A -7B–dependent endocytosis pathway mediates cellular entry of α-synuclein fibrils and polycation-bearing cargos

Qi Zhanga,1, Yue Xua,1, Juhyung Leea, Michal Jarnikb, Xufeng Wuc, Juan S. Bonifacinob, Jingshi Shend, and Yihong Yea,2

aLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Diseases, National Institutes of Health, Bethesda, MD 20892; bNeurosciences and Cellular and Structural Biology Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; cLaboratory of Cell Biology, National Heart, , and Blood Institute, National Institutes of Health, Bethesda, MD 20892; and dMolecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309

Edited by Nancy M. Bonini, University of Pennsylvania, Philadelphia, PA, and approved March 31, 2020 (received for review October 23, 2019) Cell-to-cell transmission of misfolding-prone α-synuclein (α-Syn) and oligomerized α-Syn (15, 16). On the uptake side, α-Syn PFFs has emerged as a key pathological event in Parkinson’s disease. can enter cells via clathrin-mediated endocytosis (CME) on This process is initiated when α-Syn–bearing fibrils enter cells via binding to surface receptors (17), but whether α-Syn endocytosis clathrin-mediated endocytosis, but the underlying mechanisms are involves substrate specific endocytosis regulators has not yet been unclear. Using a CRISPR-mediated knockout screen, we identify explored. This type of regulator may be particularly relevant to the SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators development of PD drugs that target α-Syn endocytosis. Another for α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key unresolved issue is the identity of relevant membrane receptors for regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is α-Syn PFFs; several studies have reported different candidates: essential for recruiting α-Syn PFFs to the cell surface because this heparan sulfate proteoglycans (HSPGs), membrane process is mediated by interactions between negatively charged LAG3 for unmodified α-Syn PFFs (18, 19), and a glycoprotein, α sugar moieties of HSPGs and clustered K-T-K motifs in -Syn PFFs. neurexin 1β, for N-terminally acetylated α-Syn PFFs (20). By contrast, MYO7B regulates α-Syn PFF cell entry by maintaining

HSPGs compose a class of cell surface and extracellular matrix CELL BIOLOGY a plasma membrane-associated network that controls mem- glycoproteins that carry one or more heparan sulfate (HS) chains brane dynamics. Without MYO7B or actin filaments, many clathrin- bearing repeated disaccharide units. This modification is found coated pits fail to be severed from the membrane, causing accumu- primarily in the syndecan and glycosylphosphatidylinositol- “ ” lation of large clathrin-containing scars on the cell surface. Intrigu- anchored glypican families (21). The biosynthesis of HSPGs ingly, the requirement for MYO7B in endocytosis is restricted to begins in the endoplasmic reticulum, but its completion requires α-Syn PFFs and other polycation-bearing cargos that enter cells via modification of the assembled sugar chains with sulfate using 3′- HSPGs. Thus, our study not only defines regulatory factors for α-Syn phosphoadenosine-5′-phosphosulfate (PAPS) as the sulfate do- PFF endocytosis, but also reveals a previously unknown endocytosis nor, which occurs in the Golgi complex (22). Because HS chains mechanism for HSPG-binding cargos in general, which requires carry negative charges, they can interact with a plethora of ligands forces generated by MYO7B and actin filaments.

α-synuclein | clathrin-mediated endocytosis | myosin-7B | heparan sulfate Significance proteoglycan | actin filament Direct cell-to-cell spreading of misfolded protein aggregates, α α n Parkinson’s disease (PD), the major proteinaceous aggregate such as -synuclein fibrils ( -Syn), is a pathological hallmark as- Iknown as a Lewy body is composed mostly of a synaptic pro- sociated with the progression of many neurodegenerative dis- tein, α-synuclein (α-Syn) (1). Coincidentally, genetic mutations in eases, but it is unclear how mammalian cells take up large α – protein aggregates to initiate this prion-like protein transmission -Syn encoding were identified as a major contributing α factor for Parkinson’s disease (2, 3). α-Syn is a small polypeptide process. Here we define the endocytosis mechanism of -Syn that is aggregation-prone. Clinical studies have shown that preformed fibrils (PFFs) using a combination of genetic, bio- chemical, and live-cell imaging techniques. Our study shows that α-Syn–positive Lewy bodies can spread in the brain during dis- α-Syn PFFs enter cells following an entry pathway that is spe- ease progression (4). Furthermore, following tissue trans- cifically tailored for cargos bearing positive charges. These car- plantation therapy, grafted tissues can accumulate Lewy body- gos bind to heparan sulfate proteoglycans on the cell surface via like aggregates over time, which does not occur if these cells are charge–charge interactions, which enable cargo internalization left intact in the donor (5, 6). These observations prompted the by a previously unknown endocytosis mechanism that requires idea that α-Syn might undergo cell-to-cell transmission analo- forces generated by myosin-7B and actin filaments. gously to a prion protein (7, 8). Indeed, in vitro and animal α α studies have shown that -Syn preformed fibrils ( -Syn PFFs) Author contributions: Q.Z., Y.X., J.L., M.J., J.S.B., J.S., and Y.Y. designed research; Q.Z., can be readily taken up by various cell types and subsequently Y.X., J.L., M.J., X.W., and Y.Y. performed research; J.S. contributed new reagents/analytic transmitted to nearby cells that have not been directly exposed to tools; Q.Z., Y.X., J.L., and Y.Y. analyzed data; and Q.Z., Y.X., J.S.B., J.S., and Y.Y. wrote α-Syn PFFs (9–13). the paper. At the cellular level, the intercellular transmission of neuro- The authors declare no competing interest. toxic like α-Syn comprises two biological events: the This article is a PNAS Direct Submission. release of α-Syn from donor cells via unconventional protein Published under the PNAS license. secretion and its internalization by recipient cells via endocytosis 1Q.Z. and Y.X. contributed equally to this work. (14). The molecular mechanisms of these processes are poorly 2To whom correspondence may be addressed. Email: [email protected]. defined. Recent studies have suggested nanotubes, exosome se- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ cretion, or a pathway termed misfolding-associated protein se- doi:10.1073/pnas.1918617117/-/DCSupplemental. cretion as potential mechanisms for the release of monomeric

