Journal: The Journal of Cell Biology

Article Title: APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

doi: 10.1083/jcb.201311117

Figure Lengend Snippet: Models of cargo trafficking through APPL and EEA1 compartments. (A) In model 1, the APPL compartment serves as an intermediate en route to EEA1-positive endosomes. Cargo first binds to receptors on the PM and is internalized via CDE or CIE. The CDE includes formation of CCPs and internalization CCVs. Some CCVs acquire APPL1 or fuse with APPL1 membranes. Other CCVs and CIV directly fuse with EEA1 endosomes. APPL1 vesicles directly (10% through an APPL1+EEA1 double-positive endocytic intermediate [A & E]) or indirectly (47%+43%) convert into EEA1 endosomes. Cargo can be recycled to the surface via recycling endosomes (RE) or transported to late endosomes (LE) and lysosomes for degradation. Blue and red arrows demark transport of Tf and EGF, respectively. Black arrows, steps that must be common for both cargos. (B) In model 2, APPL1 endosomes that were not accounted in the literature (43%) constitute a stable endocytic compartment. These endosomes sort cargo for recycling and bi-directionally exchange of cargo with EEA1 endosomes through APPL1+EEA1 double-positive endosomes. Transition to late endocytic compartment occurs through EEA1 by conversion mechanism.

Article Snippet: HeLa cells were transiently transfected using DreamFect Gold reagent (OZ BioSciences) or Effectene (Qiagen) for 24 h with plasmids pEGFP-C3-APPL1 , pEGFP-C3-Rab5 , mRFP-human APPL1 (a gift from P. De Camilli [Addgene plasmid 22202]) or tagRFP-T-EEA1 (a gift from S. Corvera, University of Massachusetts Medical School, Worcester, MA [Addgene plasmid 42635]) and analyzed 24 h after transfection.

Techniques:

Journal: The Journal of Cell Biology

Article Title: APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

doi: 10.1083/jcb.201311117

Figure Lengend Snippet: Immunoelectron microscopic localization of APPL1. HeLa cells were labeled with antibodies to APPL1, followed by a Nanogold-labeled conjugate. Gold particles were visualized by silver enhancement. (A) A low-magnification view with two APPL-positive structures circled in orange, one of which is shown at higher magnification in the inset. (B–F) A gallery of representative structures. Dense labeling is associated with small tubular profiles close to the PM or deeper inside the cell as well as with larger heterogeneous structures. Note that labeling of the PM or CCPs is very low. M, mitochondria. Bars: (A, C, and E) 500 nm; (A [inset], B, D, and F) 200 nm.

Article Snippet: HeLa cells were transiently transfected using DreamFect Gold reagent (OZ BioSciences) or Effectene (Qiagen) for 24 h with plasmids pEGFP-C3-APPL1 , pEGFP-C3-Rab5 , mRFP-human APPL1 (a gift from P. De Camilli [Addgene plasmid 22202]) or tagRFP-T-EEA1 (a gift from S. Corvera, University of Massachusetts Medical School, Worcester, MA [Addgene plasmid 42635]) and analyzed 24 h after transfection.

Techniques: Labeling

Journal: The Journal of Cell Biology

Article Title: APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

doi: 10.1083/jcb.201311117

Figure Lengend Snippet: Cargo internalization into APPL endosomes is clathrin dependent but their biogenesis is not. (A) Silencing of CHC by RNAi in HeLa cells assessed by Western blot in comparison to EEA1, APPL1, and Rab5 as controls. (B) Internalization of biotinylated Tf (b-Tf) (after 30 min of continuous uptake) is inhibited upon CHC knockdown. The amounts of b-Tf in cell lysates were quantified by electrochemiluminescence. (C ) Knockdown of CHC decreased colocalization of Tf to EEA1 (red) and APPL1 (blue). Colocalization was quantified after 3.5-min chase after 0.5-min internalization pulse of Tf. (D–F) Knockdown of clathrin inhibits Tf uptake but does not affect the number of APPL1-positive vesicles. Example images of endogenous APPL1 and fluorescent Tf at 3.5-min chase after the 30-s internalization pulse in control and clathrin-depleted cells (D). Inset presents full image, yellow rectangle depicts zoomed part. The numbers of vesicles marked by APPL1 (red), Tf (blue) or EGF (green) (E) and their integral intensities (F) are plotted (quantifications based on 80 images and ∼320,000 APPL1 endosomes). (G–I) Dynasore treatment (from 10 to 60 min) does not affect the number of APPL1-positive vesicles but progressively suppresses Tf uptake (10 min of Tf internalization). (G) Example images of HeLa cells treated with Dynasore (80 µM) for 60 min. The numbers of vesicles marked by APPL1 (red) and Tf (blue) (H) and their integral intensities (I) in cells pretreated with Dynasore for the indicated times are plotted (quantifications based on 10 images, ∼110 cells, and ∼45,000 APPL1 endosomes). Bars: (D and G, inset) 10 µm.

Article Snippet: HeLa cells were transiently transfected using DreamFect Gold reagent (OZ BioSciences) or Effectene (Qiagen) for 24 h with plasmids pEGFP-C3-APPL1 , pEGFP-C3-Rab5 , mRFP-human APPL1 (a gift from P. De Camilli [Addgene plasmid 22202]) or tagRFP-T-EEA1 (a gift from S. Corvera, University of Massachusetts Medical School, Worcester, MA [Addgene plasmid 42635]) and analyzed 24 h after transfection.

Techniques: Western Blot, Comparison, Knockdown, Electrochemiluminescence, Control

Journal: The Journal of Cell Biology

Article Title: APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

doi: 10.1083/jcb.201311117

Figure Lengend Snippet: APPL endosomes are stable structures. (A) Gallery of images showing a long-lived APPL endosome (arrow) containing internalized Tf, tracked for 12 min in HeLa cells expressing EGFP-APPL1 (see Video 1 ). (B) APPL1 (green) endosome with EGF (blue) and Tf (red) sorts Tf from EGF. Tf-positive tubule growing over time and pinched-off from APPL1 endosome (see Video 3 ). (C) Double EEA1 (blue) + APPL1 (green) endosome produces Tf- (red) and APPL1-positive, EEA1-negative tubule (see Video 4 ). Later this tubule was separated from the main endosome body.

Article Snippet: HeLa cells were transiently transfected using DreamFect Gold reagent (OZ BioSciences) or Effectene (Qiagen) for 24 h with plasmids pEGFP-C3-APPL1 , pEGFP-C3-Rab5 , mRFP-human APPL1 (a gift from P. De Camilli [Addgene plasmid 22202]) or tagRFP-T-EEA1 (a gift from S. Corvera, University of Massachusetts Medical School, Worcester, MA [Addgene plasmid 42635]) and analyzed 24 h after transfection.

