A myosin-7B–dependent endocytosis pathwaymediates cellular entry of α-synuclein fibrils andpolycation-bearing cargosQi 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 Kidney 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, NationalInstitutes of Health, Bethesda, MD 20892; cLaboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD20892; 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)has emerged as a key pathological event in Parkinson’s disease.This process is initiated when α-Syn–bearing fibrils enter cells viaclathrin-mediated endocytosis, but the underlying mechanisms areunclear. Using a CRISPR-mediated knockout screen, we identifySLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulatorsfor α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a keyregulator of heparan sulfate proteoglycan (HSPG) biosynthesis, isessential for recruiting α-Syn PFFs to the cell surface because thisprocess is mediated by interactions between negatively chargedsugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFFs.By contrast, MYO7B regulates α-Syn PFF cell entry by maintaininga plasma membrane-associated actin network that controls mem-brane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumu-lation of large clathrin-containing “scars” on the cell surface. Intrigu-ingly, the requirement for MYO7B in endocytosis is restricted toα-Syn PFFs and other polycation-bearing cargos that enter cells viaHSPGs. Thus, our study not only defines regulatory factors for α-SynPFF endocytosis, but also reveals a previously unknown endocytosismechanism for HSPG-binding cargos in general, which requiresforces generated by MYO7B and actin filaments.

α-synuclein | clathrin-mediated endocytosis | myosin-7B | heparan sulfateproteoglycan | actin filament

In Parkinson’s disease (PD), the major proteinaceous aggregateknown as a Lewy body is composed mostly of a synaptic pro-

tein, α-synuclein (α-Syn) (1). Coincidentally, genetic mutations inα-Syn–encoding genes were identified as a major contributingfactor for Parkinson’s disease (2, 3). α-Syn is a small polypeptidethat is aggregation-prone. Clinical studies have shown thatα-Syn–positive Lewy bodies can spread in the brain during dis-ease progression (4). Furthermore, following tissue trans-plantation therapy, grafted tissues can accumulate Lewy body-like aggregates over time, which does not occur if these cells areleft intact in the donor (5, 6). These observations prompted theidea that α-Syn might undergo cell-to-cell transmission analo-gously to a prion protein (7, 8). Indeed, in vitro and animalstudies have shown that α-Syn preformed fibrils (α-Syn PFFs)can be readily taken up by various cell types and subsequentlytransmitted to nearby cells that have not been directly exposed toα-Syn PFFs (9–13).At the cellular level, the intercellular transmission of neuro-

toxic proteins like α-Syn comprises two biological events: therelease of α-Syn from donor cells via unconventional proteinsecretion and its internalization by recipient cells via endocytosis(14). The molecular mechanisms of these processes are poorlydefined. Recent studies have suggested nanotubes, exosome se-cretion, or a pathway termed misfolding-associated protein se-cretion as potential mechanisms for the release of monomeric

and oligomerized α-Syn (15, 16). On the uptake side, α-Syn PFFscan enter cells via clathrin-mediated endocytosis (CME) onbinding to surface receptors (17), but whether α-Syn endocytosisinvolves substrate specific endocytosis regulators has not yet beenexplored. This type of regulator may be particularly relevant to thedevelopment of PD drugs that target α-Syn endocytosis. Anotherunresolved issue is the identity of relevant membrane receptors forα-Syn PFFs; several studies have reported different candidates:heparan sulfate proteoglycans (HSPGs), membrane proteinLAG3 for unmodified α-Syn PFFs (18, 19), and a glycoprotein,neurexin 1β, for N-terminally acetylated α-Syn PFFs (20).HSPGs compose a class of cell surface and extracellular matrix

glycoproteins that carry one or more heparan sulfate (HS) chainsbearing repeated disaccharide units. This modification is foundprimarily in the syndecan and glycosylphosphatidylinositol-anchored glypican families (21). The biosynthesis of HSPGsbegins in the endoplasmic reticulum, but its completion requiresmodification of the assembled sugar chains with sulfate using 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as the sulfate do-nor, which occurs in the Golgi complex (22). Because HS chainscarry negative charges, they can interact with a plethora of ligands

Significance

Direct cell-to-cell spreading of misfolded protein aggregates,such as α-synuclein fibrils (α-Syn), is a pathological hallmark as-sociated with the progression of many neurodegenerative dis-eases, but it is unclear how mammalian cells take up largeprotein aggregates to initiate this prion-like protein transmissionprocess. Here we define the endocytosis mechanism of α-Synpreformed fibrils (PFFs) using a combination of genetic, bio-chemical, and live-cell imaging techniques. Our study shows thatα-Syn PFFs enter cells following an entry pathway that is spe-cifically tailored for cargos bearing positive charges. These car-gos bind to heparan sulfate proteoglycans on the cell surface viacharge–charge interactions, which enable cargo internalizationby a previously unknown endocytosis mechanism that requiresforces generated by myosin-7B and actin filaments.

