Journal: bioRxiv

Article Title: Slow scission of single synaptic vesicles by Dynamin at physiological temperature

doi: 10.1101/2025.03.18.643914

Figure Lengend Snippet: a, Normalized average fluorescent traces of Dynamin I-EGFP overexpressed in Xenapses responding to a 50 AP 40 Hz stimulus at RT (black) and 37°C (red). mean ± s.e.m. of n = 19 measurements at RT vs n = 27 measurements at 37°C both from 5 biological replicates. b, Normalized average trace of single Dynamin events elicited with 1 or 2 AP. mean ± s.e.m. c, Normalized cumulative event duration distribution (pORANGE Dynamin I-EGFP knock-in) for RT (red) and 37°C (black). For 37°C, 1031 events (evoked with 1 or 2 AP) consisting of n = 16 measurements from 9 biological replicates. For RT, 119 events (evoked with 2 or 5 AP) consisting of n = 10 measurements from 2 biological replicates.

Article Snippet: These were then individually ligated into BbsI digested pORANGE cloning template vector (Addgene Plasmid #131471) enabled by the duplex having CACCG and AAAC overhangs at either end.

Techniques: Knock-In

Journal: bioRxiv

Article Title: Slow scission of single synaptic vesicles by Dynamin at physiological temperature

doi: 10.1101/2025.03.18.643914

Figure Lengend Snippet: a, Schematic depicting EGFP targetting to the Dynamin I gene by CRISPR-Cas9 based pORANGE strategy (BSE : Bundle signalling element, PRD : Proline rich domain). b, TIRF-microscopy image 3 s after 1 AP stimulus of Xenapses expressing EGFP tagged to endogenous Dynamin I with the Dynamin puncta circled in red; scale bar, 3 µm. c, Representative traces of individual evoked Dynamin recruitment and scission events occurring at various time points after the stimulus. d, An exemplar trace showing the stepwise Dynamin recruitment and annotated with measurable properties. Fit (red) obtained by applying the Autostepfinder step-detection function. Shown below are fluorescence images of the single Dynamin scission event at times indicated; scale bar, 0.5 µm. e, Event duration distribution, f, Event latency distribution and g , Event time to scission distribution. e,f,g are from the same data set of 1129 events in response to 1 or 2 AP and analyzed from n = 16 measurements from 7 biological replicates.

Article Snippet: These were then individually ligated into BbsI digested pORANGE cloning template vector (Addgene Plasmid #131471) enabled by the duplex having CACCG and AAAC overhangs at either end.

Techniques: CRISPR, Microscopy, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Slow scission of single synaptic vesicles by Dynamin at physiological temperature

doi: 10.1101/2025.03.18.643914

Figure Lengend Snippet: Representative traces of pre-stimulus spontaneous (a) and straddle (b) Dynamin recruitment and scission events. c, d, Average trace of spontaneous single Dynamin events centered in space and time along the scission frame and the corresponding average trace of Clathrin fluorescence (d) . Inset images show normalized average fluorescence at time points indicated. mean ± s.e.m. of 38 events from n = 7 measurements with both sets of data from 3 biological replicates. e, Normalized cumulative event duration distributions for Dynamin I EGFP knock-in by pORANGE for evoked (black) and spontaneous events (red, including straddle events). 1031 evoked and 190 spontaneous events both from same data set consisting of n = 16 measurements and 9 biological replicates. f, Normalized inverse cumulative distribution of scission waiting times (time course of endocytosis) for all Dynamin scission events (solid red) and excluding straddle events (dashed red). A monoexponential fit (black) to the decaying phase after 4 seconds (median duration time) yields a time constant of 9.64 seconds. 206 evoked (1 AP) events and 27 straddle events both from n = 4 measurements and biological replicates.

Article Snippet: These were then individually ligated into BbsI digested pORANGE cloning template vector (Addgene Plasmid #131471) enabled by the duplex having CACCG and AAAC overhangs at either end.

Techniques: Fluorescence, Knock-In

Journal: bioRxiv

Article Title: Slow scission of single synaptic vesicles by Dynamin at physiological temperature

doi: 10.1101/2025.03.18.643914

Figure Lengend Snippet: a, Schematic depicting EGFP insertion to the Dynamin I 3‘UTR by CRISPR-Cas9 based TKIT strategy. b, Normalized cumulative stimulus-evoked event duration for Dynamin I EGFP-tagged by pORANGE (black) and TKIT(red) knock-in strategies. 118 events from n = 6 measurements and 2 biological replicates with post-stimulus events evoked at 2 or 5 AP. c, Representative TIRFM image of a Xenapse formed on a 2.5 µm micropattern expressing pORANGE tagged Dynamin-EGFP. Several puncta of Dynamin recruitment are visible 1 s after 2 AP; scale bar, 1 µm. d, Normalized cumulative event durations including straddle events for Xenapses growing on 5 µm (black) and 2.5 µm (red) micropatterns. e , Normalized inverse cumulative distribution of waiting times of scission (time course of endocytosis) for Xenapses growing on 5 µm (black) and 2.5 µm (red) micropatterns. For pORANGE (b) and 5 µm spots (d,e) , same data set as for post-stimulus in Fig.3f . and for 2.5 µm spots (d,e) , 65 events from n = 7 measurements and 2 biological replicates with post-stimulus events evoked at 1, 2 or 5 AP.

Article Snippet: These were then individually ligated into BbsI digested pORANGE cloning template vector (Addgene Plasmid #131471) enabled by the duplex having CACCG and AAAC overhangs at either end.

