9198S; 1:500), anti-CREB1 (rabbit polyclonal, ABclonal, #A11064, RRID: AB_2758389; 1:500), anti–c-Fos (rabbit monoclonal, Cell Signaling Technology, #2250S, RRID: AB_2247211; 1:1000), anti-Ca v 1.2 α1C (rabbit polyclonal, Proteintech, #21774-1-AP, RRID: AB_2878918; 1:500), and anti-Ca v 1.3 α1D (rabbit polyclonal, Alomone Labs, #ACC-005, RRID: AB_2039775; 1:100).

Techniques: Cell Culture, Staining, Membrane

Journal: Science Advances

Article Title: Adaptable thermoresponsive polymer for long-term electrical coupling in plant electrophysiology monitoring

doi: 10.1126/sciadv.ady1400

Figure Lengend Snippet: ( A ) Procedure of drought stress and recovery experiment and photographs of plants under testing. Predrought is defined as 1 day before plants display wilting symptoms. ( B ) Amplitude of light-induced potential change for plants under 9 days of escalating drought (red) benchmarked against well-watered control plants (gray), showing an increase followed by decrease upon wilting. a.u., arbitrary units. ( C ) Light-induced potential change before and after inhibition of Ca 2+ channels in the same leaf. N = 3. ( D ) Light-induced potential change before and after inhibition of ROS production in the same leaf. N = 3. ( E ) Confocal images of intracellular Ca 2+ (top) and apoplastic ROS (bottom) during drought simulation, with quantification of the mean fluorescence intensity per region of interest (ROI) shown on the right. BF, bright field. Fluo-3 AM and 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA): dyes used for visualizing Ca 2+ and ROS, respectively. Scale bar, 50 μm. CK, control plants; DR, drought-stressed plants. ( F ) Plant EP signals under the light and dark conditions with starting potential aligned for all samples on each day. N = 6. ( G ) Mean potential calculated from signals in (F) 1 hour after light on or light off, showing opposing trends with time under light and dark conditions. Dark mean potential changes before visual symptom onset on day 7. Solid lines in (C), (D), and (F) are mean values, and shadows represent SD. Box plots in (B), (E), and (G) display first quartile, median, and third quartile. Whiskers are drawn at 1.5 interquartile range distance. Means are denoted by empty squares. * P < 0.05; *** P < 0.001; **** P < 0.0001; n.s., not significant. Two sample t test.

Article Snippet: Intracellular Ca 2+ ions were detected using the fluorescent Ca 2+ indicator Fluo-3 AM (MedChemExpress, HY-D0716).

Techniques: Control, Inhibition, Fluorescence

Journal: Nature Communications

Article Title: CYFIP1 governs the development of cortical axons by modulating calcium availability

