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

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 via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFFs to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFFs. By contrast, MYO7B regulates α-Syn PFF cell entry by maintaining a plasma membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing “scars” on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is restricted to α-Syn PFFs and other polycation-bearing cargos that enter cells via HSPGs. Thus, our study not only defines regulatory factors for α-Syn PFF endocytosis, but also reveals a previously unknown endocytosis mechanism for HSPG-binding cargos in general, which requires forces generated by MYO7B and actin filaments.

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

10.1101/2020.02.12.946137 ◽

2020 ◽

Author(s):

Qi Zhang ◽

Yue Xu ◽

Juhyung Lee ◽

Michal Jarnik ◽

Xufeng Wu ◽

...

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Actin Filaments ◽

Mammalian Cells ◽

Imaging Techniques ◽

Protein Aggregates ◽

Membrane Dynamics ◽

Coated Pits ◽

Endocytosis Pathway ◽

Underlying Mechanisms

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 via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and Myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFF). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFF to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFF. By contrast, MYO7B regulates α-Syn PFF entry by maintaining a plasma-membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing ‘scars’ on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is only restricted to α-Syn PFF and other cargos that enter cells via HSPGs. Thus, by identifying new regulatory factors for α-Syn PFF endocytosis, our study defines a mechanistically distinct clathrin-mediated endocytosis pathway that requires additional force generated by MYO7B and actin filaments.SignificanceThe spreading of misfolded protein aggregates such as α-Synuclein preformed fibrils (α-Syn PFF) from cell to cell is a pathologic hallmark associated with the progression of many neurodegenerative diseases, but it is unclear how mammalian cells take up these large protein aggregates to initiate this prion-like protein transmission process. Here we define the mechanism of α-Syn PFF endocytosis using a combination of genetic, biochemical, and live-cell imaging techniques. Our study reveals how α-Syn PFF binds to the cell surface heparan sulfate proteoglycans using two lysine-bearing motifs and then enters cells following a Myosin-7B- and actin-dependent endocytosis mechanism that is specifically tailored for polycation-bearing cargos.

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Efficiency of lamellipodia protrusion is determined by the extent of cytosolic actin assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e16-05-0334 ◽

2017 ◽

Vol 28 (10) ◽

pp. 1311-1325 ◽

Cited By ~ 31

Author(s):

Georgi Dimchev ◽

Anika Steffen ◽

Frieda Kage ◽

Vanessa Dimchev ◽

Julien Pernier ◽

...

Keyword(s):

Cell Migration ◽

Cell Surface ◽

Actin Filaments ◽

Family Members ◽

Cell Communication ◽

Actin Assembly ◽

Actin Monomer ◽

Actin Network ◽

Site Specific ◽

Actin Networks

Cell migration and cell–cell communication involve the protrusion of actin-rich cell surface projections such as lamellipodia and filopodia. Lamellipodia are networks of actin filaments generated and turned over by filament branching through the Arp2/3 complex. Inhibition of branching is commonly agreed to eliminate formation and maintenance of lamellipodial actin networks, but the regulation of nucleation or elongation of Arp2/3-independent filament populations within the network by, for example, formins or Ena/VASP family members and its influence on the effectiveness of protrusion have been unclear. Here we analyzed the effects of a set of distinct formin fragments and VASP on site-specific, lamellipodial versus cytosolic actin assembly and resulting consequences on protrusion. Surprisingly, expression of formin variants but not VASP reduced lamellipodial protrusion in B16-F1 cells, albeit to variable extents. The rates of actin network polymerization followed a similar trend. Unexpectedly, the degree of inhibition of both parameters depended on the extent of cytosolic but not lamellipodial actin assembly. Indeed, excess cytosolic actin assembly prevented actin monomer from rapid translocation to and efficient incorporation into lamellipodia. Thus, as opposed to sole regulation by actin polymerases operating at their tips, the protrusion efficiency of lamellipodia is determined by a finely tuned balance between lamellipodial and cytosolic actin assembly.