Author contributions: Q.Z., Y.X., J.L., M.J., J.S.B., J.S., and Y.Y. designed research; Q.Z.,Y.X., J.L., M.J., X.W., and Y.Y. performed research; J.S. contributed new reagents/analytictools; Q.Z., Y.X., J.L., and Y.Y. analyzed data; and Q.Z., Y.X., J.S.B., J.S., and Y.Y. wrotethe paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1Q.Z. and Y.X. contributed equally to this work.2To whom correspondence may be addressed. Email: yihongy@mail.nih.gov.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1918617117/-/DCSupplemental.

First published May 4, 2020.

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on the cell surface, either mediating their uptake or activatingdownstream signal transduction (23, 24). The known HSPG li-gands do not share any specific sequence motif for engagingHSPGs; instead, the binding energy seems to be provided byelectrostatic interactions, often involving positively charged resi-dues from random sequences in ligands (21). Along with α-SynPFFs, HSPGs have been implicated in the uptake of tau PFFs andAβ (18, 25, 26), but how HSPGs interact with these neurotoxicprotein aggregates is unclear.In this study, we used an unbiased CRISPR/Cas9 screen to

identify factors that regulate α-Syn PFF endocytosis in mam-malian cells. The screen not only isolated known endocytosisregulators of α-Syn PFFs (e.g., HSPGs), but also identified otherfactors important for α-Syn PFF uptake (e.g., an unconventional

myosin and actin). Mechanistically, our study defines the in-teraction of HSPGs with α-Syn PFFs using biochemical assaysand structural modeling. Importantly, we reveal a unique CMEmechanism for α-Syn PFFs and other polycation-bearing cargos,which, unlike conventional CME, requires myosin 7B (MYO7B)and actin filaments.

ResultsA CRISPR Screen Identifies Proteins Involved in α-Syn PFF Endocytosis.To dissect the mechanism of α-Syn PFF endocytosis, we labeledpurified α-Syn with either a stable fluorophore (Alexa Fluor 594)or a pH-sensitive dye (pHrodo). The pHrodo dye becomes ac-tivated only on its arrival at the acidic late endosome/lysosomecompartment, 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 butnot 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 for4 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 FACSanalysis. (E–G) Knockdown (KD) of SLC35B2 attenuates α-Syn PFF uptake in primary neurons. (E) Primary neurons were infected with lentivirus expressing theindicated 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 wereincubated 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-tailedt 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 cellsreexpressing 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 byconfocal microscopy (Scale bar: 5 μm.) (I) Quantification of α-Syn PFF surface level in individual cells from two independent experiments.

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We used labeled proteins to prepare α-Syn PFFs following awell-established protocol (27). Electron microscopy (EM) anal-ysis of sonicated α-Syn PFF revealed 20- to 100-nm-long particles(SI Appendix, Fig. S1B), consistent with the reported α-Syn PFFsbearing pathogenic activities (27). We tested α-Syn PFF uptakein various cell types and found that both neuronal and non-neuronal cells could efficiently internalize α-Syn PFF (SI Ap-pendix, Fig. S1C), suggesting that the core endocytosis machineryfor α-Syn PFFs is not specific to neurons. Treating cells withdynamin inhibitors, such as dynasore (28) or dynole 34-2 (29),significantly reduced α-Syn PFF uptake (SI Appendix, Fig. S1 Dand E). In addition, α-Syn PFF uptake was also dramaticallyattenuated in HeLa cells lacking the σ2 subunit of AP-2 (enco-ded by the AP2S1 gene), a cargo adaptor in CME (SI Appendix,Fig. S1F). These findings confirm CME-dependent α-Syn PFFendocytosis (17, 30).To search for α-Syn PFF endocytosis regulators, we performed

a pooled CRISPR/Cas9 screen (Fig. 1A). We chose HEK293Tcells because their fast-growing and high-DNA recombinationproperties make them well suited for a CRISPR screen. Sincepooled CRISPR screens often suffer from poor specificity andreproducibility, we used a strategy akin to the classical geneticsapproach. We treated HEK293T cells with a lentiviral GeCKOlibrary expressing single guide RNAs (sgRNAs) targeting allhuman genes. We then incubated mutagenized cells withpHrodo-labeled α-Syn PFFs. Florescence-activated cell sorting(FACS) was used to sort α-Syn PFF–negative cells into 96-wellplates at single-cell resolution. When these cells formed colonies,we rescreened them to identify cells defective in α-Syn PFFuptake. Among the 1,248 clones screened, one clone (C10)showed almost no α-Syn PFF uptake (SI Appendix, Fig. S2A),whereas 27 other clones showed reduced α-Syn PFF uptake. Inthis study, we characterized two clones, which revealed an un-expected requirement for the endocytosis of α-Syn PFF andother polycation-bearing cargos.