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

doi: 10.1083/jcb.201311117

Figure Lengend Snippet: APPL endosomes exhibit features of cargo sorting by live cell imaging. ( A) Sequential images showing an EEA1-positive APPL1-negative vesicle carrying EGF, which fuses with multiple preexisting APPL1 endosomes (see Video 5 ). (B) Sequential images depicting fusion of a double APPL1+EEA1–positive endosome with an APPL1-positive endosome (see Video 6 ). (C) Double APPL1+EEA1 endosome gradually loses APPL1 and converts to EEA1 endosome (see Video 7 ). (D) Double APPL1+EEA1–positive endosome gradually loses EEA1 and converts to APPL1 endosome (see Video 8 ). (E) Relative frequencies of individual events of interaction between APPL1- (A), EEA1- (E), and double APPL1+EEA1 (AE)–positive endosomes (error bars represent SEMs). Data were collected from 34 movies.Total number of events equals 234.

Article Snippet: HeLa cells were transiently transfected using DreamFect Gold reagent (OZ BioSciences) or Effectene (Qiagen) for 24 h with plasmids pEGFP-C3-APPL1 , pEGFP-C3-Rab5 , mRFP-human APPL1 (a gift from P. De Camilli [Addgene plasmid 22202]) or tagRFP-T-EEA1 (a gift from S. Corvera, University of Massachusetts Medical School, Worcester, MA [Addgene plasmid 42635]) and analyzed 24 h after transfection.

Techniques: Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

doi: 10.1083/jcb.201311117

Figure Lengend Snippet: Time course of cargo distribution in EEA1- and APPL1-positive structures. Fluorescently labeled Tf and EGF were internalized for 30 s and chased for the indicated periods of time (A). All experimental data are the mean of four independent experiments. The intensity of Tf and EGF colocalized to EEA1 and APPL1 was corrected for apparent (random) colocalization . (B and C) Kinetics of fluorescence intensity of Tf (B) or EGF (C) colocalized with EEA1-positive and APPL1-negative endosomes (red), APPL1-positive and EEA1-negative endosomes (blue), with double EEA1+APPL1–positive endosomes (green) and colocalized with none of them (black). The inset shows the part of the curve corresponding to the initial 3 min of the time course. (D) Time course of colocalization of EGF-with-Tf (magenta) and Tf-with-EGF (green) on all endosomes. (E) Time course of colocalization of EGF-with-Tf (magenta) and Tf-with-EGF (green) on APPL1-positive and EEA1-negative endosomes. This colocalization is defined as the ratio of the amount of EGF colocalized with Tf on APPL1 endosomes to the total amount of EGF on APPL endosomes. The same applies to Tf-with-EGF colocalization on APPL endosomes. Error bars represent SEMs.

Article Snippet: HeLa cells were transiently transfected using DreamFect Gold reagent (OZ BioSciences) or Effectene (Qiagen) for 24 h with plasmids pEGFP-C3-APPL1 , pEGFP-C3-Rab5 , mRFP-human APPL1 (a gift from P. De Camilli [Addgene plasmid 22202]) or tagRFP-T-EEA1 (a gift from S. Corvera, University of Massachusetts Medical School, Worcester, MA [Addgene plasmid 42635]) and analyzed 24 h after transfection.

Techniques: Labeling, Fluorescence

Journal: The Journal of Cell Biology

Article Title: APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

doi: 10.1083/jcb.201311117

Figure Lengend Snippet: Proposed models of cargo trafficking through APPL and EEA1 compartments. ( A) In model 2, cargo on PM follows either the clathrin pathway through CCPs to CCVs or the clathrin-independent route by CIVs. About 94% of Tf (blue) and 25% of EGF (red) follow CDE. Almost 75% of EGF and only 6% of Tf are internalized by CIVs. In line with model 1, A and B, we considered two pools of CCV that deliver cargo to APPL1-positive (36% EGF and 99.5%Tf) and EEA1-positive (64% EGF, 0.5%Tf) endosomes. The flux of Tf through CCV to EEA1 increased up to 22% by down-regulation of APPL1. The fit of model suggests that CIVs deliver cargo to APPL1+EEA1 and EEA1 endosomes. The dynamic of cargo traffic through EEA1-positive demonstrated complex behavior that cannot be explained in case of kinetically homogeneous compartments. The down-regulation of APPL1 and CHC revealed that cargo traffic consists of two components that can be separately inhibited. Therefore, we introduced in the model 2 two kinetically distinct EEA1 compartments, which we denote EEA1(ccv) and EEA1(civ) according to the main mode of cargo delivery. The corresponding double APPL1+EEA1 compartments were denoted A&E(ccv) and A&E(civ) accordingly. The sorting of cargo toward the recycling route occurs in the all three endocytic compartments APPL1, APPL1+EEA1, and EEA1. However, the delivery of EGF to the late endosomes (LE) and following degradation (∼70% of EGF degrade in 30 min) occurs only through EEA1 compartment. We denote the recycling endosomes en route to PM and perinuclear recycling endosome in accordance to the kinetic rates of either fast recycling endosomes (FRE) or recycling endosomes RE. The thin arrows denote the routes that transport less than 10% of cargo from compartment, however the removal of them makes the fit to the experimental data unsatisfactory (P < 0.01). (B–D) Tables present results of the best fit of model 1A (B), model 1B (C), and model 2 (D) to the experimental data in control and perturbed cell. X 2 /N denotes normalized χ 2 : χ 2 / N = 1 N ∑ i = 1 N + 1 ( f i − d i ) 2 σ i 2 , where f i and d i are model prediction and experimental data, σ i is SEM of experimental data, N = 167. The p-values were calculated by χ 2 distribution. (B) The probability of null hypothesis that the deviation of the model prediction from the experiment is the result of random noise (p-value) is extremely low for all four conditions. Therefore, model 1A has to be rejected. (C) The probability of null hypothesis is very low for all conditions, although the logarithm of probability of model 1B (see Materials and methods) is much higher than those of 1A. Nevertheless, model 1B has to be rejected as well. (D) The probability of null hypothesis is high. Therefore, most probably the deviation of model 2 from the experiment is the result of experimental uncertainty. The model 2 is much more probable ln( P ) > 100 than model 1B for all conditions. Therefore, the improvement of the quality of fit is statistically significant to justify three additional parameters.

Article Snippet: HeLa cells were transiently transfected using DreamFect Gold reagent (OZ BioSciences) or Effectene (Qiagen) for 24 h with plasmids pEGFP-C3-APPL1 , pEGFP-C3-Rab5 , mRFP-human APPL1 (a gift from P. De Camilli [Addgene plasmid 22202]) or tagRFP-T-EEA1 (a gift from S. Corvera, University of Massachusetts Medical School, Worcester, MA [Addgene plasmid 42635]) and analyzed 24 h after transfection.