www.pnas.org/cgi/doi/10.1073/pnas.1918617117 PNAS Latest Articles | 1of11 Downloaded by guest on September 28, 2021 on the cell surface, either mediating their uptake or activating myosin and actin). Mechanistically, our study defines the in- downstream signal transduction (23, 24). The known HSPG li- teraction of HSPGs with α-Syn PFFs using biochemical assays gands do not share any specific sequence motif for engaging and structural modeling. Importantly, we reveal a unique CME HSPGs; instead, the binding energy seems to be provided by mechanism for α-Syn PFFs and other polycation-bearing cargos, electrostatic interactions, often involving positively charged resi- which, unlike conventional CME, requires myosin 7B (MYO7B) dues from random sequences in ligands (21). Along with α-Syn and actin filaments. PFFs, HSPGs have been implicated in the uptake of tau PFFs and Aβ (18, 25, 26), but how HSPGs interact with these neurotoxic Results protein aggregates is unclear. A CRISPR Screen Identifies Proteins Involved in α-Syn PFF Endocytosis. In this study, we used an unbiased CRISPR/Cas9 screen to To dissect the mechanism of α-Syn PFF endocytosis, we labeled identify factors that regulate α-Syn PFF endocytosis in mam- purified α-Syn with either a stable fluorophore (Alexa Fluor 594) malian cells. The screen not only isolated known endocytosis or a pH-sensitive dye (pHrodo). The pHrodo dye becomes ac- regulators of α-Syn PFFs (e.g., HSPGs), but also identified other tivated only on its arrival at the acidic late endosome/lysosome factors important for α-Syn PFF uptake (e.g., an unconventional compartment, emitting red fluorescence (SI Appendix, Fig. S1A).

Fig. 1. SLC35B2 is required for endocytosis of α-Syn PFF. (A) The CRISPR screen strategy. (B–D) SLC35B2-KO cells are defective in endocytosis of α-Syn PFF but not monomeric α-Syn. (B) Validation of SLC35B2 (35B2)-KO cells by qRT-PCR. mRNA levels were normalized to the level in control cells. The error bar rep- resents SEM. n = 3. (C) Control, SLC35B2-KO, or SLC35B2-KO cells stably expressing FLAG-SLC35B2 were incubated with pHrodo-labeled α-Syn PFF (200 nM for 4 h) and analyzed by FACS. FL, fluorescence. (D) Control and SLC35B2-KO cells were treated with labeled α-Syn monomer at 800 nM overnight before FACS analysis. (E–G) Knockdown (KD) of SLC35B2 attenuates α-Syn PFF uptake in primary neurons. (E) Primary neurons were infected with lentivirus expressing the indicated shRNAs together with EGFP (driven by the synapsin promoter) at day in vitro (DIV) 3. mRNA was purified from cells at DIV8 for qRT-PCR analysis. Error bar represents SEM. n = 2 biological repeats, each with triplicate PCR analyses. (F) Control or SLC35B2-KD neurons expressing EGFP (green) at DIV8 were incubated with α-Syn PFF Alexa Fluor 594 (200 nM) (red) for 4 h, stained with DAPI (blue), and analyzed by confocal microscopy (Scale bar: 5 μm.) (G) Quantification of α-Syn PFF level in individual cells (indicated by dots) from two independent experiments. Error bar represents SEM. P value from a two-tailed t test. A.U., arbitrary unit. (H and I) SLC35B2 is required for α-Syn PFF binding to the plasma membrane. (H) Control, SLC35B2-KO, or SLC35B2-KO cells reexpressing WT SLC35B2 were incubated with α-Syn PFF Alexa Fluor 594 (200 nM) (magenta) on ice for 30 min, stained with DAPI (blue), and imaged by confocal microscopy (Scale bar: 5 μm.) (I) Quantification of α-Syn PFF surface level in individual cells from two independent experiments.

