pex yfp stim1 deltak  (Addgene inc)

Journal: bioRxiv

Article Title: Activity-Dependent Localization and Heterogeneous Dynamics of STIM1 and STIM2 at ER-PM contacts in Hippocampal Neurons

doi: 10.1101/2024.12.23.630200

Figure Lengend Snippet: A) Schematic representation of the constructs used in this study, high-lighting the locations of tags and key domains of STIM proteins. The schematic also illustrates the AAV delivery approach and HaloTag labeling methodology employed for tracking the overexpressed or knock-in constructs. The diffusion coefficients derived from tracked molecules are plotted as cumulative frequency distributions on a logarithmic scale, illustrating the global relative changes in diffusion dynamics. B) Visualization of STIM1 tracking (yellow) within axons and dendrites, juxtaposed against presynaptic synaptophysin-GCaMP labeling (magenta in axon) and post-synaptic PSD95 labeling (magenta in dendrite). Data acquired for both resting conditions at 37°C (Ringer solution with 2 mM Ca 2+ , 2 mM Mg 2+ , 10 M CNQX, 10 M APV) and store-depleted conditions (Ringer solution with 2 mM Ca 2+ , 2 mM Mg 2+ , 10 M CNQX, 10 M APV, 20 M CPA). C) Cumulative frequency distribution showing the diffusion coefficient of STIM1 within axons and dendrites. D) Median diffusion coefficient comparison of STIM1 between the resting state and store-depleted conditions. Each data point depicted on the graph is the median diffusion coefficient from a single acquisition. E) Visual representation of Halo-STIM2 tracking (yellow) within axons and dendrites under both resting and store-depleted conditions, in relation to presynaptic synaptophysin-GCaMP and post-synaptic PSD95 labeling. F) Cumulative frequency distribution demonstrating the diffusion coefficient of STIM2 within axons and dendrites. G) Comparative evaluation of median diffusion coefficient for STIM2 between the resting state and store-depleted condition. Each data point presented on the graph represents the median from a single acquisition. H) Comparison of STIM1 and STIM2 diffusion within axons and dendrites, highlighting the presence of a fast-moving fraction in STIM2 in axons while a confined STIM2 in dendrites relative to STIM1. I) Diffusion of Halo-tag control (Halo-TMC) in axons and dendrites in resting conditions. J) SPT of STIM1 and STIM2 knock-in constructs, demonstrating a consistent trend of STIM2 being more confined than STIM1 in dendrites. K) Tracking of overexpressed STIM proteins was performed following Cre-mediated knockout of endogenous Stim1 and Stim2 (in the graphs, abbreviated as S1 and S2 respectively). The terms KO-S1 and KO-S2 refer to the Cre-mediated knockout of Stim1 and Stim2, respectively. rAAV carrying Cre was delivered to neurons on DIV2, while rAAV encoding Halo-STIM1 and Halo-STIM2 was introduced on DIV7. Imaging and data acquisition were performed on DIV14-15. Error bars for all figures depict the mean ± standard deviation (SD). Statistical significance is indicated as **** P < 0.0001, *** P < 0.001, * P < 0.1. For comparisons between two groups, a parametric unpaired t-test was used, while multiple groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. Refer to the supplementary table for p -values, N, n , and trajectory counts.

Article Snippet: For STIM1 overexpression experiments, the cells were transfected with a plasmid coding for human STIM1 fused with YFP (Addgene, plasmid no. 19754) 3 days before experiments.

