BORC/Kinesin-1 Ensemble Drives Polarized Transport of Lysosomes

BORC/kinesin-1 ensemble drives polarized transport of PNAS PLUS lysosomes into the axon

Ginny G. Faríasa, Carlos M. Guardiaa, Raffaella De Pacea, Dylan J. Britta, and Juan S. Bonifacinoa,1

aCell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892

Edited by Pietro De Camilli, Howard Hughes Medical Institute, Yale University, New Haven, CT, and approved February 27, 2017 (received for review October 2, 2016) The ability of lysosomes to move within the cytoplasm is cellular processes that depend on lysosome movement, such as important for many cellular functions. This ability is particularly endocytic degradation, autophagy, cell adhesion and migration, and critical in neurons, which comprise vast, highly differentiated antigen presentation, are impaired (9, 11–15). domains such as the axon and dendrites. The mechanisms that The mechanisms of organelle distribution in polarized cells such control lysosome movement in these domains, however, remain as neurons are far more complex. Unlike nonpolarized cells, neu- poorly understood. Here we show that an ensemble of BORC, Arl8, rons have a soma, dendrites, and axon with different organelle SKIP, and kinesin-1, previously shown to mediate centrifugal trans- compositions. Whereas some organelles are distributed throughout port of lysosomes in nonneuronal cells, specifically drives lysosome the neuron (e.g., mitochondria), others are largely segregated to the transport into the axon, and not the dendrites, in cultured rat somatodendritic domain (e.g., the Golgi complex) or the axonal hippocampal neurons. This transport is essential for maintenance of domain (e.g., synaptic vesicles) (16). Specific patterns of organelle axonal growth-cone dynamics and autophagosome turnover. Our distribution in neurons are partly the result of the organelles’ ability findings illustrate how a general mechanism for lysosome dispersal to move along different microtubule tracks through coupling to in nonneuronal cells is adapted to drive polarized transport in different microtubule motors (17, 18). Neuronal microtubules are neurons, and emphasize the importance of this mechanism for acentrosomal and exhibit distinct properties in the somatodendritic critical axonal processes. and axonal domains. Dendritic microtubules are shorter and have mixed orientations, whereas axonal microtubules consist of long lysosomes | polarized organelle transport | kinesin-1 | BORC | bundles with uniform plus end-out orientation (19, 20). In addition, autophagosomes dendritic and axonal microtubules display different posttranslational modifications of tubulin (21) and microtubule-associated proteins ince their discovery by de Duve in the mid-20th century (1), (22). These distinct properties determine the specificity and di- Slysosomes have become recognized as the main site for the rection of organelle movement by different microtubule motors in degradation of a wide variety of biomacromolecules in the endo- different regions of the neuron. Of the ∼45 kinesins encoded in membrane system of animal cells (2). Indeed, lysosomes degrade mammalian genomes, many mediate axonal anterograde transport, extracellular and plasma membrane substrates delivered by way of whereas a few additionally drive transport in the dendrites (23–25). NEUROSCIENCE endocytosis, as well as cytoplasmic substrates engulfed in the pro- In contrast, there is only one cytoplasmic dynein in mammals that cess of autophagy. In recent years, lysosomes and their precursor mediates retrograde transport in the axon, as well as transport in the late endosomes (hereafter referred to indistinctly as “lysosomes”) dendrites (26, 27). Dendritic transport mediated by kinesins and have been found to participate in many other cellular processes, dynein is potentially bidirectional because of the mixed polarity of including metabolic signaling, immunity, plasma membrane repair, dendritic microtubules. exocytosis, cell adhesion and migration, and tumor invasion and Lysosomes have been previously reported to occur in all re- metastasis (2). The performance of all these functions requires that gions of the neuron and to play essential roles in general and lysosomes constantly survey the cytoplasmic space in search of targets (3). Bidirectional movement of lysosomes occurs along mi- Significance crotubules (4) and is driven by the plus end-directed kinesin (5) and minus end-directed dynein microtubule motors (6). Various en- Lysosomes are found in all neuronal domains, including the soma, sembles of small GTPases, their effectors, and other adaptor and axon, and dendrites. How neurons are transported in these do- regulatory molecules mediate coupling of lysosomes to the micro- mains, however, remains poorly understood. In the present study, tubule motors, enabling their controlled movement in response to we show that a protein ensemble comprising BORC, Arl8, SKIP, diverse cellular needs (7, 8). and kinesin-1 specifically drives lysosome transport into the axon We recently described a multiprotein complex named BORC and not the dendrites. We also show that this mechanism of ax- – (for BLOC-One Related Complex) that regulates coupling of ly- onal lysosome transport is essential for maintenance of growth- sosomes to kinesin-1, promoting their radial movement toward the cone dynamics and turnover of autophagosomes in the distal peripheral cytoplasm in nonpolarized cells (9). BORC comprises axon. These findings imply that transport of lysosomes into the eight subunits named BLOS1, BLOS2, snapin, KXD1, myrlysin axon and the dendrites occurs by different mechanisms, and (LOH12CR1), lyspersin (C17orf59), diaskedin (C10orf32), and demonstrate that BORC-regulated lysosome transport is critical for MEF2BNB (LOC729991). The eight subunits have also been axonal functions. designated BORCS1–BORCS8, respectively, by the Human Gene Nomenclature Committee. This complex associates with the cyto- Author contributions: G.G.F. and J.S.B. designed research; G.G.F., C.M.G., R.D.P., and D.J.B. performed research; G.G.F. and C.M.G. contributed new reagents/analytic tools; G.G.F., solic aspect of the lysosomal membrane in part through a myristoyl R.D.P., D.J.B., and J.S.B. analyzed data; and G.G.F., C.M.G., R.D.P., D.J.B., and J.S.B. wrote group at the N terminus of myrlysin (9). BORC does not interact the paper. with kinesin-1 directly, but does so through the small GTPase The authors declare no conflict of interest. Arl8b and its effector SKIP (8, 9). Interference with any link in this This article is a PNAS Direct Submission. chain of interactors impairs anterograde movement of lysosomes 1To whom correspondence should be addressed. Email: [email protected]. and leads to the collapse of the lysosome population to the peri- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. centrosomal region of nonpolarized cells (8–10). As a consequence, 1073/pnas.1616363114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1616363114 PNAS | Published online March 20, 2017 | E2955–E2964 Downloaded by guest on October 1, 2021 neuron-specific processes (28–33). Moreover, lysosome dys- BORC-Arl8-SKIP-kinesin-1 ensemble controls axonal but not function underlies many neurodevelopmental and neurodegen- dendritic transport of lysosomes. Indeed, interference with these erative disorders. Among these are hereditary lysosomal-storage proteins decreases lysosome transport specifically into the axon. diseases caused by mutations in lysosomal proteins, which often Conversely, its enhancement drives lysosome accumulation at affect the central nervous system (34). In addition, defective ly- axon terminals. We also show that this mechanism is essential for sosome function contributes to the pathogenesis of more com- maintenance of axonal growth-cone dynamics and autophagosome mon neurological disorders such as Alzheimer’s disease and clearance. These findings illustrate how a general mechanism for Parkinson’s disease (35). Although it is clear that lysosomes are centrifugal transport of lysosomes in nonpolarized cells is adapted essential for proper neuronal function, the segregation of the to drive polarized transport of lysosomes in neurons. neuronal cytoplasm into structurally and functionally different domains poses additional challenges to the understanding of Results lysosome distribution and dynamics. Outstanding questions are: Nonpolarized Distribution of Lysosomes in Hippocampal Neurons. As How do lysosomes traffic in different neuronal domains? Is the a first step to analyze the mechanisms that regulate lysosome dis- BORC/kinesin-1–dependent lysosome transport characterized in tribution in neurons, we examined the localization of various en- nonpolarized cells also operational in neurons, and, if so, in what dogenous and transgenic lysosomal markers in day in vitro (DIV) neuronal domains? What neuronal functions are dependent on 7 or 8 rat hippocampal neurons in primary culture. Immunofluo- this transport mechanism? rescence microscopy for the endogenous lysosome-associated In this study, we have addressed these questions by using rat membrane protein 1 (LAMP1) (36) or the late endosomal/lyso- hippocampal neurons in primary culture as a model system. We somal adaptor and MAPK and MTOR activator 4 (LAMTOR4) find that lysosomes exhibit a largely nonpolarized distribution, and (37) showed that these proteins were most concentrated in the move bidirectionally in all regions of the neuron. Remarkably, the soma, but were also present in discrete puncta along both the

