Journal: eLife

Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

doi: 10.7554/eLife.27362

Figure Lengend Snippet: ( A ) Schematic depicting inducible release constructs. Multiple copies of self-associating F M domains were fused to target proteins downstream of an ER signal peptide and upstream of a fluorescent protein (FP). DDS dissociates F M domains allowing synchronous ER exit. A furin cleavage site allows removal of the F M domains as they transit the GA. A thrombin cleavage site was included in some constructs so that the FP could be selectively removed from proteins localized at the PM. ( B ) Comparison of 3xF M -mEOS-GluA1 with the endogenous ER-marker BiP before release (left panel) and the Golgi marker GM130 1 hr after DDS addition (right panel). ( C ) Detection of GluA1 surface delivery at various time points following addition of DDS by surface labelling against the extracellular HA-tag of 3xF M -GluA1. ( D ) Quantification of GluA1 surface delivery shown in C (mean ± SEM, n = 10–12 neurons/timepoint from 2 independent experiments). All values normalized to neurons that were not treated with DDS. ( E ) Time-course of NL1 surface delivery (mean ± SEM, n = 9–10 neurons/timepoint from 2 independent experiments). ( F ) Localization of surface GluA1 after ER-release. Images taken from insets in panel C. Scale bar, 2 µm.

Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

Techniques: Construct, Comparison, Marker

Journal: eLife

Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

doi: 10.7554/eLife.27362

Figure Lengend Snippet: ER-retained 4xF M -SEP-NL1 coexpressed with an engineered ER-marker TfR-mCh-KDEL in a COS7 cell. Scale bar, 5 µm.

Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

Techniques: Marker

Journal: eLife

Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

doi: 10.7554/eLife.27362

Figure Lengend Snippet: Trafficking of 4xF M -SNAPtag-NL1 (labelled with JF646) in COS7 cells. 30 min after addition of DDS, NL1 accumulates in a perinuclear GA-like distribution. 120’ after addition of DDS, NL1 is present on the membrane and in vesicular structures located throughout the cell. Addition of thrombin eliminates the surface signal. Scale bar, 10 µm.

Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

Techniques: Membrane

Journal: eLife

Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

doi: 10.7554/eLife.27362

Figure Lengend Snippet: ( A ) Live-cell imaging of a cortical neuron expressing 3xF M -GluA1-mCh and TfR-GFP before ER-release (top panels) and 120 min after ER-release (bottom panels). Blue arrowheads indicate locations where GluA1 has redistributed to TfR-GFP positive endosomes. Scale bar, 10 µm. ( B ) Experimental paradigm to visualize individual vesicles trafficking in neurons expressing 3xF M -GluA1-mCh and a green RE marker (either GFP-Rab11 or TfR-GFP). The middle panel shows photobleaching of a dendritic segment (indicated by red square). The right panel is a frame taken 20 s after photobleaching showing endosomes entering the bleached area (blue arrows) Also see and . ( C ) Representative example of a mobile vesicle (orange arrows) associated with both Rab11 and 3xF M -GluA1. Individual frames are taken from 4 Hz dual-color imaging of a dendritic segment following photobleaching. Scale bar, 5 µm. ( D ) Kymographs of dendritic segments following photobleaching showing the movement of Rab11 and GluA1 vesicles at various times following 3xF M -mCh-GluA1 release from the ER. The amount of time elapsed between addition of DDS and imaging is indicated above the kymographs. The black arrow indicates the time of photobleaching. Blue arrowheads denote double-positive mobile vesicles. ( E ) Quantification of the percentage of GluA1 vesicles that also contain the indicated RE marker. The orange bar (+Thr) indicates the inclusion of 1 U/ml thrombin along with DDS. (mean ± SEM, n = 6–8 neurons/timepoint/marker from 3 experiments per marker; 2 independent experiments for thrombin n.s. p=0.31 by unpaired two-tailed Student’s t-test). Numbers on each bar indicate the raw number of double-positive vesicles/total GluA1 vesicles. ( F ) Schematic of soluble anterograde trafficking marker (4xF M -mCh). During vesicle exocytosis, the soluble marker is released from the cell and therefore cannot be recycled. ( G ) Kymographs showing cotrafficking between anterograde soluble marker (4xF M -mCh) and GFP-Rab11 within a segment of dendrite 150 min after DDS addition. Blue arrowheads highlight individual cotrafficking vesicles. ( H ) Quantification of the percentage of vesicles cotrafficking Rab11 and 4xF M -mCh before and 150 min after ER-release (mean ± SEM, no rel. n = 5 neurons; 150 minutes n = 8 neurons from 2 experiments). ( I ) Quantification of cotrafficking between NL1 and Rab11. Numbers on bars indicate raw numbers of vesicles positive for both markers divided by the total number positive for NL1 (mean ± SEM, n = 5–6 neurons/condition from 2 experiments). ( J ) GluA1 accumulates in a subset of spine endosomes following ER release. Shown is a stretch of dendrite from a neuron expressing 3xF M -mCh-GluA1 along with TfR-HaloTag labeled with JF646 before and 150 min following addition of DDS. Orange arrowheads denote spines containing REs. Blue arrows indicate accumulation of GluA1 in spine-resident REs. Dotted yellow outline drawn based on GFP cell fill (not shown). ( K ) Images are of a neuron coexpressing 3xF M -mCh-GluA1 and TfR-HaloTag (JF646) fixed 150 min after addition of DDS in the presence of thrombin (1 U/ml) to prevent visualization of recycled proteins. As in F, blue arrows highlight spine REs that contain trafficking GluA1. Orange arrowheads indicate GluA1-negative spine REs. The right panel is the quantification of the percentage of spines with REs that also contain GluA1 before and 150’ after release in the presence of thrombin. (mean ± SEM, n = 5 neurons/condition from 2 experiments, **p=0.006 unpaired two-tailed Student’s t-test.) Scale bar, 2 μm.

Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

Techniques: Live Cell Imaging, Expressing, Marker, Imaging, Two Tailed Test, Labeling

Journal: eLife

Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

doi: 10.7554/eLife.27362

Figure Lengend Snippet: Surface delivery of VSV-G-YFP-4xF M (left) and 4xF M -mCh-NL1 (right) was measured in dissociated cortical neurons after various combinations of temperature and BFA treatment. Experimental conditions are indicated under the bars and are the same as in . Surface intensity is expressed as a percentage of control (37 ˚C, no BFA) surface delivery. Values are reported as mean relative to control ± SEM (n = 5–8 cells/condition, n.s. = not significant by one way ANOVA/Bonferonni post hoc test). Both NL1 and VSV-G experiments were independently repeated with similar results.

Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

Techniques: Control

Journal: eLife

Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

doi: 10.7554/eLife.27362

Figure Lengend Snippet:

Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

Techniques: Transfection, Construct, Generated, Derivative Assay, Plasmid Preparation, Western Blot, Recombinant

Journal: eLife

Article Title: Golgi-independent secretory trafficking through recycling endosomes in neuronal dendrites and spines

doi: 10.7554/eLife.27362

Figure Lengend Snippet:

Article Snippet: 4xF M -NL1 was generated by insertion of NL1 ORF (Addgene #15260, a gift from Peter Schieffelle) into a construct containing 4xF M. This fusion was then subcloned into a pCAG backbone.