Techniques: Control

Journal: The Journal of Cell Biology

Article Title: APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

doi: 10.1083/jcb.201311117

Figure Lengend Snippet: Time course of Tf (A, C, and E) and EGF (B, D, and F) distribution in EEA1- and APPL1-positive structures under down-regulation of CHC, APPL1, and EEA1. Cells were transfected by siRNA for CHC (A and B), APPL1 (C and D) and EEA1 (E and F) for 48 h (see Materials and methods). Then fluorescently labeled Tf and EGF were internalized and chased as described in . Solid circles present the integral intensity of cargo colocalized with EEA1- (red), APPL1- (blue), and double APPL1+EEA1–positive endosomes (green). The control curves are presented by empty squares. Down-regulation of CHC, APPL1, and EEA1 was 95%, 70%, and 90%, respectively. Traffic of cargo in the control condition is repeatedly presented on panels A–F by empty squares. (G) Integral intensity of EGF colocalized with EEA1 under CHC knockdown (green) and APPL1 knockdown (blue). Sum of blue and green curves (sum of integral intensities of EGF colocalized with EEA1 upon APPL1 or CHC knockdown) is plotted by solid black circles. The time course of integral intensities of EGF colocalized with EEA1 for control is plotted by red squares.

Article Snippet: HeLa cells were transiently transfected using DreamFect Gold reagent (OZ BioSciences) or Effectene (Qiagen) for 24 h with plasmids pEGFP-C3-APPL1 , pEGFP-C3-Rab5 , mRFP-human APPL1 (a gift from P. De Camilli [Addgene plasmid 22202]) or tagRFP-T-EEA1 (a gift from S. Corvera, University of Massachusetts Medical School, Worcester, MA [Addgene plasmid 42635]) and analyzed 24 h after transfection.

Techniques: Transfection, Labeling, Control, Knockdown

Journal: Nature Communications

Article Title: Deterministic early endosomal maturations emerge from a stochastic trigger-and-convert mechanism

doi: 10.1038/s41467-023-40428-1

Figure Lengend Snippet: a FLIM of orientation with phosphoinositide lipid specificity. Scatter and box-and-whisker plots of PI(3)P staining intensity by endosome type; Rab5 positive and EEA1 negative ( magenta ), NT EEA1 bound ( blue ), or CT EEA1 bound ( green ). PI(3)P intensities are measured using 2xFYVE-GST labelling and normalised across each cell. Error bars on scatter plots indicate mean ± S.D. Statistical significance determined using one-way ANOVA, ** indicates p < 0.01, **** indicates p < 0.0001(EEA1 vs. NT EEA1 p = 0.0086, No EEA1 vs. CT EEA1 p = 1.32 × 10 −6 , NT EEA1 vs. CT EEA1 p = 3.45 × 10 −5 ) n = 18 cells. Single points indicate measured data, and the box plots correspond to the 25th to 75th percentile of events, with bars showing the total range. b SAR405-treated cells show reduced fusions between APPL1 and EEA1 endosomes, but not reduced APPL1 to EEA1 conversions. siRNA-INPP4A-treated cells show reduced fusions and conversions. Single points indicate measured data; the violin plots correspond to a normal distribution of all events; and the box plots correspond to the standard error of the mean, with bars showing the total range. Means are depicted by the open squares. Statistical significance was determined using one-way ANOVA (Conversions: untreated vs. SAR 405. p = 0.2936, Conversions: untreated vs SiRNA INPP4A. p = 1.45 × 10 −5 , Fusions: untreated vs SAR405 p = 0.36 × 10 −5 , Fusions: untreated vs SiRNA INPP4A. p = 0.85 × 10 −5 ; n = 5 cells per condition). c RPE1 wild-type cells and HeLa EEA1 knockout (KO) cell lines expressing wild-type EEA1 ( blue ) or N-terminal mutant deficient in binding Rab5 ( red ) were imaged using LLSM. d The total number of conversions and fusions were quantified for WT EEA1 (blue shade) and EEA1 Nt-mutant (Grey shade); these data indicate that the initial N-terminal of EEA1 is essential for endosomal conversions. ns indicates non-significant difference, * indicates p < 0.05; p = 0.0018. Each mean was compared against the others using an ordinary one-way ANOVA. In the case of HeLa EEA1 KO cells expressing EEA1 N-terminal Rab5 binding mutant, no events were detected by the analysis workflow or by visual inspection. Single points indicate measured data; the violin plots correspond to a normal distribution of all events; and the box plots correspond to the standard deviations of events, with bars showing the total range. Means are depicted by the open squares ( n = 6 cells per condition).

Article Snippet: Wild-type HeLa cells were transfected with pEGFPC1-human APPL1, a gift from Pietro De Camilli (Addgene plasmid #22198) ; EEA1 TagRFP-T, a gift from Silvia Corvera (Addgene plasmid #42635) ; EGFP-EEA1, a gift from Silvia Corvera (Addgene plasmid #42307) ; EGFP-Rab5, a gift from Marci Scidmore (Addgene plasmid #49888); and mRFP-Rab5, a gift from Ari Helenius (Addgene plasmid #14437) .

Techniques: Whisker Assay, Staining, Knock-Out, Expressing, Mutagenesis, Binding Assay

Journal: The Journal of Neuroscience

Article Title: Increased Neuronal Expression of the Early Endosomal Adaptor APPL1 Replicates Alzheimer’s Disease-Related Endosomal and Synaptic Dysfunction with Cholinergic Neurodegeneration

doi: 10.1523/JNEUROSCI.2331-24.2025

Figure Lengend Snippet: APPL1 transgene expression in the brain. A , Representative Western blot comparing APPL1 expression in various brain regions (CTX, cortex; HIP, hippocampus; CBL, cerebellum; RB, remaining brain) from 4- to 5-month-old nontransgenic littermate (non-Tg) and Thy1-APPL1 transgenic (Thy1-APPL1) mice. Overexpressed flag-tagged APPL1 proteins were detected in all brain regions in Thy1-APPL1 mice. B , Representative brain section immunolabeling with an anti-APPL1 antibody at low magnification (scale bar, 1 mm) demonstrates the APPL1 overexpression patterns in various brain regions of Thy1-APPL1 mice. C , Representative immunofluorescent images at low (40×) and high (120×) magnification show APPL1 (green) and NeuN (red) with DAPI (blue) staining in cortex layer V (scale bar, 10 µm; yellow arrows indicate neurons, white arrowheads identify non-neuronal cells; genotype as indicated). D , Western blots probed with anti-APPL1 and anti-rab5 antibodies of both non-Tg and Thy1-APPL1 brain homogenate prepared from mice of the indicated ages. E , Western blot analysis quantification showing increased APPL1 in the brain homogenates of Thy1-APPL1 mice compared with non-Tg mice at the indicated ages. ** p < 0.01, *** p < 0.001, two-tailed, unpaired t test. Data shown as mean ± SEM.