A Dendrites Dendrites

AIS AIS

Axon Axon

20 µm LAMP1-GFP + PM-RFP / AnkG

B Dendrite Proximal axon C Dendrite Proximal axon LAMP1-GFP CD63-GFP PM-RFP PM-RFP Merge Merge Distal axon Distal axon LAMP1-GFP CD63-GFP

PM-RFP PM-RFP

Merge Merge Fig. 1. Nonpolarized distribution of lysosomes in DIV 7 hippocampal neurons. (A) Fixed DIV 7 neuron expressing LAMP1-GFP (grayscale, green in merge) and D Dendrite Proximal axon E 3 CatD-GFP D/A AT/D AT/A PM-RFP (red in merge), and stained for endogenous AnkG (blue in merge). Arrow points to the AIS, and the PM-RFP 2 – Merge dashed circle shows an axon tip. (B D) Fifty-micrometer 1 segments of dendrites and proximal axons and 100-μm Distal axon

Intensity ratio segments of distal axons from fixed DIV 7 neurons CatD-GFP 0 expressing PM-RFP together with LAMP1-GFP (B), Transfected Endogenous CD63-GFP (C), or cathepsin D (CatD)-GFP (D). Fig. S1 LAMP1-GFP LAMP1 PM-RFP shows immunostaining of endogenous LAMP1 and CD63-GFP LAMTOR4 LAMTOR4 and staining with LysoTracker and Magic CatD-GFP LysoTracker Merge Red. (E) D/A, AT/D, and AT/A ratios are represented as Magic Red the mean ± SD from 25 neurons such as those in B–D F Proximal axon Proximal axon G and Fig. S1.(F) Fifty-micrometer segments of proximal LAMP1-GFP LAMP1-GFP 100 axons from DIV 7 neurons expressing LAMP1-GFP and LAMTOR4 EEA1 stained live with Magic Red or, after fixation, for en- Merge Merge 50 dogenous LAMTOR4, EEA1, or synaptophysin-1 (Syp1). Proximal axon Proximal axon (G) Quantification of colocalization of 150 LAMP1-GFP– LAMP1-GFP LAMP1-GFP LAMP1-GFP 0 positive particles from 15 neurons per condition ana-

Magic Red co-localization (%) Syp1 LAMTOR4 EEA1 lyzed with the different markers shown in F,and Merge Merge Magic Red Syp1 expressed as mean ± SD.