HSPGs Are Required for α-Syn PFF Endocytosis. Sequencing identi-fied a sgRNA in one of the clones (C10) targeting SLC35B2.Analysis of SLC35B2-knockout (KO) cells confirmed a specificrequirement for SLC35B2 in endocytosis of α-Syn PFFs, but notmonomeric α-Syn (mSyn) (Fig. 1 B–D). Furthermore, shRNA-mediated knockdown of SLC35B2 in primary neurons also re-duced α-Syn PFF uptake (Fig. 1 E–G), but the uptake defect wasless dramatic compared with that in SLC35B2 KO cells, likelybecause of partial gene silencing. The SLC35B2 gene containsfour exons. The identified sgRNA targets a sequence in exon 2(SI Appendix, Fig. S2B). SLC35B2 is known as a Golgi-localizedmembrane protein required for transporting PAPS into theGolgi complex for protein sulfation (SI Appendix, Fig. S2C) (31).We confirmed the Golgi localization of SLC35B2 by FLAGantibody immunostaining of SLC35B2-KO cells stably expressingFLAG-tagged SLC35B2 (SI Appendix, Fig. S2D). The function-ality of ectopically expressed SLC35B2 was confirmed by itsability to rescue α-Syn PFF uptake in SLC35B2-KO cells(Fig. 1C). Collectively, these results suggest a role for Golgi-localized protein sulfation in α-Syn PFF endocytosis.Cell surface binding experiments showed that SLC35B2 is

required for the recruitment of α-Syn PFFs to the plasmamembrane (Fig. 1 H and I). Given the known role of SLC35B2 inthe biosynthesis of HSPGs (31) and recent studies suggestingHSPGs as a potential receptor for α-Syn PFFs (18, 32), wepresumed that α-Syn PFFs failed to bind SLC35B2-KO cells,because these cells lacked HSPGs. Indeed, immunostaining withan antibody to HSPGs stained the plasma membrane of WT, butnot that of SLC35B2-KO cells (SI Appendix, Fig. S2 E and F).Furthermore, KO of XYLT2, another key HSPG biosyntheticenzyme (33), diminished both cell surface HSPG signals (SI

Appendix, Fig. S2G) and α-Syn PFF binding (SI Appendix, Fig.S2 H and I). Thus, our unbiased genetic screen confirms HSPGsas critical mediators of α-Syn PFF–membrane interaction, whichis essential for α-Syn PFF endocytosis.

Electrostatic Interactions Recruit α-Syn PFF to the Plasma Membrane.To better characterize the α-Syn PFF–HSPG interaction, wedeveloped a fluorescence probe that can track HSPGs in livecells. Since the oligosaccharide chains in HSPGs are enriched innegative charges (23), we reasoned that an engineered GFPvariant carrying 36 net positive charges (GFP+) (34) might be aspecific probe for HSPGs. Indeed, several lines of evidencesupport this notion. First, live-cell confocal microscopy showedthat GFP+, but not a GFP variant bearing extra negative charges(GFP−) bound efficiently to the cell surface, initially forming asmooth profile, which was rapidly converted to a punctate pat-tern reminiscent of cells treated with α-Syn PFF Alexa Fluor 594(Fig. 2A). The interaction of GFP+ with the cell surface wascompletely dependent on SLC35B2, as no GFP+ staining wasdetected in SLC35B2-KO cells (Fig. 2B, compare panel 2 withpanels 1 and 3). Furthermore, when cells were pretreated withGFP+, cell surface HSPGs could not be stained by antibodies toHSPG (Fig. 2C), suggesting that GFP+ competes with HSPGantibodies for the same binding site. Because cells treated withGFP+ and α-Syn PFF Alexa Fluor 594 showed extensive coloc-alization of these two cargos on the cell surface (Fig. 2D), andbecause pretreating α-Syn PFF with GFP− reduced the bindingof α-Syn PFF to cells (Fig. 2E), electrostatic interactions mustprovide the major energy that recruits α-Syn PFFs to the cellsurface (Fig. 2F).

Two K-T-K Motifs in Oligomerized α-Syn Enable HSPG Interactions.Previous studies have shown that two α-Syn monomers couldform either a face-to-face or back-to-back dimer. Many dimersthen stack together to form long fibrils of “rod” or “twisted”conformers (35, 36). To elucidate how these α-Syn PFF con-formers interact with HSPGs, we used the ClusPro program tomodel the interaction of α-Syn PFF conformers with heparin, anHS analog that competitively inhibits the binding of α-Syn PFFto cells (18). The modeling resulted in 14 clusters falling into 3groups, as shown in Fig. 3A and SI Appendix, Fig. S3 A and B.Interestingly, in all clusters, the interactions between α-Syn PFFand heparin were mediated by two K-T-K motifs, K43-T-K45and K58-T-K60, in α-Syn fibrils. In the twisted conformer, eachof these lysine-rich motifs lined up to form a shallow concavesurface that accommodates a di-heparin molecule bearing sixsulfate groups (SI Appendix, Fig. S3 A and B). In contrast, in therod conformation, two K-T-K motifs from distinct α-Syn mole-cules in a dimeric fibril joined together to form one deep bindinggroove for di-heparin (Fig. 3 A and B). As a result, each fibrilcould offer only two binding grooves, but within each binding sitethe heparin molecule was sandwiched by four rows of lysineresidues, forming extensive electrostatic interactions as well ashydrogen bonds with α-Syn (Fig. 3C and SI Appendix, Fig. S3C).Thus, α-Syn fibrils in the rod conformer are expected to have amuch higher affinity for HSPGs than the twisted conformer.These models explain why HSPGs are only required for theuptake of α-Syn fibrils but not monomeric α-Syn. This result alsosuggests that a twisted α-Syn oligomer containing three proto-mers is the minimum requirement for binding one heparin unit;in contrast, for the rod conformer, five or six α-Syn protomersare required to form a higher-affinity heparin-binding site.To validate our models, we generated α-Syn mutants with the