2of11 | www.pnas.org/cgi/doi/10.1073/pnas.1918617117 Zhang et al. Downloaded by guest on September 28, 2021 We used labeled proteins to prepare α-Syn PFFs following a Appendix, Fig. S2G) and α-Syn PFF binding (SI Appendix, Fig. well-established protocol (27). Electron microscopy (EM) anal- S2 H and I). Thus, our unbiased genetic screen confirms HSPGs ysis of sonicated α-Syn PFF revealed 20- to 100-nm-long particles as critical mediators of α-Syn PFF–membrane interaction, which (SI Appendix, Fig. S1B), consistent with the reported α-Syn PFFs is essential for α-Syn PFF endocytosis. bearing pathogenic activities (27). We tested α-Syn PFF uptake in various cell types and found that both neuronal and non- Electrostatic Interactions Recruit α-Syn PFF to the Plasma Membrane. α SI Ap- neuronal cells could efficiently internalize -Syn PFF ( To better characterize the α-Syn PFF–HSPG interaction, we pendix C , Fig. S1 ), suggesting that the core endocytosis machinery developed a fluorescence probe that can track HSPGs in live α for -Syn PFFs is not specific to neurons. Treating cells with cells. Since the oligosaccharide chains in HSPGs are enriched in inhibitors, such as dynasore (28) or dynole 34-2 (29), negative charges (23), we reasoned that an engineered GFP α SI Appendix D + significantly reduced -Syn PFF uptake ( , Fig. S1 variant carrying 36 net positive charges (GFP ) (34) might be a and E). In addition, α-Syn PFF uptake was also dramatically specific probe for HSPGs. Indeed, several lines of evidence attenuated in HeLa cells lacking the σ2 subunit of AP-2 (enco- support this notion. First, live-cell confocal microscopy showed ded by the AP2S1 ), a cargo adaptor in CME (SI Appendix, that GFP+, but not a GFP variant bearing extra negative charges − Fig. S1F). These findings confirm CME-dependent α-Syn PFF (GFP ) bound efficiently to the cell surface, initially forming a endocytosis (17, 30). smooth profile, which was rapidly converted to a punctate pat- To search for α-Syn PFF endocytosis regulators, we performed tern reminiscent of cells treated with α-Syn PFF Alexa Fluor 594 a pooled CRISPR/Cas9 screen (Fig. 1A). We chose HEK293T (Fig. 2A). The interaction of GFP+ with the cell surface was cells because their fast-growing and high-DNA recombination completely dependent on SLC35B2, as no GFP+ staining was properties make them well suited for a CRISPR screen. Since detected in SLC35B2-KO cells (Fig. 2B, compare panel 2 with pooled CRISPR screens often suffer from poor specificity and panels 1 and 3). Furthermore, when cells were pretreated with reproducibility, we used a strategy akin to the classical genetics GFP+, cell surface HSPGs could not be stained by to approach. We treated HEK293T cells with a lentiviral GeCKO HSPG (Fig. 2C), suggesting that GFP+ competes with HSPG library expressing single guide RNAs (sgRNAs) targeting all antibodies for the same binding site. Because cells treated with human genes. We then incubated mutagenized cells with GFP+ and α-Syn PFF Alexa Fluor 594 showed extensive coloc- pHrodo-labeled α-Syn PFFs. Florescence-activated cell sorting alization of these two cargos on the cell surface (Fig. 2D), and − (FACS) was used to sort α-Syn PFF–negative cells into 96-well because pretreating α-Syn PFF with GFP reduced the binding plates at single-cell resolution. When these cells formed colonies, of α-Syn PFF to cells (Fig. 2E), electrostatic interactions must CELL BIOLOGY we rescreened them to identify cells defective in α-Syn PFF provide the major energy that recruits α-Syn PFFs to the cell uptake. Among the 1,248 clones screened, one clone (C10) surface (Fig. 2F). showed almost no α-Syn PFF uptake (SI Appendix, Fig. S2A), whereas 27 other clones showed reduced α-Syn PFF uptake. In Two K-T-K Motifs in Oligomerized α-Syn Enable HSPG Interactions. this study, we characterized two clones, which revealed an un- α α Previous studies have shown that two -Syn monomers could expected requirement for the endocytosis of -Syn PFF and form either a face-to-face or back-to-back dimer. Many dimers other polycation-bearing cargos. then stack together to form long fibrils of “rod” or “twisted” conformers (35, 36). To elucidate how these α-Syn PFF con- HSPGs Are Required for α-Syn PFF Endocytosis. Sequencing identi- formers interact with HSPGs, we used the ClusPro program to fied a sgRNA in one of the clones (C10) targeting SLC35B2. model the interaction of α-Syn PFF conformers with heparin, an Analysis of SLC35B2-knockout (KO) cells confirmed a specific HS analog that competitively inhibits the binding of α-Syn PFF requirement for SLC35B2 in endocytosis of α-Syn PFFs, but not to cells (18). The modeling resulted in 14 clusters falling into 3 monomeric α-Syn (mSyn) (Fig. 1 B–D). Furthermore, shRNA- groups, as shown in Fig. 3A and SI Appendix, Fig. S3 A and B. mediated knockdown of SLC35B2 in primary neurons also re- Interestingly, in all clusters, the interactions between α-Syn PFF duced α-Syn PFF uptake (Fig. 1 E–G), but the uptake defect was and heparin were mediated by two K-T-K motifs, K43-T-K45 less dramatic compared with that in SLC35B2 KO cells, likely and K58-T-K60, in α-Syn fibrils. In the twisted conformer, each because of partial gene silencing. The SLC35B2 gene contains of these lysine-rich motifs lined up to form a shallow concave four exons. The identified sgRNA targets a sequence in exon 2 surface that accommodates a di-heparin molecule bearing six (SI Appendix, Fig. S2B). SLC35B2 is known as a Golgi-localized sulfate groups (SI Appendix, Fig. S3 A and B). In contrast, in the membrane protein required for transporting PAPS into the rod conformation, two K-T-K motifs from distinct α-Syn mole- Golgi complex for protein sulfation (SI Appendix, Fig. S2C) (31). cules in a dimeric fibril joined together to form one deep binding We confirmed the Golgi localization of SLC35B2 by FLAG groove for di-heparin (Fig. 3 A and B). As a result, each fibril immunostaining of SLC35B2-KO cells stably expressing could offer only two binding grooves, but within each binding site FLAG-tagged SLC35B2 (SI Appendix, Fig. S2D). The function- the heparin molecule was sandwiched by four rows of lysine ality of ectopically expressed SLC35B2 was confirmed by its residues, forming extensive electrostatic interactions as well as ability to rescue α-Syn PFF uptake in SLC35B2-KO cells hydrogen bonds with α-Syn (Fig. 3C and SI Appendix, Fig. S3C). (Fig. 1C). Collectively, these results suggest a role for Golgi- Thus, α-Syn fibrils in the rod conformer are expected to have a localized protein sulfation in α-Syn PFF endocytosis. much higher affinity for HSPGs than the twisted conformer. Cell surface binding experiments showed that SLC35B2 is These models explain why HSPGs are only required for the required for the recruitment of α-Syn PFFs to the plasma uptake of α-Syn fibrils but not monomeric α-Syn. This result also membrane (Fig. 1 H and I). Given the known role of SLC35B2 in suggests that a twisted α-Syn oligomer containing three proto- the biosynthesis of HSPGs (31) and recent studies suggesting mers is the minimum requirement for binding one heparin unit; HSPGs as a potential receptor for α-Syn PFFs (18, 32), we in contrast, for the rod conformer, five or six α-Syn protomers presumed that α-Syn PFFs failed to bind SLC35B2-KO cells, are required to form a higher-affinity heparin-binding site. because these cells lacked HSPGs. Indeed, immunostaining with To validate our models, we generated α-Syn mutants with the an antibody to HSPGs stained the plasma membrane of WT, but four lysine residues K43, K45, K58, and K60 substituted to either not that of SLC35B2-KO cells (SI Appendix, Fig. S2 E and F). glutamine (4Q) or alanine (4A) (Fig. 3D). These mutants could Furthermore, KO of XYLT2, another key HSPG biosynthetic form long fibrils similarly to WT α-Syn (Fig. 3E); however, enzyme (33), diminished both cell surface HSPG signals (SI compared with WT α-Syn PFFs, the uptake of these mutant