Techniques: Construct

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Spatial and temporal properties of synaptic protein trafficking in hippocampal neurons following global ER release. (A) Schematic of constructs used for ER release experiments in hippocampal neurons. Right: Image series of TfR-GFP-DHFR (top), DHFR-GFP-NL1 (middle), and DHFR-mNeon-GluA1 (bottom) accumulation in the GA of hippocampal neurons following light exposure at time 0. Scale bar, 10 µm. (B) Time course of TfR-GFP-DHFR (blue), DHFR-GFP-NL1 (red), and DHFR-mNeon-GluA1 (black) trafficking to the GA following global (whole cell) light-triggered ER release; mean ± SEM ( n = 11–27 cells from two or three independent experiments). (C) Comparison of the time to peak accumulation for TfR-GFP-DHFR (blue), DHFR-GFP-NL1 (red), and DHFR-mNeon-GluA1 (black) following ER release. ****, P < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test). (D) Surface expression of DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) was detected by including Alexa647-anti-GFP in the extracellular solution. Somatic regions before and 60 min and 120 min following ER release are shown (right). Scale bar, 20 µm. Inset scale bar, 10 µm. (E) Appearance of DHFR-GFP-NL1 and DHFR-GFP-GluA1 at the surface of proximal (top), intermediate (middle), and distal (bottom) dendrites. Examples show surface signal before and 60 min and 120 min following ER release. Arrowheads denote spines with surface label. Scale bar, 5 µm.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Construct, Comparison, Expressing

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Localization of DHFR fused synaptic proteins and validation of antibody cross-linking for immobilizing surface proteins. (A) DHFR does not disrupt basal trafficking. Hippocampal neurons expressing the indicated constructs for 18 h (in the absence of zapalog) were labeled with Alexa647-anti-GFP under nonpermeabilizing conditions. The image panels show representative surface labeling (magenta) for DHFR-GFP-GluA1 (left) compared with GFP-GluA1 (right). The ratios of total surface (Alexa647-anti-GFP) to total (GFP) signal (top) and spine to shaft surface signal (bottom) are plotted for DHFR-GFP-NL1 and DHFR-GFP-GluA1 and compared with constructs without DHFR. The surface/total ratio signal is normalized to control (non-DHFR fused constructs); mean ± SEM, n = at least 8 neurons/condition from two separate cultures (Student’s t test). Scale bar, 10 µm. (B) Kinetics of Alexa647-anti-GFP binding to surface cargoes. Schematic of antibody cross-linking/labeling strategy is shown (top left). Bottom right: Antibody binding/cross-linking signal (magenta) measured in live neurons expressing DHFR-GFP-GluA1 immediately before and 30 s and 180 s following antibody addition. Kinetic data (top right) were fit with a single exponential (solid red line, τ = 1.66 min, n = 6 neurons). The two-dimensional plot (bottom right) models the probability of receptor localization following surface insertion and antibody cross-linking as a function of displacement from the membrane insertion site (see Materials and methods for details). Scale bar, 10 µm. (C) Representative examples of stable antibody binding to DHFR-GFP-GluA1 as it appears at the cell surface. A neuron expressing DHFR-GFP-GluA1 and cell fill (green) was imaged at the times indicated after ER release in the continuous presence of extracellular Alexa647-anti-GFP (magenta). Note the stable, sequential appearance of new surface puncta (arrowheads) on dendrites and select spines. Two representative examples are shown from different cells. Scale bar, 2 µm. (D) Alexa647-anti-GFP labeled DHFR-GFP-GluA1 is shown before, immediately following, and 60 min after photobleaching a small region (designated by blue box). Quantification of Alexa647 signal within the photobleached region is shown below; mean ± SEM ( n = 10 dendritic regions from seven neurons). Scale bar, 5 µm. (E) The majority of cross-linked/labeled DHFR-GFP-GluA1 remains on the cell surface. DHFR-GFP-GluA1 was released from the ER and allowed to traffic to the surface for 80 min in the continuous presence of Alexa647- anti-GFP (magenta, generated in rabbit). Alexa647-anti-GFP was washed off, and Alexa568-anti-rabbit (teal) was added to label surface anti-GFP and confirm its localization at the cell surface. Magnified images of the highlighted region (white box) are shown to the right. Yellow arrowheads denote colocalized puncta. 87 ± 3% (mean ± SEM) of the Alexa647-anti-GFP puncta overlapped with Alexa568-anti-rabbit puncta. Data are averaged from five neurons. Scale bar, 10 µm (left); 2 µm (right). norm., normalized.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Biomarker Discovery, Expressing, Construct, Labeling, Control, Binding Assay, Membrane, Generated