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Cell Surface-expressed Phosphatidylserine and Annexin A5 Open a Novel Portal of Cell Entry

Journal of Biological Chemistry ◽

10.1074/jbc.m409009200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52623-52629 ◽

Cited By ~ 103

Author(s):

Heidi Kenis ◽

Hugo van Genderen ◽

Abdel Bennaghmouch ◽

Hilde A. Rinia ◽

Peter Frederik ◽

...

Keyword(s):

Cell Surface ◽

Protein Interactions ◽

Actin Polymerization ◽

Membrane Dynamics ◽

Cell Entry ◽

Protein Network ◽

Annexin A5 ◽

Vesicle Formation ◽

Two Dimensional ◽

Endocytic Vesicle

Expression of phosphatidylserine (PtdSer) at the cell surface is part of the membrane dynamics of apoptosis. Expressed phosphatidylserine functions as an “eat me” flag toward phagocytes. Here, we report that the expressed phosphatidylserine forms part of a hitherto undescribed pinocytic pathway. Annexin A5, a phosphatidylserine-binding protein, binds to and polymerizes through protein-protein interactions on membrane patches expressing phosphatidylserine. The two-dimensional protein network of annexin A5 at the surface prevents apoptotic body formation without interfering with the progression of apoptosis as demonstrated by activation of caspase-3, PtdSer exposure, and DNA fragmentation. The annexin A5 protein network bends the membrane patch nanomechanically into the cell and elicits budding, endocytic vesicle formation, and cytoskeleton-dependent trafficking of the endocytic vesicle. Annexin A1, which binds to PtdSer without forming a two-dimensional protein network, does not induce the formation of endocytic vesicles. This novel pinocytic pathway differs from macropinocytosis, which is preceded by membrane ruffling and actin polymerization. We clearly showed that actin polymerization is not involved in budding and endocytic vesicle formation but is required for intracellular trafficking. The phosphatidylserine-annexin A5-mediated pinocytic pathway is not restricted to cells in apoptosis. We demonstrated that living tumor cells can take up substances through this novel portal of cell entry. This opens new avenues for targeted drug delivery and cell entry.

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What do nanometer molecular trajectories tell us about heterogeneous cell surface domaines?

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140300 ◽

1995 ◽

Vol 53 ◽

pp. 786-787

Author(s):

Watt W. Webb

Keyword(s):

Cell Surface ◽

Lipid Membranes ◽

Surface Proteins ◽

Protein Diffusion ◽

Coated Pits ◽

Cell Surface Proteins ◽

Membrane Heterogeneity ◽

Fluorescence Photobleaching Recovery ◽

Photobleaching Recovery ◽

Plasma Membrane Heterogeneity

Plasma membrane heterogeneity is implicit in the existence of specialized cell surface organelles which are necessary for cellular function; coated pits, post and pre-synaptic terminals, microvillae, caveolae, tight junctions, focal contacts and endothelial polarization are examples. The persistence of these discrete molecular aggregates depends on localized restraint of the constituent molecules within specific domaines in the cell surface by strong intermolecular bonds and/or anchorage to extended cytoskeleton. The observed plasticity of many of organelles and the dynamical modulation of domaines induced by cellular signaling evidence evanescent intermolecular interactions even in conspicuous aggregates. There is also strong evidence that universal restraints on the mobility of cell surface proteins persist virtually everywhere in cell surfaces, not only in the discrete organelles. Diffusion of cell surface proteins is slowed by several orders of magnitude relative to corresponding protein diffusion coefficients in isolated lipid membranes as has been determined by various ensemble average methods of measurement such as fluorescence photobleaching recovery(FPR).