E2956 | www.pnas.org/cgi/doi/10.1073/pnas.1616363114 Farías et al. Downloaded by guest on October 1, 2021 dendrites and the axon (Fig. S1). Staining with the acidic-organelle PNAS PLUS dye LysoTracker or the fluorogenic cathepsin B substrate Magic A B Red, both functional markers of lysosomes, showed a similar distribution of fluorescent puncta to the soma, dendrites, and axon (Fig. S1). Expression of various GFP-tagged lysosomal proteins (i.e., LAMP1-GFP, CD63-GFP, or cathepsin D-GFP) by transfection at DIV 4 recapitulated the nonpolarized distribution of the endogenous lysosomal proteins at DIV 7/8 (Fig. 1A–D). In the axon, lysosomes were evenly distributed among the proximal, middle, and distal regions (Fig. 1A–D). Quantification of the mean fluorescence intensity of endogenous and transgenic lysosomal markers in dendrites vs. axon (D/A), axon tips vs. dendrites (AT/D), and axon tips vs. axon (AT/A) from many neurons yielded ratios in the range of LAMP1-GFP Endogenous LAMTOR4 0.8–1.5 (Fig. 1E), confirming that lysosomes have a largely nonpolarized distribution, and are not particularly concentrated in axon tips. The axonal LAMP1-GFP colocalized with endogenous LAMTOR4 and Magic Red product, but not the endogenous early endosomal antigen 1 (EEA1) and synaptic vesicle protein synaptophysin-1 (Syp1; Fig. 1 F and G), confirming that LAMP1- GFP is confined to lysosomes and does not spill over into other axonal organelles. LAMP1-GFP and LAMTOR4 also exhibited a nonpolarized distribution in more mature DIV 17 neurons (Fig. 2 A–C). Taken together, these observations demonstrated the presence of conventional lysosomes in all neuronal domains, and PM-RFP Endogenous PSD95 validated LAMP1-GFP as a faithful reporter of this distribution.

Lysosomes Move Bidirectionally in both Axons and Dendrites. To examine lysosome dynamics in hippocampal neurons, we performed live-cell imaging of LAMP1-GFP in cells coexpressing plasma membrane-targeted RFP (PM-RFP) and stained with a CF640R-conjugated antibody to the axon initial segment (AIS) protein neurofascin (i.e., NF-640R) (38). This imaging revealed bidirectional movement of lysosomes containing LAMP1-GFP in dendrites and axon (Fig. 3A and Movie S1). Motile lysosomes accounted for 74.8% of the total in dendrites (51.5% antero- +Merge AnkG MergeMerge grade, 48.5% retrograde) and 88.4% in the axon (50.4% anter- Dendrite Dendrite NEUROSCIENCE ograde, 49.6% retrograde), whereas the rest were stationary (Fig. LAMP1-GFP Endogenous LAMTOR4 3 A and B). The average run length of moving lysosomes was shorter in dendrites than in axons (Fig. 3 A and B), consistent with the different microtubule organization in the two domains. PM-RFP Endogenous PSD95 The number and run length of lysosomes moving anterogradely and retrogradely in the proximal axon (Fig. 3 A and B and Movie S1) were not significantly different from those parameters in the Merge Merge midaxon. In the distal axon, including axon tips, however, we saw an increase in the number of stationary lysosomes, particularly at Proximal axon Proximal axon the entrance to the growth cones (Fig. 3 A and B). Additional LAMP1-GFP Endogenous LAMTOR4 experiments showed comovement of LAMP1-RFP with CD63- GFP and Cathepsin D-GFP in dendrites and axon (Fig. 3 C–E and Movies S2 and S3), corroborating the identity of the moving PM-RFP Endogenous PSD95 vesicles as conventional lysosomes. These results confirmed and expanded previous observations in cortical neurons (30), demonstrating that functional lysosomes have a largely nonpolarized, Merge Merge dynamic distribution in different neuronal types.

Reduced Acidity of Lysosomes at Axon Tips. Mature lysosomes in 2 nonneuronal cells have an acidic luminal pH (4.5–5.0). To verify C that lysosomes in dendrites and axon are acidic, we fused LAMP1-GFP 1

superecliptic pHluorin (SEP) to the luminal domain of LAMP1- D/A RFP (Fig. 3F) and expressed the chimeric construct (SEP- LAMTOR4

LAMP1-RFP) in neurons. SEP fluoresces green at neutral pH intensity ratio 0 but is nonfluorescent at pH < 6 (39). In the dendrites and the proximal axon, anterogradely and retrogradely moving lysosomes Fig. 2. Nonpolarized distribution of lysosomes in DIV 17 hippocampal were positive for RFP but negative for SEP, indicating that they neurons. (A) Fixed DIV 17 neuron expressing LAMP1-GFP (green channel) were acidic (Fig. 3 G and Movie S4). In the distal axon, including and PM-RFP (red channel) and stained for AnkG (blue channel). (B) Fixed DIV 17 neuron stained for endogenous LAMTOR4 (green channel), the post- axon tips, however, we observed some puncta exhibiting both synaptic marker PSD-95 (red channel), and AnkG (blue channel). Arrows SEP and RFP fluorescence (Fig. 3G). The latter likely represent point to the AIS. (Bottom) Straightened 50-μm segments of dendrites and lysosomes that are less acidic, such as those described in the proximal axons. (C) D/A ratios are represented as the mean ± SD from periphery of nonneuronal cells (40). We also observed more 20 neurons per condition.

Farías et al. PNAS | Published online March 20, 2017 | E2957 Downloaded by guest on October 1, 2021 A B

C DE

Fig. 3. Bidirectional movement of lysosomes in dendrites and axon. (A) Single frames from Movie S1 (top three strips) and kymographs (bottom strip) of 50-μm segments of a dendrite, proximal axon, and distal axon from a DIV 8 neuron expressing LAMP1-GFP and PM-RFP. The kymographs show LAMP1-GFP moving in anterograde (lines with negative slopes) and retrograde (lines with positive slopes) directions, and in stationary foci (vertical lines), during 240 s of recording. (B) Quantification of the number and run length of LAMP1-GFP particles in dendrites, proximal axon, and distal axon expressed as mean ± SD from eight neurons. Statistical significance for all groups was calculated by one-way ANOVA followed by Dunnett’stest(*P < 0.01). (C and D) Single frames from Movies S2 and S3 showing colocalization of moving and stationary particles containing LAMP1-RFP together with CD63-GFP (C)orCatD-GFP(D)in50-μmsegmentsofadendrite and proximal axon. (E) Quantification of the colocalization of LAMP1-RFP with CD63-GFP and CatD-GFP in anterograde, retrograde, and stationary particles in dendrites and proximal axon. A total of 148 and 141 LAMP1-RFP–positive particles from five neurons per condition were analyzed for colocalization with CD63- GFP and CatD-GFP, respectively. Results are expressed as the mean ± SD. (F) Schematic representation of SEP-LAMP1-RFP. (G) Single frames from Movie S4 (top three strips) and kymographs (bottom strip) of 50-μm segments of a dendrite, proximal axon, and distal axon from a DIV 7 neuron expressing SEP-LAMP1-RFP. Note the absence of SEP fluorescence in lysosomes moving in dendrites and proximal axon, and its presence near the axon tip. Also note the more diffuse appearance of SEP fluorescence in the distal axon.