four lysine residues K43, K45, K58, and K60 substituted to eitherglutamine (4Q) or alanine (4A) (Fig. 3D). These mutants couldform long fibrils similarly to WT α-Syn (Fig. 3E); however,compared with WT α-Syn PFFs, the uptake of these mutant

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α-Syn PFFs was dramatically reduced (Fig. 3F). In contrast, theuptake of monomeric α-Syn was not affected by these mutations(SI Appendix, Fig. S3D). Furthermore, cell-binding experimentsshowed that the interaction of α-Syn PFF with the plasmamembrane was significantly inhibited by these mutations(Fig. 3G). These results strongly suggest that α-Syn binds HSPGsvia a specific sequence motif that forms binding sites only onα-Syn oligomerization.

HSPG-Mediated Endocytosis Requires MYO7B and Actin Filaments.Using a similar approach, we identified a sgRNA from a clonepartially defective in α-Syn PFF endocytosis, which targetedMYO7B, an unconventional myosin widely expressed in varioushuman tissues (37). Like for SLC35B2, the sgRNA targetingMYO7B mapped to an early exon (Fig. 4A). ReconstructedMYO7B-KO HEK293T cells or cells treated with MYO7B-specific small interfering RNA (siRNA) showed that depletionof MYO7B diminished endocytosis of α-Syn PFF (Fig. 4 B and Cand SI Appendix, Fig. S4A), while reexpression of EGFP-MYO7B in MYO7B-KO cells largely rescued the α-Syn PFFuptake phenotype (SI Appendix, Fig. S4B). An α-Syn PFF uptakedefect was similarly observed in primary neurons expressingMYO7B-specific shRNA (Fig. 4 F–H). Strikingly, the function ofMYO7B in endocytosis appeared to be restricted to HSPG-dependent cargos, because like HSPG-deficient cells (23),

MYO7B-KO cells were also defective in the uptake of GFP+ (SIAppendix, Fig. S4 C and D) and of DNA in complex with apolycation carrier (SI Appendix, Fig. S4E). Furthermore, HSPG-dependent uptake of a lentiviral reporter expressing GFP wassignificantly reduced in MYO7B-KO cells (SI Appendix, Fig. S4 Fand G). On the other hand, endocytosis of monomeric α-Syn ortransferrin was not affected in MYO7B-KO cells (Fig. 4 Dand E).To test whether HSPG-mediated endocytosis involves the

motor activity of MYO7B, we ectopically expressed MYO7Bmutants that either lack the motor domain (CT) or carry amutation that disrupts the ATPase cycle of the motor domain(N207A, I482A, or E442A) (38). If coupling the motor activity tocargo binding were required for α-Syn PFF uptake, then thesemutants should inhibit α-Syn PFF uptake in a dominant negativemanner. Indeed, cells expressing these mutants showed signifi-cantly reduced α-Syn PFF uptake compared with neighboringuntransfected cells or cells transfected with WT MYO7B(Fig. 4 I–K). Furthermore, deleting the C-terminal membrane-binding FERM domain abolished the dominant negative activityof MYO7B-CT, suggesting that membrane binding by a motor-defective MYO7B mutant accounts for the observed endocytosisdefect. These results, together with the observation that actin-binding compounds (e.g., jasplakinolide, latrunculin A) that in-terfere with the function of F-actin also inhibited α-Syn PFFendocytosis (SI Appendix, Fig. S5 A and B), strongly suggest that

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 aHEK293T 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 plasmamembrane depends on SLC35B2. Control (panel 1) or SLC35B2-KO HEK293T cells with (panel 3) or without (panel 2) the reexpression of WT SLC35B2 werestained 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 surfacewas quantified by confocal imaging. (F) An HSPG–cargo interaction model.

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optimal α-Syn PFF endocytosis requires a membrane-associatedfunctional interplay between MYO7B and actin filaments.