Techniques: CRISPR, Knock-In, Expressing

Journal: bioRxiv

Article Title: Slow scission of single synaptic vesicles by Dynamin at physiological temperature

doi: 10.1101/2025.03.18.643914

Figure Lengend Snippet: a, Event latency distribution for Dynamin I-EGFP (pORANGE knock-in) scission events in response to 1 (n = 206), 2 (n = 208) and 5 AP (n = 233). n = 4 measurement from 3 biological replicates for 1 AP and n = 6 measurements from 5 biological replicates for 2 and 5 AP. b , SEM images of the two major observed classes of Clathrin coated structures, domed and pit, and the transition between the two. c , Density of domed (8.03 ± 3.62 per mm 2 ) and pit (0.81 ± 0.36 per mm 2 ) structures from SEM images of unroofed Xenapses (n = 17).

Article Snippet: These were then individually ligated into BbsI digested pORANGE cloning template vector (Addgene Plasmid #131471) enabled by the duplex having CACCG and AAAC overhangs at either end.

Techniques: Knock-In

Journal: bioRxiv

Article Title: Multiplex labeling and manipulation of endogenous neuronal proteins using sequential CRISPR/Cas9 gene editing

doi: 10.1101/2022.01.02.474730

Figure Lengend Snippet: A. Illustration of the problem of multiplex knock-in strategies based on NHEJ. With simultaneous editing of multiple genes, donor DNAs can be integrated in either allele, leading to crosstalk. B. Proposed solution for multiplex knock-ins. By introducing a delay between two genome editing events, crosstalk can be avoided. CAKE is designed to control the delay between the two events, using Cre- or Flp-recombinase. C. CAKE strategy. The Cre OFF vector is active in the absence of Cre, leading to editing of the first target gene, and removal of the Cre OFF donor for subsequent genomic integration. Upon addition of Cre, gRNA expression from Cre OFF is deactivated, and gRNA expression from Cre ON vector is enabled for editing the second target gene. All donors contain a fluorophore or epitope tag flanked by a PAM- and target sequence (not shown, see and Willems et al ., 2020). D. Example confocal image of a Cre OFF β3-tubulin-GFP and Cre ON Halo-β-actin knock-in. 20 µL lenti-Cre was added at DIV 7, and cells were fixed at DIV 14.

Article Snippet: The Cre ON knock-in vector (pOC1) is based on pORANGE LOX from ( Willems et al ., 2020 , Addgene #139651), where CAG HA-SpCas9 was removed with XmaJI and NotI and replaced with primers AJ19164 and AJ19165 using primer ligation.

Techniques: Multiplex Assay, Knock-In, Control, Plasmid Preparation, Expressing, Sequencing

Journal: bioRxiv

Article Title: Multiplex labeling and manipulation of endogenous neuronal proteins using sequential CRISPR/Cas9 gene editing

doi: 10.1101/2022.01.02.474730

Figure Lengend Snippet: A. Overview of original CAKE knock-in mechanism introduced in ( Willems et al ., 2020 ), referred to as 2A-Cre throughout this Figure. The constitutively active knock-in labels β3-tubulin with GFP-2A-Cre. Cre expression from this allele then activates gRNA expression for the Cre ON Halo-β-actin knock-in. B. Overview of improved CAKE constructs introduced in this study (see main text) which will be referred to as Lenti-Cre throughout this Figure. Lentivirus encoding for Cre is added at DIV 7, which switches off gRNA expression for Cre OFF , and switches on gRNA expression for Cre ON . C. Example confocal images of CAKE double knock-ins using the 2A-Cre or Lenti-Cre mechanism. D. Average number of single and double knock-in cells per coverslip for 2A-Cre and Lenti-Cre CAKE. 2A-Cre n = 8 coverslips, lenti-Cre n = 7 coverslips, N = 4 independent cultures. β3-tubulin-GFP p = 0.50, Halo-β-actin p = 0.29, double knock-in p = 0.53, incorrect knock-in * p = 0.019, unpaired t -test. E. Average fluorescence intensity in proximal dendrites of single knock-ins, normalized per culture. For β3-tubulin-GFP: 2A-Cre n = 21 cells, lenti-Cre n = 25 cells, N = 3 independent cultures. For Halo-β-actin: 2A-Cre n = 18 cells, lenti-Cre n = 14 cells, N = 3 independent cultures. β3-tubulin-GFP * p = 0.001, Halo-β-actin p = 0.29, unpaired t -test. F. Example confocal image of an incorrect double knock-in. Fluorescence signal of both GFP and Halo are consistent with β3-tubulin distribution, suggesting that the Halo donor was inserted in Tubb3 .

Article Snippet: The Cre ON knock-in vector (pOC1) is based on pORANGE LOX from ( Willems et al ., 2020 , Addgene #139651), where CAG HA-SpCas9 was removed with XmaJI and NotI and replaced with primers AJ19164 and AJ19165 using primer ligation.

