LC3 binding to the scaffolding protein JIP1 regulates processive dynein-driven transport of autophagosomes

Abstract

Autophagy is essential for maintaining cellular homeostasis in neurons, where autophagosomes undergo robust unidirectional retrograde transport along axons. We find that the motor scaffolding protein JIP1 binds directly to the autophagosome adaptor LC3 via a conserved LIR motif. This interaction is required for the initial exit of autophagosomes from the distal axon, for sustained retrograde transport along the midaxon, and for autophagosomal maturation in the proximal axon. JIP1 binds directly to the dynein activator dynactin but also binds to and activates kinesin-1 in a phosphorylation-dependent manner. Following JIP1 depletion, phosphodeficient JIP1-S421A rescues retrograde transport, while phosphomimetic JIP1-S421D aberrantly activates anterograde transport. During normal autophagosome transport, residue S421 of JIP1 may be maintained in a dephosphorylated state by autophagosome-associated MKP1 phosphatase. Moreover, binding of LC3 to JIP1 competitively disrupts JIP1-mediated activation of kinesin. Thus, dual mechanisms prevent aberrant activation of kinesin to ensure robust retrograde transport of autophagosomes along the axon.

Figure 1. JIP1 Knockdown Disrupts Transport of Rab7-Positive Vesicles

(A) Representative kymographs of EGFP-Rab7-positive late endosome motility in DRGs transfected with JIP1 siRNA. Kymographs represent motion as displacement along the axon (x-axis) over time (y-axis). (B) JIP1 knockdown significantly decreases the retrograde motility of EGFP-Rab7-positive vesicles and concurrently increases the percentage of bidirectional and stationary Rab7-positive vesicles in DRG axons. Data represents 3 independent experiments (n = 13–16 neurons). (C) EGFP-Rab7 and mCherry-LC3 co-migrate in the mid-axon of DRGs. Representative kymographs of a 3-minute movie show two retrograde vesicles that are co-positive for EGFP-Rab7 and mCherry-LC3 (yellow arrowheads) and one retrograde vesicle that is only positive for EGFP-Rab7 (white arrowhead). The underlined region is highlighted in Figure 1E with time-lapse images. (D) The majority of retrograde EGFP-Rab7-positive vesicles co-migrate with mCherry-LC3 while a low percentage of bidirectional/stationary EGFP-Rab7-positive vesicles co-migrate with mCherry-LC3 (n = 7 double-transfected neurons). (E) Time-lapse images from the underlined region in Figure 1C.

Figure 2. JIP1 Associates with Autophagosomes

(A) High-molecular-weight JIP1 is enriched in purified autophagosomes. The purified autophagosome fraction (AP), which is enriched for lipidated LC3-II, contains components of the retrograde motor complex, including the p150^Glued subunit of dynactin and DIC (dynein intermediate chain). Briefly, homogenized brain post-nuclear supernantant (PNS) was subject to Nycodenz gradient centrifugation, which yielded fractions enriched for cytosol, autophagosomes and ER (AP/ER), and mitochondria and peroxisomes (Mito./Perox.). Centrifugation of the AP/ER fraction through a Percoll gradient resulted in separation into an ER fraction and a dilute autophagosome (Dil. AP) fraction, which was then concentrated in a subsequent spin to yield the autophagosome fraction (AP). Each lane contains equal protein loading. Data represents 2 independent experiments. (B) Endogenous JIP1 coimmunoprecipitates with LC3 in mouse brain homogenate. A monoclonal anti-LC3 antibody coimmunoprecipitates both 90-kD and 110-kD bands of JIP1. A monoclonal JIP1 antibody also coimmunoprecipitates LC3, though to a lesser extent. Data represents 2 independent experiments. (C,D) Endogenous JIP1 and LC3 colocalize on vesicles along axons and in distal axon tips of nontransfected DRGs (arrowheads). Representative images from 2 independent experiments show immunofluorescence staining of LC3 (green) and JIP1 (red).

