Journal: bioRxiv

Article Title: Heterotypic Endosomal Interactions Drive Emergent Early Endosomal Maturations

doi: 10.1101/2022.04.15.488498

Figure Lengend Snippet: Representative images of each stage of imaging workflow used to detect and measure endosomes at a whole cell level. a) APPL1-EGFP and EEA1-T-TagRFP are imaged using LLSM at 2.5 seconds/volume, yielding b) whole cell volumes lasting up to ∼30 minutes. c) Preliminary endosomes in each channel are identified separately by blob detection then an unsupervised pattern recognition-based routine is used to identify true endosomes, following by d) linking based on localisation and intensity values to construct complete trajectories with continuous spatial data and intensity traces. e) Trajectories of endosomes identified from opposite channels are then analysed together to identify of events of interest. (i) The intensity profile between two endosomes ( cyan : APPL1, magenta : EEA1) along the line connecting their centres of mass is shown. Dashed line indicates centre of mass; dotted lines indicate endosome boundaries determined by detection routine. Changes in surface–to–surface distances between pairs of nearby events are used to identify events, with (ii) collisions defined as the point of nearest approach, if below the threshold for surface–to–surface separation and (iii) conversions calculated by identifying regions of colocalised trajectories; (iv) such events are considered to be fusions if the EEA1 track that remains after the disappearance of the APPL1 track existed as a distinct tracked object prior to the colocalisation event.

Article Snippet: 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: Imaging, Construct

Journal: bioRxiv

Article Title: Heterotypic Endosomal Interactions Drive Emergent Early Endosomal Maturations

doi: 10.1101/2022.04.15.488498

Figure Lengend Snippet: a) Montage showing (i) a single APPL1 endosome that collides with (ii) a dim EEA1 endosome and separates, then collides with (iii) a brighter EEA1 endosome and separates, (iv) collides again with the same EEA1 endosome then (v) separates, at which point EEA1 levels are significantly elevated; by (vi) APPL1 is no longer independently detected. Scale bar = 1 μm. b) Corresponding intensity traces with events indicated by dotted vertical lines. Time is reported relative to the start of APPL1–EEA1 colocalisation; intensity is reported as the average value of each channel’s intensity over the pixels detected by APPL1 detection until the end of localisation, by EEA1 detection thereafter. c) Population-average intensity traces for wild-type (WT) ( top ) and nocodazole-treated ( bottom ) cells, aligned with the start of each conversion event. WT cells demonstrate a steady increase in EEA1 signal and decrease in APPL1 signal from the start of colocalisation, whereas nocodazole-treated cells show no clear separation between the channels, on average (i.e., detected events likely represent ‘conversions’ that subsequently revert). Up until the end of colocalisation, average intensity was calculated using the mask of the object detected in the APPL1 channel; afterward, it was calculated using the mask of the object detected in the EEA1 channel. Intensity as normalised by the value at the start of detected colocalisation. WT data represent >100 events; nocodazole-treated data represent >25 events; error range shows 95% confidence interval. d) Average intensity traces for WT cells, separated into cohorts according to duration of conversion—short (10–20 s), medium (20–30 s), and long (30–40 s)—for APPL1 ( top ) and EEA1 ( bottom ) channels. e) Heatmap of the fraction of events that fall into cohorts defined by the total duration of colocalisation (in 10-s bins) and the number of heterotypic collisions immediately preceding the event. ( Top ) All events are shown for WT ( left ) and nocodazole-treated ( right ) cells. ( Bottom ) WT data are split according to fusions ( left ) and conversions ( right ). Importantly, in WT cells, longer durations of colocalisation are notably less frequent in those cases where multiple collisions occur. Note that nocodazole events represent fusions only, as no conversions were detected for these cells. Total events for each condition are WT ( n = 127), which is further segmented into fusions ( n = 96) and conversions ( n = 32), and nocodazole-treated ( n = 18). f) Numbers of events observed in each category: unaided fusions (39%), collision-induced fusions (38%), unaided conversions (12%), and collision-induced conversions (11%). Strict criteria were applied to filter the collisions; thus, we consider the values reported here for collision-induced events to likely represent a conservative estimate. g) Size distributions of populations of APPL1 endosomes, segmented into four types of events. Endosomes that undergo unaided (direct) conversions are, on average, larger than those that undergo collision-induced conversions or fusions.

