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

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.

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

2020 ◽  
Vol 117 (20) ◽  
pp. 10865-10875 ◽  
Author(s):  
Qi Zhang ◽  
Yue Xu ◽  
Juhyung Lee ◽  
Michal Jarnik ◽  
Xufeng Wu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Actin Filaments ◽  
Membrane Dynamics ◽  
Cell Entry ◽  
Sulfate Proteoglycan ◽  
Actin Network ◽  
Coated Pits ◽  
Endocytosis Pathway ◽  
Underlying Mechanisms ◽  
Pathological Event

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.

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

2006 ◽  
Vol 80 (17) ◽  
pp. 8830-8833 ◽  
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.

scholarly journals Interaction of Chlamydia trachomatis with Mammalian Cells Is Independent of Host Cell Surface Heparan Sulfate Glycosaminoglycans

2006 ◽  
Vol 74 (3) ◽  
pp. 1795-1799 ◽  
Author(s):  
Richard S. Stephens ◽  
Jesse M. Poteralski ◽  
Lynn Olinger
Keyword(s):  
Chlamydia Trachomatis ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Host Cell ◽  
Cell Line ◽  
Mammalian Cells ◽  
Chlamydial Infection ◽  
Functional Role ◽  
A Cell ◽  
Host Cell Surface

ABSTRACT The hypothesis that host cell surface heparan sulfate is required to promote chlamydial infection was tested using a cell line (CHO-18.4) containing a single retroviral insertion and the concomitant loss of heparan sulfate biosynthesis. Tests of chlamydial infectivity of heparan sulfate-deficient CHO-18.4 cells and parental cells, CHO-22, demonstrated that both were equally sensitive to infection by Chlamydia trachomatis serovars L2 and D. These data do not support the hypothesis and demonstrate that host cell surface heparan sulfate does not serve an essential functional role in chlamydial infectivity.

scholarly journals Neuropilin-1 and heparan sulfate proteoglycans cooperate in cellular uptake of nanoparticles functionalized by cationic cell-penetrating peptides

2015 ◽  
Vol 1 (10) ◽  
pp. e1500821 ◽  
Author(s):  
Hong-Bo Pang ◽  
Gary B. Braun ◽  
Erkki Ruoslahti
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Vascular Permeability ◽  
Mammalian Cells ◽  
Cell Penetrating Peptides ◽  
Proteolytic Processing ◽  
Cell Surface Receptor ◽  
Neuropilin 1 ◽  
Cell Penetrating ◽  
Surface Receptor

Cell-penetrating peptides (CPPs) have been widely used to deliver nanomaterials and other types of macromolecules into mammalian cells for therapeutic and diagnostic use. Cationic CPPs that bind to heparan sulfate (HS) proteoglycans on the cell surface induce potent endocytosis; however, the role of other surface receptors in this process is unclear. We describe the convergence of an HS-dependent pathway with the C-end rule (CendR) mechanism that enables peptide ligation with neuropilin-1 (NRP1), a cell surface receptor known to be involved in angiogenesis and vascular permeability. NRP1 binds peptides carrying a positive residue at the carboxyl terminus, a feature that is compatible with cationic CPPs, either intact or after proteolytic processing. We used CPP and CendR peptides, as well as HS- and NRP1-binding motifs from semaphorins, to explore the commonalities and differences of the HS and NRP1 pathways. We show that the CendR-NRP1 interaction determines the ability of CPPs to induce vascular permeability. We also show at the ultrastructural level, using a novel cell entry synchronization method, that both the HS and NRP1 pathways can initiate a macropinocytosis-like process and visualize these CPP-cargo complexes going through various endosomal compartments. Our results provide new insights into how CPPs exploit multiple surface receptor pathways for intracellular delivery.

scholarly journals Extracellular vesicle formation mediated by local phosphatidylserine exposure promotes efficient cell extrusion

