A Mobile Endocytic Network Connects Clathrin-Independent Receptor Endocytosis to Recycling and Promotes T Cell Activation

Component: Cell Types; Description: • Jurkat T cells (E6.1 clone) and derivatives (JCaM1, P116)<br>• LAT knockout Jurkat (CRISPR/Cas9)<br>• Flotillin1/2 double knockout Jurkat (CRISPR/Cas9)<br>• Raji B cells (antigen-presenting cells)<br>• Primary CD4+ T cells from WT and flotillin2 KO mice

Component: Key Proteins Tracked; Description: • TCRζ - fused to photoactivatable mCherry (PA-mCherry) or PS-CFP2<br>• Lck - PA-mCherry fusion<br>• Flotillin-1 and -2 - EGFP or PA-mCherry fusions<br>• Clathrin - EGFP or PA-mCherry fusions

Component: Endocytic Cargo Controls; Description: • Transferrin-Alexa488 (clathrin-mediated endocytosis control)<br>• AP180-C-GFP (clathrin coat marker)

Component: Activation Conditions; Description: • Non-activating: anti-CD90 coated surfaces<br>• Activating: anti-CD3ε + anti-CD28 coated surfaces, or SEE-pulsed Raji B cells

Component: Pharmacological Inhibitors; Description: • Pitstop2 (6 μM) - clathrin inhibitor<br>• Dynasore (80 μM) - dynamin inhibitor<br>• CK666 (100 μM) - Arp2/3 inhibitor

Component: Optogenetic Tool; Description: Cry2Clust-mediated clustering of flotillin-positive endosomes

Parameter: Primary Live-Cell Imaging; Details: Photoactivation of PA-mCherry-tagged proteins at plasma membrane regions (405 nm laser); vesicle tracking over 250-450 s; confocal microscopy at 37°C

Parameter: Cell Lines & Mutants; Details: • Jurkat WT, JCaM1 (Lck-deficient), P116 (Zap70-deficient), LAT KO<br>• Flotillin1/2 double KO generated by CRISPR/Cas9 (confirmed by western blot)

Parameter: Internalization Assays; Details: • Photoactivation: vesicle count over time after localized photoactivation<br>• Flow cytometry: surface biotinylated anti-CD3ε labeled, internalization at 37°C for 5-20 min, remaining surface detected with fluorescent streptavidin

Parameter: Recycling Assays; Details: Antibody feeding: label TCR with biotinylated anti-CD3ε (40 min, 37°C), block surface with unlabeled streptavidin, detect recycled TCR at 0-20 min with fluorescent streptavidin

Parameter: Single-Molecule Localization Microscopy; Details: PALM with TCRζ-PS-CFP2 on fixed cells; DBSCAN clustering analysis; cluster density, size, and radius distributions

Parameter: Signaling Analysis; Details: • Flow cytometry: phospho-TCR, phospho-Zap70, phospho-ERK in fixed/permeabilized cells<br>• Immunofluorescence: phospho-TCR, phospho-Zap70, NFAT, TSAd nuclear translocation (T-B cell conjugates)<br>• Primary CD4 T cells: CD69 and CD25 surface expression at 20 and 36 h post-activation

Parameter: Conjugation Assays; Details: Jurkat T cells + SEE-pulsed Raji B cells; flow cytometry (T cells = Fixable Violet Dead Stain, B cells = CFDA) and fixed-cell imaging

Parameter: Replicates; Details: Data obtained from three or more independent experiments; at least five cells per experiment for imaging

Analysis Category: Endocytosis Dynamics; Methods: Custom MATLAB vesicle tracking (PAVesT); vesicle count per frame; track length quantification; cross-channel nearest-neighbor distance analysis (320 nm threshold)

Analysis Category: Pharmacological Inhibition Validation; Methods: Clathrin-EGFP vesicle count with pitstop2; transferrin-Alexa488 internalization; dynamin and Arp2/3 inhibitor effects on vesicle mobility

Analysis Category: Recycling Quantification; Methods: Flow cytometry-based antibody feeding (BD FACSCanto II / LSR II); mean fluorescence intensity (MFI) over time

Analysis Category: TCR Spatial Organization; Methods: • Fixed-cell confocal: intensity ratios (synapse/endosomes vs. distal region)<br>• TIRF/PALM: intensity compactness metric (0 = evenly distributed, 1 = single pixel)<br>• DBSCAN clustering (ε = 3 neighbors, r = 20 nm)

Analysis Category: Signaling Readouts; Methods: • Flow cytometry: phospho-TCR, phospho-Zap70, phospho-ERK<br>• Immunofluorescence: nuclear NFAT/TSAd co-localization with Hoechst<br>• Western blot (primary cells): flotillin1/2 expression

Analysis Category: Optogenetic Clustering; Methods: Cry2Clust-mediated flotillin clustering with 470 nm blue light (10 s on/off pulses, 10 min prior to activation); flow cytometry for phospho-TCR