Techniques: Knock-In, Expressing, Construct, Fluorescence

Journal: bioRxiv

Article Title: Multiplex labeling and manipulation of endogenous neuronal proteins using sequential CRISPR/Cas9 gene editing

doi: 10.1101/2022.01.02.474730

Figure Lengend Snippet: A. Overview of pORANGE ( Willems et al ., 2020 ) and the CAKE template vectors introduced in this study. All vectors contain a multiple cloning site for addition of the donor DNA. B. Cloning guide to create CAKE knock-in constructs. The example shows a step-by-step protocol for pOC2 Cre OFF knock-in vectors, but identical cloning steps apply to all pOC vectors shown in A. #1 shows the empty pOC2 template vector. After digestion with BbsI, a primer pair encoding for the gRNA target sequence is added to obtain the intermediate construct (#2). The donor DNA, which always contains a protospacer adjacent motif (PAM) and target sequence ( Suzuki et al ., 2016 ) is obtained with standard PCR techniques. of Willems et al ., (2020) contains a detailed description on design of the donor DNA. The donor is cloned using HindIII and MluI restriction sites to obtain the final pOC2 knock-in construct (#3). Most of the knock-in constructs used in this study contain BmtI and AfeI restriction sites around the fluorophore, for universal exchange of donors between knock-in vectors, without the need of PCR (shown in #4, optional). The entire knock-in cassette, containing U6-driven gRNA expression, Lox551 and/or Frt sites and the donor DNA, can be removed from pOC vectors using PscI and MluI restriction sites.

Article Snippet: The Cre ON knock-in vector (pOC1) is based on pORANGE LOX from ( Willems et al ., 2020 , Addgene #139651), where CAG HA-SpCas9 was removed with XmaJI and NotI and replaced with primers AJ19164 and AJ19165 using primer ligation.

Techniques: Cloning, Knock-In, Construct, Plasmid Preparation, Sequencing, Clone Assay, Expressing

Journal: bioRxiv

Article Title: Periodic ER-plasma membrane junctions support long-range Ca 2+ signal integration in dendrites

doi: 10.1101/2024.05.27.596121

Figure Lengend Snippet: A) Schematic illustration of a primary pyramidal hippocampal neuron emphasizing distinct dendritic regions: proximal dendrites (0.5 to 4.1 μm in diameter), medial dendrites (0.3 to 1.2 μm in diameter), and distal dendrites (0.1 to 0.76 μm in diameter). B) Lattice-SIM image portrays the organization of dendritic ER (expression of HaloTag-Sec61β labeled with JF635 HaloTag-ligand) in primary rat hippocampal neuron at 21 DIV. In proximal (P) and medial (M) dendritic segments, we observed a ladder-like organization of the ER, as highlighted in the corresponding insets. Scale bar: 5 μm main panel, 0.5 μm insets. C) Lattice-SIM images of proximal, medial and distal neuronal dendrites expressing mEmerald-Sec61β (ER; grayscale) and mScarlet-CAAX2 (PM; blue). Scale bar: 1 μm. D) Endogenous distribution of JPH3 (gene targeted insertion of mEmerald) in proximal, medial and distal dendrites. The blue outline highlights the outer PM signal (mTagBFP2-CAAX2). Gray arrowheads indicate the position of endogenous JPH3 clusters. Scale bar: 1 μm. E) Lattice-SIM images of proximal, medial and distal neuronal dendrites expressing HaloTag-Junctophilin-3 (HT-JPH3, labeled with JF635 HaloTag-ligand), mEmerald-Sec61β, mScarlet-CAAX2. The blue outline in each panel represents the outer PM signal (mScarlet-CAAX2). Scale bar: 1 μm. F) Average HT-JPH3 contact sites interval along the dendritic plasma membrane in proximal (0.82 ± 0.12 μm), medial (0.81 ± 0.11 μm) and distal (0.95 ± 0.15 μm) dendritic regions (mean ± SD, n = 20 neurons, N = 3 animals). G) Average interval between adjacent transversal ER tubular connections in proximal (0.74 ± 0.13 μm) and medial (0.76 ± 0.16 μm) dendrites (mean ± SD, n = 20 neurons, N = 3 animals). H) Orthogonal views of complete dendritic cross-sections captured with high-resolution Airyscan z-stacks. The dotted line delineates the process boundary. Scale bar: 500 nm. I) Plot illustrating the relationship between ER organization complexity and dendritic process diameter. In proximal and larger medial dendrites (>1.61 ± 0.63 μm in diameter, n = 165, N = 20 cells) the ER is organized as a complex scaffold. In medial dendrites, ranging between 0.35 and 1.58 μm (n = 113, N = 20 cells) in diameter, the ER is organized as a simple ladder-like scaffold. In distal dendrites, with a diameter of approximately 0.34 ± 0.12 μm, the ER appears as a single tubule (n = 113, N = 20 cells) (N= 3 animals). J) Orthogonal and longitudinal views of ER (green), plasma membrane (blue) and contact sites (magenta) segmented from FIB-SEM datasets of mouse hippocampus medial dendrites. Magenta arrowheads indicate contact sites anchoring ER tubular connections (black arrow). Asterisks indicate flattened ER cisternae. Scale bar: 1 μm. K) Box-plot of contact sites interval measured in 3 FIB-SEM segmentations of mouse hippocampus medial dendrites. (Dendrite 1 (D1) = 0.78 ± 0.43 μm, n = 16; Dendrite 2 (D2) = 0.87 ± 0.53 μm, n= 11; Dendrite 3 (D3) = 1.07 ± 0.60 μm n = 22; mean ± SD). L) FIB-SEM orthoslice (top panel) and the segmentation of endoplasmic reticulum (ER, green), plasma membrane (PM, blue) and contact sites (CS, magenta arrowheads) (bottom panel) of mouse hippocampus medial dendrite showing ER tubules anchored to opposing sides of the dendritic PM. Junctional sites contain electron-dense material. (m, mitochondrion). Scale bar: 500 nm.