2021 ◽  
Author(s):  
Akihito Kira ◽  
Machiko Murata ◽  
Keisuke Saito ◽  
Ichiko Tatsutomi ◽  
Izumi Hattori ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Extracellular Vesicle ◽  
Membrane Dynamics ◽  
Vesicle Formation ◽  
Cell Competition ◽  
Tissue Integrity ◽  
Underlying Mechanisms ◽  
A Site ◽  
Multicellular Organisms ◽  
Cell Extrusion

Abstract Numerous unwanted cells are removed from epithelial and endothelial tissues—in which cells are tightly connected to one another—without disturbing tissue integrity and homeostasis. Cell extrusion is a unique mode of cell removal from tissues, and it plays an important role in regulating cell numbers and the eliminating unwanted cells, such as apoptotic cells, cancer cells, and cells with a lower fitness in cell competition . During this process, cells delaminate from the cell layer, to which they initially used to adhere, through communication with the neighboring cells. Defects in cell extrusion are believed to associate with inflammation and cancer in epithelium as well as blood vessel dysfunction. However, the correlation between them has not yet been evaluated owing to a lack of knowledge of the underlying mechanisms. In particular, the process whereby the cell exit from the tissue remains to be elucidated. Here, we report a novel and conserved execution mechanism of cell extrusion—common to mammalian cells and Drosophila epithelia—i.e., spatiotemporally regulated extracellular vesicle formation in extruding cells at a site opposite to the direction of extrusion. Particularly, we found that a lipid-scramblase‒mediated local exposure of phosphatidylserine is responsible for extracellular vesicle formation and is crucial for the execution of cell extrusion, and inhibition of this process disrupted prompt cell delamination as well as tissue homeostasis. Furthermore, we revealed the mechanism underlying vesicle formation. Importantly, our results reveal that membrane dynamics is the driving force by a “rocket launch”-like mechanism behind the extrusion of cells from tissues, a fundamental cell behavior in multicellular organisms that is also observed in other contexts including cancer cell invasion and neural cell differentiation. Our understanding of this new mechanism of cell extrusion enables us to examine the relationship between cell extrusion abnormalities and the onset of various diseases.

Coated pits with pinocytosis in Tetrahymena

1983 ◽  
Vol 63 (1) ◽  
pp. 209-222 ◽  
Author(s):  
J.R. Nilsson ◽  
B. van Deurs
Keyword(s):  
Cell Surface ◽  
Growth Temperature ◽  
Mammalian Cells ◽  
Inorganic Salt ◽  
Normal Growth ◽  
Cationized Ferritin ◽  
Salt Medium ◽  
Coated Pits ◽  
Food Vacuoles ◽  
Section Analysis

The possible role in pinocytosis of coated pits at the parasomal sacs of Tetrahymena has been studied using cationized ferritin (CF; pI = 8.5) as a marker of membrane and content. It is shown that CF binds evenly to the surface, including the coated pits, of Tetrahymena in an inorganic salt medium (to avoid formation of food vacuoles) at the normal growth temperature. Moreover, CF is internalized by coated vesicles (shown to be truly free by thin serial-section analysis) and delivered initially (1-5 min of incubation) to cisterna near the cell surface. Later (5-10 min) CF occurs also in autophagic vacuoles, formed as a result of starvation, and eventually (15-90 min) it is present in preformed (old) food vacuoles. These observations indicate that the coated pits at the parasomal sacs of Tetrahymena function in adsorptive pinocytosis in much the same manner as coated pits at the surface of mammalian cells.

scholarly journals Autographa californica Multicapsid Nucleopolyhedrovirus Efficiently Infects Sf9 Cells and Transduces Mammalian Cells via Direct Fusion with the Plasma Membrane at Low pH

2010 ◽  
Vol 84 (10) ◽  
pp. 5351-5359 ◽  
Author(s):  
Sicong Dong ◽  
Manli Wang ◽  
Zhijuan Qiu ◽  
Fei Deng ◽  
Just M. Vlak ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Heterologous Gene Expression ◽  
Specific Inhibitor ◽  
Low Ph ◽  
Autographa Californica ◽  
Transduction Efficiency ◽  
Sf9 Cells ◽  
Endocytosis Pathway ◽  
Direct Fusion