Figure 3. JIP1 Binds to the Autophagosome Adaptor LC3

(A) JIP1 binds to LC3. Lysates from COS7 cells co-transfected with GFP-LC3 and myc-JIP1 fragments were immunoprecipitated with an anti-myc antibody. Both full-length myc-JIP1, myc-JIP1[1-390] and myc-JIP1[307-711] coimmunoprecipitated GFP-LC3. Asterisk denotes antibody light chain. (B) JIP1 binds directly to GST-LC3. GST control and GST-LC3 bound to glutathione beads were incubated with recombinant untagged JIP1. GST did not bind to JIP1 while GST-LC3 saturated binding of JIP1. The ~43-kD band in the GST eluate lane may represent spillover of protein from the adjacent lane. (C) JIP1 contains a predicted F-type LIR (LC3-interacting region) motif. This EEEEGFDCL motif is conserved in mammalian JIP1 and contains three components that define LIR motifs and function to mediate binding to LC3 – a central aromatic phenylalanine residue, a leucine residue, and several flanking acidic residues. (D) Crystal structure of LC3 highlighting LIR-interacting residues. The 2.05-Angstrom crystal structure of rat LC3B-I (PDB code U1GM) (Sugawara et al., 2004) was visualized using the PyMOL PDB viewer. Sticks highlight key residues in the hydrophobic pockets of LC3 (K49, K51) that interact with the LIR aromatic residue and leucine as well as the basic residues in N-terminal LC3 (R10, R11) that interact with the acidic residues preceding the LIR aromatic residue. (E) Sequence logo of several F-type LIR motifs shows the degree of conservation at each residue. The net height of letters indicates the degree of conservation at each position while the heights of individual letters indicates that relative frequency of certain residues at each position. F-type LIR motifs of JIP1, optineurin, FYCO1, ULK1, ATG13, FIP200, and TBC1D5 (Birgisdottir et al., 2013) were plotted using the WebLogo server (http://weblogo.berkeley.edu/logo.cgi). (F) Truncation or mutation of LIR decreases JIP1 binding to LC3. Mutant myc-JIP1-ΔLIR lacks AA327-341 (PSISEEEEGFDCLSS). Lysates from COS7 cells co-transfected with GFP-LC3 and either wildtype or mutant myc-JIP1 were immunoprecipitated with an anti-myc antibody. Densitometry of coimmunoprecipitated GFP-LC3 bands shows that lysates from cells expressing myc-JIP1-ΔLIR or myc-JIP1-F336A have ~20% of the GFP-LC3 binding ability of wildtype myc-JIP1.

Figure 4. Efficient Exit of Autophagosomes from the Distal Axon Requires JIP1

(A) Schematic of four distinct axonal regions: the distal axon tip, the distal axon, the mid-axon, and the proximal axon. (B) Representative time-lapse confocal images highlight autophagosome biogenesis in the distal axon tip of a DRG cultured from the GFP-LC3 mouse and transfected with fluorescent JIP1 siRNA. Newly formed autophagosomes are small punctate structures that gradually enlarge into a ring-like structure (arrowheads) on the timescale of 2–3 minutes. (C-D) JIP1 knockdown does not perturb the number of autophagosomes in the distal axon tip. The density of autophagosomes in GFP-LC3 DRGs transfected with fluorescent JIP1 siRNA did not differ significantly from control neurons (p = 0.23). Data represents 3 independent experiments (n = 18–22 neurons). (E) Retrograde mCherry-LC3 autophagosomes selectively co-migrate with EGFP-JIP1 in the distal axon. Wildtype DRGs were co-transfected with mCherry-LC3 and EGFP-JIP1. Representative kymographs were generated from a 10-minute movie and pseudo-colored traces of mCherry-LC3 motility (bottom panel) highlight EGFP-JIP1-positive (yellow) and EGFP-JIP1-negative (red) autophagosomes. (F) A greater percentage of EGFP-JIP1-positive autophagosomes than EGFP-JIP1-negative autophagosomes move in the retrograde direction in the distal axon. For each neuron, mCherry-LC3-positive autophagosomes were binned as JIP1-positive or JIP1-negative and separately analyzed for direction of transport. Data represents 3 independent experiments (n = 5 neurons, 19–29 autophagosomes). (G) Representative kymographs of GFP-LC3 motility in the distal axon of DRGs transfected with fluorescent JIP1 siRNA. Data from Figures 5C–5E represent data from 3 independent experiments (n = 11–12 neurons). (H) The mean number of GFP-LC3 autophagosomes in the distal axon increases upon JIP1 knockdown. (I) The percentage of retrograde GFP-LC3 autophagosomes in the distal axon decreases upon JIP1 knockdown.

Figure 5. Processive Retrograde Autophagosome Transport in the Mid-Axon Requires Nonphosphorylated JIP1