MYO7B Maintains Membrane Dynamics at Clathrin-EnrichedMembrane Domains. To elucidate the mechanism of MYO7B-mediated endocytosis, we used total internal reflection fluores-cence microscopy (TIRF) to examine the localization of MYO7Bin U2OS cells stably expressing mCherry-tagged clathrin lightchain (mCh-CLC) to label clathrin-coated pits (CCPs) togetherwith either GFP-WT MYO7B or GFP-MYO7B-CT. Consistentwith the reported localization of MYO7B to plasma membrane-derived microvilli (38), GFP-MYO7B also bound to the plasmamembrane in U2OS cells, forming discrete dots (Fig. 5A). Time-lapse microscopy showed that MYO7B-positive signals fluctu-ated rapidly, displaying a flickering pattern (Fig. 5B and MovieS1). Interestingly, the MYO7B-CT mutant appeared to formmore stable interactions with the membranes (Fig. 5A), resultingin bright foci that remained constant for a prolonged period(Movie S2). We concluded that the MYO7B C-tail domain canbind to the plasma membrane, while its actin-binding domaincontributes to the dynamic property of this interaction.When colocalization of MYO7B with CCPs was examined by

two-color TIRF, we found limited overlapping signals (Fig. 5C,Insets). However, we noticed that MYO7B was not uniformlydistributed on the plasma membrane. Instead, certain areas had

more MYO7B dots than others (Fig. 5C). Intriguingly, MYO7B-high plasma membrane domains were often enriched in CCPs(Fig. 5 C and D). Therefore, we postulated that dynamic inter-actions of MYO7B within certain plasma membrane domainsenable membrane flexibility and deformability, which might inturn facilitate the maturation of CCPs located in the samemembrane domain.To measure plasma membrane dynamics, we used GFP+ to

label the cell surface and then took time-lapse videos. InWT cells, the plasma membrane usually undergoes dynamic ex-pansion and retraction, generating a ruffling motion (SI Appen-dix, Fig. S5C and Movie S3). In contrast, the membranes ofMYO7B-KO cells had significantly reduced mobility (Movie S4and Fig. 5E). Furthermore, TIRF microscopy showed thatMYO7B-KO cells had a significantly reduced amount of actinfilaments underneath the plasma membranes (Fig. 5F). Theseresults suggest that MYO7B may regulate actin assembly tomaintain membrane dynamics.If MYO7B promotes endocytosis via membrane dynamics at

cargo-binding sites, other agents capable of increasing mem-brane stiffness should similarly inhibit α-Syn PFF uptake. To testthis idea, we treated cells with the cholesterol-depleting drugmethyl-β-cyclodextrin, which increases membrane stiffness inmammalian cells (39). Consistent with our hypothesis, methyl-β-cyclodextrin also reduced α-Syn PFF uptake to a similar degreeas depletion of MYO7B (Fig. 5G).

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. (Aand 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 along 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 thecharge 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 FACSanalysis. (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 AlexaFluor 594 at 400 nM in the presence or absence of heparin (4 μM) on ice. **P < 0.01, two-tailed Student’s t test.

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MYO7B Facilitates the Maturation of Clathrin-Coated Pits. Sinceα-Syn PFF endocytosis requires CME, we characterized the ef-fect of MYO7B inactivation on CCP morphology using TIRFmicroscopy. In WT cells transfected with GFP-CLC, TIRF

detected small CLC-positive fluorescence puncta at the basalmembranes (Fig. 6A, panel 1). In time-lapse videos, these CLC-positive puncta frequently detached from the membranes, van-ishing into the cytosol due to endocytosis (Fig. 6B, Upper).

Fig. 4. HSPG-mediated endocytosis requires MYO7B and actin filaments. (A) Mapping the identified MYO7B sgRNA. (B) Validation of MYO7B-KO cells byqRT-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) MYO7BKO 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 orMYO7B-KO cells were incubated with the α-Syn monomer (800 nM) overnight beforeFACS analysis. (E) MYO7B is not required for transferrin uptake. Control or MYO7B-KO cells were incubated with fluorescein-labeled transferrin (50 μg/mL for3 h), washed, and analyzed with a fluorometer. Error bars represent SEM. n = 3. (F–H) KD ofMYO7B in primary neurons reduces α-Syn PFF endocytosis withoutaffecting its binding to the plasma membrane. (F) Primary neurons infected with the indicated shRNA-expressing lentivirus together with Synapsin_EGFPlentivirus 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 indicatethe 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 MYO7Bplasmids 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 tonormalize uptake. N, nuclei. (J) Graph showing the domain structure of the truncated MYO7B mutants. (K) Quantification of α-Syn PFF fluorescence (FL) inindividual cells transfected (t) with the indicated MYO7B mutants normalized by the signal in untransfected cells (u).