Article Snippet: We synthesized an AAV knock-in template vector (pAAV KI Cloning Vector_hSyn mTagBFP2-CAAX2) using pAAV hSyn intron Psam4GlyR m3m4 HA (Addgene, plasmid #196041) as backbone containing: 1) a U6-driven expression cassette for the guide RNA (gRNA) targeting the genomic locus of interest that can be inserted at BbsI sites; 2) HindIII and XhoI sites where it is possible to subclone the donor sequence containing the (fluorescent) tag (the template for this region was the Addgene plasmid #131497, pORANGE Tubb3-GFP KI); 3) a 448 bp human synapsin-1 promoter element driving the neuronal specific expression of 4) the fluorescent protein mTagBFP2 (Addgene plasmid #191566 pLKO.1-TRC mTagBFP2) fused to the plasma membrane targeting sequence CAAX2 (Addgene plasmid #162247, eMagAF-EGFP-PM).

Techniques: Expressing, Labeling, Membrane

Journal: bioRxiv

Article Title: Periodic ER-plasma membrane junctions support long-range Ca 2+ signal integration in dendrites

doi: 10.1101/2024.05.27.596121

Figure Lengend Snippet: A) The N-terminal domain of JPH3 is predicted to contain 8 membrane occupation recognition nexus (MORN) repeats (orange) interrupted by a joining region (purple), capable of interacting with the inner surface of the plasma membrane. Following this domain, an α-helical rich domain (blue) is succeeded by a divergent region characterized by low sequence complexity and predicted to be intrinsically disordered. Finally, a short C-terminal segment traverses the ER membrane (T, gray). This domain organization suggests that JPH3 could be an ER-PM tethering protein. B) Primary sequence organization of JPH3 showing the 8 MORN repeats (orange) interrupted by a joining region (purple), a pseudo-MORN repeat (orange) preceding the α-helical–rich stretch (blue), followed by the divergent region where the RNA guide targets the insertion of mEmerald (green) ending in a short transmembrane helix (T, gray). C) HITI-mediated gene targeting consists of a unique 20-nucleotide RNA guide that directs SpCas9 to the genomic locus of interest. Simultaneously, a donor sequence housing the knock-in gene (mEmerald) is poised for insertion into the targeted genomic locus. In our experiments, we co-infected primary rat neurons using two viral vectors. The first, a lentiviral vector (pLenti_SIV hSyn HA-NLS-SpCas9-NLS WPRE), expressed SpCas9 under control of the human Synapsin 1 promoter. The second vector, an AAV vector (pAAV Jph3gRNA mEmerald hSyn mTagBFP2-CAAX2), included all the elements required for targeted CRISPR/Cas9-based genome editing: a U6-driven expression cassette for the 20 bp guide RNA (gRNA) targeting exon 4 of the rat Jph3 gene, the donor sequence containing the tag of interest (mEmerald) flanked by genomic target sequence homology arms. The gRNA serves a dual purpose: it triggers a genomic double-strand break (DSB) and facilitates the removal of donor DNA from the plasmid, enabling its integration into the genome. Importantly, the orientation of the target sequence and protospacer adjacent motif (PAM) sites flanking the donor is inverted as compared to the genomic sequence guaranteeing that integration occurs in the correct orientation. Additionally, this AAV vector included a plasma membrane marker (mTagBFP2-CAAX2) regulated by the human Synapsin 1 promoter. D) Knock-in construct design for rat Jph3 and endogenous locus sequence after integration. The gRNA targets the sequence in the positive genomic strand of Jph3 exon 4. The target sequence is indicated in magenta, the protospacer adjacent motif (PAM) sequence is in blue. Amino acid translation is shown under the sequences. Dashed lines indicate position of Cas9 cleavage and sites of integration. Asterisks indicate nucleotide added to preserve the open reading frame. E) The endogenous tagging efficiency of our co-infection system was estimated by calculating the ratio between JPH3-mEmerald positive cells and mTagBFP2-CAAX2 expressing neurons (JPH3-mEmerald+/ mTagBFP2-CAAX+) at 21-23 DIV to be 1.03 ± 0.25% (mean ± SEM, N = 3). F) To ensure precise integration of the mEmerald tag into the targeted genomic locus, we conducted next-generation sequencing (NGS) analysis. Our findings indicate successful tagging of JPH3 revealed by a high frequency of in-frame integration in the targeted locus as shown by the heatmap summarizing the sequencing results for 5’ (73.4 ± 1.6%) and 3’ (68.3 ± 0.7%) junction amplicons obtained in 3 independent replicates (mean ± SEM). With correct integration, we observed short out-of-frame insertions or deletions (indels) causing the expression of cytosolic non-fluorescent fragments of the protein or soluble fluorescent fragments due to the loss of the C-terminal transmembrane domain. These observations align with the rarity of off-target integration observed, which mainly occurred as soluble mEmerald expression. Heatmap is color-coded for the frequency of indel size, as analyzed using CRIS.py.

Article Snippet: We synthesized an AAV knock-in template vector (pAAV KI Cloning Vector_hSyn mTagBFP2-CAAX2) using pAAV hSyn intron Psam4GlyR m3m4 HA (Addgene, plasmid #196041) as backbone containing: 1) a U6-driven expression cassette for the guide RNA (gRNA) targeting the genomic locus of interest that can be inserted at BbsI sites; 2) HindIII and XhoI sites where it is possible to subclone the donor sequence containing the (fluorescent) tag (the template for this region was the Addgene plasmid #131497, pORANGE Tubb3-GFP KI); 3) a 448 bp human synapsin-1 promoter element driving the neuronal specific expression of 4) the fluorescent protein mTagBFP2 (Addgene plasmid #191566 pLKO.1-TRC mTagBFP2) fused to the plasma membrane targeting sequence CAAX2 (Addgene plasmid #162247, eMagAF-EGFP-PM).