Techniques: Construct, Labeling, Knock-In, Diffusion-based Assay, Derivative Assay, Comparison, Control, Knock-Out, Imaging, Standard Deviation

Journal: bioRxiv

Article Title: Activity-Dependent Localization and Heterogeneous Dynamics of STIM1 and STIM2 at ER-PM contacts in Hippocampal Neurons

doi: 10.1101/2024.12.23.630200

Figure Lengend Snippet: A) Confocal imaging of knock-in Halo-STIM1 in Cas9-expressing hippocampal cultures, co-labeled with the presynaptic marker RIM. The merged image additionally displays AnkyrinG staining, not included in grayscale images and marked in yellow. Scale: 20 m. B) Pearson correlation coefficients for STIMs with RIM1. C) Manders’ M1 coefficients (co-localization of STIMs with RIM). D) Manders’ M2 coefficients (co-localization of RIM with STIMs) calculated for both STIM1 and STIM2 under resting and store-depleted conditions (1 M TG). E, G, I) Visualization of STIM1 ( E ), STIM2 ( G ), and Halo-control ( I ) tracks (yellow) in axons co-labeled with presynaptic synaptophysin-GCaMP. Representative 0.91 m 2 regions of interest (ROIs) are marked as synaptic (red) and extrasynaptic (white). Scale: 10 m for full images, 2 m for magnified views. F, H, J) Median diffusion coefficients of STIM1 ( F ), STIM2 ( H ), and Halo-control ( J ) plotted under resting conditions at 37°C (Ringer’s solution with 2 mM Ca 2+ , 2 mM Mg 2+ , 10 M CNQX, 10 M APV) and store-depleted conditions (Ringer’s solution with 20 M CPA). Each data point represents the median diffusion coefficient from a single acquisition encompassing multiple presynaptic locations. Error bars indicate mean ± SD. Refer to the supplementary table for p -values, N, n , and trajectory counts.

Article Snippet: For STIM1 overexpression experiments, the cells were transfected with a plasmid coding for human STIM1 fused with YFP (Addgene, plasmid no. 19754) 3 days before experiments.

Techniques: Imaging, Knock-In, Expressing, Labeling, Marker, Staining, Control, Diffusion-based Assay

Journal: bioRxiv

Article Title: Activity-Dependent Localization and Heterogeneous Dynamics of STIM1 and STIM2 at ER-PM contacts in Hippocampal Neurons

doi: 10.1101/2024.12.23.630200

Figure Lengend Snippet: A-C) Visualization of Halo-STIM1 ( A ), STIM2 ( B ), and Halo-control ( C ) tracks (yellow) in dendrites co-labeled with the postsynaptic marker PSD-95. Acquisitions were performed under resting conditions at 37 C (Ringer’s solution with 2 mM Ca 2+ , 2 mM Mg 2+ , 10 M CNQX, 10 M APV). The grayscale images represent maximum projections of SPT movies without synaptic labels in magnified dendritic sections. Scale bars: 10 m (large images), 2 m (magnified views). D) Confocal imaging of knock-in Halo-STIM1 in Cas9-expressing hippocampal cultures, co-labeled with the post-synaptic marker Homer. Scale 20 m. E) Pearson correlation coefficients of STIMs with Homer, F) Manders’ M 1 coefficients (co-localization of STIMs with Homer), and G) Manders’ M 2 coefficients (co-localization of Homer with STIMs) calculated for both STIM1 and STIM2 under resting and store-depleted conditions (TG). Error bars in graphs indicate the mean ± SD. Refer to supplementary table for p-values, N, n , and trajectory counts.

Article Snippet: For STIM1 overexpression experiments, the cells were transfected with a plasmid coding for human STIM1 fused with YFP (Addgene, plasmid no. 19754) 3 days before experiments.

Techniques: Control, Labeling, Marker, Imaging, Knock-In, Expressing

Journal: bioRxiv

Article Title: Activity-Dependent Localization and Heterogeneous Dynamics of STIM1 and STIM2 at ER-PM contacts in Hippocampal Neurons