Article Snippet: After >48 h of fixation, 40-μm-thick vibratome brain tissue sections from various brain regions were collected and washed three times with antibody dilution buffer containing bovine serum albumin (BSA; 1%; Sigma), saponin (0.05%; Sigma), and normal horse serum (NHS; 1%; Thermo Fisher) in Tris-buffered saline (TBS), pH 7.4 and then blocked with 20% NHS in TBS for 1 h at room temperature before incubation with commercial antibodies against rab5 (Abcam, catalog #ab18211, RRID:AB_470264; 1:500; ; ), rab5-GTP (active rab5: NewEast Biosciences, catalog #26911, RRID:AB_2617182; 1:50; ; ), ChAT (Millipore, catalog #AB144P, RRID:AB_2079751; 1:250; ; ), APPL1 (Proteintech, catalog #12639-1-AP, RRID:AB_2289669; 1:500; ), and the Flag-tag (Thermo Fisher Scientific, catalog #MA1-91878-D488, RRID:AB_2537621; 1:500), NeuN (Millipore, catalog #MAB377, RRID:AB_2298772; 1:2000; ), either alone or in combination and visualized with either biotinylated secondary antibodies (Vector Laboratories; catalog #BA-9200, BA-1000 and BA-6000; 1:500) or fluorescence-conjugated secondary antibodies (Thermo Fisher; catalog #A10037, A21206 and A21099; 1:500), as described in our previous studies ( ; ).

Techniques: Expressing, Western Blot, Transgenic Assay, Immunolabeling, Over Expression, Staining, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Increased Neuronal Expression of the Early Endosomal Adaptor APPL1 Replicates Alzheimer’s Disease-Related Endosomal and Synaptic Dysfunction with Cholinergic Neurodegeneration

doi: 10.1523/JNEUROSCI.2331-24.2025

Figure Lengend Snippet: Early endosome alterations in Thy1-APPL1 mice. A , Representative immunofluorescent images of active, GTP-bound rab5 (rab5-GTP; red) and APPL1 (green) in neurons (layer V, prefrontal cortex) of non-Tg and Thy1-APPL1 mice at 12–13 months of age. Shown are images at lower magnification (40×, merged). The white arrow indicating the neuron shown at higher magnification for individual and merged immunofluorescent signal. Scale bar, 10 µm. B , Quantification of the average number, size, and total area of Rab5-GTP immunolabeled endosomes per cortical neuron in non-Tg versus Thy1-APPL1 mice at the indicated ages. Representative electron microscopy images containing dendritic profiles in layer V of prefrontal cortex ( C ) and hippocampus ( D ) regions and ( E ) quantification of average size and circumference of endosomes in both cortex and hippocampi showing the enlargement in Thy1-APPL1 compared with non-Tg mice aged at 12–13 months (red asterisk indicating endosome; 7 mice each genotype for cortex and 4 mice each genotype for hippocampi were used for quantification. Scale bar, 500 nm). Sixty images/mouse were quantified in E ; for cortex sections, a total of 2,635 endosomes from 14 mice were counted, averaging 188 endosomes per mouse; for hippocampal sections, a total of 1,594 endosomes were counted from 8 mice, with an average of 199 endosomes per mouse. F , Representative immunoelectron microscopy images containing dendritic profiles in layer V of prefrontal cortex labeled with rabbit anti-rab5 and mouse anti-APPL1 followed by gold-conjugated secondary anti-rabbit (6 nm, green arrow) and anti-mouse (10 nm, red arrow) in non-Tg and Thy1-APPL1 mice. Scale bar, 100 nm. G , Percentage of rab5/APPL1 double-positive endosomes versus total rab5-positive endosomes in the dendritic region of the cortex and hippocampus significantly increased in Thy1-APPL1 mice (3 mice per genotype. 35–40 images/mouse were used for counting; for cortex sections, a total of 130 endosomes were counted from 6 mice, with an average of 22 endosomes per mouse; for hippocampal sections, a total of 121 endosomes were counted from 6 mice, with an average of 20 endosomes per mouse). * p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed, unpaired t test. Data is shown as mean ± SEM.

Article Snippet: After >48 h of fixation, 40-μm-thick vibratome brain tissue sections from various brain regions were collected and washed three times with antibody dilution buffer containing bovine serum albumin (BSA; 1%; Sigma), saponin (0.05%; Sigma), and normal horse serum (NHS; 1%; Thermo Fisher) in Tris-buffered saline (TBS), pH 7.4 and then blocked with 20% NHS in TBS for 1 h at room temperature before incubation with commercial antibodies against rab5 (Abcam, catalog #ab18211, RRID:AB_470264; 1:500; ; ), rab5-GTP (active rab5: NewEast Biosciences, catalog #26911, RRID:AB_2617182; 1:50; ; ), ChAT (Millipore, catalog #AB144P, RRID:AB_2079751; 1:250; ; ), APPL1 (Proteintech, catalog #12639-1-AP, RRID:AB_2289669; 1:500; ), and the Flag-tag (Thermo Fisher Scientific, catalog #MA1-91878-D488, RRID:AB_2537621; 1:500), NeuN (Millipore, catalog #MAB377, RRID:AB_2298772; 1:2000; ), either alone or in combination and visualized with either biotinylated secondary antibodies (Vector Laboratories; catalog #BA-9200, BA-1000 and BA-6000; 1:500) or fluorescence-conjugated secondary antibodies (Thermo Fisher; catalog #A10037, A21206 and A21099; 1:500), as described in our previous studies ( ; ).

Techniques: Immunolabeling, Electron Microscopy, Immuno-Electron Microscopy, Labeling, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Increased Neuronal Expression of the Early Endosomal Adaptor APPL1 Replicates Alzheimer’s Disease-Related Endosomal and Synaptic Dysfunction with Cholinergic Neurodegeneration

doi: 10.1523/JNEUROSCI.2331-24.2025

Figure Lengend Snippet: Age-related impairments of synaptic plasticity and hippocampal-dependent memory deficit in Thy1-APPL1 mice. A–C , Non-Tg and Thy1-APPL1 mice at 7–9 months of age ( n = 5 for both genotype), input/output relationship plots ( A ), LTP by theta-burst stimulation (TBS) in the Schaffer collateral synapses (CA3-CA1) of hippocampal slices ( B ), and averages of fEPSP slopes at 1, 40, and 80 min following tetanic stimulation ( C ) of the hippocampal slices showing no differences between non-Tg and Thy1-APPL1 mice. D–F , Non-Tg and Thy1-APPL1 mice at 12–13 months of age ( n = 5 for both genotype), input/output relationship plots ( D ), LTP induced by TBS in the Schaffer collateral synapses (CA3-CA1) in hippocampal slices ( E ), and averages of fEPSP slopes at 1, 40, and 80 min ( F ) showing significant reduction in Thy1-APPL1 mice ( F (1,236) = 6.696, p = 0.0103 for the slope, linear regression). G–I , Non-Tg and Thy1-APPL1 mice at 12–13 months of age ( n = 5 for both genotypes), input/output relationship plots ( G ), LTD induced by low-frequency stimulation (LFS; H ) in hippocampal slices of Thy1-APPL1 ( F (1,127) = 14.83, p = 0.0002 for the intercepts, linear regression), and averages of fEPSP slopes at 50 min following LFS induction ( I ) in the hippocampal slices did not show expected reduction in Thy1-APPL1 mice. J , Recognition index at 3 h after familiarization indicated the memory deficit in Thy1-APPL1 mice at 12–13 months of age ( t (18) = 2.187, p = 0.042). * p < 0.05, *** p < 0.001, two-tailed, unpaired t test. Data is shown as mean ± SEM.