Zhang et al. PNAS Latest Articles | 3of11 Downloaded by guest on September 28, 2021 Fig. 2. α-Syn PFF interacts with cells via electrostatic interactions. (A)GFP+ binds to the plasma membrane. Panels 1 and 2 show two confocal sections of a HEK293T cell stained with 200 nM GFP+ (green) and DAPI (blue) for 5 min. (Scale bar: 5 μm.) Panels 3 and 4 show U2OS cells incubated with 200 nM of GFP+ − (panel 3) or GFP (panel 4) and then stained with DAPI. (Inset) Enlarged view of the box in panel 3 (Scale bars: 10 μm.) (B) Interaction of GFP+ with the plasma membrane depends on SLC35B2. Control (panel 1) or SLC35B2-KO HEK293T cells with (panel 3) or without (panel 2) the reexpression of WT SLC35B2 were stained with GFP+ and DAPI (Scale bar: 5 μm.) (C)GFP+ inhibits the binding of HSPG antibodies to the cell surface. U2OS cells were either pretreated with GFP+ (200 nM) (Right) for 10 min or left untreated (Left) and then stained with antibodies to HSPG (red) and DAPI (blue) (Scale bar: 5 μm.) (D) Colocalization of GFP+ with α-Syn PFF in cells. Cells incubated with α-Syn PFF Alexa Fluor 594 and GFP+ were imaged. (E)GFP− attenuates α-Syn PFF binding to the plasma membrane. Cells were incubated with α-Syn PFF Alexa Fluor 594 (200 nM) in the presence of the indicated concentrations of GFP−. α-Syn PFF binding to the cell surface was quantified by confocal imaging. (F) An HSPG–cargo interaction model.

α-Syn PFFs was dramatically reduced (Fig. 3F). In contrast, the MYO7B-KO cells were also defective in the uptake of GFP+ (SI uptake of monomeric α-Syn was not affected by these mutations Appendix, Fig. S4 C and D) and of DNA in complex with a (SI Appendix, Fig. S3D). Furthermore, cell-binding experiments polycation carrier (SI Appendix, Fig. S4E). Furthermore, HSPG- showed that the interaction of α-Syn PFF with the plasma dependent uptake of a lentiviral reporter expressing GFP was membrane was significantly inhibited by these mutations significantly reduced in MYO7B-KO cells (SI Appendix, Fig. S4 F (Fig. 3G). These results strongly suggest that α-Syn binds HSPGs and G). On the other hand, endocytosis of monomeric α-Syn or via a specific sequence motif that forms binding sites only on transferrin was not affected in MYO7B-KO cells (Fig. 4 D α-Syn oligomerization. and E). To test whether HSPG-mediated endocytosis involves the motor activity of MYO7B, we ectopically expressed MYO7B HSPG-Mediated Endocytosis Requires MYO7B and Actin Filaments. mutants that either lack the motor domain (CT) or carry a Using a similar approach, we identified a sgRNA from a clone α mutation that disrupts the ATPase cycle of the motor domain partially defective in -Syn PFF endocytosis, which targeted (N207A, I482A, or E442A) (38). If coupling the motor activity to MYO7B, an unconventional myosin widely expressed in various α SLC35B2 cargo binding were required for -Syn PFF uptake, then these human tissues (37). Like for , the sgRNA targeting mutants should inhibit α-Syn PFF uptake in a dominant negative MYO7B A mapped to an early exon (Fig. 4 ). Reconstructed manner. Indeed, cells expressing these mutants showed signifi- MYO7B MYO7B -KO HEK293T cells or cells treated with - cantly reduced α-Syn PFF uptake compared with neighboring specific small interfering RNA (siRNA) showed that depletion untransfected cells or cells transfected with WT MYO7B of MYO7B diminished endocytosis of α-Syn PFF (Fig. 4 B and C (Fig. 4 I–K). Furthermore, deleting the C-terminal membrane- and SI Appendix, Fig. S4A), while reexpression of EGFP- binding FERM domain abolished the dominant negative activity MYO7B in MYO7B-KO cells largely rescued the α-Syn PFF of MYO7B-CT, suggesting that membrane binding by a motor- uptake phenotype (SI Appendix, Fig. S4B). An α-Syn PFF uptake defective MYO7B mutant accounts for the observed endocytosis defect was similarly observed in primary neurons expressing defect. These results, together with the observation that actin- MYO7B-specific shRNA (Fig. 4 F–H). Strikingly, the function of binding compounds (e.g., jasplakinolide, latrunculin A) that in- MYO7B in endocytosis appeared to be restricted to HSPG- terfere with the function of F-actin also inhibited α-Syn PFF dependent cargos, because like HSPG-deficient cells (23), endocytosis (SI Appendix, Fig. S5 A and B), strongly suggest that

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Fig. 3. Two K-T-K motifs in α-Syn PFF enable specific interactions with heparin. (A–C) A model of the α-Syn PFF rod conformer in complex with heparin. (A and B) A model representing five clusters. In this model, each α-Syn PFF dimeric fibril contains two heparin-binding pockets (sites 1 and 2) that each forms a long groove to accommodate a heparin molecule. (C) Heparin is sandwiched by four rows of lysine residues in α-Syn fibril. Dashed lines in C label comple- mentarily charged atoms within 3 Å. (D) Purified WT α-Syn and charge mutants (4A and 4Q) analyzed by SDS/PAGE and Coomassie blue staining. (E) Rep- resentative negative stained EM images showing PFFs formed by WT α-Syn and the 4Q α-Syn mutant. (F and G) α-Syn PFFs Alexa Fluor 594 formed by the charge mutants are defective in endocytosis (F) and cell surface binding (G). (F) HEK293T cells were treated with α-Syn PFF Alexa Fluor 594 for 3 h before FACS analysis. (G) Whisker plot showing the relative level of α-Syn PFF binding to cells as determined by confocal imaging of U2OS cells treated with α-Syn PFF Alexa Fluor 594 at 400 nM in the presence or absence of heparin (4 μM) on ice. **P < 0.01, two-tailed Student’s t test.