Techniques: Membrane, Sequencing, Knock-In, Infection, Plasmid Preparation, CRISPR, Expressing, Marker, Construct, Next-Generation Sequencing

Journal: bioRxiv

Article Title: Periodic ER-plasma membrane junctions support long-range Ca 2+ signal integration in dendrites

doi: 10.1101/2024.05.27.596121

Figure Lengend Snippet: A) JPH3 endogenously tagged with mEmerald in the cell body of a primary hippocampal neuron. Endogenous JPH3 accumulates in puncta juxtaposed to the plasma membrane (mTagBFP2-CAAX2). Scale bar: 5 μm. B) Normalized intensity plot profile of endogenously tagged JPH3 hotspots drawn along the dendritic plasma membrane. Arrows indicate well-separated, evenly spaced peaks detected based on an unbiased minimum prominence threshold. C) JPH3 puncta are localized at evenly spaced intervals of ∼1 μm (0.88 ± 0.12 μm, n = 10 neurons, N = 5 animals, mean ± SD) along the entire dendritic arbor. D) Box plot showing endogenous JPH3 contact sites intervals regularly spaced at ∼1 μm in all neurons analyzed (43 to 122 contact intervals were analyzed in each neuron). E) Overexpression of HaloTag(HT)-JPH3 (labeled with JF647 HaloTag-ligand) in primary rat neurons at 21 DIV showed accumulation at ER-PM contact sites similar to endogenously tagged JPH3. Overexpression conditions resulted in a brighter, more consistent JPH3 signal and enlarged ER-PM junctions. Scale bar: 5 μm, inset scale bar: 1 μm. F) Average contact sites interval measured as the distance between adjacent HT-JPH3 clusters in intensity plot profiles drawn along the dendritic plasma membrane, as shown in panel B (0.82 ± 0.10 μm, n = 20 neurons, N = 3 animals, mean ± SD) G) Box plot showing regular HT-JPH3 contact sites spacing of ∼1 μm in all neurons analyzed (56 to 118 contacts were analyzed in each neuron). H,L) Upon detecting peaks in the line scans of both endogenous JPH3 (H) and overexpressed HT-JPH3 (L), we noticed that the intervals between the junctions were spaced at very regular intervals (panels C,D for endogenous JPH3 and F,G for HT-JPH3), suggesting some degree of periodicity. To evaluate the degree of periodicity, we synthesized a localization signal with maxima at the detected peaks and cross-correlated them with periodic functions of comparable frequencies (the periodic Gaussian signal with the matched detected frequency is plotted in magenta in H for endogenous JPH3 and in L for HT-JPH3). We found that the localization signals significantly correlated with the frequency-matched periodic functions when compared to a localization signal generated by a white noise process (as shown in I and M for endogenous and HT-JPH3, respectively, where the dashed magenta lines indicate the 95% confidence intervals as described in the Methods). The normalized Fourier power spectrum of the periodic signal and the synthetic signal with Gaussians placed at the detected peaks is shown in (J) for endogenous and (N) for HT-JPH3. Raincloud plots of the contact sites intervals in dendrites detected from Fourier analysis are shown in (K) for endogenous and (O) for overexpressed JPH3. While not purely periodic in the mathematical sense due to biological variability in the intervals, these results show that the junction membrane complexes marked by JPH3 occur at regular intervals distinct from pure chance illustrating a quasi-periodic distribution. P) Lattice-SIM images illustrate the localization of contact sites (CS), marked by accumulation of HaloTag-JPH3 (labeled with JF635 HaloTag-ligand) relative to dendritic spines (visualized with the plasma membrane (PM) marker mScarlet-CAAX2), with or without ER (mEmerald-Sec61β) accumulation in the spine head. Scale bars: 1 µm. Q) Analysis of contact sites distance from the spine head reveals that when ER tubules protrude in the spine head, contact sites are located approximately 363 ± 116 nm from the spine neck (n = 154 spines). In spines with no ER presence, the closest contact site is localized approximately 566 ± 191 nm from the spine neck (n = 203 spines). Data were analyzed in 10 neurons from 3 independent experiments and represented as mean ± SD.

Article Snippet: We synthesized an AAV knock-in template vector (pAAV KI Cloning Vector_hSyn mTagBFP2-CAAX2) using pAAV hSyn intron Psam4GlyR m3m4 HA (Addgene, plasmid #196041) as backbone containing: 1) a U6-driven expression cassette for the guide RNA (gRNA) targeting the genomic locus of interest that can be inserted at BbsI sites; 2) HindIII and XhoI sites where it is possible to subclone the donor sequence containing the (fluorescent) tag (the template for this region was the Addgene plasmid #131497, pORANGE Tubb3-GFP KI); 3) a 448 bp human synapsin-1 promoter element driving the neuronal specific expression of 4) the fluorescent protein mTagBFP2 (Addgene plasmid #191566 pLKO.1-TRC mTagBFP2) fused to the plasma membrane targeting sequence CAAX2 (Addgene plasmid #162247, eMagAF-EGFP-PM).