diffuse SEP fluorescence in the distal axon, including the growth kinesin-1, a heterotetramer composed of two kinesin heavy chains cone (Fig. 3G and Movie S4). This fluorescence could result (KIF5) and two kinesin light chains (KLC) (47). Neurons express from fusion of lysosomes with the plasma membrane, as pre- three KIF5 paralogs encoded by different genes: the neuron- viously reported in other cells (29, 41–43). specific KIF5A and KIF5C and the ubiquitous KIF5B (48). Treatment with shRNAs targeting all three KIF5 proteins caused a Interference with the BORC–Arl8b–SKIP–kinesin-1 Ensemble Specifically drastic depletion of lysosomes from the axon but not the dendrites Impairs Lysosome Transport into the Axon. ABORC–Arl8b–SKIP– (Fig. 4B vs.Fig.4C). Likewise, overexpression of a KLC1-DN kinesin-1 ensemble was previously shown to drive centrifugal construct encoding the cargo-recognition tetratricopeptide-repeat transport of lysosomes in nonpolarized cells (8, 10, 12, 44–46) (Fig. (TPR) but not the KIF5-interacting heptad-repeat domain (49) 4A). Given the polarized organization of neurons, we asked specifically reduced the presence of lysosomes in the axon (Fig. whether this mechanism was involved in moving lysosomes into the 4D). Similar observations were made upon treatment with shRNAs dendrites and/or the axon. To address this question, we performed to SKIP, Arl8b, or the myrlysin or lyspersin subunits of BORC (Fig. shRNA-mediated knockdown (KD) or dominant-negative (DN) 4 E–H). The depletion of LAMP-1-RFP–labeled lysosomes from interference of the different components of this machinery, and the axon in myrlysin- or lyspersin-KD neurons could be rescued by examined the effect of these manipulations on the distribution of expression of shRNA-resistant, epitope-tagged forms of the cor- LAMP1-RFP in neurons. KD was confirmed by immunostaining, responding proteins (Fig. S3). Quantification by image analysis of confocal microscopy, and image analysis of the endogenous targets many neurons showed that interference with the BORC–Arl8b– for each shRNA (Fig. S2). We first tested the involvement of SKIP–kinesin-1 complex increased the dendrite/axon polarity index

E2958 | www.pnas.org/cgi/doi/10.1073/pnas.1616363114 Farías et al. Downloaded by guest on October 1, 2021 A PNAS PLUS Lysosome SKIP BORC KLC Arl8 KIF5

B Control shRNA C KIF5A+B+C shRNAs

20 m 20 m LAMP1-RFP GFP LAMP1-RFP GFP D KLC1 DN E SKIP shRNA

20 m 20 m LAMP1-RFP GFP TPR LAMP1-RFP GFP F Arl8b shRNA G Myrlysin shRNA

20 m 20 m LAMP1-RFP GFP LAMP1-RFP GFP

H Lyspersin shRNA I D/A AT/D AT/A NEUROSCIENCE 12 * 2 4 * * 8 * * * 1 2 4 * * * * * *

Intensity ratio 0 0 0 Control shRNA Arl8b shRNA KIF5A/B/C shRNA Myrlysin shRNA 20 m KLC1 DN Lyspersin shRNA LAMP1-RFP GFP SKIP shRNA

Fig. 4. Reduced localization of lysosomes to the axon caused by interference with components of the BORC/kinesin-1 machinery. (A) Schematic representation of the BORC–Arl8–SKIP–kinesin-1 ensemble (Left) and of the domains involved in interactions (Right). (B–H) DIV 7 neurons coexpressing LAMP1-RFP together with GFP plus control shRNA (B), KIF5A+B+CshRNA(C), KLC1-DN (D), SKIP shRNA (E), Arl8b shRNA (F), myrlysin shRNA (G), or lyspersin shRNA (H). All shRNAs used in this study were shown to be effective at depleting the target proteins (Fig. S2). Arrows point to the AIS, and arrowheads indicate the axon. (I) D/A, AT/D, and AT/A ratios expressed as mean ± SD from 25 neurons analyzed as in B–H. Statistical significance for all groups, including KIF5A+B+C shRNA, KLC1-DN, SKIP shRNA, Arl8b shRNA, myrlysin shRNA, and lyspersin shRNA, was calculated by one-way ANOVA followed by Dunnett’stest(*P < 0.01 vs. control). The effects of myrlysin- and lyspersin-shRNAs could be rescued by expression of the corresponding shRNA-resistant constructs (Fig. S3).

of lysosomes from ∼1 in control neurons to 4–8 in the treated GFP-KIF5A with HA-KLC1 caused only a modest increase in the neurons (Fig. 4I and Fig. S3). Depletion of lysosomes was uniform amount of lysosomes at axon tips relative to the rest of the axon throughout the axon, as shown by the largely unchanged ratio of (Fig. 5A). Additional expression of myc-SKIP, however, resulted in axon tips to the rest of the axon (Fig. 4I and Fig. S3). These ob- strong accumulation of lysosomes at axon tips (Fig. 5B), as reflected servations thus demonstrated a requirement for all of the members by an AT/A ratio of ∼6(Fig.5A). For comparison, we showed that – – – of the BORC Arl8b SKIP kinesin-1 ensemble for polarized forced coupling of lysosomes to the KIF5A motor domain by movement of lysosomes into the axon, but not the dendrites. coexpression of chimeric constructs comprising the motor domain Kinesin-1 Requires SKIP for Its Ability to Promote Axonal Transport of of KIF5A fused to streptavidin and mCherry (mCh-KIF5A-Strep), Lysosomes. To analyze in more detail the activity of the different and LAMP1 fused to the streptavidin-binding peptide and GFP components of the BORC/kinesin-1 machinery in moving lysosomes (LAMP1-SBP-GFP), caused massive accumulation of lysosomes at to the axon, we examined the effect of overexpressing different axon tips (Fig. 5A and Fig. S4). In these experiments, D/A ratios did combinations of motor and adaptor proteins on the distribution of not change significantly (Fig. 5A), but this was because lysosome LAMP1-RFP. We observed that overexpression of GFP-KIF5A accumulation occurred at axon tips and not along the axon. Im- had no effect on lysosome distribution (Fig. 5A). Coexpression of portantly, GFP-KIF5A, HA-KLC1, and myc-SKIP colocalized on