doi: 10.1101/2024.12.23.630200

Figure Lengend Snippet: Neurons were exposed to 20 M glutamate (Glu) in Mg 2+ -free Ringer solution prior to imaging, followed by wash and acquisition in Ringer solution (including Mg 2+ ) supplemented with 10 M APV and 10 M CNQX at 37 ° C. Representative maximum projection images of 500 frames extracted from STIM1 ( A ), STIM2 ( D ), and STIM1K ( G ) SPT movies showing dendrites under control conditions, glutamate treatment, and NMDA receptor blockade with APV. B, E) Cumulative frequency distribution plot of STIM1 ( B ) and STIM2 ( E ) diffusion coefficients ( N = 3 for both). C, F) Comparative evaluation of median diffusion coefficients for STIM1 ( C ) and STIM2 ( F ) across conditions, with each data point representing the median from a single acquisition. G) Maximum projection images of STIM1K under control conditions and after treatment with glutamate. H) Median diffusion coefficients of STIM1K, including comparisons with the diffusion data for wild-type STIM1. I) Statistical analysis and comparison of diffusion coefficients of both STIM1 and STIM2 after 24 h MK-801 treatment. J) Immunostaining of endogenously labeled STIM1 in Cas9 neurons, comparing STIM1 cluster morphology under store depletion with TG treatment and NMDA treatment. K) Cluster size quantification of STIM knock-ins under Control, CPA, and NMDA treatments, analyzed from 4–5 cells per condition. L) SPT analysis of STIM1, comparing the impact of glutamate on STIM1 dynamics. Cumulative frequency distribution plot showing the diffusion coefficients of STIM1 during treatments with glutamate (20 M) and CPA (20 M). M) ER-GCaMP Ca 2+ imaging in hippocampal neurons treated with CPA or NMDA to induce Ca 2+ release from the ER at t = 100 s. The figure compares the amplitude of ER Ca 2+ release and the kinetics of Ca 2+ release ( τ ) between the two treatments. Error bars for all figures depict the mean ± standard deviation. Statistical significance is indicated as **** P < 0.0001, *** P < 0.001, * P < 0.1. For comparisons between two groups, a parametric unpaired t -test was used, while multiple groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. Refer to supplementary table for p -values, N, n , and trajectory counts.

Article Snippet: For STIM1 overexpression experiments, the cells were transfected with a plasmid coding for human STIM1 fused with YFP (Addgene, plasmid no. 19754) 3 days before experiments.

Techniques: Imaging, Control, Diffusion-based Assay, Comparison, Immunostaining, Labeling, Standard Deviation

Journal: bioRxiv

Article Title: Activity-Dependent Localization and Heterogeneous Dynamics of STIM1 and STIM2 at ER-PM contacts in Hippocampal Neurons

doi: 10.1101/2024.12.23.630200

Figure Lengend Snippet: A) Maximum intensity projection images from STIM1 SPT movies under control conditions, NMDA treatment, and NMDA + nimodipine treatment. NMDA stimulation involved 50 M NMDA in Mg 2+ - and APV-free Ringer solution for 1 minute, followed by washout. For nimodipine treatment, cells were pre-incubated with 50 M nimodipine (nim) for 3 minutes prior to and during NMDA activation. Imaging was performed in Ringer solution containing 2 mM Mg 2+ , 2 mM Ca 2+ , and 10 M CNQX at 37 ° C. B) Cumulative frequency distribution plot and statistical analysis of median diffusion coefficients per acquisition for STIM1. C) ER calcium imaging using ER-GCaMP-150, with NMDA added at t = 100 s for both control and nimodipine pre-treated conditions (see Methods). D–E) Experimental timeline for assessing the temporal dynamics of STIMs following NMDA treatment. D) Cumulative distribution plot of diffusion coefficients and median diffusion coefficients per acquisition for STIM1 under NMDA treatments. E) Corresponding data for the nimodipine pre-treatment condition. Control recordings primarily included data from the first 1–5 minutes of acquisition. For NMDA- and nimodipine + NMDA-treated cells, continuous recordings were performed for 10 minutes, with data separated and plotted as two distinct time intervals: 1–5 minutes and 5–10 minutes post-NMDA treatment, collected from the same coverslip. F) Representative maximum projection images from STIM2 SPT movies under the same treatments and conditions as described for STIM1. G) Cumulative frequency distribution plot and statistical analysis of median diffusion coefficients per acquisition for STIM2. H) Temporal dynamics of STIM2 following NMDA treatment, with data separated and plotted as two distinct time intervals: 1–5 minutes and 5–10 minutes post-NMDA treatment, obtained from the same coverslip. I , J) Cytosolic calcium levels were measured using Fura-2 in hippocampal neurons with Stim1 ( I ) and Stim2 ( J ) knockouts. Experiments were performed at 37 ° C in Ringer’s solution, with a high-potassium pulse introduced at t = 120 s. Data were collected from neurons at DIV 14–16. K , L) Whole-cell patch-clamp recordings from HEK293T cells stably expressing L-type 1.2 calcium channels (see Methods). K) Measurement of calcium currents following store depletion induced by 1 M TG or with vehicle (DMSO). L) Measurement of calcium currents in cells overexpressing EYFP-STIM1 in comparison to control. Refer to supplementary table for p -values, N, n , and trajectory counts.