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Kinetics of somatic GA accumulation for different cargoes. Shown are hippocampal neurons expressing FKBP-mCh-KDEL (channel not displayed) and either TfR-GFP-DHFR (left), DHFR-GFP-NL1 (center), or DHFR-mNeon-GluA1 (right) before and after full-field 405-nm illumination (indicated by the white dot) delivered at time 0. The duration of the videos is 67 min with a baseline acquisition rate of 1 frame/min (first 5 frames) and a post-release acquisition rate of 1 frame/2 min. Time stamp displays the time elapsed following full-field 405-nm illumination. Scale bar, 10 µm. Playback speed, 2 min between frames.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Surface accumulation of cargoes following whole-cell illumination in neurons. Shown are hippocampal neurons expressing FKBP-mCh-KDEL (channel not displayed) and either DHFR-GFP-NL1 (top) or DHFR-GFP-GluA1 (bottom) before and after full-field. 405-nm illumination in the presence of extracellular Alexa647-anti-GFP (magenta). The first frame after photoexcitation is denoted by the appearance of the white circle (upper left corner). Left: The surface signal. Right: The merge between the cell fill (green) and surface signal (magenta). Time stamp displays the time elapsed following full-field 405-nm illumination. Scale bar, 20 µm. Playback speed, 2 min between frames.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Subcellular distribution of NL1 and GluA1 surface presentation following global ER release. (A) The timing and location of surface trafficking for DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) are shown following global light-triggered ER release. Surface signal with a shorter latency of appearance is rendered in warmer colors. Scale bars, 20 µm. (B) Time courses of DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) surface delivery at different cellular domains following global ER release. Shown are the normalized intensities for surface signal at the soma (pink line) and proximal (peach line; up to 40 µm from the soma) or distal regions of the dendritic arbor (lavender line; 40–200 µm from the soma); mean ± SEM (NL1: n = 10 neurons/time point from four independent experiments; GluA1: n = 11 neurons/time point from two independent experiments). (C) Comparison of DHFR-GFP-NL1 (gray) and DHFR-GFP-GluA1 (black) surface accumulation (from B) at distal dendrites (40–200 µm from the soma) following ER release. The yellow shaded region denotes P < 0.05 (two-way ANOVA, Bonferroni’s multiple comparisons test). (D) Time to 10% of total surface accumulation is plotted for DHFR-GFP-NL1 (gray) and DHFR-GFP-GluA1 (black) in different cellular domains; mean ± SEM; **, P < 0.01 (Student's t test). (E) Representative images of DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) surface signal (magenta, Alexa647-anti-GFP) in spines 90 min following ER release. Solid arrowheads denote cargo-positive spines. Open arrowheads mark spines that lack detectable surface cargo. The outline of the cell (dashed line) was drawn based on the cell fill (green signal in merge). Scale bar, 2 µm. (F) Time course of the fraction of spines in proximal (circles) and distal (triangles) dendrites with detectable DHFR-GFP-NL1 (gray) or DHFR-GFP-GluA1 (black) signal following ER release. A comparison of the fraction of DHFR-GFP-NL1– and DHFR-GFP-GluA1–positive spines at 90 and 120 min is shown on the right; mean ± SEM; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (Student's t test; n = 10 neurons/time point for NL1 and GluA1). (G) Plotted is the ratio of the total spine surface signal to the total dendritic shaft surface signal for DHFR-GFP-NL1 (gray) or DHFR-GFP-GluA1 (black) in proximal and distal dendrites, 90 and 120 min following ER release; mean ± SEM; **, P < 0.01; ***, P < 0.001 (Student's t test; n = 8–10 neurons/time point [NL1], n = 7–9 neurons/time point [GluA1]). dist., distal; norm., normalized; prox., proximal.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Comparison