Techniques: Membrane, Over Expression, Labeling, Synthesized, Generated, Marker

Journal: bioRxiv

Article Title: Periodic ER-plasma membrane junctions support long-range Ca 2+ signal integration in dendrites

doi: 10.1101/2024.05.27.596121

Figure Lengend Snippet: A) Time-lapse acquired using 2D lattice-SIM in burst mode of HaloTag-Sec61β (labeled with JF585 HaloTag-ligand) expressing neurons revealing ER tubule dynamicity with the persistent presence of ER-PM junctional sites (marked with magenta asterisks, see Movie 3). Scale bar: 0.5 μm. B) Neuronal dendrite expressing mCherry-JPH3, mEmerald-Sec61β and mTagBFP2-CAAX2 (blue outline) following hypotonic swelling (∼63 mOsM solution). Scale bar: 1 μm, inset 0.5 μm. C) Average mCherry-JPH3 contact sites interval after treatment with strong hypotonic solution (0.97 ± 0.15 μm, n = 16, N =2, mean ± SD). D) Time-lapse series acquired using Lattice-SIM of mEmerald-Sec61β expressing neurons. Lysosomes were stained using Lysview650. Lysosomes move through ladder-like ER arrays (see arrow and Movie 4). Scale bar: 0.5 μm. E) 3D renderings of ER (green), plasma membrane (blue) and mitochondria (pink) in medial dendrites of mouse hippocampus. Black arrows indicate ER rungs. Scale bar: 1 μm. F-G) 3D renderings of ER (green), plasma membrane (blue) and mitochondria (pink) in Drosophila MBON1 neuron segmented from FIB-SEM datasets. The white box frames a mitochondrion extending through multiple ER rungs. Scale bars: 1 μm. H) Orthogonal and longitudinal views of the 3D renderings of ER (green), plasma membrane (blue) mitochondria (pink) and microtubules (tan) in Drosophila MBON1 neuron segmented from FIB-SEM datasets. Scale bars: 1 μm. I) ) Primary hippocampal neurons were transiently transfected with pORANGE Tubb3-GFP KI, to endogenously tag tubulin, allowing visualization of the microtubule cytoskeleton. Images show microtubule organization in untreated cells (Control) and nocodazole treated cells (5 μM nocodazole for 2-3 h, microtubule destabilization is evident in cell bodies and proximal dendrites). Even after prolonged nocodazole-induced destabilization of the microtubule cytoskeleton, the laddered ER organization remained unchanged. The ER was imaged in the same neurons also expressing HaloTag-Sec61β (labeled with JF585 HaloTag-ligand). Scale bar: 5 μm in cell body and 0.5 μm in dendrites. J) Lattice-SIM image of dendritic actin rings (SirActin labeling) and ladder-like ER (HaloTag-Sec61β labeled with JF549 HaloTag-ligand). Arrowheads show ER tubules protruding between dendritic actin rings to establish contacts with the PM. Scale bar: 1 μm.

Article Snippet: We synthesized an AAV knock-in template vector (pAAV KI Cloning Vector_hSyn mTagBFP2-CAAX2) using pAAV hSyn intron Psam4GlyR m3m4 HA (Addgene, plasmid #196041) as backbone containing: 1) a U6-driven expression cassette for the guide RNA (gRNA) targeting the genomic locus of interest that can be inserted at BbsI sites; 2) HindIII and XhoI sites where it is possible to subclone the donor sequence containing the (fluorescent) tag (the template for this region was the Addgene plasmid #131497, pORANGE Tubb3-GFP KI); 3) a 448 bp human synapsin-1 promoter element driving the neuronal specific expression of 4) the fluorescent protein mTagBFP2 (Addgene plasmid #191566 pLKO.1-TRC mTagBFP2) fused to the plasma membrane targeting sequence CAAX2 (Addgene plasmid #162247, eMagAF-EGFP-PM).

Techniques: Labeling, Expressing, Staining, Membrane, Transfection

Journal: bioRxiv

Article Title: Periodic ER-plasma membrane junctions support long-range Ca 2+ signal integration in dendrites

doi: 10.1101/2024.05.27.596121

Figure Lengend Snippet: A) Live-cell imaging of mEmerald-Ca V 2.1 (glow LUT) and mScarlet-CAAX2 (cyan LUT) in primary rat hippocampal neurons showed mEmerald-Ca V 2.1 accumulation in nerve terminals and dendrites. Scale bar: 5 μm. B-C) Both mEmerald-Ca V 2.1 and mEmerald-Ca V 2.2 accumulate in small clusters in neuronal cell bodies and dendrites. In large proximal dendrites, Ca V 2.1 and Ca V 2.2 clusters are rare, but they are often symmetrically distributed on opposite sides of the dendritic membrane (examples are indicated with arrowheads and shown in insets). Scale bar: 5 μm. D) Immunostaining experiments revealed endogenous Ca V 2.1 puncta positioned at opposite sides of ER rungs visualized using the ER marker HaloTag-Sec61β (labeled with JF647 HaloTag-ligand). Scale bar: 0.5 μm. E) mEmerald-Ca V 2.1 and mEmerald-Ca V 2.2 colocalized with HaloTag-JPH3 (labeled with JF635 HaloTag ligand) in neuronal cell bodies. Scale bar: 5 μm. F) ShRNA knockdown of JPH3 significantly reduced protein levels in Western blot experiments. Relative JPH3 expression measured after knockdown = 0.45 ± 0.11, N = 3. Data analyzed with unpaired t-test G) RT-PCR shows significant downregulation of JPH3 transcripts upon JPH3 shRNA expression. Relative JPH3 expression = 0.45 ± 0.05, n=7, N=3. Data were analyzed with the Mann-Whitney-test. H) Knockdown of JPH3 (neurons expressing shRNA for JPH3 were identified by cytosolic expression of mTagBFP2) results in the loss of mEmerald-Ca V 2.1 and mEmerald-Ca V 2.2 clusters in cell bodies. Scale bar: 5 μm. All data are mean ± SEM; ** p<0.01, *** p<0.001.