Article Snippet: After >48 h of fixation, 40-μm-thick vibratome brain tissue sections from various brain regions were collected and washed three times with antibody dilution buffer containing bovine serum albumin (BSA; 1%; Sigma), saponin (0.05%; Sigma), and normal horse serum (NHS; 1%; Thermo Fisher) in Tris-buffered saline (TBS), pH 7.4 and then blocked with 20% NHS in TBS for 1 h at room temperature before incubation with commercial antibodies against rab5 (Abcam, catalog #ab18211, RRID:AB_470264; 1:500; ; ), rab5-GTP (active rab5: NewEast Biosciences, catalog #26911, RRID:AB_2617182; 1:50; ; ), ChAT (Millipore, catalog #AB144P, RRID:AB_2079751; 1:250; ; ), APPL1 (Proteintech, catalog #12639-1-AP, RRID:AB_2289669; 1:500; ), and the Flag-tag (Thermo Fisher Scientific, catalog #MA1-91878-D488, RRID:AB_2537621; 1:500), NeuN (Millipore, catalog #MAB377, RRID:AB_2298772; 1:2000; ), either alone or in combination and visualized with either biotinylated secondary antibodies (Vector Laboratories; catalog #BA-9200, BA-1000 and BA-6000; 1:500) or fluorescence-conjugated secondary antibodies (Thermo Fisher; catalog #A10037, A21206 and A21099; 1:500), as described in our previous studies ( ; ).

Techniques: Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Increased Neuronal Expression of the Early Endosomal Adaptor APPL1 Replicates Alzheimer’s Disease-Related Endosomal and Synaptic Dysfunction with Cholinergic Neurodegeneration

doi: 10.1523/JNEUROSCI.2331-24.2025

Figure Lengend Snippet: NGF retrograde signaling and involvement of APPL1 in healthy neurons and after a rise in neuronal APPL1 levels related to Alzheimer's disease in APPL1 overexpressing neurons. Schematic diagram depicts ( A ) the endocytosis of APP, NGF, and its receptor TrkA into a rab5 early endosome. TrkB mediation of BDNF signaling by APPL1 (data not shown) is considered to follow a similar sequence. B , Normal NGF signaling is facilitated by recruitment of APPL1, a direct TrkA ligand and adaptor for other signaling molecules mediating retrograde transport of a maturing endosome carrying the NGF signal to the nucleus to activate a neurotrophic transcriptional program supporting functioning of ChAT neurons and other NGF targets. In AD (not shown; see main text), abnormally elevated APP-βCTF levels arising via multiple possible mechanisms raise levels of the activated form of rab5 (rab5-GTP) on endosomal membranes, in part by recruiting more APPL1 to the endosome via the phosphotyrosine binding (PTB) domain of APPL1 . APPL1's greater affinity for rab5-GTP prolongs association of this activated form on the endosome, thus promoting a pathogenic rab5 hyperactivation leading to increased endocytosis, early endosomal fusion, and endosome enlargement, as depicted in C . C , Moderately elevating APPL1 selectively in neurons of Thy1-APPL1 mice phenocopies mouse models of APP-βCTF elevation or rab5 overexpression with respect to rab5 hyperactivation, abnormal endosome enlargement, stasis of endosome transport, synaptic plasticity deficits, and basal forebrain cholinergic neurodegeneration. These pathological effects, as observed in AD brain, also reflect impaired NGF/TrkA signaling and decreased expression of genes for neuronal survival, growth and differentiation ( ; ). Further information is provided in the text and in more detail in reviews ( ; ).

Article Snippet: After >48 h of fixation, 40-μm-thick vibratome brain tissue sections from various brain regions were collected and washed three times with antibody dilution buffer containing bovine serum albumin (BSA; 1%; Sigma), saponin (0.05%; Sigma), and normal horse serum (NHS; 1%; Thermo Fisher) in Tris-buffered saline (TBS), pH 7.4 and then blocked with 20% NHS in TBS for 1 h at room temperature before incubation with commercial antibodies against rab5 (Abcam, catalog #ab18211, RRID:AB_470264; 1:500; ; ), rab5-GTP (active rab5: NewEast Biosciences, catalog #26911, RRID:AB_2617182; 1:50; ; ), ChAT (Millipore, catalog #AB144P, RRID:AB_2079751; 1:250; ; ), APPL1 (Proteintech, catalog #12639-1-AP, RRID:AB_2289669; 1:500; ), and the Flag-tag (Thermo Fisher Scientific, catalog #MA1-91878-D488, RRID:AB_2537621; 1:500), NeuN (Millipore, catalog #MAB377, RRID:AB_2298772; 1:2000; ), either alone or in combination and visualized with either biotinylated secondary antibodies (Vector Laboratories; catalog #BA-9200, BA-1000 and BA-6000; 1:500) or fluorescence-conjugated secondary antibodies (Thermo Fisher; catalog #A10037, A21206 and A21099; 1:500), as described in our previous studies ( ; ).

Techniques: Sequencing, Binding Assay, Over Expression, Expressing

Journal: The Journal of Neuroscience

Article Title: Increased Neuronal Expression of the Early Endosomal Adaptor APPL1 Replicates Alzheimer’s Disease-Related Endosomal and Synaptic Dysfunction with Cholinergic Neurodegeneration

doi: 10.1523/JNEUROSCI.2331-24.2025

Figure Lengend Snippet: Western blot analysis and synaptic vesicle endocytosis (SVE) revealed the abnormalities in hippocampal synaptosomes of Thy1-APPL1 mice. A , Representative Western blots from one of the two experiments showing various protein markers in hippocampal homogenates and hippocampal synaptosome preparations from non-Tg and Thy1-APPL1 mice ( n = 6 each genotype). Quantitation of these Western blots ( B , C ) shows significantly higher levels of APPL1, βCTF, PHF1, and the ratio of PHF1/total tau in synaptosomes of Thy1-APPL1 compared with non-Tg mice at 12–13 months of age. D , Representative fluorescent images of internalized FM4-64 (red) with corresponding CMFDA labeling of total synaptosomes (green) using hippocampal synaptosomes prepared from non-Tg and Thy1-APPL1 mice at 12–13 months of age. E , The ratio of internalized FM4-64 to CMDFA was significantly increased in hippocampal synaptosomes of Thy1-APPL1 mice at 12–13 months, but not in 7 months of age, indicating elevated endocytosis in older Thy1-APPL1 mice compared with non-Tg. F , Representative images of CMFDA (green) labeling followed by anti-rab5 (red) immunolabeling and ( G ) quantification of the intensity of rab5 to CMFDA showing an increase in rab5 immunosignal per CMFDA-labeled synaptosome in hippocampal synaptosomes of Thy1-APPL1 mice at 12–13 months of age. Scale bar, 5 µm, * p < 0.05, *** p < 0.001, two-tailed, unpaired t test. Data shown as mean ± SEM. Western blots of hippocampal synaptosome (10 µg of synaptosome protein, input; H ), and GTP-agarose pull-down of hippocampal synaptosome (200 µg of synaptosome protein) from non-Tg and Thy1-APPL1 mice ( I ) probed with anti-APPL1 and anti-rab5 antibodies, total protein stained by Revert 700 Total Protein Stain are also shown at the bottom of H and I . The APPL1 and rab5 band density in both input and pull-down against total protein in relation with non-Tg are presented in J , * p < 0.05, *** p < 0.001, one-way ANOVA. Data is shown as mean ± SEM.

