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• W2036768594 abstract "•Retrograde axonal transport of autophagosomes requires the scaffolding protein JIP1•JIP1 binds directly to the autophagosome protein LC3 via a conserved LIR motif•LC3-binding to JIP1 blocks the JIP1-mediated activation of kinesin-1 motor activity•JIP1 dephosphorylation by organelle-bound MKP1 may also block kinesin-1 activation 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. 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. Protein quality control and degradation play important roles in cellular homeostasis. In addition to the ubiquitin-proteasome pathway, cells utilize macroautophagy (henceforth referred to as autophagy) to selectively target misfolded or aggregated proteins and defective organelles for removal. Autophagy is essential in neurons as knockout of genes in this pathway leads to neuronal cell death (Hara et al., 2006Hara T. Nakamura K. Matsui M. Yamamoto A. Nakahara Y. Suzuki-Migishima R. Yokoyama M. Mishima K. Saito I. Okano H. Mizushima N. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice.Nature. 2006; 441: 885-889Crossref PubMed Scopus (3122) Google Scholar, Komatsu et al., 2006Komatsu M. Waguri S. Chiba T. Murata S. Iwata J. Tanida I. Ueno T. Koike M. Uchiyama Y. Kominami E. Tanaka K. Loss of autophagy in the central nervous system causes neurodegeneration in mice.Nature. 2006; 441: 880-884Crossref PubMed Scopus (2843) Google Scholar). Defects in autophagy are associated with neurodegenerative diseases including Alzheimer’s (Lee et al., 2011Lee S. Sato Y. Nixon R.A. Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy.J. Neurosci. 2011; 31: 7817-7830Crossref PubMed Scopus (339) Google Scholar), Parkinson’s (Ashrafi and Schwarz, 2013Ashrafi G. Schwarz T.L. The pathways of mitophagy for quality control and clearance of mitochondria.Cell Death Differ. 2013; 20: 31-42Crossref PubMed Scopus (994) Google Scholar, Dagda et al., 2009Dagda R.K. Cherra 3rd, S.J. Kulich S.M. Tandon A. Park D. Chu C.T. Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission.J. Biol. Chem. 2009; 284: 13843-13855Crossref PubMed Scopus (758) Google Scholar, Lynch-Day et al., 2012Lynch-Day M.A. Mao K. Wang K. Zhao M. Klionsky D.J. The role of autophagy in Parkinson’s disease.Cold Spring Harbor Perspect. Med. 2012; 2: a009357Crossref Scopus (285) Google Scholar, Narendra et al., 2008Narendra D. Tanaka A. Suen D.F. Youle R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.J. Cell Biol. 2008; 183: 795-803Crossref PubMed Scopus (2858) Google Scholar), Huntington’s (Martinez-Vicente et al., 2010Martinez-Vicente M. Talloczy Z. Wong E. Tang G. Koga H. Kaushik S. de Vries R. Arias E. Harris S. Sulzer D. Cuervo A.M. Cargo recognition failure is responsible for inefficient autophagy in Huntington’s disease.Nat. Neurosci. 2010; 13: 567-576Crossref PubMed Scopus (622) Google Scholar, Wong and Holzbaur, 2014Wong Y.C. Holzbaur E.L. The regulation of autophagosome dynamics by huntingtin and HAP1 is disrupted by expression of mutant huntingtin, leading to defective cargo degradation.J. Neurosci. 2014; 34: 1293-1305Crossref PubMed Scopus (247) Google Scholar), and amyotrophic lateral sclerosis (ALS) (Ravikumar et al., 2005Ravikumar B. Acevedo-Arozena A. Imarisio S. Berger Z. Vacher C. O’Kane C.J. Brown S.D. Rubinsztein D.C. Dynein mutations impair autophagic clearance of aggregate-prone proteins.Nat. Genet. 2005; 37: 771-776Crossref PubMed Scopus (372) Google Scholar, Rubino et al., 2012Rubino E. Rainero I. Chiò A. Rogaeva E. Galimberti D. Fenoglio P. Grinberg Y. Isaia G. Calvo A. Gentile S. et al.TODEM Study GroupSQSTM1 mutations in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Neurology. 