Analysis Category: Statistical Analysis; Methods: GraphPad Prism; Mann-Whitney t-test; ANOVA; p-values: p ≤ 0.05, p < 0.01, p < 0.001, p < 0.0001; ns = not significant

Finding: TCR internalization; Detail: Constitutive in resting cells; rapidly increased upon activation (most molecules internalized within 20 s)

Finding: Lck internalization; Detail: Significantly less than TCR; strictly retained at surface in activated cells

Finding: Kinase requirement; Detail: Lck and Zap70 required for activation-induced TCR endocytosis; LAT knockout did NOT impair internalization

Parameter: Co-localization with TCR vesicles; Clathrin: 11% (not above random); Flotillin: 50.7% (TCR with flotillin2) / 54.6% (flotillin2 with flotillin1)

Parameter: Transferrin co-localization; Clathrin: 41.7%; Flotillin: No correlation above random

Parameter: Pitstop2 effect; Clathrin: ✓ Inhibited; Flotillin: ✓ Inhibited (TCR similarly affected)

Parameter: Arp2/3 requirement; Clathrin: Not required; Flotillin: Required (CK666 caused immobile puncta)

Parameter: Vesicle persistence; Clathrin: Decreased with time (coat dissociation); Flotillin: Stable for ≥250 s (and up to 450 s)

Parameter: Network dynamics; Clathrin: Short-lived; Flotillin: Mobile network moving between periphery and center of synapse

Assay: TCR internalization (photoactivation); WT: Normal; Flotillin1/2 KO: No difference; Significance: ns

Assay: TCR internalization (flow cytometry); WT: ~40% decrease at 20 min; Flotillin1/2 KO: No difference; Significance: ns

Assay: TCR recycling (flow cytometry); WT: Gradual increase; Flotillin1/2 KO: Significant reduction at 10-20 min; Significance: p < 0.01

Assay: TCR vesicle accumulation (10 min post-PA); WT: Baseline; Flotillin1/2 KO: Higher vesicle count; Significance: -

Assay: TCR at synapse (T-B conjugates); WT: Normal; Flotillin1/2 KO: 5-fold less; Significance: -

Assay: TCR cluster density (PALM); WT: ~600 clusters/μm²; Flotillin1/2 KO: ~200 clusters/μm²; Significance: -

Assay: TCR intensity compactness (TIRF); WT: More concentrated; Flotillin1/2 KO: More dispersed; Significance: p < 0.0001

Readout: Phospho-TCR (activation); WT: Robust increase; Flotillin1/2 KO: Significant reduction

Readout: Phospho-Zap70; WT: Robust increase; Flotillin1/2 KO: Significant reduction

Readout: Phospho-ERK; WT: Robust increase; Flotillin1/2 KO: Significant reduction

Readout: NFAT nuclear translocation; WT: Present; Flotillin1/2 KO: Abolished

Readout: TSAd nuclear translocation; WT: Present; Flotillin1/2 KO: Abolished

Readout: Conjugate formation (T-B); WT: Normal; Flotillin1/2 KO: Reduced

Activation Marker: CD69 expression (Flot2 KO vs. WT); 20 h: Significantly reduced; 36 h: -

Activation Marker: CD25 expression (Flot2 KO vs. WT); 20 h: -; 36 h: Significantly reduced

1. Mechanistic gaps: "Many questions remain as to which molecular mechanisms drive the clathrin-independent endocytosis of TCR, how the mobile endocytic network demarked by flotillin functions, and how TCR recycling actually supports signaling events and the nuclear translocation of transcription factors."

2. Direct binding not established: TCR lacks the canonical cytosolic consensus sequences (dileucine motifs) that flotillins typically bind; interaction occurs through cholesterol-enriched membrane domains rather than direct protein-protein binding—the exact molecular connection remains undefined.

3. Primary cell limitations: Primary T cells isolated from flotillin2 KO mice were effectively double KO (flotillin1 undetectable), but full mechanistic studies (photoactivation, PALM, optogenetics) were performed exclusively in Jurkat cell lines.

4. Tissue context: All experiments performed in vitro on antibody-coated surfaces or with artificial antigen-presenting cells (Raji B cells + SEE); no in vivo T cell activation or immune response studies.

5. Pharmacological inhibitor specificity: Pitstop2, dynasore, and CK666 have known off-target effects; the authors partially addressed this with co-localization and genetic approaches (CRISPR KO).

6. Endosome morphology: The manuscript describes the flotillin network as having "distinct morphology and dynamics" but does not fully characterize the ultrastructure (e.g., electron microscopy of these compartments).

7. Cargo specificity: The study focuses exclusively on TCR; whether other surface receptors (e.g., integrins, co-stimulatory molecules) use the same flotillin-mediated recycling pathway in T cells was not investigated.

8. Species differences: Human Jurkat cells and mouse primary T cells were used; potential species-specific differences in flotillin function were not systematically compared.

Report prepared based on the published Nature Communications article. For full experimental details, supplementary figures, and raw data, please refer to the original publication and accompanying supplementary information.