Article Snippet: After >48 h of fixation, 40-μm-thick vibratome brain tissue sections from various brain regions were collected and washed three times with antibody dilution buffer containing bovine serum albumin (BSA; 1%; Sigma), saponin (0.05%; Sigma), and normal horse serum (NHS; 1%; Thermo Fisher) in Tris-buffered saline (TBS), pH 7.4 and then blocked with 20% NHS in TBS for 1 h at room temperature before incubation with commercial antibodies against rab5 (Abcam, catalog #ab18211, RRID:AB_470264; 1:500; ; ), rab5-GTP (active rab5: NewEast Biosciences, catalog #26911, RRID:AB_2617182; 1:50; ; ), ChAT (Millipore, catalog #AB144P, RRID:AB_2079751; 1:250; ; ), APPL1 (Proteintech, catalog #12639-1-AP, RRID:AB_2289669; 1:500; ), and the Flag-tag (Thermo Fisher Scientific, catalog #MA1-91878-D488, RRID:AB_2537621; 1:500), NeuN (Millipore, catalog #MAB377, RRID:AB_2298772; 1:2000; ), either alone or in combination and visualized with either biotinylated secondary antibodies (Vector Laboratories; catalog #BA-9200, BA-1000 and BA-6000; 1:500) or fluorescence-conjugated secondary antibodies (Thermo Fisher; catalog #A10037, A21206 and A21099; 1:500), as described in our previous studies ( ; ).

Techniques: Western Blot, Quantitation Assay, Labeling, Immunolabeling, Two Tailed Test, Staining

Journal: Science Advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: HeLa cells grown on 100-nm nanostructures and treated with siRNAs [(A) to (C) and (G) to (I)] and/or transfected with constructs [(D), (E), and (G) to (I)]. Quantifications of actin [(B), (E), and (G)] or mCherry (H) fluorescence around 100-nm deformations and corresponding representative Airyscan images [(A), (D), and (I)]. ( A and B ) Effect of CDC42, CIP4, TOCAs (CIP4, FBP17, and FNBP1L), Rac1, clathrin heavy chain, or μ2-adaptin depletion with siRNAs on actin enrichment around deformations. Regions marked by dashed squares, expanded below with individual channels displayed (A, bottom). ( C ) Immunoblots of CDC42, CIP4, FBP17, FNBP1L, Rac1, clathrin heavy chain, and μ2-adaptin document siRNA knockdown efficiency (uncropped blots, see fig. S13). ( D and E ) Effect of transient expression of GFP or individual TOCA proteins (mCherry-CIP4, mCherry-FBP17, or GFP-FNBP1L) on actin enrichment around deformations. ( F ) CIP4 truncation mutants (mCherry-tagged). FL, full-length protein; F-BAR, BAR domain only; F-BAR+, BAR domain with basic region; ∆SH3, lacking SH3 domain; ∆REM1, lacking basic region and REM1 domain; ∆Basic, lacking basic region. ( G to I ) Rescue experiment. Transient expression of free mCherry, mCherry-tagged full-length CIP4, or truncation mutants in cells where endogenous TOCA proteins are depleted. Number of independent experiments, three [(A), (B), and (G) to (I)] or four [(D) and (E)]. Number of deformations ( n ) depicted on each graph. Data are means ± SEM; **** P < 0.0001; *** P < 0.001; ** P < 0.01 [one-way ANOVA with Dunnett’s [(B), (E), and (G)] or Tukey’s (H) multiple comparison test]. White arrowheads, colocalization [(A), (D), and (I)]. Scale bars, 10 μm (A) and 2 μm [(D) and (I)].

Article Snippet: APPL1 , Mouse , pmCherry-C1 , CMV , N-ter mCherry , Addgene #27683 , .

Techniques: Transfection, Construct, Fluorescence, Western Blot, Knockdown, Expressing, Comparison

Journal: Science Advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: ( A to D ) Representative Airyscan time lapses and corresponding quantifications of enrichment ratios of the following pairs around 100-nm deformations over time: (A) GFP-CDC42 and LifeAct-mCherry, (B) GFP-CDC42 and mCherry-CIP4, (C) LifeAct-GFP and mCherry-CIP4, and (D) LifeAct-GFP and PalMyr-mCherry (PalMyr). Thirty-minute time-lapses with 15-s intervals between frames. Region size, 0.85 μm. ( E ) Correlation coefficients between the pairs of proteins shown in (A) to (D). Number of deformations: PalMyr/LifeAct, n = 65; CIP4/CDC42, n = 118; CIP4/LifeAct, n = 83; LifeAct/CDC42, n = 145. Three independent experiments. ( F and G ) Monitoring of CDC42-dependent actin polymerization upon deformation of the plasma membrane by FluidFM. (F) Quantification of normalized LifeAct-mCherry fluorescence intensity around FluidFM tip over time upon coexpression of GFP (control, black), GFP-CDC42-T17N (red), GFP-CDC42-WT (blue), or GFP-CDC42-Q61L (green). Ten-minute time-lapses with 9-s intervals between frames. Number of cells: GFP, n = 20; GFP-CDC42-T17N, n = 18; GFP-CDC42-WT, n = 15; GFP-CDC42-Q61L, n = 17. Two independent experiments. (G) Representative image of FluidFM/confocal experiments upon coexpression of GFP-CDC42-Q61L and LifeAct-mCherry. Region marked by a dashed square surrounding the bead, expanded below at the indicated time points; t = 0 min, initial deformation of the cell membrane. TL, transmitted light (FluidFM cantilever) (representative images of GFP, GFP-CDC42-T17N, and GFP-CDC42-WT in fig. S11, F to H). Data are means ± SEM; **** P < 0.0001 (E, one-way ANOVA with Dunnett’s multiple comparison test). Scale bar, 10 μm (G).

Article Snippet: APPL1 , Mouse , pmCherry-C1 , CMV , N-ter mCherry , Addgene #27683 , .

Techniques: Clinical Proteomics, Membrane, Fluorescence, Control, Comparison

Journal: Science Advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: Plasmids containing BAR domain protein sequences used in this study.

Article Snippet: APPL1 , Mouse , pmCherry-C1 , CMV , N-ter mCherry , Addgene #27683 , .

Techniques:

Journal: Science Advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: Other plasmids used in this study.

Article Snippet: APPL1 , Mouse , pmCherry-C1 , CMV , N-ter mCherry , Addgene #27683 , .