2012; 79: 1556-1562Crossref PubMed Scopus (225) Google Scholar). The fidelity of the autophagic process may be particularly relevant in neurons for two reasons. First, accumulation of misfolded proteins and organelles may be especially toxic to neurons because they are postmitotic. Second, neuronal processes may present a spatial challenge for efficient clearance of proteins and organelles via autophagy. In the axon, autophagosomes undergo long-range microtubule-based retrograde transport that is coupled to compartment maturation (Hollenbeck, 1993Hollenbeck P.J. Products of endocytosis and autophagy are retrieved from axons by regulated retrograde organelle transport.J. Cell Biol. 1993; 121: 305-315Crossref PubMed Scopus (232) Google Scholar, Lee et al., 2011Lee S. Sato Y. Nixon R.A. Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy.J. Neurosci. 2011; 31: 7817-7830Crossref PubMed Scopus (339) Google Scholar, Maday et al., 2012Maday S. Wallace K.E. Holzbaur E.L. Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons.J. Cell Biol. 2012; 196: 407-417Crossref PubMed Scopus (440) Google Scholar). Thus, autophagosome transport dynamics along the axon are spatially regulated. In primary dorsal root ganglion (DRG) neurons, autophagosomes form preferentially in the distal axon tip and initially undergo inefficient back-and-forth bidirectional transport followed by a switch to unidirectional retrograde motility. In the midaxon, autophagosomes rarely exhibit pauses or switches in direction (Maday et al., 2012Maday S. Wallace K.E. Holzbaur E.L. Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons.J. Cell Biol. 2012; 196: 407-417Crossref PubMed Scopus (440) Google Scholar). In cortical neurons, autophagosomes also exhibit unidirectional retrograde transport along the axon (Lee et al., 2011Lee S. Sato Y. Nixon R.A. Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy.J. Neurosci. 2011; 31: 7817-7830Crossref PubMed Scopus (339) Google Scholar). The pronounced unidirectional transport characteristic of autophagosomes requires the microtubule motor dynein (Maday et al., 2012Maday S. Wallace K.E. Holzbaur E.L. Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons.J. Cell Biol. 2012; 196: 407-417Crossref PubMed Scopus (440) Google Scholar) and differs from the movement of other organelles in the axon, such as lysosomes (Moughamian and Holzbaur, 2012Moughamian A.J. Holzbaur E.L. Dynactin is required for transport initiation from the distal axon.Neuron. 2012; 74: 331-343Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), amyloid precursor protein (APP) vesicles (Fu and Holzbaur, 2013Fu M.M. Holzbaur E.L. JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors.J. Cell Biol. 2013; 202: 495-508Crossref PubMed Scopus (173) Google Scholar, Stokin et al., 2005Stokin G.B. Lillo C. Falzone T.L. Brusch R.G. Rockenstein E. Mount S.L. Raman R. Davies P. Masliah E. Williams D.S. Goldstein L.S. Axonopathy and transport deficits early in the pathogenesis of Alzheimer’s disease.Science. 2005; 307: 1282-1288Crossref PubMed Scopus (972) Google Scholar), mitochondria (Morris and Hollenbeck, 1993Morris R.L. Hollenbeck P.J. The regulation of bidirectional mitochondrial transport is coordinated with axonal outgrowth.J. Cell Sci. 1993; 104: 917-927Crossref PubMed Google Scholar), and RNA granules (van Niekerk et al., 2007van Niekerk E.A. Willis D.E. Chang J.H. Reumann K. Heise T. Twiss J.L. Sumoylation in axons triggers retrograde transport of the RNA-binding protein La.Proc. Natl. Acad. Sci. USA. 2007; 104: 12913-12918Crossref PubMed Scopus (88) Google Scholar), which exhibit both anterograde and retrograde motility. Surprisingly, however, live-cell imaging and organelle isolation indicate that both dynein and the anterograde motor kinesin remain bound to axonal autophagosomes (Maday et al., 2012Maday S. Wallace K.E. Holzbaur E.L. Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons.J. Cell Biol. 2012; 196: 407-417Crossref PubMed Scopus (440) Google Scholar). Thus, retrograde axonal transport of autophagosomes must be tightly regulated in order to promote dynein motor activity while inhibiting the activity of associated kinesins. A candidate coordinator of dynein and kinesin-1 activity is the motor scaffolding protein JIP1, known to regulate the highly processive transport of APP (Fu and Holzbaur, 2013Fu M.M. Holzbaur E.L. JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors.J. Cell Biol. 2013; 202: 495-508Crossref PubMed Scopus (173) Google Scholar, Muresan and Muresan, 2005Muresan Z. Muresan V. Coordinated transport of phosphorylated amyloid-beta precursor protein and c-Jun NH2-terminal kinase-interacting protein-1.J. Cell Biol. 2005; 171: 615-625Crossref PubMed Scopus (93) Google Scholar). Direct binding of JIP1 to kinesin heavy chain (KHC), the motor subunit of kinesin-1, is sufficient to activate KHC motility. JIP1 also directly binds to the p150Glued subunit of the dynein activator dynactin. Importantly, JIP1 cannot simultaneously bind to both KHC and dynactin and instead forms either an anterograde or retrograde motor complex. In the retrograde complex, binding of dynactin to JIP1 disrupts the ability of JIP1 to activate KHC motility. Phosphorylation of JIP1 at S421 acts as a directional switch by increasing the affinity of JIP1 for KHC, favoring the formation of the anterograde complex and thus preferentially enhances anterograde over retrograde motility (Fu and Holzbaur, 2013Fu M.M. Holzbaur E.L. JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors.J. Cell Biol. 2013; 202: 495-508Crossref PubMed Scopus (173) Google Scholar). Here, we demonstrate that JIP1 functions more broadly in axonal transport, also regulating the unidirectional retrograde transport of autophagosomes in a cargo-specific manner. JIP1 associates with autophagosomes via direct binding to the autophagosome adaptor LC3. While knockdown of JIP1 does not affect autophagosome biogenesis in the distal axon tip, JIP1 depletion inhibits the ability of autophagosomes to exit from the distal axon, to sustain retrograde transport along the midaxon, and to mature normally in the proximal axon. Expression of phosphodeficient JIP1-S421A rescues retrograde autophagosome transport induced by JIP1 depletion, while expression of phosphomimetic JIP1-S421D leads to aberrant anterograde autophagosome transport, suggesting that sustained retrograde motility requires continued inhibition of KHC activation by JIP1. The phosphatase MKP1 colocalizes with JIP1 on autophagosomes and may function to prevent aberrant phosphorylation of JIP1. Furthermore, we find that direct binding of LC3 to JIP1 disrupts the ability of JIP1 to activate KHC motility in vitro. Thus, LC3 binding to JIP1 functions not only to mediate autophagosome association, but also as a cargo-specific mechanism to regulate motor coordination and to sustain efficient retrograde motility. These experiments establish JIP1 as a versatile scaffolding protein capable of regulating the transport of diverse cargos along the axon. JIP1 regulates the transport of diverse classes of vesicular cargo, including synaptic vesicles and mitochondria in Drosophila neurons (Horiuchi et al., 2005Horiuchi D. Barkus R.V. Pilling A.D. Gassman A. Saxton W.M. APLIP1, a kinesin binding JIP-1/JNK scaffold protein, influences the axonal transport of both vesicles and mitochondria in Drosophila.Curr. Biol. 2005; 15: 2137-2141Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) and APP-positive vesicles in mammalian neurons (Fu and Holzbaur, 2013Fu M.M. Holzbaur E.L. JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors.J. Cell