Article Snippet: We synthesized an AAV knock-in template vector (pAAV KI Cloning Vector_hSyn mTagBFP2-CAAX2) using pAAV hSyn intron Psam4GlyR m3m4 HA (Addgene, plasmid #196041) as backbone containing: 1) a U6-driven expression cassette for the guide RNA (gRNA) targeting the genomic locus of interest that can be inserted at BbsI sites; 2) HindIII and XhoI sites where it is possible to subclone the donor sequence containing the (fluorescent) tag (the template for this region was the Addgene plasmid #131497, pORANGE Tubb3-GFP KI); 3) a 448 bp human synapsin-1 promoter element driving the neuronal specific expression of 4) the fluorescent protein mTagBFP2 (Addgene plasmid #191566 pLKO.1-TRC mTagBFP2) fused to the plasma membrane targeting sequence CAAX2 (Addgene plasmid #162247, eMagAF-EGFP-PM).

Techniques: Live Cell Imaging, Membrane, Immunostaining, Marker, Labeling, shRNA, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: bioRxiv

Article Title: Periodic ER-plasma membrane junctions support long-range Ca 2+ signal integration in dendrites

doi: 10.1101/2024.05.27.596121

Figure Lengend Snippet: A-B) Airyscan images showing mEmerald-CaV2.1 (A) and mEmerald-CaV2.2 (B) localize at the plasma membrane (visualized with mScarlet-CAAX2) of dendrites in primary rat hippocampal neurons. mEmerald-CaV2.1 and mEmerald-CaV2.2 accumulated in regularly interspaced puncta (inset). Scale bar: 5 μm, inset: 1 μm. C) mEmerald-CaV2.1 and mEmerald-CaV2.2 colocalized with HaloTag-JPH3 contacts in neuronal dendrites. Scale bar: 1 μm. D) mEmerald-CaV2.1 and mEmerald-CaV2.2 puncta were observed in approximately 70.7 ± 2.7% (n =18, N= 5) and 68.0 ± 3.2% (n =16, N= 4) of JPH3-positive contacts, respectively, along the dendritic tree. E) Knockdown of JPH3 resulted in the loss of mEmerald-CaV2.1 and mEmerald-CaV2.2 clusters in dendrites. Neurons expressing shRNA for JPH3 were identified by cytosolic expression of mTagBFP2. Scale bars: 2 μm. F) mEmerald-CaV2.1 hotspots at contact sites per dendritic surface unit was approximately 0.46 ± 0.04 clusters/μm2 (n=13, N=5). Co-overexpression with HaloTag-JPH3 significantly increased the density of mEmerald-CaV2.1 clusters (0.87 ± 0.06%, n = 18, N = 5) while JPH3 knock down significantly reduced mEmerald-CaV2.1 clusters density (0.13 ± 0.02 clusters/μm2, n = 14, N = 4). The density of mEmerald-CaV2.2 was 0.66 ± 0.05 clusters/μm2 (n=13, N=4). While the co-overexpression of HaloTag-JPH3 did not significantly increase the density of mEmerald-CaV2.2 clusters (0.70 ± 0.05 clusters/μm2, n = 15, N = 4); JPH3 knock down significantly decreased mEmerald-CaV2.2 clusters density (0.08 ± 0.02 clusters/μm2, n = 14, N = 3). G) HaloTag-JPH3 significantly increased the size of mEmerald-CaV2.1 and mEmerald-CaV2.2 clusters at contact sites (mEmerald-CaV2.1 = 0.070 ± 0.004 μm2, n = 13, N =5; mEmerald-CaV2.1 + JPH3 OE = 0.122 ± 0.011 μm2, n = 18, N = 5; mEmerald-CaV2.1 + JPH3 KD = 0.036 ± 0.002, n=12, N =4; mEmerald-CaV2.2 = 0.067 ± 0.005, n = 13, N = 4; mEmerald-CaV2.2 + JPH3 OE = 0.120 ± 0.006 μm2, n = 16, N =4; mEmerald-CaV2.2 + JPH3 KD = 0.051 ± 0.007 μm2, n=13, N =3). H) Morphology and distribution of HaloTag-JPH3 junctions in neuronal dendrites after 12-hour treatment with TTX (0.5 μM), Bicuculline (Bic, 20 μM), and Kainic Acid (KA, 250 nM). Scale bar: 1 μm. I) HaloTag-JPH3 contact site sizes in Ctrl conditions (0.084 ± 0.005 μm2, n = 17) and after treatments with TTX (0.082 ± 0.006 μm2, n = 12), Bic (0.131 ± 0.009 μm2, n = 17), and KA (0.132 ± 0.006 μm2, n = 19); N =3. J) Comparison of HaloTag-JPH3 contact site intervals in control (Ctrl) conditions (0.91 ± 0.04 μm, n = 17) and after treatments with TTX (0.85 ± 0.03 μm, n = 12), Bic (0.80 ± 0.03 μm, n = 17), and KA (0.82 ± 0.04 μm, n = 19); N = 3. K-L) mEmerald-CaV2.1 (K) and mEmerald-CaV2.2 (L) plasma membrane puncta colocalizing with mCherry-JPH3 increased size upon KA treatment. Scale bars: 1 μm. M) mEmerald-CaV2.1 clusters size at contacts in Ctrl (0.060 ± 0.003 μm2, n = 8) and after KA incubation (0.099 ± 0.007 μm2, n = 12), mEmerald-CaV2.2 clusters size at contacts in Ctrl (0.058 ± 0.004 μm2, n = 9) and after KA incubation (0.103 ± 0.008 μm2, n = 10); N =3. N) Comparison of mEmerald-CaV2.1 clusters density in control (Ctrl) conditions (1.02 ± 0.08 clusters/ μm2, n = 8) and after KA treatment (0.99 ± 0.07 clusters/μm2, n = 12) and mEmerald-CaV2.2 clusters density in control (Ctrl) conditions (0.98 ± 0.08 clusters/μm2, n = 9) and upon KA treatment (1.01 ± 0.11 clusters/μm2, n = 10); N = 3. O) Schematic illustrating how changes in ER-PM contact site size and modulation of JPH3 accumulation affect voltage-dependent Ca2+ channel subdomains on the plasma membrane. n = neurons, N = independent experiments. All data are mean ± SEM and were analyzed with one-way ANOVA and Tuckey’s multiple comparison tests; * p<0.05, *** p<0.001, **** p<0.0001.