Techniques:

Journal: bioRxiv

Article Title: Rapid Whole Cell Imaging Reveals A Calcium-APPL1-Dynein Nexus That Regulates Cohort Trafficking of Stimulated EGF Receptors

doi: 10.1101/481796

Figure Lengend Snippet: (a) Cells transfected with APPL1-eGFP (green) were imaged using LLSM, during which time EGF-A647 (magenta) was pulse injected. The inserts correspond to APPL1 and EGFA647 from left to right for the denoted time points. Rectangles indicate zoomed versions for each time point. Time is in seconds. Arrows indicate APPL1 and EGFA647 colocalization. Scale bar = 10 µm. (b) Similar experiment with fluorescently labeled transferrin. No colocalization was observed. Scale bar = 15 µm. (c) Schematic of tracking and analysis workflow. All identified tracks, or tracks filtered on the basis of co-trafficking by presence of both channels within a determined radius sphere at each time point, were selected for ensemble MSD analysis. Based on the MSD analysis of the tracks, each track was characterized as constrained, diffusive or undergoing directed motion (Methods) and the tracks colored correspondingly (bottom) or exported for grouped statistical analysis. (d) Graph of fraction of cargo tracks with time (seconds) following EGF addition to HeLa cells transfected with APPL1 EGFP. Graphs show all the EGF tracks and the fraction of EGF tracks positive for APPL1. Error bars indicate standard deviation (n = 13 cells). (e) Percentages of APPL1 tracks categorized as constrained (yellow), diffusive (green) and directed (magenta) motions as a function of time by MSD analysis in a single cell. In addition, the blue trace shows the subset of APPL1 tracks positive for EGF that displayed directed motion, demonstrating that most APPL1 tracks with directed motions were also positive for the EGF. Error bars correspond to the standard deviation. (f) Percentage of APPL1 tracks undergoing constrained (yellow), diffusive (green), directed (magenta) motions and directed motions of EGF bearing APPL1 endosomes (blue) that were grouped in time as pre-cargo addition, 0-5 mins and 5-15 mins post addition based on example data presented in (e). Error bars correspond to the standard deviation where applicable (n = 9 cells). (g) Graph of the fraction of cargo tracks with time (seconds) following transferrin addition to HeLa cells transfected with APPL1 EGFP. Graphs show all the transferrin tracks and the fraction of transferrin tracks positive for APPL1. Error bars indicate standard deviation (n = 8 cells). (h) Percentages of APPL1 tracks categorized as constrained (yellow), diffusive (green) and directed (magenta) motions as a function of time by MSD analysis in a single cell. In addition, the graphs show the subset of APPL1 tracks positive for transferrin that display directed motion (grey), demonstrating that only a subset of APPL1 tracks showed the presence of transferrin in the initial time points post addition. Error bars correspond to the standard deviation. (i) Percentages of APPL1 tracks undergoing constrained (yellow), diffusive (green), directed (magenta) motions and directed motions of transferrin bearing APPL1 endosomes (grey) that are grouped in time as pre-cargo addition, 0-5 mins and 5-15 mins post addition based on example data presented in (e). Error bars correspond to the standard deviation where applicable (n = 7 cells).

Article Snippet: Cells were transfected with pEGFPC1-human APPL1, a gift from Pietro De Camilli (Addgene plasmid #22198) (26), GFP-APPL1-ΔPTB, a gift from Donna Webb (Addgene plasmid #59768) (34), DsRed-p150 217-548, a gift from Trina Schroer (Addgene plasmid #51146) (29), EEA1 TagRFP-T (Addgene plasmid #42635), a gift from Silvia Corvera.

Techniques: Transfection, Injection, Labeling, Standard Deviation

Journal: bioRxiv

Article Title: Rapid Whole Cell Imaging Reveals A Calcium-APPL1-Dynein Nexus That Regulates Cohort Trafficking of Stimulated EGF Receptors

doi: 10.1101/481796

Figure Lengend Snippet: (a) Scatter plot of calculated trajectory lengths in microns against maximum velocity in um/s of APPL1 endosomes in unstimulated cells (black) and EGF-bearing APPL1 endosomes 2-7 mins following injection (magenta). Line of best fit plotted following linear regression. (b) Schematic of single plane lattice illumination (dotted red line) of cells micropatterned in 5 µm patterns (blue). This elongation of the cells accentuates retrograde motility towards the PNR as indicated by the arrow. (c) Percentages of APPL1 endosomes which underwent anterograde (grey), retrograde (red) or no net movement (white) in micropatterned HeLa cells pre-EGF stimulation, 0-5 mins and beyond 10 mins post-EGF stimulation. Error bars indicate standard deviation. (d) Scatter plots of the distance in microns of EGF endosomes from a selected centroid in HeLa cells, stimulated with 100 nM EGF, at 0-2 mins and 5-7 mins post addition. The grey plot represents the total amount of EGF bearing endosomes and the blue plots indicate APPL1 positive endosomes bearing EGF. The inner box of the box plot represents the standard deviation, the inner bar the median and the dot the mean, the ‘x’ represents the counts within 1-99% of the sample and the horizontal bars the range. (e) Kymographs of APPL1 endosomes in HeLa cells pre-EGF stimulation (left) and 0-5mins post-EGF stimulation (right). The top graphs show a zoomed-out view, scale bar = 5 µm y-axis, 5 seconds x-axis. The overlaid lines show the area of the kymograph corresponding to the perinuclear region as seen in the oblique slice insert. The bottom graphs show the area of the dotted box zoomed 5x, ‘+’ indicates plus-end directed motions ‘-’ indicates minus-end directed motions. (f) Percentages of EGF bearing endosome tracks which showed confined (yellow), diffusive (green) and directed (magenta) motion in HeLa cells 5-15 mins post stimulation with 100 nM EGF. The cells were either uninhibited, treated with 50 µM ciliobrevin or transfected with DsRed p150 cc. The error bars represent standard deviation. (g) Schematic of endosomal distribution calculation. Euclidian distances between endosomal positions (circles) and the centroid of all the positions (squares) were calculated within a time interval of 2 mins immediately post EGF stimulation (0 – 2 mins) and 5 mins later (5 – 7 mins). (h) Scatter plots of the endosomal distances from centroid in microns of EGF endosomes in HeLa cells stimulated with 100 nM EGF, at 0-2 mins and 5-7 mins post addition. The dotted box and whisker plots represent cells treated with 50 µM ciliobrevin and the stripped plots cells transfected with DsRed p150 cc. The inner box of the box plot represents the standard deviation, the inner bar the median and the dot the mean, the ‘x’ represents the counts within 1-99% of the sample and the horizontal bars the range. (i) A bar graph of the percentage of APPL1 (right) and APPL1 + EGF (left) bearing endosome tracks which show confined (yellow), diffusive (green) and directed (magenta) motion in HeLa cells treated with 50 µM ciliobrevin, 5-15 mins post stimulation with 100 nM EGF. The error bars represent standard deviation.

Article Snippet: Cells were transfected with pEGFPC1-human APPL1, a gift from Pietro De Camilli (Addgene plasmid #22198) (26), GFP-APPL1-ΔPTB, a gift from Donna Webb (Addgene plasmid #59768) (34), DsRed-p150 217-548, a gift from Trina Schroer (Addgene plasmid #51146) (29), EEA1 TagRFP-T (Addgene plasmid #42635), a gift from Silvia Corvera.