Biol. 2013; 202: 495-508Crossref PubMed Scopus (173) Google Scholar, Muresan and Muresan, 2005Muresan Z. Muresan V. Coordinated transport of phosphorylated amyloid-beta precursor protein and c-Jun NH2-terminal kinase-interacting protein-1.J. Cell Biol. 2005; 171: 615-625Crossref PubMed Scopus (93) Google Scholar). Immunostaining of primary DRG neurons shows colocalization of JIP1 with APP along the axon, but also suggests that JIP1 may localize to other organelle compartments (Fu and Holzbaur, 2013Fu M.M. Holzbaur E.L. JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors.J. Cell Biol. 2013; 202: 495-508Crossref PubMed Scopus (173) Google Scholar). To determine whether JIP1 mediates the axonal transport of additional organelles in mammalian neurons, we asked if the transport of Rab7-positive late endosomes (Deinhardt et al., 2006Deinhardt K. Salinas S. Verastegui C. Watson R. Worth D. Hanrahan S. Bucci C. Schiavo G. Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway.Neuron. 2006; 52: 293-305Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar) is dependent on JIP1. We knocked down endogenous mouse JIP1 in primary DRG neurons using a targeted siRNA that depletes >90% of JIP1 without detectable off-target effects (Fu and Holzbaur, 2013Fu M.M. Holzbaur E.L. JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors.J. Cell Biol. 2013; 202: 495-508Crossref PubMed Scopus (173) Google Scholar). Following electroporation of red fluorescent JIP1 small interfering RNA (siRNA) and an EGFP-Rab7 construct, we imaged transport in the midaxon and quantitated motility by generating kymographs (Figure 1A). In control neurons, the majority of Rab7-positive vesicles exhibit bidirectional or stationary motility defined by net displacement <10 μm (72.5% ± 2.3%). Motile Rab7-positive vesicles, defined by net displacement >10 μm over our imaging period, moved predominantly in the retrograde direction (26.6% ± 2.4%) and rarely in the anterograde direction (0.9% ± 0.7%; Figure 1B). JIP1 knockdown did not affect the density of Rab7-positive vesicles along the axon (control: 0.33 ± 0.02 vesicles per μm; JIP1 knockdown: 0.36 ± 0.02 vesicles per μm; p = 0.30), but did alter their motility. JIP1 depletion decreased the percentage of retrograde-moving vesicles by 68% (p < 0.001) and commensurately increased the percentage of bidirectional/stationary vesicles (p < 0.001, Figure 1B). The subpopulation of Rab7-positive vesicles that continue to move in the retrograde direction in JIP1-depleted neurons displayed unaltered motility with no significant changes in net displacement (control: 22.4 ± 1.3 μm; JIP1 knockdown: 23.2 ± 2.4 μm; p = 0.79) or net speed (control: 0.43 ± 0.03 μm/s; JIP1 knockdown: 0.50 ± 0.07 μm/s; p = 0.37). Thus, there are two subpopulations of Rab7-positive vesicles moving along axons; while the movement of one subpopulation is independent of JIP1, we find that JIP1 depletion arrests a distinct subpopulation of retrograde Rab7-positive organelles. Classic experiments in chick DRG axons showed that retrograde phase-dense vesicles are both endosomal and autophagosomal in nature (Hollenbeck, 1993Hollenbeck P.J. Products of endocytosis and autophagy are retrieved from axons by regulated retrograde organelle transport.J. Cell Biol. 1993; 121: 305-315Crossref PubMed Scopus (232) Google Scholar). In axons of DRGs and cortical neurons, autophagosomes comigrate with LAMP1, a marker for late endosomes (Lee et al., 2011Lee S. Sato Y. Nixon R.A. Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy.J. Neurosci. 2011; 31: 7817-7830Crossref PubMed Scopus (339) Google Scholar, Maday et al., 2012Maday S. Wallace K.E. Holzbaur E.L. Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons.J. Cell Biol. 2012; 196: 407-417Crossref PubMed Scopus (440) Google Scholar), suggesting that autophagosomes fuse with endosomes to form amphisomes in the axon. Moreover, the speed and displacement of the JIP1-dependent retrograde Rab7-positive vesicles resemble the characteristic unidirectional retrograde motility of autophagosomes (Maday et al., 2012Maday S. Wallace K.E. Holzbaur E.L. Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons.J. Cell Biol. 2012; 196: 407-417Crossref PubMed Scopus (440) Google Scholar). Thus, we hypothesized that this subpopulation of retrograde Rab7-positive vesicles affected by JIP1 knockdown are autophagosomes. To verify this, we cotransfected DRGs with EGFP-Rab7 and the autophagosome marker mCherry-LC3 (Figures 1C and 1E; Movie S1 available online). The majority of retrograde Rab7-positive vesicles with net displacement >10 μm comigrate with mCherry-LC3 (64.7% ± 7.5%) in the midaxon. In contrast, few bidirectional/stationary Rab7-positive vesicles are positive for mCherry-LC3 (22.6% ± 8.6%, Figure 1D). Of note, the percentage of retrograde Rab7-positive vesicles that are LC3-positive (65%) is similar to the 68% decrease in retrograde Rab7-positive vesicles observed following JIP1 knockdown (Figure 1B), suggesting that LC3-positive organelles are preferentially susceptible to JIP1 depletion. To follow up on this observation, we examined the possible association of JIP1 with autophagosomes. First, we purified autophagosomes from wild-type mouse brain by differential centrifugation (Maday et al., 2012Maday S. Wallace K.E. Holzbaur E.L. Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons.J. Cell Biol. 2012; 196: 407-417Crossref PubMed Scopus (440) Google Scholar, Strømhaug et al., 1998Strømhaug P.E. Berg T.O. Fengsrud M. Seglen P.O. Purification and characterization of autophagosomes from rat hepatocytes.Biochem. J. 1998; 335: 217-224Crossref PubMed Scopus (138) Google Scholar). Whereas cytosolic LC3-I (∼18 kDa) is enriched in brain homogenate, membrane-bound lipidated LC3-II (∼16 kDa) is enriched in the autophagosome fraction (Kabeya et al., 2000Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5457) Google Scholar). A comparison of the initial brain homogenate and cytosolic fractions indicates that JIP1 is also enriched in the autophagosome fraction (Figure 2A). Interestingly, high-molecular-weight JIP1 (∼110 kDa) was enriched in the autophagosome fraction while low-molecular-weight JIP1 (∼90 kDa) was not, suggesting that differentially spliced JIP1 isoforms may preferentially associate with different organelles. To further explore this association, we used an anti-LC3 antibody to immunoprecipitate LC3 from mouse brain homogenate and found that endogenous JIP1 coimmunoprecipitated with this fraction (Figure 2B), suggesting that endogenous LC3 and JIP1 form a complex. Next, we stained nontransfected wild-type DRG neurons for JIP1 and LC3 and observed that large JIP1-positive puncta colocalized with LC3 along the axon (Figure 2C). Quantification shows that 72% ± 2% (n = 10 neurons) of LC3-positive puncta were also JIP1-positive. We also noted some colocalization of JIP1 and LC3 at axon tips, although to a lesser extent than was observed along the axon (Figure 2D). One mechanism for motor adaptor association with autophagosomes is via direct binding to LC3; the adaptors FYCO1 and optineurin, which have been proposed to regulate kinesin and myosin activity, respectively, both bind to LC3 via a conserved LIR (LC3-interacting region) motif (Pankiv et al., 2010Pankiv S. Alemu E.A. Brech A. Bruun J.A. Lamark T. Overvatn A. Bjørkøy G. Johansen T. FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport.J. Cell Biol. 2010; 188: 253-269Crossref PubMed Scopus (448) Google Scholar, Wild et al., 2011Wild P. Farhan H. McEwan D.G. Wagner S. Rogov V.V. Brady N.R. Richter B. Korac J. Waidmann O. Choudhary C. et al.Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth.Science. 2011; 333: 228-233Crossref PubMed Scopus (959) Google Scholar). To test for an association between JIP1 and LC3, we immunoprecipitated lysates from COS7 cells cotransfected with GFP-LC3 and myc-JIP1. Indeed, an anti-myc antibody coimmunoprecipitated full-length myc-JIP1 as well as GFP-LC3 (Figure 3A). Next, we used purified recombinant proteins to confirm that JIP1 binds directly to LC3. GST-JIP1 expressed in Escherichia coli was purified and subsequently cleaved to generate untagged JIP1. JIP1 was loaded onto glutathione columns with either GST or GST-LC3 bound to the matrix; bound proteins were then eluted with glutathione. In the control condition, JIP1 did not bind to GST and the majority of JIP1 was found in the flow-through fraction. In contrast, JIP1 bound robustly to GST-LC3 with no detectable JIP1 remaining in the flow-through (Figure 3B). To further map the binding site for LC3 on JIP1, we performed coimmunoprecipitations from lysates of COS7 cells cotransfected with GFP-LC3 and truncated myc-JIP1 constructs. C-terminal myc-JIP1[445–565] and myc-JIP1[554–711] did not coimmunoprecipitate GFP-LC3. However, N-terminal myc-JIP1[307–711] robustly coimmunoprecipitated GFP-LC3, as did myc-JIP1[1–390], though to a lesser extent (Figure 3A). Thus, the region spanning amino acid (aa) 307–390, where these two constructs overlap, may be a putative LC3-binding domain. This region of JIP1 contains a predicted LIR motif, EEEEGFDCL (Figure 3C). LIRs contain the core consensus sequence [W/F/Y]xx[L/I/V] and acidic residues frequently flank the aromatic residue (Birgisdottir et al., 2013Birgisdottir A.B. Lamark T. Johansen T. The LIR motif - crucial for selective autophagy.J. Cell Sci. 2013; 126: 3237-3247Crossref PubMed Scopus (568) Google Scholar). The intermolecular interactions underlying LIR binding to LC3 are well characterized. The LIR of the autophagosome cargo adaptor p62 (DDDWTHL) binds inside a narrow groove in LC3 (Figure 3D), with the aromatic residue and the leucine binding to hydrophobic pockets in LC3 and the three consecutive aspartic acids interacting with basic residues in the N terminus of LC3 (Ichimura et al., 2008Ichimura Y. Kumanomidou T. Sou Y.S. Mizushima T. Ezaki J. Ueno T. Kominami E. Yamane T. Tanaka K. Komatsu M. Structural basis for sorting mechanism of p62 in selective autophagy.J. Biol. Chem. 2008; 283: 22847-22857Crossref PubMed Scopus (601) Google Scholar, Noda et al., 2008Noda N.N. Kumeta H. Nakatogawa H. Satoo K. Adachi W. Ishii J. Fujioka Y. Ohsumi Y. Inagaki F. Structural basis of target recognition by Atg8/LC3 during selective autophagy.Genes Cells. 2008; 13: 1211-1218Crossref PubMed Scopus (297) Google Scholar). Similar to FYCO1 and optineurin, JIP1 contains a F-type LIR with a phenylalanine residue at the aromatic position. Multiple sequence alignments of F-type LIRs (Figure 3E) show that these residues are conserved in mammalian JIP1 LIRs (Figure 3C). We generated JIP1 mutants lacking the LIR (ΔLIR) or containing a mutation in the phenylalanine residue (F336A). In coimmunoprecipitations from COS7 cells cotransfected with myc-JIP1 and GFP-LC3 constructs, myc-JIP1-ΔLIR and F336A exhibited significantly decreased binding to GFP-LC3 as compared to wild-type myc-JIP1 (Figure 3F). Thus, the LIR motif we identified in JIP1 is necessary for robust binding to LC3. Because both JIP1 LIR mutants contain intact SH3 homodimerization domains (Kristensen et al., 2006Kristensen O. Guenat S. Dar I. Allaman-Pillet N. Abderrahmani A. Ferdaoussi M. Roduit R. Maurer F. Beckmann J.S. Kastrup J.S. et al.A unique set of SH3-SH3 interactions controls IB1 homodimerization.EMBO J. 2006; 25: 