Endocytosis and the Src family of non-receptor tyrosine kinases

AbstractThe regulated intracellular transport of nutrient, adhesion, and growth factor receptors is crucial for maintaining cell and tissue homeostasis. Endocytosis, or endocytic membrane trafficking, involves the steps of intracellular transport that include, but are not limited to, internalization from the plasma membrane, sorting in early endosomes, transport to late endosomes/lysosomes followed by degradation, and/or recycling back to the plasma membrane through tubular recycling endosomes. In addition to regulating the localization of transmembrane receptor proteins, the endocytic pathway also controls the localization of non-receptor molecules. The non-receptor tyrosine kinase c-Src (Src) and its closely related family members Yes and Fyn represent three proteins whose localization and signaling activities are tightly regulated by endocytic trafficking. Here, we provide a brief overview of endocytosis, Src function and its biochemical regulation. We will then concentrate on recent advances in understanding how Src intracellular localization is regulated and how its subcellular localization ultimately dictates downstream functioning. As Src kinases are hyperactive in many cancers, it is essential to decipher the spatiotemporal regulation of this important family of tyrosine kinases.

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Clathrin Hub Expression Affects Early Endosome Distribution with Minimal Impact on Receptor Sorting and Recycling

Molecular Biology of the Cell ◽

10.1091/mbc.12.9.2790 ◽

2001 ◽

Vol 12 (9) ◽

pp. 2790-2799 ◽

Cited By ~ 27

Author(s):

Elizabeth M. Bennett ◽

Sharron X. Lin ◽

Mhairi C. Towler ◽

Frederick R. Maxfield ◽

Frances M. Brodsky

Keyword(s):

Plasma Membrane ◽

Early Endosome ◽

Dominant Negative ◽

Endocytic Pathway ◽

Endocytic Trafficking ◽

Minimal Impact ◽

Recycling Endosomes ◽

Receptor Mediated Endocytosis ◽

Early Endosomes ◽

Endosomal Membranes

Clathrin-coated vesicles execute receptor-mediated endocytosis at the plasma membrane. However, a role for clathrin in later endocytic trafficking processes, such as receptor sorting and recycling or maintaining the organization of the endocytic pathway, has not been thoroughly characterized. The existence of clathrin-coated buds on endosomes suggests that clathrin might mediate later endocytic trafficking events. To investigate the function of clathrin-coated buds on endosomal membranes, endosome function and distribution were analyzed in a HeLa cell line that expresses the dominant-negative clathrin inhibitor Hub in an inducible manner. As expected, Hub expression reduced receptor-mediated endocytosis at the plasma membrane. Hub expression also induced a perinuclear aggregation of early endosome antigen 1-positive early endosomes, such that sorting and recycling endosomes were found tightly concentrated in the perinuclear region. Despite the dramatic redistribution of endosomes, Hub expression did not affect the overall kinetics of receptor sorting or recycling. These data show that clathrin function is necessary to maintain proper cellular distribution of early endosomes but does not play a prominent role in sorting and recycling events. Thus, clathrin's role on endosomal membranes is to influence organelle localization and is distinct from its role in trafficking pathways at the plasma membrane and trans-Golgi network.

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Glycosylation Modulates Plasma Membrane Trafficking of CD24 in Breast Cancer Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22158165 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8165

Author(s):

Amanda Chantziou ◽

Kostas Theodorakis ◽

Hara Polioudaki ◽

Eelco de Bree ◽

Marilena Kampa ◽

...

Keyword(s):

Breast Cancer ◽

Plasma Membrane ◽

Subcellular Localization ◽

Cancer Cells ◽

Membrane Trafficking ◽

Breast Cancer Cells ◽

Endocytic Pathway ◽

Cell Periphery ◽

Wild Type ◽

Mcf 7

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

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Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

Journal of Cell Science ◽

10.1242/jcs.103.4.1139 ◽

1992 ◽

Vol 103 (4) ◽

pp. 1139-1152

Author(s):

J.W. Kok ◽

K. Hoekstra ◽

S. Eskelinen ◽

D. Hoekstra

Keyword(s):

Plasma Membrane ◽

Intracellular Ph ◽

Brefeldin A ◽

Fluid Phase ◽

Endocytic Pathway ◽

Late Endosomes ◽

Early Endosomes ◽

Recycling Pathway ◽

Golgi Network ◽

Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

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Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane

Journal of Cell Science ◽

10.1242/jcs.114.10.1893 ◽

2001 ◽

Vol 114 (10) ◽

pp. 1893-1900 ◽

Cited By ~ 3

Author(s):

S. Lusa ◽

T.S. Blom ◽

E.L. Eskelinen ◽

E. Kuismanen ◽

J.E. Mansson ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Interactions ◽

Mammalian Cells ◽

Intracellular Transport ◽

Ldl Cholesterol ◽

Normal Cells ◽

Recycling Endosomes ◽

Regulate Protein ◽

Niemann Pick ◽

Accumulation Characteristic

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

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Rab7 involves Vps35 to mediate AQP2 sorting and apical trafficking in collecting duct cells

AJP Renal Physiology ◽

10.1152/ajprenal.00297.2019 ◽

2020 ◽

Vol 318 (4) ◽

pp. F956-F970 ◽

Cited By ~ 4

Author(s):

Wei-Ling Wang ◽

Shih-Han Su ◽

Kit Yee Wong ◽

Chan-Wei Yang ◽

Chin-Fu Liu ◽

...

Keyword(s):

Plasma Membrane ◽

Apical Membrane ◽

Collecting Duct ◽

Water Channel ◽

Small Gtpase ◽

Apical Plasma Membrane ◽

Water Reabsorption ◽

Recycling Endosomes ◽

Early Endosomes ◽

Vacuolar Protein

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for osmotic water reabsorption by kidney collecting ducts. In response to vasopressin, AQP2 traffics from intracellular vesicles to the apical plasma membrane of collecting duct principal cells, where it increases water permeability and, hence, water reabsorption. Despite continuing efforts, gaps remain in our knowledge of vasopressin-regulated AQP2 trafficking. Here, we studied the functions of two retromer complex proteins, small GTPase Rab7 and vacuolar protein sorting 35 (Vps35), in vasopressin-induced AQP2 trafficking in a collecting duct cell model (mpkCCD cells). We showed that upon vasopressin removal, apical AQP2 returned to Rab5-positive early endosomes before joining Rab11-positive recycling endosomes. In response to vasopressin, Rab11-associated AQP2 trafficked to the apical plasma membrane before Rab5-associated AQP2 did so. Rab7 knockdown resulted in AQP2 accumulation in early endosomes and impaired vasopressin-induced apical AQP2 trafficking. In response to vasopressin, Rab7 transiently colocalized with Rab5, indicative of a role of Rab7 in AQP2 sorting in early endosomes before trafficking to the apical membrane. Rab7-mediated apical AQP2 trafficking in response to vasopressin required GTPase activity. When Vps35 was knocked down, AQP2 accumulated in recycling endosomes under vehicle conditions and did not traffic to the apical plasma membrane in response to vasopressin. We conclude that Rab7 and Vps35 participate in AQP2 sorting in early endosomes under vehicle conditions and apical membrane trafficking in response to vasopressin.

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CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, β1 integrin recycling, and cell migration

Science Signaling ◽

10.1126/scisignal.aav5938 ◽

2019 ◽

Vol 12 (579) ◽

pp. eaav5938 ◽

Cited By ~ 6

Author(s):