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Intriguingly, in MYO7B-KO cells, only a fraction of CCPs be-haved like those in WT cells, while in ∼80% of the cells, wedetected large CCP clusters that were completely immobile(Fig. 6A, panel 2 and B, Lower). The CCP clustering phenotype

was also observed in GFP-CLC–stable U2OS cells transfectedwith a MYO7B-specific siRNA (Fig. 6A, panel 4 vs. panel 3). Inaddition, cells treated with jasplakinolide, dynole 34-2, ormethyl-β-cyclodextrin (MBCD) also accumulated clustered CCP

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 MYO7Bsignal on the plasma membrane in a time-lapse video. Shown are fluorescence intensities of three randomly chosen spots measured by Fiji. A.U., arbitraryunit. (C and D) MYO7B binds to the plasma membrane in clathrin-enriched domains. (C) TIRF microscopy analysis of cells stably expressing EGFP-WT MYO7Band mCh-CLC. (Insets) Close-up views of cells revealing limited colocalization between MYO7B and CLC. The dashed lines indicate surface regions eitherlacking clathrin (white) or enriched in clathrin (yellow). (D) Correlation between CLC and MYO7B fluorescence (FL) intensity in cell surface domains as outlinedin 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 velocitydetermined 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 cellstransfected with EGFP-tractin. Note the presence of actin-containing aggregates in MYO7B-KO cells (indicated by arrows) (Scale bar: 10 μm.) (G) Depletion ofcholesterol 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 pHrodoand FACS analysis.

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puncta at the basal membranes, coincident with α-Syn PFF en-docytosis inhibition (Fig. 6 C and D). These results suggest thatMYO7B-dependent membrane dynamics might facilitate CCPfunction in HSPG-mediated endocytosis. When CCPs fail tomature, these endocytosis-defective pits accumulate at the basalplasma membranes and form clusters.To test the foregoing hypothesis, we used transmission EM to

examine the morphology of CCPs at the apical and lateralmembranes where HSPG-mediated endocytosis occurs. To

capture defects associated with α-Syn PFF uptake, we first in-cubated cells with α-Syn PFF at 37 °C for 1 h to initiate PFFuptake. EM analyses identified approximately four CCPs per cellsection in WT cells, but only one CPP per section on the plasmamembrane of SLC35B2-KO cells (SI Appendix, Fig. S6 A and B),suggesting that the detected CCPs were mostly linked to HSPG-mediated endocytosis. Although the number of CCPs inMYO7B-KO cells was comparable to that of WT cells, we found thatcompared with WT cells, MYO7B-KO cells had a ∼9% increase

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) orMYO7B (panel 4) siRNA. Approximately 80% of MYO7B-KO cells show the phenotype shown in panel 2. (Insets) Enlarged views of the boxed regions. (Scalebar: 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 thecytosol 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 ofpits analyzed. ****P < 0.0001, one-way ANOVA. (E–G) Transmission EM analysis of CCP morphology in control and MYO7B-KO cells. Control and MYO7B-KOcells 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-shapedpits 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 twoindependent experiments. ***P < 0.001, unpaired Student’s t test. NS, not significant.

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in the number of U- or V-shaped pits and a concomitant re-duction of omega-shaped pits (Fig. 6 E and F). In addition, forthe U- or V-shaped pits, the average neck diameter was signifi-cantly increased in MYO7B-KO cells, whereas the neck diameterfor omega-shaped pits was comparable in WT and MYO7B-KOcells (Fig. 6G). Since U- and V-shaped CCPs are thought to beprecursors of omega-shaped CCPs, these results support thenotion that MYO7B is required for optimal CCP function.

DiscussionRole of HSPGs in Endocytosis. HSPGs have been implicated inendocytosis of a variety of cargos (23), and the substrate diversityhas been attributed to nonspecific electrostatic interactions be-tween cargos and HSPGs. Consistent with this notion, in thisstudy, we have shown that a GFP variant bearing net positivecharges (GFP+) can be used as a probe to label HSPGs inmammalian cells. However, our study also shows that certaincargos like α-Syn PFFs can interact with HSPGs via specific se-quence motifs. Interestingly, among the 15 lysine residues inα-Syn, 4 have been confirmed to mediate the interaction withHSPGs. These residues by themselves do not form the bindingsite, but when α-Syn assembles into stacked oligomers, they canline up to form long binding grooves that accommodate thelinear oligosaccharide chains of HSPGs. Interaction with α-SynPFFs does not require the protein core of HSPGs, meaning thatany of the 16 members of the HSPG family could possibly serveas a recruiter for α-Syn PFFs. Importantly, our findings alsosuggest that the two previously established α-Syn PFF con-formers have different affinities to HSPGs, which may corre-spond to the reported α-Syn PFF strains bearing differentpathogenic activities (40, 41). Although we cannot exclude theinvolvement of other α-Syn motifs in cell surface binding, iden-tification of the K-T-K motifs in α-Syn as a major binding ele-ment for HSPGs may help design specific inhibitors that targetα-Syn PFF entry for PD.Despite its essential function in recruiting α-Syn PFFs to the

cell surface, HSPGs alone might not be sufficient for linkingHSPG cargos to downstream CME effectors such as clathrin,because many HSPG family members do not have a cytoplasmicdomain that is required for recruiting cytosolic endocytosis ef-fectors. Another membrane protein capable of interacting withclathrin or other endocytosis regulators may assist HSPGs byproviding the missing link. Along this line, a recent study sug-gested LAG3 as a neuronal-specific membrane receptor forα-Syn PFFs (19). We propose that HSPGs may cooperate withdistinct membrane receptors in a tissue-specific manner to jointlymediate α-Syn PFF endocytosis. Recruitment of α-Syn PFFs tothe plasma membrane by HSPGs should increase their localconcentration, and thus the avidity for a downstream coreceptor.