optimal α-Syn PFF endocytosis requires a membrane-associated more MYO7B dots than others (Fig. 5C). Intriguingly, MYO7B- functional interplay between MYO7B and actin filaments. high plasma membrane domains were often enriched in CCPs (Fig. 5 C and D). Therefore, we postulated that dynamic inter- MYO7B Maintains Membrane Dynamics at Clathrin-Enriched actions of MYO7B within certain plasma membrane domains Membrane Domains. To elucidate the mechanism of MYO7B- enable membrane flexibility and deformability, which might in mediated endocytosis, we used total internal reflection fluores- turn facilitate the maturation of CCPs located in the same cence microscopy (TIRF) to examine the localization of MYO7B membrane domain. To measure plasma membrane dynamics, we used GFP+ to in U2OS cells stably expressing mCherry-tagged clathrin light label the cell surface and then took time-lapse videos. In chain (mCh-CLC) to label clathrin-coated pits (CCPs) together WT cells, the plasma membrane usually undergoes dynamic ex- with either GFP-WT MYO7B or GFP-MYO7B-CT. Consistent pansion and retraction, generating a ruffling motion (SI Appen- with the reported localization of MYO7B to plasma membrane- dix, Fig. S5C and Movie S3). In contrast, the membranes of derived microvilli (38), GFP-MYO7B also bound to the plasma MYO7B A -KO cells had significantly reduced mobility (Movie S4 membrane in U2OS cells, forming discrete dots (Fig. 5 ). Time- and Fig. 5E). Furthermore, TIRF microscopy showed that lapse microscopy showed that MYO7B-positive signals fluctu- MYO7B-KO cells had a significantly reduced amount of actin B ated rapidly, displaying a flickering pattern (Fig. 5 and Movie filaments underneath the plasma membranes (Fig. 5F). These S1). Interestingly, the MYO7B-CT mutant appeared to form results suggest that MYO7B may regulate actin assembly to A more stable interactions with the membranes (Fig. 5 ), resulting maintain membrane dynamics. in bright foci that remained constant for a prolonged period If MYO7B promotes endocytosis via membrane dynamics at (Movie S2). We concluded that the MYO7B C-tail domain can cargo-binding sites, other agents capable of increasing mem- bind to the plasma membrane, while its actin-binding domain brane stiffness should similarly inhibit α-Syn PFF uptake. To test contributes to the dynamic property of this interaction. this idea, we treated cells with the cholesterol-depleting drug When colocalization of MYO7B with CCPs was examined by methyl-β-cyclodextrin, which increases membrane stiffness in two-color TIRF, we found limited overlapping signals (Fig. 5C, mammalian cells (39). Consistent with our hypothesis, methyl- Insets). However, we noticed that MYO7B was not uniformly β-cyclodextrin also reduced α-Syn PFF uptake to a similar degree distributed on the plasma membrane. Instead, certain areas had as depletion of MYO7B (Fig. 5G).

Zhang et al. PNAS Latest Articles | 5of11 Downloaded by guest on September 28, 2021 Fig. 4. HSPG-mediated endocytosis requires MYO7B and actin filaments. (A) Mapping the identified MYO7B sgRNA. (B) Validation of MYO7B-KO cells by qRT-PCR. mRNAs extracted from two clones of MYO7B KO cells and control (Ctrl.) clone were analyzed by qRT-PCR. Error bars indicate SEM. n = 3. (C) MYO7B KO cells are defective in α-Syn PFF uptake. Control (Ctrl.) and MYO7B-KO clones were incubated with pHrodo-labeled α-Syn PFF for 4 h before FACS analyses. (D) MYO7B is not required for endocytosis of α-Syn monomer. Control or MYO7B-KO cells were incubated with the α-Syn monomer (800 nM) overnight before FACS analysis. (E) MYO7B is not required for transferrin uptake. Control or MYO7B-KO cells were incubated with fluorescein-labeled transferrin (50 μg/mL for 3 h), washed, and analyzed with a fluorometer. Error bars represent SEM. n = 3. (F–H)KDofMYO7B in primary neurons reduces α-Syn PFF endocytosis without affecting its binding to the plasma membrane. (F) Primary neurons infected with the indicated shRNA-expressing lentivirus together with Synapsin_EGFP lentivirus at DIV3 were incubated with α-Syn PFF Alexa Fluor 594 (200 nM) for 4 h and then imaged at DIV8. (Insets) Enlarged views of the box. Arrows indicate the perinuclear enrichment of α-Syn PFF–positive vesicles in control cells. (G) The ratio of internalized α-Syn PFF relative to total α-Syn PFF in individual cells. (H) qRT-PCR evaluation of MYO7B mRNA levels using the same batch of cells. Error bars represent SEM. n = two biological repeats with triplicated PCR analysis. (I–K) MYO7B dominant-negative mutants inhibit α-Syn PFF uptake. (I) Representative confocal images of COS7 cells transfected with the indicated MYO7B plasmids and treated with α-Syn PFF Alexa Fluor 594 (400 nM) for 3 h. Arrows indicate normal α-Syn PFF uptake in untransfected cells, which was used to normalize uptake. N, nuclei. (J) Graph showing the domain structure of the truncated MYO7B mutants. (K) Quantification of α-Syn PFF fluorescence (FL) in individual cells transfected (t) with the indicated MYO7B mutants normalized by the signal in untransfected cells (u).

MYO7B Facilitates the Maturation of Clathrin-Coated Pits. Since detected small CLC-positive fluorescence puncta at the basal α-Syn PFF endocytosis requires CME, we characterized the ef- membranes (Fig. 6A, panel 1). In time-lapse videos, these CLC- fect of MYO7B inactivation on CCP morphology using TIRF positive puncta frequently detached from the membranes, van- microscopy. In WT cells transfected with GFP-CLC, TIRF ishing into the cytosol due to endocytosis (Fig. 6B, Upper).