ABSTRACT The budded virus (BV) of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue the infectivity of AcMNPV in the presence of endocytosis pathway inhibitors. A colocalization assay of the major capsid protein VP39 with the early endosome marker EEA1 showed that at low pH, AcMNPV entered Sf9 cells via an endosome-independent pathway. Using a fluorescent probe (R18), we showed that at low pH, the viral nucleocapsid entered Sf9 cells via direct fusion at the cell surface. By using the myosin-specific inhibitor 2,3-butanedione monoxime (BDM) and the microtubule inhibitor nocodazole, the low pH-triggered direct fusion was demonstrated to be dependent on myosin-like proteins and independent of microtubules. The reverse transcription-PCR of the IE1 gene as a marker for viral entry showed that the kinetics of AcMNPV in cells triggered by low pH was similar to that of the normal entry via endocytosis. The low pH-mediated infection assay and VP39 and EEA1 colocalization assay also demonstrated that AcMNPV could efficiently transduce mammalian cells via direct membrane fusion at the cell surface. More importantly, we found that a low-pH trigger could significantly improve the transduction efficiency of AcMNPV in mammalian cells, leading to the potential application of this method when using baculovirus as a vector for heterologous gene expression and for gene therapy.

scholarly journals Ligand binding to heparan sulfate proteoglycans induces their aggregation and distribution along actin cytoskeleton.

1996 ◽  
Vol 7 (11) ◽  
pp. 1771-1788 ◽  
Author(s):  
R G Martinho ◽  
S Castel ◽  
J Ureña ◽  
M Fernández-Borja ◽  
R Makiya ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Cell Surface ◽  
Actin Cytoskeleton ◽  
Heparan Sulfate ◽  
Lipoprotein Lipase ◽  
Actin Filaments ◽  
Fibroblast Cell ◽  
Heparan Sulfate Proteoglycans ◽  
Heparin Binding ◽  
Triton X

Cell surface heparan sulfate proteoglycans (HSPGs) participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study demonstrates that soluble heparin-binding proteins or cross-linking antibodies induce the aggregation of cell surface HSPGs and their distribution along underlying actin filaments. Immunofluorescence and confocal microscopy and immunogold and electron microscopy indicate that, in the absence of ligands, HSPGs are irregularly distributed on the fibroblast cell surface, without any apparent codistribution with the actin cytoskeleton. In the presence of ligand (lipoprotein lipase) or antibodies against heparan sulfate, HSPGs aggregate and colocalize with the actin cytoskeleton. Triton X-100 extraction and immunoelectron microscopy have demonstrated that in this condition HSPGs were clustered and associated with the actin filaments. Crosslinking experiments that use biotinylated lipoprotein lipase have revealed three major proteoglycans as binding sites at the fibroblast cell surface. These cross-linked proteoglycans appeared in the Triton X-100 insoluble fraction. Platinum/carbon replicas of the fibroblast surface incubated either with lipoprotein lipase or antiheparan sulfate showed large aggregates of HSPGs regularly distributed along cytoplasmic fibers. Quantification of the spacing between HSPGs by confocal microscopy confirmed that the nonrandom distribution of HSPG aggregates along the actin cytoskeleton was induced by ligand binding. When cells were incubated either with lipoprotein lipase or antibodies against heparan sulfate, the distance between immunofluorescence spots was uniform. In contrast, the spacing between HSPGs on fixed cells not incubated with ligand was more variable. This highly organized spatial relationship between actin and proteoglycans suggests that cortical actin filaments could organize the molecular machinery involved in signal transduction and molecular movements on the cell surface that are triggered by heparin-binding proteins.

scholarly journals A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

1998 ◽  
Vol 72 (2) ◽  
pp. 1577-1585 ◽  
Author(s):  
Che-Sheng Chung ◽  
Jye-Chian Hsiao ◽  
Yuan-Shau Chang ◽  
Wen Chang
Keyword(s):  
Virus Infection ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Mammalian Cells ◽  
Myxoma Virus ◽  
Cowpox Virus ◽  
Virus Binding ◽  
Vaccinia Virus Infection

ABSTRACT Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.

Sign in / Sign up

Export Citation Format

Share Document