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

2004 ◽  
Vol 279 (50) ◽  
pp. 52623-52629 ◽  
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.

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 A Dynamic Actin Cytoskeleton Functions at Multiple Stages of Clathrin-mediated Endocytosis

2005 ◽  
Vol 16 (2) ◽  
pp. 964-975 ◽  
Author(s):  
Defne Yarar ◽  
Clare M. Waterman-Storer ◽  
Sandra L. Schmid
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Actin Polymerization ◽  
Cell Surface Receptor ◽  
Actin Dynamics ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Actin Assembly ◽  
Electron Microscopic ◽  
Surface Receptor

Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-coated structure (CCS) dynamics. Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and internalization. In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface. Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells.

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

2007 ◽  
Vol 179 (5) ◽  
pp. 1067-1082 ◽  
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.

Retargeting of the mitochondrial protein p32/gC1Qr to a cytoplasmic compartment and the cell surface

2001 ◽  
Vol 114 (11) ◽  
pp. 2115-2123
Author(s):  
Hans C. van Leeuwen ◽  
Peter O’Hare
Keyword(s):  
Cell Surface ◽  
Protein Interactions ◽  
Mitochondrial Protein ◽  
Physiological Role ◽  
Surface Expression ◽  
Transport Processes ◽  
Cell Surface Expression ◽  
Bacterial Proteins ◽  
Er Chaperone ◽  
Cellular Compartments

p32/gC1qR is a small acidic protein that has been reported to have a broad range of distinct functions and to associate with a wide array of cellular, viral and bacterial proteins. It has been found in each of the main cellular compartments including mitochondria, nucleus and cytoplasm and is also thought to be located at the plasma membrane and secreted into the extracellular matrix. The true physiological role(s) of p32 remains controversial because it has been difficult to reconcile all of the findings on protein interactions and the seemingly disparate observations on compartmentalisation. However, it has been proposed that p32 is somehow involved in transport processes connecting diverse cellular compartments and the cell surface. Here we show that native p32 appears to be localised mainly in the mitochondria and is not detectable on the cell surface. However, addition of a short tag to the N-terminus of p32 appears to block its mitochondrial targeting, resulting in redirection into a cytoplasmic vesicular pattern, overlapping with the endoplasmic reticulum. The redirection of p32 results in an alteration in and co-localisation with ER markers including calreticulin, a lumenal ER chaperone. Furthermore, we show both by immunofluorescence and cross-linking studies that this also results in cell-surface expression of p32. These results indicate that, at least under certain circumstances, p32 can be retargeted and may help to provide an explanation for the diverse observations on its localization.

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