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BORC/Kinesin-1 Ensemble Drives Polarized Transport of Lysosomes

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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 September 26, 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
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