785-797Crossref PubMed Scopus (37) Google Scholar), their residual binding to LC3 may be attributed to dimerization with endogenous wild-type JIP1. The observation that JIP1 directly binds to LC3 via its LIR motif, together with immunostaining, coimmunoprecipitation, and cofractionation results, establishes JIP1 as an autophagosome adaptor. In order to elucidate the function of JIP1, we asked how JIP1 may affect autophagosome biology in four distinct axonal regions: the distal axon tip, the distal axon, the midaxon, and the proximal axon. In the CAD neuronal cell line and in DRGs, JIP1 accumulates in distal neurites or axon tips (Fu and Holzbaur, 2013Fu M.M. Holzbaur E.L. JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors.J. Cell Biol. 2013; 202: 495-508Crossref PubMed Scopus (173) Google Scholar, Muresan and Muresan, 2005Muresan Z. Muresan V. Coordinated transport of phosphorylated amyloid-beta precursor protein and c-Jun NH2-terminal kinase-interacting protein-1.J. Cell Biol. 2005; 171: 615-625Crossref PubMed Scopus (93) Google Scholar, Verhey et al., 2001Verhey K.J. Meyer D. Deehan R. Blenis J. Schnapp B.J. Rapoport T.A. Margolis B. Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules.J. Cell Biol. 2001; 152: 959-970Crossref PubMed Scopus (497) Google Scholar). Moreover, a limited number of JIP1-positive puncta colocalize with LC3 at the distal axonal tip (Figure 2D). Because biogenesis of axonal autophagosomes in primary DRGs occurs exclusively in distal axonal tips (Maday et al., 2012Maday S. Wallace K.E. Holzbaur E.L. Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons.J. Cell Biol. 2012; 196: 407-417Crossref PubMed Scopus (440) Google Scholar), we asked whether JIP1 is necessary for autophagosome formation. Using time-lapse live-cell confocal microscopy of primary DRGs cultured from GFP-LC3 mice (Mizushima et al., 2004Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1937) Google Scholar), we observed continuous autophagosome biogenesis in the distal axon tip (Figure 4A). Initially, small punctate LC3-positive structures become visible and then gradually enlarge to form a ring-like structure up to ∼1 μm in diameter. These events occur on the timescale of several minutes (Figure 4B; Movie S2), consistent with previously published observations (Maday et al., 2012Maday S. Wallace K.E. Holzbaur E.L. Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons.J. Cell Biol. 2012; 196: 407-417Crossref PubMed Scopus (440) Google Scholar). JIP1 depletion did not significantly decrease the density of autophagosomes in the axon tip (control: 0.135 ± 0.018 autophagosomes per μm2; JIP1 knockdown: 0.162 ± 0.013 autophagosomes per μm2; p = 0.24; Figures 4C and 4D), further confirming that biogenesis of this organelle does not depend on JIP1. Further, the lack of accumulation of autophagosomes in JIP1-depleted axon tips suggests that JIP1 is not required for the exit of autophagosomes from the axon tip into the distal axon, which we define as a distinct region immediately proximal to the axon tip (Figure 3A). In primary DRGs, most autophagosomes in the distal axon exhibit bidirectional/stationary motility, while autophagosomes in the midaxon exhibit robust retrograde motility (Maday et al., 2012Maday S. Wallace K.E. Holzbaur E.L. Autophagosomes initiate distally" @default.
• W2036768594 created "2016-06-24" @default.
• W2036768594 creator A5012415712 @default.
• W2036768594 creator A5075390520 @default.
• W2036768594 creator A5080686828 @default.
• W2036768594 date "2014-06-01" @default.
• W2036768594 modified "2023-10-16" @default.
• W2036768594 title "LC3 Binding to the Scaffolding Protein JIP1 Regulates Processive Dynein-Driven Transport of Autophagosomes" @default.
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