Mallika Ghosh ◽

Robin Lo ◽

Ivan Ivic ◽

Brian Aguilera ◽

Veneta Qendro ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Migration ◽

Cell Adhesion ◽

Cell Attachment ◽

Leading Edge ◽

Small Gtpase ◽

Β1 Integrin ◽

Recycling Endosomes ◽

Cellular Processes ◽

Early Endosomes

Cell attachment to the extracellular matrix (ECM) requires a balance between integrin internalization and recycling to the surface that is mediated by numerous proteins, emphasizing the complexity of these processes. Upon ligand binding in various cells, the β1 integrin is internalized, traffics to early endosomes, and is returned to the plasma membrane through recycling endosomes. This trafficking process depends on the cyclical activation and inactivation of small guanosine triphosphatases (GTPases) by their specific guanine exchange factors (GEFs) and their GTPase-activating proteins (GAPs). In this study, we found that the cell surface antigen CD13, a multifunctional transmembrane molecule that regulates cell-cell adhesion and receptor-mediated endocytosis, also promoted cell migration and colocalized with β1 integrin at sites of cell adhesion and at the leading edge. A lack of CD13 resulted in aberrant trafficking of internalized β1 integrin to late endosomes and its ultimate degradation. Our data indicate that CD13 promoted ARF6 GTPase activity by positioning the ARF6-GEF EFA6 at the cell membrane. In migrating cells, a complex containing phosphorylated CD13, IQGAP1, GTP-bound (active) ARF6, and EFA6 at the leading edge promoted the ARF6 GTPase cycling and cell migration. Together, our findings uncover a role for CD13 in the fundamental cellular processes of receptor recycling, regulation of small GTPase activities, cell-ECM interactions, and cell migration.

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TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling

Cellular and Molecular Life Sciences ◽

10.1007/s00018-020-03656-y ◽

2020 ◽

Author(s):

Anna Ciesielska ◽

Marta Matyjek ◽

Katarzyna Kwiatkowska

Keyword(s):

Plasma Membrane ◽

Inflammatory Responses ◽

Adaptor Proteins ◽

Human Diseases ◽

Recycling Endosomes ◽

Lysosomal Compartment ◽

E Coli ◽

Early Endosomes ◽

Endosome Maturation

Abstract Toll-like receptor (TLR) 4 belongs to the TLR family of receptors inducing pro-inflammatory responses to invading pathogens. TLR4 is activated by lipopolysaccharide (LPS, endotoxin) of Gram-negative bacteria and sequentially triggers two signaling cascades: the first one involving TIRAP and MyD88 adaptor proteins is induced in the plasma membrane, whereas the second engaging adaptor proteins TRAM and TRIF begins in early endosomes after endocytosis of the receptor. The LPS-induced internalization of TLR4 and hence also the activation of the TRIF-dependent pathway is governed by a GPI-anchored protein, CD14. The endocytosis of TLR4 terminates the MyD88-dependent signaling, while the following endosome maturation and lysosomal degradation of TLR4 determine the duration and magnitude of the TRIF-dependent one. Alternatively, TLR4 may return to the plasma membrane, which process is still poorly understood. Therefore, the course of the LPS-induced pro-inflammatory responses depends strictly on the rates of TLR4 endocytosis and trafficking through the endo-lysosomal compartment. Notably, prolonged activation of TLR4 is linked with several hereditary human diseases, neurodegeneration and also with autoimmune diseases and cancer. Recent studies have provided ample data on the role of diverse proteins regulating the functions of early, late, and recycling endosomes in the TLR4-induced inflammation caused by LPS or phagocytosis of E. coli. In this review, we focus on the mechanisms of the internalization and intracellular trafficking of TLR4 and CD14, and also of LPS, in immune cells and discuss how dysregulation of the endo-lysosomal compartment contributes to the development of diverse human diseases.

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A presynaptic endosomal trafficking pathway controls synaptic growth signaling

The Journal of Cell Biology ◽

10.1083/jcb.201009052 ◽

2011 ◽

Vol 193 (1) ◽

pp. 201-217 ◽

Cited By ~ 57

Author(s):

Avital A. Rodal ◽

Aline D. Blunk ◽

Yulia Akbergenova ◽

Ramon A. Jorquera ◽

Lauren K. Buhl ◽

...

Keyword(s):

Endocytic Pathway ◽

Physical Interaction ◽

Endosomal Trafficking ◽

Synaptic Growth ◽

Recycling Endosomes ◽

Sorting Nexin ◽

Trafficking Pathway ◽

Early Endosomes ◽

Protein Receptors ◽

Escrt Complex

Structural remodeling of synapses in response to growth signals leads to long-lasting alterations in neuronal function in many systems. Synaptic growth factor receptors alter their signaling properties during transit through the endocytic pathway, but the mechanisms controlling cargo traffic between endocytic compartments remain unclear. Nwk (Nervous Wreck) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila melanogaster. In this paper, we show that Nwk acts through a physical interaction with sorting nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated bone morphogenic protein receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwk-containing recycling endosomes. We identify an alternative signal termination pathway in the absence of Snx16 that is controlled by endosomal sorting complex required for transport (ESCRT)–mediated internalization of receptors into the endosomal lumen. Our results define a presynaptic trafficking pathway mediated by SNX16, NWK, and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments.