Article Snippet: 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:

Journal: bioRxiv

Article Title: Heterotypic Endosomal Interactions Drive Emergent Early Endosomal Maturations

doi: 10.1101/2022.04.15.488498

Figure Lengend Snippet: a) 3D PALM imaging of Dendra2-EEA1 shown as a maximum intensity projection ( top ) and as a 3D volume ( bottom ). Intensity is colour-coded to z position. Scale bar = 1 μm. b) Montage of live SRRF experiment showing dynamic APPL1 ( cyan ) and EEA1 ( magenta ) clustering. Scale bar = 1 μm. c) Kymograph of line intensity plot of normalised APPL1 ( cyan ) and EEA1 ( magenta ) intensity of a converting APPL1 endosome imaged with SRRF, after processing. Circumference position in degrees is plotted on the y axis, as indicated in the schematic ( left ), and time in seconds is plotted on the x axis.

Article Snippet: 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: Imaging

Journal: bioRxiv

Article Title: Heterotypic Endosomal Interactions Drive Emergent Early Endosomal Maturations

doi: 10.1101/2022.04.15.488498

Figure Lengend Snippet: a) Schematic diagram of EEA1 FLIM experiment logic. Shorter fluorescence lifetime ( right ) indicates a FRET interaction between EEA1-EGFP and Rab5-RFP and therefore indicates EEA1 is bound via its N-terminal binding domain. Correspondingly, longer fluorescence lifetime ( left ) indicates no FRET interaction and thus that EEA1 is bound via its C-terminal binding domains to the membrane. b) Normalised frequency histograms of the detected fluorescence lifetimes of EEA1-EGFP photons measured in peripheral endosomes ( blue ) and perinuclear endosomes ( green ); bars represent standard errors of the mean; dashed curves show Gaussian fits for reference. Mean lifetimes calculated from these data are 1.87 ns and 2.10 ns for the peripheral and perinuclear curves, respectively. Endosomes were measured across n = 6 cells. c) Representative FLIM-FRET experiments of RPE1 cells transfected with EEA1-EGFP and Rab5-RFP. Coloured scale bar represents donor lifetime ranging from 1.8 ns ( blue ) to 2.3 ns ( red ). Left panel shows the FLIM image of EEA1 (donor) lifetime, middle panel shows EEA1 fluorescence intensity and right panel shows Rab5 fluorescence intensity; boxes indicate the regions of the zoomed insert. Scale bar = 10 μm. Zoomed insert scale bar = 1 μm.

Article Snippet: 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: Fluorescence, Binding Assay, Membrane, Transfection

Journal: bioRxiv

Article Title: Heterotypic Endosomal Interactions Drive Emergent Early Endosomal Maturations

doi: 10.1101/2022.04.15.488498

Figure Lengend Snippet: a) Representative montage showing the appearance of N-terminal EEA1 following collision-conversion, imaged by live FLIM-FRET as described in . EEA1-EGFP fluorescence lifetime has been fit with a two-component regression (τ 1 = 1.006 ns, τ 2 = 2.600 ns). The image has been pseudo-coloured by the relative photon contribution from each component; the shorter N-terminal EEA1 component ( blue ), the longer C-terminal EEA1 component ( green ), and Rab5-RFP fluorescence ( grey ). Arrow indicates nascent converting endosome. Scale bar = 2.5 μm, time is measured in seconds. b) Normalised mean intensity plot of converting endosomes following seeding at time = 0. Line graphs show mean intensity of N-terminally bound EEA1 ( blue ) and C-terminally bound EEA1 ( green ), area fill indicates 95% confidence interval at each time-point. Time is measured in seconds. n = 20 conversion events. c) Fusions are categorised by the participating endosomes; N-terminal bound (N), C-terminal bound (C) and EEA1 absent, Rab5-positive (A). Plots show mean, error bars indicate S.D. Each coloured shape indicates a different cell, n = 13. ns indicates non-significant difference, **** indicates p<0.0001. Each mean was compared against the others using an ordinary one-way ANOVA. d) Representative montage of EEA1 N-to C-terminally bound conversion. N-terminally bound EEA1 ( blue ), C-terminally bound EEA1 ( green ). Arrows indicate points of fusion as numbered in panel e, asterisks indicate ‘main’ endosome corresponding to the intensity trace, hashes show incident endosomes prior to fusion. Scale bar = 2.5 μm, time is measured in seconds. e) Intensity trace of N-to C-terminally bound conversion corresponding to montage in panel d. Lines indicate mean relative lifetime amplitudes of N-terminal EEA1 ( blue ), C-terminal EEA1 ( green ) and the N:C intensity ratio ( magenta ), bold lines indicate 3-frame moving average; intensities were manually measured for each endosomal pixel. Dotted lines indicate successive fusion time-points. Time is measured in seconds.

Article Snippet: 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: Fluorescence

Journal: bioRxiv

Article Title: Heterotypic Endosomal Interactions Drive Emergent Early Endosomal Maturations

doi: 10.1101/2022.04.15.488498

Figure Lengend Snippet: 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 and the total number of conversions and fusions were quantified. ns indicates non-significant difference, * indicates p<0.05. 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.