doi: 10.1038/s41467-025-65801-0

Figure Lengend Snippet: a CYFIP1 RNA immunoprecipitation (RNA-IP) from DIV 3 WT cortical neurons. Histogram showing relative enrichment of the mRNAs over the non-specific IgG, measured by RT-qPCR of the eluate. The values were normalized for the input and mHprt1 mRNA and expressed as fold change over the non-specific IgG of each mRNA ( n = 4 embryos; mean ± SEM; One-Way ANOVA p < 0.0001; mMap1b mRNA p = 0.0390, mCacna1c mRNA p = 0.0054, mCacna1e mRNA p = 0.0078, mCacna1i mRNA p = 0.0009, mCacng2 mRNA p = 0.9997, mCacnb3 mRNA p = 0.9983). b Total mRNA levels of the Ca 2+ channels in DIV 3 WT and Cyfip1 +/- cortical neurons. Histograms represent mCacna1c , mCacna1e , mCacna1i , mCacng2, mCacnb3 and mCyfip1 mRNA levels, normalized to mH3f3 levels and expressed as a fold change over WT (WT n = 6/7 embryos, Cyfip1 +/- n = 7 embryos; mean ± SEM; Two-tailed Multiple Mann-Whitney test, mCacna1c mRNA p = 0.0766, mCacna1e mRNA p = 0.0435, mCacna1i mRNA p = 0.0202, mCacng2 mRNA p = 0.6282, mCacnb3 mRNA p = 0.5343, mCyfip1 mRNA p = 0.0034). c Left, representative Western Blot showing CYFIP1, Ca V 1.2 (CACNA1C), Ca V 2.3 (CACNA1E), Ca V 3.3 (CACNA1I), Ca V γ2 (CACNG2/Stargazin) and Ca V β3 (CACNB3) in membrane-enriched fractions from WT and Cyfip1 +/- DIV 3 cortical neurons. The molecular weight of each protein is indicated in kDa. Right, histogram representing Ca V 1.2, Ca V 2.3, Ca V 3.3, Ca V γ2, Ca V β3 and CYFIP1 protein expression levels in membrane-enriched fractions from WT and Cyfip1 +/- DIV 3 cortical neurons. Protein levels were normalized to Coomassie staining (WT n = 4 embryos, Cyfip1 +/- n = 7/8 embryos; mean ± SEM; Two-tailed Multiple unpaired t -test, Ca V 1.2 p = 0.0338, Ca V 2.3 p = 0.0281, Ca V 3.3 p = 0.0129, Ca V γ2 p = 0.2574, Ca V β3 p = 0.6259, CYFIP1 p = 0.0137). d–f Representative images from WT and Cyfip1 +/- DIV 3 cortical neurons stained for Ca V 1.2, Ca V 2.3, Ca V 3.3 (magenta) and βIII-Tubulin (green) (scale bar 20 μm). Histograms show the fluorescence intensity of each calcium channel normalized to βIII-Tubulin in the total neuron (left) and in the axon (right), expressed as a percentage over WT (Ca V 1.2: WT n = 4 embryos, Cyfip1 +/- n = 5 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.1111, axon p = 0.4127; Ca V 2.3: WT n = 5 embryos, Cyfip1 +/- n = 4 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.0635, axon p = 0.0159; Ca V 3.3: WT n = 4 embryos, Cyfip1 +/- n = 4 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.0286, axon p = 0.0286). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: mouse anti-βIII Tubulin (1:200, BioLegend, #801201), rabbit anti-Ca V 1.2 (CACNA1C) (1:100, Alomone Labs, #ACC003), rabbit anti-Ca V 2.3 (CACNA1E) (1:100, Alomone Labs, #ACC006), and rabbit anti-Ca V 3.3 (CACNA1I) (1:100, Alomone Labs, #ACC009).

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Western Blot, Membrane, Molecular Weight, Expressing, Staining, Fluorescence

Journal: Nature Communications

Article Title: CYFIP1 governs the development of cortical axons by modulating calcium availability

doi: 10.1038/s41467-025-65801-0

Figure Lengend Snippet: CYFIP1, potentially interacting with the RNA binding proteins HuD and HuR (previously identified as CYFIP1 interactors), is implicated in regulating the mRNA stability of calcium channel subunits Cacna1c , Cacna1e and Cacna1i . In Cyfip1 +/- neurons, reduced CYFIP1 levels result in a decrease protein abundance of the regulated calcium channel subunits, consequently leading to a decrease in intracellular and mitochondrial calcium concentration. Low levels of calcium ions may affect mitochondria polarity and motility, both of which we found impaired in Cyfip1 +/- axons. The decreased calcium concentration and the mitochondrial defects concur in reducing axonal growth observed in Cyfip1 +/- neurons. By restoring the intracellular calcium homeostasis, both the axonal growth and mitochondrial defects are rescued. Created in BioRender. https://BioRender.com/xquv8cy .

Article Snippet: The following primary antibodies were used: mouse anti-βIII Tubulin (1:200, BioLegend, #801201), rabbit anti-Ca V 1.2 (CACNA1C) (1:100, Alomone Labs, #ACC003), rabbit anti-Ca V 2.3 (CACNA1E) (1:100, Alomone Labs, #ACC006), and rabbit anti-Ca V 3.3 (CACNA1I) (1:100, Alomone Labs, #ACC009).

Techniques: RNA Binding Assay, Quantitative Proteomics, Concentration Assay

Journal: Nature Communications

Article Title: CYFIP1 governs the development of cortical axons by modulating calcium availability