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New Perspectives on SNARE Function in the Yeast Minimal Endomembrane System

Genes ◽

10.3390/genes11080899 ◽

2020 ◽

Vol 11 (8) ◽

pp. 899 ◽

Cited By ~ 2

Author(s):

James H. Grissom ◽

Verónica A. Segarra ◽

Richard J. Chi

Keyword(s):

Plasma Membrane ◽

Membrane Trafficking ◽

Mammalian Cells ◽

Budding Yeast ◽

Endocytic Pathway ◽

Model Organisms ◽

Endosomal Trafficking ◽

Endomembrane System ◽

Recycling Endosome ◽

New Findings

Saccharomyces cerevisiae is one of the best model organisms for the study of endocytic membrane trafficking. While studies in mammalian cells have characterized the temporal and morphological features of the endocytic pathway, studies in budding yeast have led the way in the analysis of the endosomal trafficking machinery components and their functions. Eukaryotic endomembrane systems were thought to be highly conserved from yeast to mammals, with the fusion of plasma membrane-derived vesicles to the early or recycling endosome being a common feature. Upon endosome maturation, cargos are then sorted for reuse or degraded via the endo-lysosomal (endo-vacuolar in yeast) pathway. However, recent studies have shown that budding yeast has a minimal endomembrane system that is fundamentally different from that of mammalian cells, with plasma membrane-derived vesicles fusing directly to a trans-Golgi compartment which acts as an early endosome. Thus, the Golgi, rather than the endosome, acts as the primary acceptor of endocytic vesicles, sorting cargo to pre-vacuolar endosomes for degradation. The field must now integrate these new findings into a broader understanding of the endomembrane system across eukaryotes. This article synthesizes what we know about the machinery mediating endocytic membrane fusion with this new model for yeast endomembrane function.

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Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells

The Journal of Cell Biology ◽

10.1083/jcb.122.4.789 ◽

1993 ◽

Vol 122 (4) ◽

pp. 789-807 ◽

Cited By ~ 657

Author(s):

M Sargiacomo ◽

M Sudol ◽

Z Tang ◽

MP Lisanti

Keyword(s):

Plasma Membrane ◽

Membrane Trafficking ◽

Tyrosine Kinases ◽

Mdck Cells ◽

Apical Cell ◽

Signaling Molecules ◽

Specific Marker ◽

Serine Kinase ◽

Cellular Origin ◽

Protein Components

GPI-linked protein molecules become Triton-insoluble during polarized sorting to the apical cell surface of epithelial cells. These insoluble complexes, enriched in cholesterol, glycolipids, and GPI-linked proteins, have been isolated by flotation on sucrose density gradients and are thought to contain the putative GPI-sorting machinery. As the cellular origin and molecular protein components of this complex remain unknown, we have begun to characterize these low-density insoluble complexes isolated from MDCK cells. We find that these complexes, which represent 0.4-0.8% of the plasma membrane, ultrastructurally resemble caveolae and are over 150-fold enriched in a model GPI-anchored protein and caveolin, a caveolar marker protein. However, they exclude many other plasma membrane associated molecules and organelle-specific marker enzymes, suggesting that they represent microdomains of the plasma membrane. In addition to caveolin, these insoluble complexes contain a subset of hydrophobic plasma membrane proteins and cytoplasmically-oriented signaling molecules, including: (a) GTP-binding proteins--both small and heterotrimeric; (b) annex II--an apical calcium-regulated phospholipid binding protein with a demonstrated role in exocytic fusion events; (c) c-Yes--an apically localized member of the Src family of non-receptor type protein-tyrosine kinases; and (d) an unidentified serine-kinase activity. As we demonstrate that caveolin is both a transmembrane molecule and a major phospho-acceptor component of these complexes, we propose that caveolin could function as a transmembrane adaptor molecule that couples luminal GPI-linked proteins with cytoplasmically oriented signaling molecules during GPI-membrane trafficking or GPI-mediated signal transduction events. In addition, our results have implications for understanding v-Src transformation and the actions of cholera and pertussis toxins on hetero-trimeric G proteins.

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