Article Snippet: 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: Knock-Out, Expressing, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Heterotypic Endosomal Interactions Drive Emergent Early Endosomal Maturations

doi: 10.1101/2022.04.15.488498

Figure Lengend Snippet: Schematic of agent and node logic used in modelling. a) The endosome is simulated as a multi-layered surface of a sphere, where different layers are occupied by different agents and nodes, as shown. The sphere is initially dominated by APPL1 attachments ( cyan ), which over time are replaced by N-terminal EEA1 ( dark blue ), and finally C-terminal EEA1 ( green ). b) APPL1 and EEA1 stochastically bind and unbind, competing for Rab5 ( grey ) binding availability. (i) APPL1 requires both Rab5 and an adjacent PI(3,4)P2 ( orange ) to attach. (ii) N-terminal EEA1 replaces APPL1 in binding to Rab5 and frees up PI(3,4)P2. (iii) INPP4A ( yellow ) converts free PI(3,4)P2 to PI(3)P ( red ). C-terminal EEA1 requires Rab5 and an adjacent PI(3)P to bind.

Article Snippet: 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: Binding Assay

Journal: bioRxiv

Article Title: Heterotypic Endosomal Interactions Drive Emergent Early Endosomal Maturations

doi: 10.1101/2022.04.15.488498

Figure Lengend Snippet: a) Surface of the simulated endosome at different time-points (marked by dashed lines in panels b–d), showing the attached APPL1 ( cyan ), N-terminal EEA1 ( dark blue ), and C-terminal EEA1 ( green ). Initially, the endosome is dominated by APPL1 (i), while N-terminal EEA1 attach in clusters (ii). Upon collision (which takes place at 31.5 s in this instance), there is a large influx of EEA1 N-terminal attachments (iii), which shortly detach due to cluster detachment (iv). However, this duration is sufficient to displace a large number of APPL1, allowing INPP4A to accelerate conversion of PI(3,4)P2 to PI(3)P. Eventually, C-terminal EEA1 attach in clusters (v) and ultimately dominate over N-terminal attachments (vi). b) Time series of numbers of attached APPL1, INPP4A and total EEA1 (including both N- and C-terminal attachments). c) Time series of numbers of PI(3,4)P2 and PI(3)P molecules on the endosome’s surface. PI(3)P molecules are formed by a 1:1 conversion from PI(3,4)P2 by INPP4A. d) Time series of numbers of N- and C-terminal attachments of EEA1. Initially, most attachments are through N-terminal EEA1, while over the course of endosomal conversion, C-terminal attachments eventually become a majority. Collision mainly introduces new N-terminal attachments. For a dynamic version of this figure, refer to Supplementary Movie 9.

Article Snippet: 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:

Journal: bioRxiv

Article Title: Heterotypic Endosomal Interactions Drive Emergent Early Endosomal Maturations

doi: 10.1101/2022.04.15.488498

Figure Lengend Snippet: a) Conversion time distributions of simulated isolated endosomes. If the clustering mechanism of EEA1 molecules is removed from the simulation, the conversion time distribution shifts to the right (from grey to cyan), indicating slower conversions. If the isolated endosome is made to undergo collision (which takes place at t = 31.5 s for these simulations), the conversion time distribution shifts to the left (from grey to orange) and conversion time is reduced by almost one third. b) Conversion time distributions of a simulated cell in which endosomes collide randomly at a given collision frequency. The grey curve shows the case for no collisions. Upon increasing the collision frequency, the weight of the conversion time distribution shifts further to the left (towards red). There are multiple modes in the conversion time distribution corresponding to the number of collisions the endosome experienced before conversion. The leftmost mode lying between 20–50 s corresponds to two collisions before conversion, the middle mode lying between 60–100 s to a single collision, and the rightmost mode lying between 130–200 s to zero collisions.

Article Snippet: 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: Isolation

Journal: bioRxiv

Article Title: Heterotypic Endosomal Interactions Drive Emergent Early Endosomal Maturations

doi: 10.1101/2022.04.15.488498

Figure Lengend Snippet: Very early endosomes formed at the cell periphery (endosome 1) have PI(3,4)P2 ( orange ) containing membranes and APPL1 ( cyan ) bound to Rab5 ( grey ). These vesicles collide with mature EEA1 vesicles (endosome 0), seeding N-terminally bound EEA1 and triggering the conversion process. This enables the production of PI(3)P ( red ) and the binding of C-terminal EEA1. These vesicles can trigger conversions on nascent APPL1 vesicles (endosome 2) and participate in canonical endosomal tethering and fusion processes (bottom endosomes).

Article Snippet: 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: Binding Assay