(A) Representative 3-minute kymographs of GFP-LC3 autophagosome motility in DRGs transfected with red fluorescent siRNA targeted to mouse JIP1 and rescued with a bidirectional construct co-expressing resistant human wildtype or mutant JIP1 (S421A or S421D) and the transfection marker BFP. (B) JIP1 knockdown significantly decreased the percentage of retrograde autophagosomes and increased the percentage of bidirectional/stationary autophagosomes. Both wildtype human JIP1 and phosphodeficient JIP1-S421A restore retrograde autophagosome transport, but phosphomimetic JIP1-S421D cannot restore the percentage of retrograde autophagosomes and instead increases the percentage of anterograde autophagosomes. Data in Figs. 5B–5E represent 3 independent experiments (n = 7–16 neurons). (C) JIP1 knockdown decreased the net displacement of retrograde autophagosomes. Rescue with the S421A mutant restored net retrograde displacement while expression of the S421D mutant decreased net retrograde displacement. (D) JIP1 knockdown decreased the net speed of retrograde autophagosomes. Rescue with the S421A mutant restored net retrograde speed. (E) Quantification of the frequency of directional switches in the mid-axon of DRGs. Absolute numbers of directional switches were quantified for individual retrograde autophagosomes and normalized against the net displacement. Note that these trends are not statistically significant. (F) Endogenous JIP1 and MKP1 colocalize on autophagosomes along the axon. Representative images and linescans of nontransfected DRG neurons immunostained with a mouse monoclonal antibody against LC3 (green), a rabbit antibody against MKP1 (red), and a sheep monoclonal antibody against JIP1 (blue). Scale bar = 5 μm.

Figure 6. LC3 Binding to JIP1 Blocks Activation of Kinesin in vitro and is Necessary for Retrograde Transport of Autophagosomes in the Mid-Axon

(A) Schematic of in vitro TIRF motility assays. COS7 cells transfected with KHC-Halo were incubated with membrane-permeable red TMR-conjugated HaloTag ligand. This lysate from KHC-Halo-expressing cells was combined with lysate from myc-JIP1-expressing cells and applied to flow chambers containing blue AMCA-labeled fluorescent microtubules, which were immobilized on glass coverslips with an anti-tubulin antibody. (B) Time-lapse images acquired from a flow chamber containing KHC-Halo (red) and myc-JIP1 lysates show a motile binding event (arrowheads, left panel) to a microtubule (blue). Images from a chamber containing KHC-Halo, myc-JIP1, and GFP-LC3 lysates show a shorter run (arrowheads, right panel). (C) Activation of KHC-Halo by JIP1 is abrogated upon addition of LC3. Each representative kymograph shows 100 total frames (~33 seconds). (D–F) Addition of LC3 decreases the run frequency, run length, and speed of KHC-Halo in the presence of JIP1. The absolute number of runs was normalized to the length of each microtubule. Data from Figs. 6D–6F represent 3 independent experiments (n = 77–99 microtubules, 74–214 runs) and statistical comparisons were made to the KHC-Halo alone condition unless otherwise indicated. (G) Representative 3-minute kymographs of GFP-LC3 autophagosome motility in DRGs transfected with siRNA targeted to mouse JIP1 and rescued with a bidirectional construct co-expressing resistant human wildtype JIP1 or mutant JIP1-ΔLIR/F336A as well as the fluorescent transfection marker BFP. (H) DRGs expressing the JIP1-ΔLIR or F336A mutant display decreased percentage of retrograde autophagosomes when compared to control neurons. Data in Figures 6G–6J represent 3 independent experiments (n = 9–13 neurons); statistical comparisons were made against the control condition. (I) Retrograde autophagosomes in DRGs expressing the JIP1-ΔLIR or F336A mutant display decreased net retrograde displacement. (J) No statistically significant changes in net retrograde speed of autophagosomes from DRGs expressing JIP1 LIR mutants were observed.

Figure 7. JIP1 Binding to LC3 is Necessary for Autophagosome Acidification in the Proximal Axon

(A,B) Representative kymographs of mCherry-GFP-LC3 motility in the distal and proximal axon of DRGs knocked down and rescued with JIP1-ΔLIR. Data from Figures 7A–7D represent 3 independent experiments (n = 10–12 neurons). (C) Total number of autophagosomes in the distal axon increases in JIP1-ΔLIR-expressing DRGs. Data from Figures 7C and 7D were analyzed with t-tests comparing wildtype versus mutant neurons from each axonal region and thus are plotted on discontinuous x-axes. (D) The percentage of immature mCherry- and GFP-positive autophagosomes increases in the proximal axon of JIP1-ΔLIR-expressing DRGs. (E) Model for the effect of JIP1 recruitment on autophagosome motility and lysosomal fusion. The majority of autophagosomes in the distal axon move in a bidirectional or stationary manner (left). Upon JIP1 recruitment via binding to LC3, autophagosomes begin to exit out of the distal axon and continue into the mid-axon and the proximal (middle). Because LC3 binding to JIP1 reduces the ability of JIP1 to activate kinesin, JIP1 remains bound to p150^Glued and sustains retrograde autophagosome transport in the mid-axon. Robust retrograde autophagosome transport is important for fusion with lysosomes and efficient cargo degradation.