Farías et al. PNAS | Published online March 20, 2017 | E2959 Downloaded by guest on October 1, 2021 lysosomes in the axon (Fig. 5 C–E), consistent with their effects with HA-KLC1 and myc-SKIP, on the contrary, endows the excess being the consequence of a direct association. GFP-Rigor-KIF5A with the ability to bind, and thus immobilize, As another means of demonstrating the importance of SKIP for lysosomes independently of endogenous KIF5–KLC–SKIP com- the effect of kinesin-1 on lysosomes, we examined the effect of plexes. All these experiments were consistent with SKIP being overexpressing SKIP together with HA-KLC1 and a GFP-KIF5A critical for the ability of kinesin-1 to associate with lysosomes and to G235A “rigor” mutant (Rigor-KIF5A), which can bind to its pre- drive them down the axon. ferred microtubules but not “walk” along them (50). As previously reported (38, 51), under these conditions, GFP-Rigor-KIF5A dec- Arl8b and SKIP Are Limiting Factors in the Coupling of Lysosomes to orated microtubule tracks spanning the initial part of the axon, Kinesin-1 in Neurons. Arl8b and BORC function upstream of SKIP corresponding to the preaxonal exclusion zone (38) and AIS (Fig. to couple lysosomes to kinesin-1 (8, 9) (Fig. 4A). Single over- 5F). Importantly, whereas expression of GFP-Rigor-KIF5A alone expression of Arl8b or SKIP showed association of these proteins had little or no effect on lysosome motility in the axon (Fig. 4G), with axonal lysosomes (Movie S5), but caused only a modest coexpression with HA-KLC1 and myc-SKIP arrested lysosomes in concentration of lysosomes in axon tips (Fig. 6A). When over- the initial part of the axon (Fig. 5 F and H). The failure of GFP- expressed together, however, Arl8b and SKIP promoted robust Rigor-KIF5A alone to exert a DN effect on axonal lysosome accumulation of lysosomes in axon tips (Fig. 6 A–C), comparable transport could be the result of incomplete replacement of this to that resulting from coexpression of mCh-KIF5A-Strep with protein for endogenous KIF5 isoforms in complex with KLC and LAMP1-SBP-GFP (Fig. 6A and Fig. S4). The strong accumula- SKIP over the time course of the experiments. The coexpression tion of lysosomes at axon tips in neurons overexpressing a

A B D/A AT/D AT/A

1.5 15 * 15 *

* 1 10 10 *

0.5 5 5 Intensity ratio * * LAMP1-RFP GFP-KIF5A 0 0 0 + GFP + GFP-KIF5A + HA-KLC1 + GFP-KIF5A HA-KLC1 + GFP-KIF5A HA-KLC1 myc-SKIP 20 m LAMP1-SBP-GFP + mCh-KIF5A-Strep HA-KLC1 myc-SKIP Distal axon C Dendrite Proximal axon D LAMP1-RFP LAMP1-RFP 200 GFP-KIF5A GFP-KIF5A HA-KLC1 HA-KLC1 Fig. 5. Kinesin-1 requires SKIP to drive axonal lyso- myc-SKIP myc-SKIP 100 some transport. (A) Quantification of the effects of overexpressing different combinations of GFP-KIF5A, Merge Intensity KLC1-HA, and myc-SKIP. D/A, AT/D, and AT/A ratios Distal axon 0 ± 0 20 40 60 80 100 are expressed as the mean SD from 25 neurons. LAMP1-RFP E 80 Statistical significance for all groups was calculated by one-way ANOVA followed by Dunnett’stest(*P < GFP-KIF5A 60 0.01 vs. control). GFP was used as a control for uniform distribution, and a combination of LAMP1-SBP-GFP 40 HA-KLC1 with mCh-KIF5A-streptavidin (Fig. S4) as a control for Intensity 20 extreme redistribution to axon tips. (B)DIV7neuron myc-SKIP expressing LAMP1-RFP together with GFP-KIF5A, 0 30 40 50 60 70 80 KLC1-HA, and myc-SKIP. Arrows point to the AIS, Merge Distance ( m) and the dashed circle shows an axon tip. (C)Fifty- micrometer segments of a dendrite and the proximal F LAMP1/ Rigor-KIF5A / KLC1 / SKIP LAMP1 Rigor-KIF5A Merge axon and 100-μm-length segment of the distal axon of 20 µm G the neuron shown in B.(D and E) Fluorescence line intensity plots show the accumulation of all proteins at the axon tip (D), and their colocalization in vesicles along the distal axon (E). (F)DIV7neuronexpressing 240 s LAMP1-RFP plus KLC1-HA, myc-SKIP, and a Rigor- KIF5A mutant able to interact with but not walk LAMP1 KLC1 + KLC-SKIP along microtubules. (G and H)Singleimagesofthe H initial part of the axon (Top) and kymographs (Bot- Rigor-KIF5A SKIP tom) from live DIV8 neurons expressing LAMP1-RFP LAMP1-RFP plus GFP-Rigor-KIF5A (G), or these proteins in combi- 200 Rigor-GFP-KIF5A nation with KLC1-HA and myc-SKIP (H). Note that ex-

HA-KLC1 240 s pression of Rigor-KIF5A alone does not affect the 100 myc-SKIP

Intensity movement of lysosomes into the axon, whereas Rigor- 0 KIF5A plus KLC1 and SKIP immobilize lysosomes in the 50 m 50 m 50 m 50 m initial part of the axon.