Article Snippet: We synthesized an AAV knock-in template vector (pAAV KI Cloning Vector_hSyn mTagBFP2-CAAX2) using pAAV hSyn intron Psam4GlyR m3m4 HA (Addgene, plasmid #196041) as backbone containing: 1) a U6-driven expression cassette for the guide RNA (gRNA) targeting the genomic locus of interest that can be inserted at BbsI sites; 2) HindIII and XhoI sites where it is possible to subclone the donor sequence containing the (fluorescent) tag (the template for this region was the Addgene plasmid #131497, pORANGE Tubb3-GFP KI); 3) a 448 bp human synapsin-1 promoter element driving the neuronal specific expression of 4) the fluorescent protein mTagBFP2 (Addgene plasmid #191566 pLKO.1-TRC mTagBFP2) fused to the plasma membrane targeting sequence CAAX2 (Addgene plasmid #162247, eMagAF-EGFP-PM).

Techniques: Membrane, Expressing, shRNA, Over Expression, Comparison, Incubation

Journal: bioRxiv

Article Title: Periodic ER-plasma membrane junctions support long-range Ca 2+ signal integration in dendrites

doi: 10.1101/2024.05.27.596121

Figure Lengend Snippet: A) Protocols used to detect pCaMKII and CaMKIIα at ER-PM junctions using a paradigm that maximizes the contribution of voltage-gated Ca 2+ channels to depolarization-induced Ca 2+ entry. B) Dendrites expressing mTagBFP2-CAAX2 to visualize dendritic plasma membrane (blue outline) and mCherry-JPH3 to visualize ER-PM junctions were promptly fixed and stained for pCaMKII under different conditions: 5 mM K + , stimulation with 90 mM K + , and 90 mM K + with Ca V blockers: the L-type Ca V blocker nimodipine (10 μM), the P/Q-type Ca V blocker ω -Agatoxin IVA (500 nM), and ω -Conotoxin GVIA (2 μM). The background signal of pCaMKII staining was subtracted to highlight hotspots (see Methods section). Scale bars: 1 μm. C) pCaMKII hotspots per μm of dendritic area in 5 mM K + = 0.02 ± 0.01, 90 mM K + = 1.2 ± 0.04%, 90 mM K + + CaV Block = 0.36 ± 0.06 hotspots/μm , n=12, N=3. D) JPH3 contacts density in dendrites in each stimulation paradigm: 5 mM K + = 1.08 ± 0.08 μm , 90 mM K + = 1.14 ± 0.06, 90 mM K + + Ca V Block = 1.03 ± 0.06 contacts/μm , n=12, N=3. E) Fraction of HaloTag-JPH3 contacts colocalizing with pCaMKII hotspots in 5 mM K + = 0.67 ± 0.49%, 90 mM K + = 62.44 ± 4.02%, 90 mM K + + Ca V Block = 3.88 ± 0.98%, n=12, N=3. All data are mean ± SEM and were analyzed with one-way ANOVA and Tuckey’s multiple comparison tests; **** p<0.0001.

Article Snippet: We synthesized an AAV knock-in template vector (pAAV KI Cloning Vector_hSyn mTagBFP2-CAAX2) using pAAV hSyn intron Psam4GlyR m3m4 HA (Addgene, plasmid #196041) as backbone containing: 1) a U6-driven expression cassette for the guide RNA (gRNA) targeting the genomic locus of interest that can be inserted at BbsI sites; 2) HindIII and XhoI sites where it is possible to subclone the donor sequence containing the (fluorescent) tag (the template for this region was the Addgene plasmid #131497, pORANGE Tubb3-GFP KI); 3) a 448 bp human synapsin-1 promoter element driving the neuronal specific expression of 4) the fluorescent protein mTagBFP2 (Addgene plasmid #191566 pLKO.1-TRC mTagBFP2) fused to the plasma membrane targeting sequence CAAX2 (Addgene plasmid #162247, eMagAF-EGFP-PM).

Techniques: Expressing, Membrane, Staining, Blocking Assay, Comparison