MYO7B and Actin Filaments in HSPG-Mediated Endocytosis. It is wellestablished that mechanical force generated by actin polymeri-zation can drive membrane invagination in CME in yeast and inmammalian cells bearing high membrane tension (42–44).However, the function of myosin motors in CME is much lessclear, particularly in higher eukaryotes. In Saccharomyces cer-evisiae, the type I myosins (Myo3p and Myo5p) can promote therecruitment of actin assembly factors to the site of endocytosis,causing spatially restricted actin polymerization to deform themembranes (45, 46). The mammalian class I myosin-1E was alsofound at CCPs, coincident with a burst of actin assembly; how-ever, depletion of myosin-1E had no discernible effect ontransferrin uptake under normal growth conditions (47). An-other study suggested a role for a class VI myosin in CME basedon its localization to CCPs/vesicles (48); however, a more recentstudy showed that myosin-6 is localized only to uncoated vesicles(49). On a related note, genetic studies have implicated MYO7A

as a regulator of Listeria phagocytosis (50, 51), but this process isindependent of CME. Thus, whether mammalian CME involvesmyosin(s) and if so, in what capacity, has been unclear.Our study now provides compelling evidence that MYO7B

serves a specific function in CME, at least for certain polycation-bearing cargos that enter cells via HSPGs. Live-cell imagingshowed that on binding to the cell surface, GFP+ is rapidlyclustered into patches (Fig. 2), suggesting that HSPGs may un-dergo oligomerization on ligand binding. Because membraneinvagination during CME is expected to bring cargos and HSPGsclose to each other, which would increase the repellent forcebetween molecules bearing the same charge, we reason thatMYO7B and actin filaments may provide the extra energy toovercome this membrane-bending barrier. Because actin wasfound both under the plasma membrane and on the surface ofsome α-Syn PFF–bearing endocytic vesicles (SI Appendix, Fig.S6C), we propose that MYO7B either promotes actin assemblyor stabilizes actin filaments at membranes near oligomerizedHSPG patches, which in turn facilitates membrane protrusions.These protrusions eventually wrap around cargos, formingomega-shaped CCPs with actin filaments surrounding the CCPneck. Membrane severance by dynamin subsequently releasesthe vesicles into the cytoplasm, leaving an F-actin “scar” at-tached to it (SI Appendix, Fig. S6D). Additional studies areneeded to reveal the mechanistic details of MYO7B-mediatedCME, as well as additional mechanisms that contribute to α-SynPFF endocytosis. Nevertheless, our study has linked the functionof MYO7B and actin filaments to HSPG-mediated endocytosisof α-Syn PFF and other polycation-bearing cargos. This uniquefeature may be exploited to develop drugs that prevent thetransmission of α-Syn pathology in PD.

Materials and MethodsCell Lines and DNA Transfection. All reagents are listed in SI Appendix, TableS1. HEK293T, U2OS, COS7, and HeLa cells were purchased from AmericanType Culture Collection. HEK293FT cells were obtained from Invitrogen. Cellswere maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning)containing 10% FBS and antibiotics (penicillin/streptomycin, 10 U/mL). Allcell lines were maintained at 37 °C in a 5% CO2 humidified atmosphere. Cellsstably expressing GFP-CLC or mCh-CLC or GFP-tagged MYO7B WT and the CTmutant were generated using U2OS cells. Specifically, these cells wereseeded in a six-well plate and transfected with the reporter constructs usingLipofectamine 2000. At 2 wk posttransfection, cells stably expressing thereporters were sorted by FACS. Stable KO cells were generated by infectingHEK293T cells with lentivirus expressing sgRNAs targeting the gene of in-terest. Cells were selected by 0.35 μg/mL puromycin for 4 d to generate cellsstably expressing the sgRNAs. Single cell-derived KO clones were obtainedby sequential dilution or by FACS sorting.

Cell transfection was performed using TransIT-293 reagent (Mirus) forHEK293T cells or Lipofectamine 2000 (Invitrogen) for COS7, HeLa, and U2OScells following the manufacturer’s instructions. For RNAi-mediated gene si-lencing, Lipofectamine RNAiMAX (Invitrogen) was used according to themanufacturer’s protocol. U2OS cells were seeded in four-well chambers andtransfected with 60 pmol siRNA per well in antibiotic-free medium for 48 hbefore imaging analyses.