E2960 | www.pnas.org/cgi/doi/10.1073/pnas.1616363114 Farías et al. Downloaded by guest on October 1, 2021 combination of Arl8b and SKIP depleted lysosomes from den- Reduced Growth Cone Dynamics and Aberrant Autophagosomes in PNAS PLUS drites and axon shafts, explaining why the D/A ratio in these Axons from BORC-KD Neurons. What is the physiological impor- neurons was higher than in those overexpressing Arl8b or SKIP tance of BORC-dependent lysosome transport into axons? As alone, in which accumulation occurred all along the axon (Fig. expected, expression of myrlysin shRNA reduced transport of 6A). These results indicated that Arl8b and its effector SKIP are LAMP1-RFP into the axon (Fig. 7 A and B and Movie S7). In- limiting factors for the coupling of lysosomes to kinesin-1 in terestingly, in the same neurons, we noticed a decrease in the neurons. size of axonal growth cones upon depletion of myrlysin (Fig. 7 C Myrlysin-GFP was also found on lysosomes moving in both and D and Movie S7). Similar decreases in growth cone size were directions, as well as in stationary foci (Movie S6). Interestingly, observed in lyspersin- and Arl8b-shRNA–treated neurons (Fig. overexpression of myrlysin-FOS or HA-lyspersin caused accu- 7D). Moreover, the shape of the growth cones did not change mulation of lysosomes in axon tips, albeit to a lesser extent rel- over time in myrlysin-KD cells, whereas it was highly dynamic, ative to Arl8b plus SKIP (Fig. 6 D–F). These observations particularly in the peripheral region, in control cells (Fig. 7C and indicated that myrlysin and lyspersin have an intrinsic ability to Movie S7). The decreased size and dynamics of the growth cones activate downstream effectors of lysosome movement, although in myrlysin-KD cells indicates a requirement of lysosomes for full activity may require the other subunits of BORC. growth cone structure and function.

A D/A AT/D AT/A D D/A AT/D AT/A 1.5 15 * * 15 * 1.5 * 15 15 * 1 * 10 10 1 10 10 * * * * * * 0.5 5 5 0.5 5 5 Intensity ratio Intensity ratio 0 0 0 0 0 0 Control + Arl8b-mCh + HA-Lyspersin Control + myc-SKIP + Arl8b-mCh myc-SKIP + Myrlysin-FOS LAMP1-SBP-GFP + mCh-KIF5A-Strep B E NEUROSCIENCE

LAMP1-GFP myc-SKIP LAMP1-RFP HA-Lyspersin

20 µm 20 µm Arl8b-mCh LAMP1 / SKIP / Arl8b CFP LAMP1 / Lyspersin C F Dendrite Proximal axon Dendrite Proximal axon LAMP1-GFP LAMP1-RFP Arl8b-mCh HA-Lyspersin myc-SKIP Merge Merge CFP CFP + CFP Distal axon Distal axon LAMP1-GFP LAMP1-RFP

Arl8b-mCh HA-Lyspersin

myc-SKIP Merge

Merge CFP

CFP + CFP

Fig. 6. Arl8b-SKIP or BORC subunit overexpression promotes accumulation of lysosomes in axon tips. (A) Quantification of the effects of overexpressing myc-SKIP or Arl8b-mCherry alone, or both proteins together, on the distribution of LAMP1-GFP. (B and C) DIV 7 neuron expressing LAMP1-GFP and SKIP-Arl8b (B) and enlarged regions of a dendrite, proximal axon, and distal axon of this neuron (C). (D) Quantification of the effects of overexpressing HA-lyspersin or myrlysin-FOS on the distribution of LAMP1-GFP. (E and F) DIV 7 neuron expressing LAMP1-RFP, HA-lyspersin, and CFP. In A and D, LAMP1 intensity ratios are the mean ± SD from 25 neurons. Statistical significance for all groups was calculated by one-way ANOVA followed by Dunnett’stest(*P < 0.01vs.control).InB and E, arrows point to the AIS and dashed circles show axon tips. Comovement of Arl8b-GFP or GFP-SKIP with LAMP-1-RFP is shown in Movie S5. Comovement of myrlysin-GFP with LAMP-1-RFP is shown in Movie S6.