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Segmented surface signal displaying where and when NL1 and GluA1 appear on the cell surface following global release. Newly appearing surface signal (anti-GFP binding to DHFR-GFP-NL1 or DHFR-GFP-GluA1) was masked, segmented, and displayed only during the first frame of appearance and for three subsequent frames (even though the signal was persistent) to visualize where and when surface signal appeared (rendered in green; see Materials and methods). The cells were exposed to full-field 405-nm illumination at time 0. The cell outlines (purple) were drawn based on a cell fill mask. The first frame after photoexcitation is denoted by the appearance of the white circle (upper right corner). The duration of the videos is 130 min with an acquisition rate of 1 frame/2 min. Scale bar, 20 µm. Playback speed, 2 min between frames.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Binding Assay

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: The AIS is a surface trafficking hotspot for specific cargoes. (A) Representative images of DHFR-GFP-NL1 (left) and DHFR-GFP-GluA1 (right) expressing neurons expressing the AIS marker ankyrinG-mCh (not shown) and a cell fill (green). The axon is indicated by the white arrowhead. Magnified images to the right (taken from the yellow boxes) display surface signal at the AIS for DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) before and 60 min after ER release. Note that Alexa647-anti-GFP was present in the extracellular solution following ER release to continuously trap and label proteins as they surfaced. Red arrowheads denote accumulated cargo at the surface of the AIS. Scale bars, 20 µm; inset, 10 µm. (B) AIS surface signal (expressed as a fraction of total surface signal throughout the entire cell) for DHFR-GFP-NL1 (orange) or DHFR-GFP-GluA1 (blue) 60 min after ER release; mean ± SEM; ****, P < 0.0001 (Student's t test, n = 8 from two independent experiments for NL1 and GluA1). (C) Surface trafficking at the AIS occurs early following ER release. Confocal image (left) and heatmap displaying the timing and location of DHFR-GFP-NL1 surface appearance (right). Insets show the AIS and proximal and distal dendrites. Scale bars, 20 µm. Inset scale bars, 5 µm. (D) Shown are mean NL1 surface intensities (normalized to their maximum values) at the AIS (teal line), soma (pink line), proximal dendrites (5–40 µm from the soma; peach line) or distal dendrites (40–200 µm from the soma; lavender line); mean ± SEM ( n = 10 neurons/time point from two independent experiments). (E) Time to reach 10% of maximum DHFR-GFP-NL1 surface signal is plotted for each subcellular compartment; mean ± SEM; *, P < 0.05; ****, P < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test; n = 10 neurons from two independent experiments). (F) ER release can be titrated by decreasing photoexcitation power. DHFR-GFP-NL1 accumulation in the GA is plotted following illumination with decreasing 405-nm light intensities (912 µW, 194 µW, and 97 µW); mean ± SEM ( n = 7–14 neurons/condition from at least two independent experiments). (G) DHFR-GFP-NL1 appears at the surface of the AIS even when decreasing amounts are released from the ER. GluA1 surface signal at the AIS following exposure to a saturating light intensity (912 µW) is shown for comparison; mean ± SEM; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test; n = 5–8 neurons/condition from two independent experiments). (H) DHFR-GFP-NL1 traffics to the surface of the AIS when network activity is elevated (Bic) or suppressed (TTX). Student's t test; n = 7 neurons/condition from three independent experiments. norm., normalized; prox., proximal.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Expressing, Marker, Comparison, Activity Assay

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: NL1 is inserted into the plasma membrane of the AIS. Shown is a hippocampal neuron expressing DHFR-GFP-NL1 along with unlabeled FKBP-KDEL, ankyrinG-mCh (red), and a GFP cell fill (green) before and after full-field 405-nm illumination in the presence of Alexa647-anti-GFP (surface signal is shown in grayscale). The first frame after photoexcitation is denoted by the appearance of the white circle (upper right corner). The duration of the video is 50 min with a baseline acquisition rate of 1 frame/2 min and a post-release acquisition rate of 1 frame/2.5 min. Time stamp displays the time elapsed following full-field 405-nm illumination at time 0. Scale bar, 10 µm. Playback speed, 2.5 min between frames.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Clinical Proteomics, Membrane, Expressing