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Molecular cloning of the major cell surface heparan sulfate proteoglycan from rat liver.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)50610-x ◽

1992 ◽

Vol 267 (6) ◽

pp. 3894-3900

Author(s):

A Pierce ◽

M Lyon ◽

I.N. Hampson ◽

G.J. Cowling ◽

J.T. Gallagher

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Molecular Cloning ◽

Rat Liver ◽

Heparan Sulfate Proteoglycan ◽

Sulfate Proteoglycan

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Functional Entry of Baculovirus into Insect and Mammalian Cells Is Dependent on Clathrin-Mediated Endocytosis

Journal of Virology ◽

10.1128/jvi.00880-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8830-8833 ◽

Cited By ~ 106

Author(s):

Gang Long ◽

Xiaoyu Pan ◽

Richard Kormelink ◽

Just M. Vlak

Keyword(s):

Electron Microscopy ◽

Mammalian Cells ◽

Low Ph ◽

Coated Pits ◽

Immuno Electron Microscopy ◽

Endocytosis Pathway ◽

Ph Dependent ◽

Budded Virus

ABSTRACT Entry of the budded virus form of baculoviruses into insect and mammalian cells is generally thought to occur through a low-pH-dependent endocytosis pathway, possibly through clathrin-coated pits. This insight is primarily based on (immuno)electron microscopy studies but requires biochemical support to exclude the use of other pathways. Here, we demonstrate using various inhibitors that functional entry of baculoviruses into insect and mammalian cells is primarily dependent on clathrin-mediated endocytosis. Our results further suggest that caveolae are somehow involved in baculovirus entry in mammalian cells. A caveolar endocytosis inhibitor, genistein, enhances baculovirus transduction in these cells considerably.

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Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

The Journal of Cell Biology ◽

10.1083/jcb.200702151 ◽

2007 ◽

Vol 179 (5) ◽

pp. 1067-1082 ◽

Cited By ~ 54

Author(s):

Valeria R. Caiolfa ◽

Moreno Zamai ◽

Gabriele Malengo ◽

Annapaola Andolfo ◽

Chris D. Madsen ◽

...

Keyword(s):

Cell Membrane ◽

Cell Surface ◽

Plasminogen Activator ◽

Fluorescent Protein ◽

Surface Protein ◽

Basal Membrane ◽

Membrane Dynamics ◽

Live Cells ◽

Cell Surface Protein ◽

Protein Assemblies

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents.

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Transient expression of a cell surface heparan sulfate proteoglycan (syndecan) during limb development

Developmental Biology ◽

10.1016/0012-1606(90)90055-n ◽

1990 ◽

Vol 140 (1) ◽

pp. 83-92 ◽

Cited By ~ 78

Author(s):

Michael Solursh ◽

Rebecca S. Reiter ◽

Karen L. Jensen ◽

Masato Kato ◽

Merton Bernfield

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Transient Expression ◽

Limb Development ◽

Heparan Sulfate Proteoglycan ◽

Sulfate Proteoglycan ◽

A Cell

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Receptor-mediated endocytosis of alpha 2-macroglobulin in cultured fibroblasts.

Journal of Histochemistry & Cytochemistry ◽

10.1177/28.8.6160180 ◽

1980 ◽

Vol 28 (8) ◽

pp. 818-823 ◽

Cited By ~ 22

Author(s):

M C Willingham ◽

F R Maxfield ◽

I Pastan

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Coated Pits ◽

Cultured Fibroblasts ◽

Receptor Mediated Endocytosis ◽

Cytoplasmic Vesicles

Alpha 2-macroglobulin is internalized into cultured fibroblasts by receptor-mediated endocytosis. This ligand binds initially to diffusely distributed receptors on the cell surface which cluster rapidly into bristle-coated pits. Within a few minutes at 37 degrees C, these complexes are internalized into uncoated cytoplasmic vesicles, called receptosomes, which move about in the cell by saltatory motion. These vesicles interact with the Golgi-endoplasmic reticulum-lysosome system in the cell to deliver the ligand to newly formed lysosomes within 30--60 min.

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