Farías et al. PNAS | Published online March 20, 2017 | E2961 Downloaded by guest on October 1, 2021 We also observed that control neurons exhibited small vesicles centrifugal transport in nonpolarized cells (8–10, 12, 44–46, 54). labeled with the autophagy marker LC3, some that were stationary Here we show that this ensemble also plays a role in lysosome and others that moved mainly in the retrograde direction, as pre- transport in neurons, but only in the axon and not the dendrites. We viously reported (30, 31, 52, 53) (Fig. 7 E–H). In contrast, neurons also show that BORC/kinesin-1–dependent anterograde transport of depleted of myrlysin, lyspersin, myrlysin plus lyspersin, or Arl8b lysosomes is required for axonal growth cone structure and dy- exhibited an accumulation of LC3 in large, immobile structures in namics, as well as autophagosome mobilization and disposal. the distal axon (Fig. 7 E and F). Analysis of mCherry-LC3 and LAMP1-GFP colocalization revealed that most stationary and ret- BORC–Arl8–SKIP–Kinesin-1 Specifically Drives Lysosome Transport into rograde mCherry-LC3 vesicles also contained LAMP1-GFP (Fig. the Axon. In agreement with previous studies (28–33, 55), fluores- 7G)orLAMP1-BFP2(Fig.7H) in control neurons. In contrast, the cence microscopy of fixed neurons showed the presence of lyso- aberrant mCherry-LC3–labeled structures in myrlysin-KD neurons somes in the soma, dendrites, and axon (Figs. 1 and 2 and Fig. S1). were largely devoid of LAMP1-BFP2 (Fig. 7H). From these ex- Furthermore, live-cell imaging revealed that lysosomes move bi- periments we concluded that BORC-dependent anterograde directionally in all neuronal domains (Fig. 3). These organelles were transport of lysosomes is important for mobilization and disposal of identified as conventional lysosomes because they contained the autophagosomes from the distal axon. This conclusion is consistent lysosomal integral membrane proteins LAMP1 and CD63, the lu- with the notion that fusion of lysosomes with autophagosomes minal acid hydrolase cathepsin D, and the LAMTOR4 subunit of contributes to the dynein-dependent retrograde transport of the the ragulator complex that mediates mTORC1 signaling at the ly- resulting autolysosomes, in addition to the degradation of their sosome surface. They also contained catalytically active cathepsin B, contents (31, 53). as revealed by using the Magic Red substrate, and most had an acidic pH, as evidenced by staining with LysoTracker and absence Discussion of SEP fluorescence (Figs. 1, 2, and 3 and Fig. S1). We cannot rule Previous studies demonstrated that BORC, Arl8b, and SKIP func- out, however, that some of these axonal organelles are late endo- tion together to couple lysosomes to kinesin-1, promoting their somes, as these represent a stage in the maturation to lysosomes,

A Distal axon B 15 * C Ctrl shRNA Myr shRNA Control shRNA Myrlysin shRNA Ctrl shRNA LAMP1-RFP 10 0 s Myr shRNA GFP 5 LAMP1 Merge transport 0 30 s D 1.5 ** 60 s 1 * 90 s

240 s 0.5

GC area 0 Normalized 120 s Ctrl shRNA Lys shRNA 100 µm 100 µm Myr shRNA Arl8b shRNA Fig. 7. Aberrant growth cones and autophagosomes E Control shRNA Myrlysin shRNA Lyspersin shRNA Myr + Lys shRNA Arl8b shRNA in the axon of myrlysin-KD cells. (A)Singleimages(top mCh-LC3 three strips) and kymographs (bottom strip) from Movie S7 of a 100-μm segment of the distal axon from a DIV 8 neuron expressing LAMP1-RFP together with

240 s control-shRNA/GFP (Left) or myrlysin-shRNA/GFP (Right). (B) Quantification of the total number of lysosomes moving in the distal axon from neurons transfected as in 100 µm 100 µm 100 µm 100 µm 100 µm A and expressed as the mean ± SD from seven neurons F 6 6 * per condition. (C) Images of an axonal growth cone at * * * * * Ctrl shRNA Myr + Lys shRNA different times from the neurons expressing control or 4 * 4 * Myr shRNA Arl8b shRNA myrlysin-shRNA in A and Movie S7.(D) Quantification of LC3 2 2 the growth cone area expressed as normalized mean ± transport Lys shRNA Normalized

LC3 intensity SD from 50 growth cones per condition relative to control 0 0 shRNA. Ctrl, control; Lys, lyspersin; Myr, myrlysin. (E)Sin- G H Control shRNA Myrlysin shRNA gle images (Top) and kymographs (Bottom)frommovies LAMP1-GFP LAMP1-BFP2 of a 100-μm-length segment of the distal axon from DIV mCh-LC3 mCh-LC3 8 neurons expressing mCherry-LC3 plus control-, myrlysin-, myrlysin+lyspersin-, or Arl8b-shRNA. (F) Quantification of the number of LC3 particles moving retrogradely in 240 s 240 s the distal axon and intensity levels of LC3 in the distal axon in neurons transfected as in E. LC3 transport is LAMP1 LAMP1 LAMP1 expressed as the mean ± SD from seven neurons per condition. Intensity levels of LC3 are expressed as normalized mean ± SD from 15 neurons per condition. 240 s 240 s (G and H) Single images (top two strips) and kymographs μ LC3 LC3 LC3 (bottom strip) from movies of 100- m-length segment of 100 µm 100 µm 100 µm the distal axon from DIV 8 neurons expressing mCherry- LC3plusLAMP1-GFP(G) or LAMP1-BFP2 in the presence

Antero Retro Stat Antero Retro Stat 10 10 of control-shRNA (H, Left) or myrlysin-shRNA (H, Right). * *n.s 8 n.s 8 * Total LC3 levels and LC3/LAMP1 colocalization events are 6 n.s 6 * n.s * expressed as the mean ± SD from seven neurons per

LC3 * LC3 4 4 condition. Statistical significance for groups (D, F,andH) (# events)

(# events) 2 2 colocalization colocalization 0 0 was calculated by one-way ANOVA followed by Dun- Total LC3 LC3 / LAMP1 shRNAs Ctrl Myr Ctrl Myr Ctrl Myr nett’s test. Significance between group pairs (B and G) colocalization Total LC3 LC3 / LAMP1 colocalization was calculated by Student’s t test (*P < 0.01 vs. control).