6of11 | www.pnas.org/cgi/doi/10.1073/pnas.1918617117 Zhang et al. Downloaded by guest on September 28, 2021 CELL BIOLOGY

Fig. 5. MYO7B maintains plasma membrane dynamics at domains enriched in clathrin. (A) TIRF microscopy analysis of plasma membrane binding by EGFP- WT MYO7B and EGFP-CT MYO7B in cells stably expressing low levels of these proteins (Scale bar: 5 μm.) (B) Quantification of representative EGFP-WT MYO7B signal on the plasma membrane in a time-lapse video. Shown are fluorescence intensities of three randomly chosen spots measured by Fiji. A.U., arbitrary unit. (C and D) MYO7B binds to the plasma membrane in clathrin-enriched domains. (C) TIRF microscopy analysis of cells stably expressing EGFP-WT MYO7B and mCh-CLC. (Insets) Close-up views of cells revealing limited colocalization between MYO7B and CLC. The dashed lines indicate surface regions either lacking clathrin (white) or enriched in clathrin (yellow). (D) Correlation between CLC and MYO7B fluorescence (FL) intensity in cell surface domains as outlined in Fig. 5C (R2 = 0.956 by linear regression). (E) MYO7B inactivation affects plasma membrane dynamics. Control and MYO7B-KO cells were stained with GFP+ and imaged by time-lapse confocal microscopy (SI Appendix, Fig. S5C and Movies S3 and S4). The whisker graph shows the plasma membrane ruffling velocity determined by the Nikon Element analysis of videos taken from three independent experiments. Control, n = 6 cells; MYO7B-KO, n = 12 cells. P values by two- tailed unpaired Student’s t test. (F) MYO7B-KO cells have reduced actin fibers under the plasma membrane. TIRF-SIM analysis of WT or MYO7B-KO cells transfected with EGFP-tractin. Note the presence of actin-containing aggregates in MYO7B-KO cells (indicated by arrows) (Scale bar: 10 μm.) (G) Depletion of cholesterol inhibits α-Syn PFF endocytosis. Cells were treated with methyl-β-cyclodextrin (5 mM) or DMSO for 30 min before incubation with α-Syn PFF pHrodo and FACS analysis.

Intriguingly, in MYO7B-KO cells, only a fraction of CCPs be- was also observed in GFP-CLC–stable U2OS cells transfected haved like those in WT cells, while in ∼80% of the cells, we with a MYO7B-specific siRNA (Fig. 6A, panel 4 vs. panel 3). In detected large CCP clusters that were completely immobile addition, cells treated with jasplakinolide, dynole 34-2, or (Fig. 6A, panel 2 and B, Lower). The CCP clustering phenotype methyl-β-cyclodextrin (MBCD) also accumulated clustered CCP

Zhang et al. PNAS Latest Articles | 7of11 Downloaded by guest on September 28, 2021 Fig. 6. MYO7B inactivation affects the maturation of CCPs. (A and B) MYO7B depletion causes CCPs to cluster. (A) TIRF microscopy analysis of CCPs in control (panel 1) or MYO7B-KO (panel 2) HEK293T cells transiently transfected with GFP-CLC, or in GFP-CLC–stable U2OS cells transfected with control (panel 3) or MYO7B (panel 4) siRNA. Approximately 80% of MYO7B-KO cells show the phenotype shown in panel 2. (Insets) Enlarged views of the boxed regions. (Scale bar: 5 μm.) (B) Images from time-lapse videos showing CCP dynamics on the cell surface. Boxes and Insets show examples of CCP (arrow) vanishing into the cytosol in a WT cell (Scale bars: 2 μm.) (C and D) Effect of jasplakinolide (JASP), dynole 34-2 (Dynole), and MBCD on CCP morphology analyzed by TIRF in GFP- CLC stable U2OS cells. (C) Representative images of DMSO-treated, JASP-treated (100 nM for 1 h), and dynole-treated (18 μM for 2 h) cells. (Scale bar: 5 μm.) (D) Violin plot showing the relative CCP sizes under different drug-treated conditions. The numbers indicate the percentage of clustered CCPs. n, number of pits analyzed. ****P < 0.0001, one-way ANOVA. (E–G) Transmission EM analysis of CCP morphology in control and MYO7B-KO cells. Control and MYO7B-KO cells were incubated with α-Syn PFF (400 nM for 1 h), and then fixed for EM analysis. (E) Representative CCPs. (F) The relative abundance of U- and V-shaped pits vs. omega-shaped pits in control and MYO7B-KO cells. n, number of pits analyzed. (G) Violin plot showing the neck length of CCPs measured from two independent experiments. ***P < 0.001, unpaired Student’s t test. NS, not significant.

puncta at the basal membranes, coincident with α-Syn PFF en- capture defects associated with α-Syn PFF uptake, we first in- docytosis inhibition (Fig. 6 C and D). These results suggest that cubated cells with α-Syn PFF at 37 °C for 1 h to initiate PFF MYO7B-dependent membrane dynamics might facilitate CCP uptake. EM analyses identified approximately four CCPs per cell function in HSPG-mediated endocytosis. When CCPs fail to section in WT cells, but only one CPP per section on the plasma mature, these endocytosis-defective pits accumulate at the basal membrane of SLC35B2-KO cells (SI Appendix, Fig. S6 A and B), plasma membranes and form clusters. suggesting that the detected CCPs were mostly linked to HSPG- To test the foregoing hypothesis, we used transmission EM to mediated endocytosis. Although the number of CCPs in MYO7B- examine the morphology of CCPs at the apical and lateral KO cells was comparable to that of WT cells, we found that membranes where HSPG-mediated endocytosis occurs. To compared with WT cells, MYO7B-KO cells had a ∼9% increase