Plasmid DNA, Molecular Biology, and Virus Production. A CRISPRv2 constructbearing sgRNA-targeting genes of interest was generated as follows. First,5 μg of CRISPRv2 was digested with BsmBI at 37 °C for 30 min. The digestedvector was then gel-purified and eluted in pure water. Each pair of sgDNAoligos was phosphorylated and annealed by incubating 1 μL of sgRNA oligo1 or 2 (100 μM) with 1 μL of 10× T4 ligation buffer in the presence of 0.5 μLof T4 polynucleotide kinase in 10 μL. The reaction was placed in a thermo-cycler using the following parameters: 37 °C for 30 min, 95 °C for 5 min, andthen a ramp down to room temperature at 5 °C/min. The annealed sgDNAoligo was then ligated with the BsmBI-digested CRISPRv2 to create CRISPRv2-sgDNA constructs targeting SLC35B2 and MYO7B. KO lentivirus was pro-duced by transfecting 1 million HEK293FT cells in a 3.5-cm dish with 0.4 μg ofpVSV-G, 0.6 μg of psPAX2, and 0.8 μg of CRISPRv2-sgRNA. Transfected cellswere incubated with 3 mL of fresh DMEM for 72 h, followed by harvesting ofvirus. shRNA knockdown virus was produced in a similar manner, except that

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a pair of shRNA-coding oligos were annealed and ligated with Age1-EcoR1–digested pLKO.1 construct. SLC35B2 rescue plasmid was generatedby PCR-amplifying the SLC35B2 cDNA sequence with a FLAG tag-coding se-quence and a NheI site appended at the 5′ end and an EcoRI site at the 3′end. The PCR fragment was digested with NheI and EcoRI and further pu-rified. The purified DNA fragment was then inserted into pLJM1. Five non-sense mutations were introduced at the sgRNA targeting sequence by site-directed mutagenesis to avoid Cas9 recognition of the rescue DNA. PCRmutagenesis was performed using the QuikChange II site-directed muta-genesis kit (Agilent) according to the manufacturer’s protocol.

Protein Purification, Labeling, and PFF Production. Human α-Syn protein wasexpressed in BL21(DE3)RIL-competent Escherichia coli and purified followingan established protocol (27). In brief, 1 L of E. coli culture was induced toexpress α-Syn with 0.5 mM isopropyl β-d-1-thiogalactopyranoside at 20 °Covernight. 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 mMEDTA) with protease inhibitors and 1 mM PMSF (50 mL for 1 L of culture).Resuspended cells were sonicated with a 0.25-inch probe tip at 60% powerfor a total time of 10 min. Sonicated cell lysate was boiled for 15 min toprecipitate unwanted proteins and then cooled on ice for 20 min. The lysatewas then centrifuged at 6,000 × g for 20 min. The supernatant containingα-Syn was collected and dialyzed in TNE buffer (10 mM Tris pH 7.6, 25 mMNaCl, and 1 mM EDTA). Isolated proteins were further fractionated with aBio-Rad NGS chromatography system first through a size exclusion column inTNE buffer, followed by an ion-exchange Mono-Q column. Protein waseluted in TNE buffer containing increasing concentrations of NaCl (25 mM to

1 M). Purified monomeric α-Syn was dialyzed extensively in PBS, followed bylabeling with a threefold molar excess of pH-Rodo Red or Alexa Fluor 596succinimidyl ester fluorescence dye (Thermo Fisher Scientific) for 4 h at roomtemperature. Unconjugated dye was removed by dialysis in PBS. The labeledα-Syn was adjusted to 5 mg/mL in a volume of 500 μL and then placed in aThermoMixer shaker (Eppendorf) at 1,000 rpm at 37 °C for 7 d to generateprotein fibrils. Preformed α-Syn PFFs were stained with 3% uranyl acetateand examined by EM to verify the formation of α-Syn PFF, then stored ina −80 °C freezer in small aliquots. GPF+ was expressed and purified accordingto a previously published protocol (34). Additional experimental proceduresand methods are presented in SI Appendix.

Data Availability. All data, associated protocols, methods, and sources ofmaterials are available in the main text or SI Appendix.

ACKNOWLEDGMENTS. We thank Drs. R. Tycko and U. Ghosh (NationalInstitute of Diabetes and Digestive and Kidney Diseases [NIDDK]) forassistance with protein negative staining, J. Reece at the NIDDK imagingcore for imaging assistance, D. Lu for assistance with data processing, H.Meyer (University of Duisburg Essen) for critical reading of the manuscript,and M. Tyska (Vanderbilt University) for MYO7B WT and mutant cDNAs.Q.Z., Y.X., J.L., and Y.Y. are supported by the NIDDK intramural researchprogram (DK075143); X.W. is supported by the National Heart, Lung, andBlood Institute intramural research program; M.J. and J.S.B. are supportedby the Eunice Kennedy Shriver National Institute of Child Health and HumanDevelopment intramural research program (ZIA HD001607); and J.S. is sup-ported by NIH Grants AG061829 and GM126960.

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