RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether

Long-distance RNA transport enables local protein synthesis at metabolicallyactive sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules “hitchhike” on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS) mutations impair RNA granule transport in neurons by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS

JNK-Interacting Protein 3 Mediates the Retrograde Transport of Activated c-Jun N-Terminal Kinase and Lysosomes

<div><p>Retrograde axonal transport requires an intricate interaction between the dynein motor and its cargo. What mediates this interaction is largely unknown. Using forward genetics and a novel <i>in vivo</i> imaging approach, we identified JNK-interacting protein 3 (Jip3) as a direct mediator of dynein-based retrograde transport of activated (phosphorylated) c-Jun N-terminal Kinase (JNK) and lysosomes. Zebrafish <i>jip3</i> mutants (<i>jip3^nl7</i>) displayed large axon terminal swellings that contained high levels of activated JNK and lysosomes, but not other retrograde cargos such as late endosomes and autophagosomes. Using <i>in vivo</i> analysis of axonal transport, we demonstrated that the terminal accumulations of activated JNK and lysosomes were due to a decreased frequency of retrograde movement of these cargos in <i>jip3^nl7</i>, whereas anterograde transport was largely unaffected. Through rescue experiments with Jip3 engineered to lack the JNK binding domain and exogenous expression of constitutively active JNK, we further showed that loss of Jip3–JNK interaction underlies deficits in pJNK retrograde transport, which subsequently caused axon terminal swellings but not lysosome accumulation. Lysosome accumulation, rather, resulted from loss of lysosome association with dynein light intermediate chain (dynein accessory protein) in <i>jip3^nl7</i>, as demonstrated by our co-transport analyses. Thus, our results demonstrate that Jip3 is necessary for the retrograde transport of two distinct cargos, active JNK and lysosomes. Furthermore, our data provide strong evidence that Jip3 in fact serves as an adapter protein linking these cargos to dynein.</p> </div

pJNK failed to accumulate distal to injury.

<p>(A) Schematic and time-line of the injury model experiment and fluorescent intensity quantification. pLL nerve (identified using the <i>neurod:EGFP</i> transgene) was severed using finely pulled glass capillaries. DIC image of a representative injury illustrates peripheral tissue remained mostly intact. Three hours post-injury, larvae were fixed and stained for GFP (to identify the nerve) and either pJNK or tJNK. Thirty µm areas immediately proximal or distal to the injury were imaged and the mean fluorescent intensity of pJNK or tJNK was determined in summed projected stacks through the nerve only in areas that overlapped with GFP expression (outlined by dotted lines in B–I). Background mean fluorescent intensity was determined in adjacent tissue. (B–I) Proximal and distal nerve (dotted outline) adjacent to site of injury (dashed line) in wildtype and <i>jip3^nl7</i> larvae immunolabled for pJNK (B–E) and tJNK (F–I). (J) Levels of pJNK were decreased distal to nerve injury in <i>jip3^nl7</i> but proximal levels were comparable to wildtype (ANOVA, post-hoc contrasts; *-<i>p</i><0.05). (K) tJNK levels trended towards a decrease proximal to the site of injury in <i>jip3^nl7</i> (ANOVA, post-hoc contrasts; <i>p</i><0.1790) but were not different in the retrograde pool, distal to axonal severing.</p

Retrograde JNK3 transport frequency was decreased in <i>jip3^nl7</i> mutants.

<p>(A) Immunolabeling for pJNK in an axon expressing JNK3-mEos showed a high degree of colocalization (arrowheads) indicating that a large percentage of axonal JNK3-mEos is activated. (B,C) Representative stills from a live imaging session showing axonal transport of JNK3-mEos in a pLL axon of a wildtype (B) and <i>jip3^nl7</i> mutant (C) at 2 dpf (see Videos S6 and S7). Pink arrowhead denotes anterograde movement, yellow retrograde movement. (D,E) Kymographs generated from these imaging sessions. (F) Number of retrograde JNK3-mEos puncta (corrected for size of analyzed region and time of imaging session) was decreased in <i>jip3^nl7</i> (ANOVA, post-hoc contrasts; *-<i>p</i><0.05). Distance of individual retrograde movement bouts (G) and velocity (H) were unaltered in <i>jip3^nl7</i>. Anterograde transport distance was decreased (*-<i>p</i><0.05; Ant = anterograde; Ret = retrograde). Scale bars = 10 µm.</p

Model of Jip3's role in retrograde transport of lysosomes and pJNK.

<p>Our data support a model in which Jip3 (green star) serves as a necessary adapter for retrograde transport of pJNK (yellow hexagon) and lysosomes (red oval). This interaction serves to attach these cargoes to the dynein motor complex and, in the case of lysosomes, likely requires interaction with dynein light intermediate chain (DLIC). Global retrograde transport initiation is unaffected with loss of Jip3 as dynein heavy chain, dynactin and other dynein cargos (late endosomes and autophagosomes) do not accumulate in <i>jip3^nl7</i> mutant axon terminals.</p

Increased levels of pJNK did not cause lysosome accumulation in <i>jip3^nl7</i>.