Techniques: Injection, Standard Deviation, Transfection, Whisker Assay

Journal: bioRxiv

Article Title: Rapid Whole Cell Imaging Reveals A Calcium-APPL1-Dynein Nexus That Regulates Cohort Trafficking of Stimulated EGF Receptors

doi: 10.1101/481796

Figure Lengend Snippet: (a) Representative maximum intensity projections of 100 mM EGF stimulation of HeLa cells expressing APPL1-eGFP and EEA1 TagRFP-T (Magenta). Hollow arrows point at few examples of APPL1 and EGF colocalization and the solid arrows point at APPL1-EEA-EGF1 triple or EEA1-EGF double positive endosomes. Scale bar = 15 µm (b) A zoomed view of APPL1-EEA1-EGF triple positive endosomes in the boxed region in (a). Scale bar = 0.5 µm (c) An EGF-APPL1 double positive endosome undergoing maturation by conversion to EGF-EEA1 positive endosome. Scale bar = 0.2 µm (d) Graph of fraction EGF tracks positive for APPL1 and EEA1. Error bars indicate standard deviation (n = 6 cells). (e) Same plot as (d) for cells treated with 50 µM Ciliobrevin. Double lines on the X-axis indicate breaks in time.

Article Snippet: Cells were transfected with pEGFPC1-human APPL1, a gift from Pietro De Camilli (Addgene plasmid #22198) (26), GFP-APPL1-ΔPTB, a gift from Donna Webb (Addgene plasmid #59768) (34), DsRed-p150 217-548, a gift from Trina Schroer (Addgene plasmid #51146) (29), EEA1 TagRFP-T (Addgene plasmid #42635), a gift from Silvia Corvera.

Techniques: Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Rapid Whole Cell Imaging Reveals A Calcium-APPL1-Dynein Nexus That Regulates Cohort Trafficking of Stimulated EGF Receptors

doi: 10.1101/481796

Figure Lengend Snippet: (a) Time-lapse montage of cross-sections across a single cell displaying desorption of APPL1-eGFP following addition of 100 nM EGF. Scale bar = 4 µm. (b) Example image depicting segmentation of APPL1 eGFP signal in the PNR using Imaris and its loss upon EGF stimulation. Scale bar: 5 µm. (c) Change in relative intensity of APPL1-GFP in the perinuclear region of HeLa cells following addition of an EGF pulse of either 100 nM (blue) or 20 nM (green). Time is recorded in seconds following EGF binding, error bars represent standard deviation. (d) Maximum intensity projection of images of APPL1-eGFP (top, green in Merge) and R-GECO (middle, magenta in Merge) in HeLa cells stimulated with 100 nM pulse of ionomycin. Time recorded in seconds from ionomycin addition. Scale bar = 10 µm. Inserts correspond to zoomed sections (unbroken and dotted squares respectively) at each time point, scale bar = 1 µm. (e) Timeline of relationships between EGF binding, R-GECO peaks and APPL1 desorption. Measured R-GECO fluorescence peak and APPL1 desorption (grey dots), each dot represents a single experiment, with R-GECO peaks overlaid to mean of EGF induced R-GECO peaks. 100 nM EGF was used to determine both the time of APPL1 desorption (blue) and R-GECO peak (orange).

Article Snippet: Cells were transfected with pEGFPC1-human APPL1, a gift from Pietro De Camilli (Addgene plasmid #22198) (26), GFP-APPL1-ΔPTB, a gift from Donna Webb (Addgene plasmid #59768) (34), DsRed-p150 217-548, a gift from Trina Schroer (Addgene plasmid #51146) (29), EEA1 TagRFP-T (Addgene plasmid #42635), a gift from Silvia Corvera.

Techniques: Binding Assay, Standard Deviation, Fluorescence

Journal: bioRxiv

Article Title: Rapid Whole Cell Imaging Reveals A Calcium-APPL1-Dynein Nexus That Regulates Cohort Trafficking of Stimulated EGF Receptors

doi: 10.1101/481796

Figure Lengend Snippet: (a) Representative maximum intensity projection of LLSM imaging of WT-APPL1-GFP (green) in HeLa cells treated with erlotinib 5 min post 100 nM EGF-647 (magenta) stimulation. Scale bar = 4 µm. Left and right panels indicate zoomed sections corresponding to unbroken and dotted rectangles respectively, scale bar = 4 µm. (b) Percentage of EGF bearing endosome tracks which showed confined (yellow), diffusive (green) or directed (magenta) motility in HeLa cells transfected with APPL1-eGFP (no treatment), APPL1-ΔPTB eGFP or APPL1-eGFP and treated with 10 µM erlotinib. Error bars correspond to the standard deviation. (c) Scatter plots of EGF distance in microns from centroid in HeLa cells treated with 100 nM EGF at 0-2 mins and 5-7-mins post addition as described in . The Hela cells were either transfected with APPL1-GFP (blank plot), APPL1-ΔPTB GFP (diagonal lined plot) or APPL1-GFP and treated with 10 µM erlotinib (crossed plot). The inner box of the box plot represents the standard deviation, the inner bar the median and the dot the mean, the ‘x’ represents the counts within 1-99% of the sample and the horizontal bars the range.

Article Snippet: Cells were transfected with pEGFPC1-human APPL1, a gift from Pietro De Camilli (Addgene plasmid #22198) (26), GFP-APPL1-ΔPTB, a gift from Donna Webb (Addgene plasmid #59768) (34), DsRed-p150 217-548, a gift from Trina Schroer (Addgene plasmid #51146) (29), EEA1 TagRFP-T (Addgene plasmid #42635), a gift from Silvia Corvera.

Techniques: Imaging, Transfection, Standard Deviation

Journal: bioRxiv

Article Title: Rapid Whole Cell Imaging Reveals A Calcium-APPL1-Dynein Nexus That Regulates Cohort Trafficking of Stimulated EGF Receptors

doi: 10.1101/481796

Figure Lengend Snippet: In steady state cells, APPL1 (yellow semi-circles) is dispersed throughout the cell. EGF binding leads to dimerization and phosphorylation of EGFR and an increase in intracellular Ca 2+ (blue background). The increase in intracellular Ca 2+ impairs APPL1 PH binding to phosphoinositide causing APPL1 desorption. The increased cytosolic APPL1 results in APPL1 binding to activated EGFR via a PTB domain. APPL1 positive EGF bearing endosomes undergo dynein (purple figure) mediated motility to the perinuclear region which is rich in ER.

Article Snippet: Cells were transfected with pEGFPC1-human APPL1, a gift from Pietro De Camilli (Addgene plasmid #22198) (26), GFP-APPL1-ΔPTB, a gift from Donna Webb (Addgene plasmid #59768) (34), DsRed-p150 217-548, a gift from Trina Schroer (Addgene plasmid #51146) (29), EEA1 TagRFP-T (Addgene plasmid #42635), a gift from Silvia Corvera.

Techniques: Binding Assay