doi: 10.1038/s41467-025-65801-0

Figure Lengend Snippet: a CYFIP1 RNA immunoprecipitation (RNA-IP) from DIV 3 WT cortical neurons. Histogram showing relative enrichment of the mRNAs over the non-specific IgG, measured by RT-qPCR of the eluate. The values were normalized for the input and mHprt1 mRNA and expressed as fold change over the non-specific IgG of each mRNA ( n = 4 embryos; mean ± SEM; One-Way ANOVA p < 0.0001; mMap1b mRNA p = 0.0390, mCacna1c mRNA p = 0.0054, mCacna1e mRNA p = 0.0078, mCacna1i mRNA p = 0.0009, mCacng2 mRNA p = 0.9997, mCacnb3 mRNA p = 0.9983). b Total mRNA levels of the Ca 2+ channels in DIV 3 WT and Cyfip1 +/- cortical neurons. Histograms represent mCacna1c , mCacna1e , mCacna1i , mCacng2, mCacnb3 and mCyfip1 mRNA levels, normalized to mH3f3 levels and expressed as a fold change over WT (WT n = 6/7 embryos, Cyfip1 +/- n = 7 embryos; mean ± SEM; Two-tailed Multiple Mann-Whitney test, mCacna1c mRNA p = 0.0766, mCacna1e mRNA p = 0.0435, mCacna1i mRNA p = 0.0202, mCacng2 mRNA p = 0.6282, mCacnb3 mRNA p = 0.5343, mCyfip1 mRNA p = 0.0034). c Left, representative Western Blot showing CYFIP1, Ca V 1.2 (CACNA1C), Ca V 2.3 (CACNA1E), Ca V 3.3 (CACNA1I), Ca V γ2 (CACNG2/Stargazin) and Ca V β3 (CACNB3) in membrane-enriched fractions from WT and Cyfip1 +/- DIV 3 cortical neurons. The molecular weight of each protein is indicated in kDa. Right, histogram representing Ca V 1.2, Ca V 2.3, Ca V 3.3, Ca V γ2, Ca V β3 and CYFIP1 protein expression levels in membrane-enriched fractions from WT and Cyfip1 +/- DIV 3 cortical neurons. Protein levels were normalized to Coomassie staining (WT n = 4 embryos, Cyfip1 +/- n = 7/8 embryos; mean ± SEM; Two-tailed Multiple unpaired t -test, Ca V 1.2 p = 0.0338, Ca V 2.3 p = 0.0281, Ca V 3.3 p = 0.0129, Ca V γ2 p = 0.2574, Ca V β3 p = 0.6259, CYFIP1 p = 0.0137). d–f Representative images from WT and Cyfip1 +/- DIV 3 cortical neurons stained for Ca V 1.2, Ca V 2.3, Ca V 3.3 (magenta) and βIII-Tubulin (green) (scale bar 20 μm). Histograms show the fluorescence intensity of each calcium channel normalized to βIII-Tubulin in the total neuron (left) and in the axon (right), expressed as a percentage over WT (Ca V 1.2: WT n = 4 embryos, Cyfip1 +/- n = 5 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.1111, axon p = 0.4127; Ca V 2.3: WT n = 5 embryos, Cyfip1 +/- n = 4 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.0635, axon p = 0.0159; Ca V 3.3: WT n = 4 embryos, Cyfip1 +/- n = 4 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.0286, axon p = 0.0286). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: mouse anti-βIII Tubulin (1:200, BioLegend, #801201), rabbit anti-Ca V 1.2 (CACNA1C) (1:100, Alomone Labs, #ACC003), rabbit anti-Ca V 2.3 (CACNA1E) (1:100, Alomone Labs, #ACC006), and rabbit anti-Ca V 3.3 (CACNA1I) (1:100, Alomone Labs, #ACC009).

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Western Blot, Membrane, Molecular Weight, Expressing, Staining, Fluorescence

Journal: Nature Communications

Article Title: CYFIP1 governs the development of cortical axons by modulating calcium availability

doi: 10.1038/s41467-025-65801-0

Figure Lengend Snippet: CYFIP1, potentially interacting with the RNA binding proteins HuD and HuR (previously identified as CYFIP1 interactors), is implicated in regulating the mRNA stability of calcium channel subunits Cacna1c , Cacna1e and Cacna1i . In Cyfip1 +/- neurons, reduced CYFIP1 levels result in a decrease protein abundance of the regulated calcium channel subunits, consequently leading to a decrease in intracellular and mitochondrial calcium concentration. Low levels of calcium ions may affect mitochondria polarity and motility, both of which we found impaired in Cyfip1 +/- axons. The decreased calcium concentration and the mitochondrial defects concur in reducing axonal growth observed in Cyfip1 +/- neurons. By restoring the intracellular calcium homeostasis, both the axonal growth and mitochondrial defects are rescued. Created in BioRender. https://BioRender.com/xquv8cy .

Article Snippet: The following primary antibodies were used: mouse anti-βIII Tubulin (1:200, BioLegend, #801201), rabbit anti-Ca V 1.2 (CACNA1C) (1:100, Alomone Labs, #ACC003), rabbit anti-Ca V 2.3 (CACNA1E) (1:100, Alomone Labs, #ACC006), and rabbit anti-Ca V 3.3 (CACNA1I) (1:100, Alomone Labs, #ACC009).

Techniques: RNA Binding Assay, Quantitative Proteomics, Concentration Assay