8of11 | www.pnas.org/cgi/doi/10.1073/pnas.1918617117 Zhang et al. Downloaded by guest on September 28, 2021 in the number of U- or V-shaped pits and a concomitant re- as a regulator of Listeria phagocytosis (50, 51), but this process is duction of omega-shaped pits (Fig. 6 E and F). In addition, for independent of CME. Thus, whether mammalian CME involves the U- or V-shaped pits, the average neck diameter was signifi- myosin(s) and if so, in what capacity, has been unclear. cantly increased in MYO7B-KO cells, whereas the neck diameter Our study now provides compelling evidence that MYO7B for omega-shaped pits was comparable in WT and MYO7B-KO serves a specific function in CME, at least for certain polycation- cells (Fig. 6G). Since U- and V-shaped CCPs are thought to be bearing cargos that enter cells via HSPGs. Live-cell imaging precursors of omega-shaped CCPs, these results support the showed that on binding to the cell surface, GFP+ is rapidly notion that MYO7B is required for optimal CCP function. clustered into patches (Fig. 2), suggesting that HSPGs may un- dergo oligomerization on ligand binding. Because membrane Discussion invagination during CME is expected to bring cargos and HSPGs Role of HSPGs in Endocytosis. HSPGs have been implicated in close to each other, which would increase the repellent force endocytosis of a variety of cargos (23), and the substrate diversity between molecules bearing the same charge, we reason that has been attributed to nonspecific electrostatic interactions be- MYO7B and actin filaments may provide the extra energy to tween cargos and HSPGs. Consistent with this notion, in this overcome this membrane-bending barrier. Because actin was study, we have shown that a GFP variant bearing net positive found both under the plasma membrane and on the surface of charges (GFP+) can be used as a probe to label HSPGs in some α-Syn PFF–bearing endocytic vesicles (SI Appendix, Fig. mammalian cells. However, our study also shows that certain S6C), we propose that MYO7B either promotes actin assembly cargos like α-Syn PFFs can interact with HSPGs via specific se- or stabilizes actin filaments at membranes near oligomerized quence motifs. Interestingly, among the 15 lysine residues in HSPG patches, which in turn facilitates membrane protrusions. α-Syn, 4 have been confirmed to mediate the interaction with These protrusions eventually wrap around cargos, forming HSPGs. These residues by themselves do not form the binding omega-shaped CCPs with actin filaments surrounding the CCP site, but when α-Syn assembles into stacked oligomers, they can neck. Membrane severance by dynamin subsequently releases line up to form long binding grooves that accommodate the the vesicles into the cytoplasm, leaving an F-actin “scar” at- linear oligosaccharide chains of HSPGs. Interaction with α-Syn tached to it (SI Appendix, Fig. S6D). Additional studies are PFFs does not require the protein core of HSPGs, meaning that needed to reveal the mechanistic details of MYO7B-mediated any of the 16 members of the HSPG family could possibly serve CME, as well as additional mechanisms that contribute to α-Syn as a recruiter for α-Syn PFFs. Importantly, our findings also PFF endocytosis. Nevertheless, our study has linked the function suggest that the two previously established α-Syn PFF con- of MYO7B and actin filaments to HSPG-mediated endocytosis formers have different affinities to HSPGs, which may corre- of α-Syn PFF and other polycation-bearing cargos. This unique CELL BIOLOGY spond to the reported α-Syn PFF strains bearing different feature may be exploited to develop drugs that prevent the pathogenic activities (40, 41). Although we cannot exclude the transmission of α-Syn pathology in PD. involvement of other α-Syn motifs in cell surface binding, iden- Materials and Methods tification of the K-T-K motifs in α-Syn as a major binding ele- ment for HSPGs may help design specific inhibitors that target Cell Lines and DNA Transfection. All reagents are listed in SI Appendix, Table α-Syn PFF entry for PD. S1. HEK293T, U2OS, COS7, and HeLa cells were purchased from American α Type Culture Collection. HEK293FT cells were obtained from Invitrogen. Cells Despite its essential function in recruiting -Syn PFFs to the were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning) cell surface, HSPGs alone might not be sufficient for linking containing 10% FBS and antibiotics (penicillin/streptomycin, 10 U/mL). All