Article Snippet: For STIM1 overexpression experiments, the cells were transfected with a plasmid coding for human STIM1 fused with YFP (Addgene, plasmid no. 19754) 3 days before experiments.

Techniques: Control, Incubation, Activation Assay, Imaging, Diffusion-based Assay, Patch Clamp, Stable Transfection, Expressing, Comparison

Journal: bioRxiv

Article Title: Activity-Dependent Localization and Heterogeneous Dynamics of STIM1 and STIM2 at ER-PM contacts in Hippocampal Neurons

doi: 10.1101/2024.12.23.630200

Figure Lengend Snippet: A) Fixed immunolabeling of Kv2.1 (green) and STIM1 (magenta) under control conditions in hippocampal neurons. B-D) Quantification of colocalization between Kv2.1 and STIMs with Manders’ overlap coefficients ( M 1 and M 2 ) and Pearson’s correlation coefficient before and after store depletion induced by 1 µM thapsigargin. B) M 1 represents the fraction of the STIMs signal that overlaps with the Kv2.1 signal. C) M 2 quantifies the fraction of the Kv2.1 signal that overlaps with the STIMs signal. D) Pearson’s correlation coefficient measuring the linear correlation between the STIM1 and Kv2.1 signals. E) Fixed immunolabeling of Kv2.1 and MAPPER: representative images of hippocampal neurons immunolabeled for Kv2.1 (green) and MAPPER (magenta). MAPPER-GFP was overexpressed and delivered via rAAV. Scale bars: 20 m for the full image and 2 m for the enlarged section. F) Line plot analysis of Kv2.1 and MAPPER signal intensity across the dendritic region. G) Size comparison of Kv2.1 and MAPPER clusters from two cultures (10 neurons each). H-K) Tracking and Diffusion Analysis of STIM1 and STIM2 at ER-PM Junctions Labeled by MAPPER. Trajectories of STIM1 ( H ) and STIM2 ( J ) molecules (yellow tracks) in control and NMDA-treated conditions, overlaid on MAPPER-labeled ER-PM junctions (magenta). I, K) Distribution of diffusion coefficients for STIM molecules within MAPPER spots, comparing control and NMDA-treated conditions. Statistical analysis of STIM1 ( I ) and STIM2 ( K ) diffusion coefficients before and after NMDA treatment, with each data point on scatter plot representing the median diffusion coefficient from a single acquisition encompassing multiple MAPPER spots from one neuron. Statistical significance was determined by parametric unpaired t -test and is indicated as ** P < 0.01, * P < 0.1. Error bars for all figures depict the mean ± SD. Refer to supplementary table for p -values, N, n , and trajectory counts.

Article Snippet: For STIM1 overexpression experiments, the cells were transfected with a plasmid coding for human STIM1 fused with YFP (Addgene, plasmid no. 19754) 3 days before experiments.

Techniques: Immunolabeling, Control, Comparison, Diffusion-based Assay, Labeling