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Control experiments for surface NL1 accumulation at the AIS. (A) Antibody-labeled DHFR-GFP-NL1 signal is localized to the cell surface. DHFR-GFP-NL1 was released from the ER and continuously cross-linked and labeled with Alexa647-anti-GFP as it appeared at the cell surface for 50 min after ER release. Left: The timing and distribution of accumulated Alexa647-anti-GFP (generated in rabbit) immediately before addition of Alexa568-anti-rabbit secondary to label cell surface Alexa647-anti-GFP. The center panel shows the same neuron 10 min following addition of Alexa568-anti-rabbit (cyan). Insets to the right show the AIS, taken from the yellow box in the image to the left. The robust colocalization of Alexa647-anti-GFP and Alexa568-anti-rabbit (arrowheads) confirms accumulated DHFR-GFP-NL1 is on the cell surface. Scale bars, 10 µm. Inset scale bar, 5 µm. (B) Comparison of the fraction of total surface cargo at the AIS for retained/released DHFR-GFP-NL1 in the continuous presence of cross-linking antibody (orange) versus three trafficking controls: SEP-GluA1 (dark gray), nonretained (no zapalog) DHFR-GFP-NL1 (gray), and DHFR-GFP-NL1 that was released and allowed to traffic for 3 h before addition of cross-linking antibody (light gray). The retained/released DHFR-GFP-NL1 is the same data shown in with Alexa647-anti-GFP present for the duration of the experiment; mean ± SEM; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA, Tukey’s multiple comparisons test; n = 8 [retained/released DHFR-GFP-NL1]; n = 6 [SEP-NL1]; n = 7 [nonretained DHFR-GFP-NL1]; n = 6 [3 h delayed addition cntrl]; n = number of neurons). cntrl, control.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Control, Labeling, Generated, Comparison

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Local ER release from dendrites reveals the rate, spatial dynamics, and activity dependence of remote secretory trafficking. (A) Schematic of experimental strategy. NL1 and GluA1 were locally released from a user-defined dendritic branch in the presence of either TTX to suppress or Bic to elevate neuronal activity. (B) Representative image (left) and time series of intracellular DHFR-HaloTag-NL1 (labeled with JF646) signal at the soma (center) and dendrites (right) shortly following local dendritic ER release (pink rectangle). Blue arrowheads mark the appearance of vesicular structures following illumination. The neighboring unstimulated control branch (white rectangle) is shown for comparison. At the end of the experiment, the cell was exposed to global full-field illumination and imaged 10 min later. Note the robust DHFR-GFP-NL1 accumulation in the somatic GA following global but not local dendritic release (middle). Scale bar, 10 µm; magnified panels, 5 µm. (C) Images showing cellular morphology (cell fill, left), DHFR-GFP-NL1 surface signal (center), and timing/location of surface trafficking (right) following local dendritic ER release. The photoactivated region is denoted by the dashed pink rectangle. The black and pink (solid lines) boxes denote the dendritic regions shown in G. Scale bars, 10 µm. (D) Images showing cellular morphology (cell fill, left), surface DHFR-GFP-GluA1 signal (center), and timing/location of surface trafficking (right) following local dendritic ER release (pink dashed rectangle). The black and pink (solid lines) boxes denote the dendritic regions shown in G. Scale bars, 10 µm. (E) Time course of DHFR-GFP-NL1 surface signal inside (in) and outside (out) the ER release zone in the presence of TTX (left) or Bic (right); mean ± SEM. No significant differences were found at any time point (two-way ANOVA, Bonferroni’s multiple comparisons test). The plots to the right show the ratio of signal in versus out of the release zone and the ratio of total dendritic to somatic signal 30 and 60 min following dendritic ER release in the presence of TTX (orange) or Bic (green); mean ± SEM (Student's t test; n = 4–7 from at least three independent experiments). (F) Time course of DHFR-GFP-GluA1 surface signal inside (in) and outside (out) the ER release zone in the presence of TTX (left) or Bic (right). No significant differences were found at any time point (two-way ANOVA, Bonferroni’s multiple comparisons test); mean ± SEM. The plots to the right show the ratio of signal in versus out of the release zone and the ratio of total dendritic to somatic signal 30 and 60 min following dendritic ER release in the presence of TTX (orange) or Bic (green); mean ± SEM (Student's t test; n = 3–7 from at least two independent experiments). (G) Image series of surface-labeled DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) inside and outside the photoactivated region depicted in C and D. Solid and open arrowheads denote cargo-positive and -negative spines, respectively. Scale bars, 2 µm. (H) Comparison of the fraction of spines with DHFR-GFP-GluA1 or DHFR-GFP-NL1 within the release zone (purple) versus randomly selected regions of the same size in separate control dendrites (gray) 60 min following local dendritic ER release in the presence of TTX (left) or Bic (right); mean ± SEM; *, P < 0.05 (paired t test; n = 4–6 from at least three independent experiments). dend., dendritic; surf., surface.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Activity Assay, Labeling, Control, Comparison