HSPG cargos to downstream CME effectors such as clathrin, cell lines were maintained at 37 °C in a 5% CO2 humidified atmosphere. Cells because many HSPG family members do not have a cytoplasmic stably expressing GFP-CLC or mCh-CLC or GFP-tagged MYO7B WT and the CT domain that is required for recruiting cytosolic endocytosis ef- mutant were generated using U2OS cells. Specifically, these cells were fectors. Another membrane protein capable of interacting with seeded in a six-well plate and transfected with the reporter constructs using clathrin or other endocytosis regulators may assist HSPGs by Lipofectamine 2000. At 2 wk posttransfection, cells stably expressing the providing the missing link. Along this line, a recent study sug- reporters were sorted by FACS. Stable KO cells were generated by infecting HEK293T cells with lentivirus expressing sgRNAs targeting the gene of in- gested LAG3 as a neuronal-specific membrane receptor for μ α terest. Cells were selected by 0.35 g/mL puromycin for 4 d to generate cells -Syn PFFs (19). We propose that HSPGs may cooperate with stably expressing the sgRNAs. Single cell-derived KO clones were obtained distinct membrane receptors in a tissue-specific manner to jointly by sequential dilution or by FACS sorting. mediate α-Syn PFF endocytosis. Recruitment of α-Syn PFFs to Cell transfection was performed using TransIT-293 reagent (Mirus) for the plasma membrane by HSPGs should increase their local HEK293T cells or Lipofectamine 2000 (Invitrogen) for COS7, HeLa, and U2OS concentration, and thus the avidity for a downstream coreceptor. cells following the manufacturer’s instructions. For RNAi-mediated gene si- lencing, Lipofectamine RNAiMAX (Invitrogen) was used according to the manufacturer’s protocol. U2OS cells were seeded in four-well chambers and MYO7B and Actin Filaments in HSPG-Mediated Endocytosis. It is well transfected with 60 pmol siRNA per well in antibiotic-free medium for 48 h established that mechanical force generated by actin polymeri- before imaging analyses. zation can drive membrane invagination in CME in yeast and in mammalian cells bearing high membrane tension (42–44). Plasmid DNA, Molecular Biology, and Virus Production. A CRISPRv2 construct However, the function of myosin motors in CME is much less Saccharomyces cer- bearing sgRNA-targeting genes of interest was generated as follows. First, clear, particularly in higher . In 5 μg of CRISPRv2 was digested with BsmBI at 37 °C for 30 min. The digested evisiae, the type I (Myo3p and Myo5p) can promote the vector was then gel-purified and eluted in pure water. Each pair of sgDNA recruitment of actin assembly factors to the site of endocytosis, oligos was phosphorylated and annealed by incubating 1 μL of sgRNA oligo causing spatially restricted actin polymerization to deform the 1 or 2 (100 μM) with 1 μLof10× T4 ligation buffer in the presence of 0.5 μL membranes (45, 46). The mammalian class I myosin-1E was also of T4 polynucleotide kinase in 10 μL. The reaction was placed in a thermo- found at CCPs, coincident with a burst of actin assembly; how- cycler using the following parameters: 37 °C for 30 min, 95 °C for 5 min, and ever, depletion of myosin-1E had no discernible effect on then a ramp down to room temperature at 5 °C/min. The annealed sgDNA transferrin uptake under normal growth conditions (47). An- oligo was then ligated with the BsmBI-digested CRISPRv2 to create CRISPRv2- sgDNA constructs targeting SLC35B2 and MYO7B. KO lentivirus was pro- other study suggested a role for a class VI myosin in CME based duced by transfecting 1 million HEK293FT cells in a 3.5-cm dish with 0.4 μgof on its localization to CCPs/vesicles (48); however, a more recent pVSV-G, 0.6 μg of psPAX2, and 0.8 μg of CRISPRv2-sgRNA. Transfected cells study showed that myosin-6 is localized only to uncoated vesicles were incubated with 3 mL of fresh DMEM for 72 h, followed by harvesting of (49). On a related note, genetic studies have implicated MYO7A virus. shRNA knockdown virus was produced in a similar manner, except that

Zhang et al. PNAS Latest Articles | 9of11 Downloaded by guest on September 28, 2021 a pair of shRNA-coding oligos were annealed and ligated with Age1- 1 M). Purified monomeric α-Syn was dialyzed extensively in PBS, followed by EcoR1–digested pLKO.1 construct. SLC35B2 rescue plasmid was generated labeling with a threefold molar excess of pH-Rodo Red or Alexa Fluor 596 by PCR-amplifying the SLC35B2 cDNA sequence with a FLAG tag-coding se- succinimidyl ester fluorescence dye (Thermo Fisher Scientific) for 4 h at room quence and a NheI site appended at the 5′ end and an EcoRI site at the 3′ temperature. Unconjugated dye was removed by dialysis in PBS. The labeled end. The PCR fragment was digested with NheI and EcoRI and further pu- α-Syn was adjusted to 5 mg/mL in a volume of 500 μL and then placed in a rified. The purified DNA fragment was then inserted into pLJM1. Five non- ThermoMixer shaker (Eppendorf) at 1,000 rpm at 37 °C for 7 d to generate sense mutations were introduced at the sgRNA targeting sequence by site- protein fibrils. Preformed α-Syn PFFs were stained with 3% uranyl acetate directed mutagenesis to avoid Cas9 recognition of the rescue DNA. PCR and examined by EM to verify the formation of α-Syn PFF, then stored in mutagenesis was performed using the QuikChange II site-directed muta- + a −80 °C freezer in small aliquots. GPF was expressed and purified according genesis kit (Agilent) according to the manufacturer’s protocol. to a previously published protocol (34). Additional experimental procedures and methods are presented in SI Appendix. Protein Purification, Labeling, and PFF Production. Human α-Syn protein was expressed in BL21(DE3)RIL-competent Escherichia coli and purified following Data Availability. All data, associated protocols, methods, and sources of an established protocol (27). In brief, 1 L of E. coli culture was induced to materials are available in the main text or SI Appendix. express α-Syn with 0.5 mM isopropyl β-d-1-thiogalactopyranoside at 20 °C overnight. Cells were pelleted down at 5,000 rpm for 20 min, then resus- pended with high-salt buffer (750 mM NaCl, 10 mM Tris pH 7.6, and 1 mM ACKNOWLEDGMENTS. We thank Drs. R. Tycko and U. Ghosh (National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK]) for EDTA) with protease inhibitors and 1 mM PMSF (50 mL for 1 L of culture). assistance with protein negative staining, J. Reece at the NIDDK imaging Resuspended cells were sonicated with a 0.25-inch probe tip at 60% power core for imaging assistance, D. Lu for assistance with data processing, H. for a total time of 10 min. Sonicated cell lysate was boiled for 15 min to Meyer (University of Duisburg Essen) for critical reading of the manuscript, precipitate unwanted proteins and then cooled on ice for 20 min. The lysate and M. Tyska (Vanderbilt University) for MYO7B WT and mutant cDNAs. × was then centrifuged at 6,000 g for 20 min. The supernatant containing Q.Z., Y.X., J.L., and Y.Y. are supported by the NIDDK intramural research α -Syn was collected and dialyzed in TNE buffer (10 mM Tris pH 7.6, 25 mM program (DK075143); X.W. is supported by the National Heart, Lung, and NaCl, and 1 mM EDTA). Isolated proteins were further fractionated with a Blood Institute intramural research program; M.J. and J.S.B. are supported Bio-Rad NGS chromatography system first through a size exclusion column in by the Eunice Kennedy Shriver National Institute of Child Health and Human TNE buffer, followed by an ion-exchange Mono-Q column. Protein was Development intramural research program (ZIA HD001607); and J.S. is sup- eluted in TNE buffer containing increasing concentrations of NaCl (25 mM to ported by NIH Grants AG061829 and GM126960.

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