E2962 | www.pnas.org/cgi/doi/10.1073/pnas.1616363114 Farías et al. Downloaded by guest on October 1, 2021 and thus share many properties. In line with previous studies (30, toward the proximal axon, they become more acidic and acquire bi- PNAS PLUS 55, 56), we did observe some heterogeneity among axonal lyso- directional movement, probably as a result of fusion with ante- somes. For example, in the distal axon, lysosomes were less mobile, rogradely moving lysosomes (53). Our observations are consistent and some were less acidic (Fig. 3). This heterogeneity resembles with the requirement of anterograde lysosome movement for fusion that recently reported for lysosomes in nonneuronal cells (40). with autophagosomes and degradation of their contents, including Lysosomes in the proximal and distal parts of the axon depend LC3. Moreover, fusion with lysosomes appears to confer on auto- on the BORC–Arl8–SKIP–kinesin-1 complex for anterograde phagosomes the ability to move retrogradely. This phenomenon was transport. Indeed, silencing or inactivation of components of this previously attributed to the delivery of a snapin-dynein complex from complex depleted lysosomes from the axon (Fig. 4), whereas late endosomes to autophagosomes during fusion (31). The fact that their overexpression drove lysosomes to axon tips (Figs. 5 and 6). snapin is a subunit of BORC (9) raises the additional possibility that This latter effect was particularly dramatic for coexpression of snapin is required for anterograde lysosome transport as part of the Arl8 and SKIP (Fig. 6), suggesting that this GTPase-effector pair BORC–Arl8–SKIP–kinesin-1 ensemble. Fusion with lysosomes then is a limiting factor in the chain of interactors. Moreover, we likely enables acquisition of other adaptor or signaling molecules for observed that Rigor-KIF5A, in conjunction with KLC and SKIP, recruitmentofdyneintotheresulting autolysosomes. arrested lysosome movement at the beginning of the axon (Fig. 5 G and H), consistent with the special role of kinesin-1 in the How Are Lysosomes Transported in Dendrites? Unlike axonal transport of lysosomes into the axon. transport, dendritic transport of lysosomes does not appear to Studies by other groups that used Caenorhabditis elegans as a depend on kinesin-1 (Figs. 4–6). In fact, many of the ∼45 mam- model system showed that Arl8 (57) and the myrlysin subunit of malian kinesins exclusively mediate axonal transport, and only a BORC (known as SAM-4 in this organism) (58) regulate the handful, including the kinesin-3 proteins KIF1A, KIF1Bβ, KIF1C, transport of synaptic vesicle precursors in the axon, although, in and KIF16B and the kinesin-4 proteins KIF21A and KIF21B, this case, through coupling to the kinesin-3 UNC-104. These mediate organelle transport to axons and dendrites (24, 25, 67). Of findings suggest a broader role for BORC and Arl8 in axonal these, KIF1A and KIF1Bβ have been reported to move late transport of different organelles. endosomes and lysosomes in nonneuronal cells (54, 68, 69). It β BORC-Dependent Lysosome Transport Promotes Axonal Growth-Cone would thus be of interest to test if KIF1A and KIF1B mediate Dynamics. Axonal growth cones are highly motile structures in- lysosome transport in dendrites. Alternatively, lysosome transport volved in axon outgrowth and guidance (59, 60). They comprise a in dendrites could be mediated by dynein, as shown in previous central region enriched in microtubules and transport vesicles and studies (20, 33, 70). Further studies will be needed to assess the a peripheral region with a high concentration of actin filaments relative contributions of different microtubule motors to lysosome that organize lamellipodia and filopodia. The dynamics of growth transport in dendrites. cones are dependent on cooperation between the microtubule and actin cytoskeletons, as well as on the delivery of transport vesicles Concluding Remarks to the leading edge. We observed that lysosomes normally reached Our findings illustrate how a mechanism that mediates radial the central region of the growth cone (Figs. 1 and 3). Importantly, dispersal of lysosomes in nonpolarized cells is adapted to pro-

impairment of lysosome transport by myrlysin, lyspersin, or Arl8b mote polarized transport of lysosomes into the axon of hippo- NEUROSCIENCE KD caused a dramatic reduction in the size and dynamics of campal neurons. The specific role of kinesin-1 in this process is growth cones (Fig. 7 A–D). These effects suggest that lysosome likely derived from its preference for microtubules enriched in transport is required to maintain the structure and function of acetylated and GTP-bound tubulin (51, 54, 71), which are par- growth cones. Lysosomes could deliver adhesion or signaling ticularly abundant in the axon (51, 72). Alterations in the ex- molecules to the growth cone, as they do to focal adhesions in pression of BORC and kinesin-1 subunits have been linked to nonpolarized cells (61–63). This explanation is consistent with the psychiatric and neurological disorders (73–76). In light of our requirement of lysosome exocytosis for the extension and arbori- findings, it would be of interest to investigate if aberrant trans- zation of neurites in sympathetic neurons (29). Alternatively, ly- port of lysosomes in the axon contributes to the pathogenesis of sosomes could function to degrade adhesion or signaling these disorders. molecules in the growth cone. In this regard, ubiquitination of the neuronal L1/NgCAM adhesion molecule was shown to target it Materials and Methods for degradation in lysosomes, influencing neurite outgrowth and Recombinant DNA constructs (Table S1), antibodies and other probes (Table S2), cell adhesion (64). In addition, Robo receptors for the repulsive culture and transfection of rat hippocampal neurons, fluorescence microscopy, axon guidance protein Slit were found to be down-regulated by live-cell imaging, image analysis, and statistical analysis are described in SI degradation in late endosomes and lysosomes (65, 66). Materials and Methods. All animal experiments were performed in accordance with National Institutes of Health and US government regulations. Research Requirement of BORC-Dependent Lysosome Transport for Autophagosome was conducted under Animal Study Proposals 13–011 and 16–011 approved by Mobilization and Disposal. An additional neuronal function that re- the National Institute of Child Health and Human Development Animal Care quires BORC-dependent lysosome transport is the turnover of axonal and Use Committee. autophagosomes, as evidenced by the accumulation of large, immobile LC3-positive structures in the distal axon of neurons depleted of ACKNOWLEDGMENTS. We thank R. Bagshaw, J. H. Brumell, R. Jia, J. Lippincott- Schwartz, S. Méresse, W. Mothes, J. Pu, D. Rubinsztein, and K. Verhey for gifts of myrlysin and/or lyspersin or Arl8b (Fig. 7 E and F). Autophagosomes reagents and X. Zhu for expert technical assistance. This work was funded by the form constitutively in the distal axon and subsequently undergo Intramural Program of National Institute of Child Health and Human Develop- dynein-mediated retrograde transport (30, 31, 52, 53). As they move ment, NIH Grant ZIA HD001607.

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