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Following local dendritic ER release, cargo can be transported out of the release zone. Shown are hippocampal neurons expressing either TfR-GFP-DHFR (top) or DHFR-Halo-NL1 (bottom, labeled with JaneliaFluor 646) along with FKBP-mCh-KDEL (signal not shown). ER-retained cargo was released at time 0 using focally directed 405-nm excitation. The timing and location of ER release are shown by purple rectangles. Arrows denote examples of mobile carriers exiting the release zone. The dimensions of the top panel (TfR) are 13 µm × 45 µm; bottom panel (NL1), 20 µm × 45 µm. Playback speed, 15 sec between frames.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Expressing, Labeling

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Local release from the cell body ER reveals direct, long range trafficking to dendrites. (A) Schematic of experimental strategy. DHFR-GFP-NL1 and DHFR-GFP-GluA1 were locally released from the soma in the presence of TTX to suppress or Bic to elevate neuronal activity. (B) Example of DHFR-GFP-NL1 intracellular localization before (left) and 14 min after (right) somatic ER release (pink circle). The magnified images to the right show the soma (top) and a section of dendrite (bottom) before and after ER release. Note the absence of vesicular structures appearing in the dendrites shortly following somatic release. Scale bar, 6 µm. Top inset scale bar, 6 µm. Bottom inset scale bar, 3 µm. (C) Merged confocal images showing cell fill (green) and surface signal (Alexa647-anti-GFP, magenta) for DHFR-GFP-NL1 (left) and DHFR-GFP-GluA1 (right) 90 min following local ER release (white circles). Surface signal is shown in the heatmap images. Insets show the soma, proximal, and distal dendrites 90 min after release. Scale bars, 20 µm. Soma inset scale bars, 5 µm. Dendrite inset scale bars, 2 µm. (D) Time to 10% surface accumulation following somatic ER release in the presence of TTX (orange bars) or Bic (green bars) for NL1 and GluA1. Mean ± SEM; **, P < 0.01 (Student's t test; n = 5–8 neurons from at least three independent experiments). (E) Time course of DHFR-GFP-NL1 surface trafficking (over the entire cell) following somatic ER release in the presence of TTX (orange line) or Bic (green line). Surface signal at 90 min is shown to the right; mean ± SEM (Student's t test; n = 5 neurons/condition from three independent experiments). (F) Time course of DHFR-GFP-GluA1 surface trafficking (over the entire cell) following somatic ER release in the presence of TTX (orange line) or Bic (green line). The gray shaded regions designate P < 0.05, two-way ANOVA, Bonferroni’s multiple comparisons test; n = 8 neurons/condition from two independent experiments. Surface signal at 90 min is shown on the right; mean ± SEM; *, P < 0.05 (Student's t test). (G) Time course of DHFR-GFP-GluA1 surface trafficking at the soma (left), proximal dendrites (<40 µm from the soma; center), and distal dendrites (40–200 µm from the soma; right) in the presence of TTX or Bic; mean ± SEM. The gray shaded region designates P < 0.05, two-way ANOVA, Bonferroni’s multiple comparisons test. (H) The ratio of total dendritic to somatic signal is plotted at different time points following local ER release from the soma (blue) or from dendrites (teal) for DHFR-GFP-NL1 (left) and DHFR-GFP-GluA1 (right); mean ± SEM; *, P < 0.05 (Student's t test; n = 9–16 neurons from at least two independent experiments). dist., distal; prox., proximal.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Activity Assay