<p>(A) Induction of caJNK3-EGFP at 4 dpf increased the level of pJNK immunofluorescence (middle) in a subset of axon terminals but did not lead to lysosome accumulation as compared to control (B). Scale bars = 10 µm. (C, D) This result was confirmed by Lysotracker red labeling. Surrounding, non-caJNK3-EGFP positive axons show similar numbers, size and density of lysosomes both 4 hours and 13 hours after induction of caJNK3. The pLL nerve was visualized by phase contrast optics and is outlined. Arrowhead indicates axonal swellings caused by high levels of activated JNK. HC denotes neuromast hair cells that strongly label with Lysotracker red. (E) Whole embryo expression of Jip3 and Jip3ΔJNK by mRNA injection partially suppressed the accumulation of lysosomes in <i>jip3^nl7</i> mutant axon terminals at 3 dpf as assayed by expression of Lamp1-mTangerine in pLL neurons. Wildtype – Lamp1-mTangerine positive small puncta only; Mild – small puncta and aggregates visible; Severe - few to no small puncta apparent and large aggregations of Lamp1-mTangerine. (F–I) Injection of 10 pg of a DNA construct encoding Jip3ΔJNK-mCherry rescued lysosome accumulation in <i>jip3^nl7</i> axon terminals. Larvae that expressed Jip3ΔJNK-mCherry (red) in pLL axons and carried the <i>neurod:EGFP</i> transgene were first imaged live (F,H) to identify expressing axon terminals. They were then individually fixed, stained for pJNK (pseudo-colored magenta) and Lamp1 (white), and subsequently the same axon terminals were reimaged (G,I). Arrowheads point to axon terminals in wildtype (F,G; NM1) and <i>jip3^nl7</i> (H,I; NM5) that express Jip3ΔJNK-mCherry (red) at 5 dpf. Arrows point to axon terminals in the same NMs that did not express this construct. Note that expression of Jip3ΔJNK-mCherry in <i>jip3^nl7</i> completely rescued lysosome accumulation (yellow arrowheads in I″) but failed to rescue high levels of pJNK (yellow arrowheads in I′).</p

Jip3 scaffolds lysosomes to DLIC for retrograde transport.

<p>(A,B) Stills from a wildtype imaging session at 3 dpf in which Lamp1-EGFP (A) and Jip3-mCherry (B) co-transport was analyzed (Video S10). Pink and yellow arrowheads point to two retrograde Jip3/Lamp1 positive cargos. (C,D) Kymographs generated from this imaging session for individual cargos. (E) Schematized kymograph of co-transport. Yellow lines denote Jip3-positive lysosomes moving in the retrograde direction. (F,G) mTangerine-DLIC expression in a wildtype (F) and <i>jip3^nl7</i> mutant (G) NM1 axon terminal at 3 dpf. (H,I) Stills from analysis of Lamp1 (H) and DLIC (I) co-transport at 3 dpf in a wildtype (Video S11). Green arrow-anterograde co-labeled puncta. Yellow arrowhead-DLIC positive lysosome undergoing retrograde transport. (J) The ratio of DLIC positive lysosomes moving in the retrograde direction was significantly decreased in <i>jip3^nl7</i> mutants (ANOVA, *-<i>p</i><0.05; Anterograde-Ant; Retrograde-Ret). (K–M) Kymographs from this imaging session and schematized kymograph depicting co-labeled anterograde lysosomes in green and retrograde in yellow.</p

Jip3 interaction with JNK was necessary for pJNK clearance and the prevention of axon swellings.

<p>(A–C) Axon terminal swellings and pJNK accumulation were rescued by Jip3 but not by Jip3ΔJNK. Axonal swellings were visualized live by <i>neurod:EGFP</i> transgene expression; following live imaging, pJNK was assayed by individual larva immunolabeling at 4 dpf. White arrowheads mark axon terminals expressing the DNA constructs. Yellow arrow points to a swelling in an axon not expressing Jip3-mCherry. Red arrowhead denotes an underlying pJNK positive cell not expressing Jip3-mCherry. (D) Ratio of axon terminal swellings in each class (mild = small swellings, severe = large swellings) show rescue of axonal swellings by full-length Jip3 but not Jip3ΔJNK. <i>DNA</i> indicates the rescue construct injected; Jip3 = full-length Jip3-mCherry; ΔJNK = Jip3ΔJNK-mCherry; Con = uninjected control. Embryo genotype, determined by control axon terminal morphology, is indicated below each bar. (E) Ratio of pJNK levels in Jip3 or Jip3ΔJNK expressing axon terminals to those not expressing the rescue construct at 4 dpf. Jip3, but not Jip3ΔJNK, suppressed increased pJNK levels in <i>jip3^nl7</i> (Wilcoxon rank-sum; *-<i>p</i><0.01). (F) Induction of constitutively active JNK3 tagged with EGFP (caJNK3; green) for 15 hours at 4 dpf increased the level of pJNK immunofluorescence concomitant with the induction of swellings shown by both the caJNK3-EGFP fill and Tag1 immunolabeling of neuronal membranes. Arrowhead points to a caJNK3-EGFP expressing axon. Yellow arrow indicates an axon terminal in the same NM not expressing this construct. (G) Axon terminal swellings were absent in axon terminals expressing an inactive form of the same construct (caJNK3-IA), indicating that JNK activation was necessary to induce swellings. (H,I) Efficacy of both caJNK3 and caJNK3-IA were assayed by Western blot analysis of phospho-cJun, a downstream target of active JNK. While whole embryo overexpression of caJNK3-EGFP by RNA injection induced elevated levels of phospho-cJun at 24 hpf (H), similar expression of the inactive form of caJNK3-EGFP (caJNK3-IA) failed to induce a similar increase (I). Scale bars = 10 µm.</p