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Subcellular distribution of NL1 and GluA1 surface accumulation following somatic ER release. (A) Images of DHFR-GFP-NL1 (top) and DHFR-GFP-GluA1 (bottom) surface accumulation at the soma (left), proximal dendrites (middle), and distal dendrites (right) before and 90 min following somatic ER release in the presence of TTX or Bic. Merged images show the Alexa647-anti-GFP signal (magenta) and the cell fill (green). White arrowheads indicate spines with detectable levels of surface signal. Scale bars, 5 µm (somatic images); 2 µm (dendritic images). (B) Time course of the fraction of spines in proximal (circles) and distal (triangles) dendrites that contain surface NL1 (gray) or GluA1 (black) following somatic ER release in the presence of TTX. A comparison of the fraction of NL1- and GluA1-positive spines at 90 min and 120 min is shown on the right; mean ± SEM; *, P < 0.05; **, P < 0.01 (unpaired t test; n = 5 neurons [NL1], n = 7 neurons [GluA1] from at least two independent experiments). (C) Time course of the fraction of spines in proximal (circles) and distal (triangles) dendrites that contain surface NL1 (gray) or GluA1 (black) following somatic ER release in the presence of Bic. A comparison of the fraction of NL1- and GluA1-positive spines at 90 min and 120 min is shown on the right; mean ± SEM; *, P < 0.05; ***, P < 0.001 (unpaired t test; n = 5 neurons [NL1], n = 8 neurons [GluA1] from at least two independent experiments). dist., distal; prox., proximal.

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Comparison

Journal: The Journal of Cell Biology

Article Title: zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways

doi: 10.1083/jcb.202103186

Figure Lengend Snippet: Contribution of lateral diffusion/recycling to protein localization in distal dendrites and spines following global and somatic ER release. (A) Experimental schematic. Cargoes are released from the ER in the absence of cross-linking antibody and allowed to traffic for 90 min before adding Alexa647-anti-GFP to label surface protein. (B) Distribution of surface DHFR-GFP-NL1 and DHFR-GFP-GluA1 following global ER release under cross-linking (anti-GFP present throughout the experiment) and non–cross-linking (anti-GFP added 90 min post-ER release) conditions. The ratio of total surface signal in dendrites (>40 µm from the soma) versus soma is shown in the middle panel. The fraction of spines with each cargo is plotted to the right. n values correspond to the number of cells, from at least two independent experiments (**, P < 0.01; ****, P < 0.0001; Student's t test). (C) Same experiment as in B, except cargoes were locally released from the somatic ER. n values correspond to the number of cells, from at least two independent experiments (*, P < 0.05; ***, P < 0.001; Student's t test).

Article Snippet: The DHHC2-mCh plasmid was a gift from Dr. Mark Dell’Acqua (University of Colorado Anschutz Medical Campus, Aurora, CO); the mEmerald-GalT (aa 1–82 Golgi targeting sequence of human GalT) plasmid was a gift from Dr. Michael Davidson (Addgene plasmid #54108); the mCh-Sec61 plasmid was a gift from Dr. Jennifer Lippincott-Schwartz (Janelia Farm Research Campus, Ashburn, VA; Addgene plasmid #90994); the AnkyrinG-mCherry plasmid was a gift from Dr. Katharine R. Smith (University of Colorado Anschutz Medical Campus, Aurora, CO); the cDNAs for GluA1 and TfR were gifts from Dr. Michael Ehlers (Duke University, Durham, NC); the NL1 open reading frame was a gift from Dr. Peter Scheiffele (University of Basel, Basel, Switzerland; Addgene clone #15262); and the Rab11a reading frame was a gift from Dr. Richard Pagano’s laboratory (Addgene clone #12674).

Techniques: Diffusion-based Assay