Polarised Clathrin-Mediated Endocytosis of EGFR During Chemotactic Invasion

Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin‐mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA‐MB‐231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin‐mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin‐mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin‐mediated endocytosis to directed cell motility.

[1]  D. Horsfall,et al.  The significance of oncogene amplification in primary breast cancer , 1989, International journal of cancer.

[2]  S. Schmid,et al.  Control of EGF Receptor Signaling by Clathrin-Mediated Endocytosis , 1996, Science.

[3]  R. Rubin.  The war on cancer. , 1996, U.S. news & world report.

[4]  S. Schmid,et al.  Dynamin GTPase, a force‐generating molecular switch , 1996, BioEssays : news and reviews in molecular, cellular and developmental biology.

[5]  D. Lauffenburger,et al.  Cell Migration: A Physically Integrated Molecular Process , 1996, Cell.

[6]  M. Sheetz,et al.  Cell migration: regulation of force on extracellular-matrix-integrin complexes. , 1998, Trends in cell biology.

[7]  A. Sorkin,et al.  Endocytosis of Functional Epidermal Growth Factor Receptor-Green Fluorescent Protein Chimera* , 1998, The Journal of Biological Chemistry.

[8]  E. Gold,et al.  Dynamin 2 Is Required for Phagocytosis in Macrophages , 1999, The Journal of experimental medicine.

[9]  P. De Camilli,et al.  A Functional Link between Dynamin and the Actin Cytoskeleton at Podosomes , 2000, The Journal of cell biology.

[10]  J. Hinshaw.  Dynamin and Its Role in Membrane Fission , 2022 .

[11]  M. McNiven,et al.  Regulated Interactions between Dynamin and the Actin-Binding Protein Cortactin Modulate Cell Shape , 2000, The Journal of cell biology.

[12]  J. Segall,et al.  Epidermal growth factor receptor distribution during chemotactic responses. , 2000, Molecular biology of the cell.

[13]  Y. Yarden,et al.  Untangling the ErbB signalling network , 2001, Nature Reviews Molecular Cell Biology.

[14]  K. Beningo,et al.  Nascent Focal Adhesions Are Responsible for the Generation of Strong Propulsive Forces in Migrating Fibroblasts , 2001, The Journal of cell biology.

[15]  A. Sorkin,et al.  Effect of Tyrosine Kinase Inhibitors on Clathrin-coated Pit Recruitment and Internalization of Epidermal Growth Factor Receptor* , 2002, The Journal of Biological Chemistry.

[16]  R. Goldman,et al.  Microfilament‐dependent movement of the β3 integrin subunit within focal contacts of endothelial cells , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[17]  G. Borisy,et al.  Cell Migration: Integrating Signals from Front to Back , 2003, Science.

[18]  J. Harborth,et al.  Effect of Clathrin Heavy Chain- and α-Adaptin-specific Small Inhibitory RNAs on Endocytic Accessory Proteins and Receptor Trafficking in HeLa Cells* , 2003, Journal of Biological Chemistry.

[19]  S. Simon,et al.  Real-time analysis of clathrin-mediated endocytosis during cell migration , 2003, Journal of Cell Science.

[20]  S. Lemeer,et al.  The AP-2 Complex Is Excluded from the Dynamic Population of Plasma Membrane-associated Clathrin* , 2003, Journal of Biological Chemistry.

[21]  S. Simon,et al.  Movement of Plasma‐Membrane‐Associated Clathrin Spots Along the Microtubule Cytoskeleton , 2003, Traffic.

[22]  W. Marshall,et al.  Analysis of Clathrin-mediated Endocytosis of Epidermal Growth Factor Receptor by RNA Interference*[boxs] , 2004, Journal of Biological Chemistry.

[23]  J. Segall,et al.  Chemotaxis of metastatic tumor cells: Clues to mechanisms from the Dictyostelium paradigm , 1992, Cancer and Metastasis Reviews.

[24]  R. Pardi,et al.  Dynamic Partitioning into Lipid Rafts Controls the Endo-Exocytic Cycle of the αL/β2 Integrin, LFA-1, during Leukocyte Chemotaxis , 2005 .

[25]  P. Transidico,et al.  Clathrin-independent endocytosis of ubiquitinated cargos. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[26]  R. Pardi,et al.  Dynamic partitioning into lipid rafts controls the endo-exocytic cycle of the alphaL/beta2 integrin, LFA-1, during leukocyte chemotaxis. , 2005, Molecular biology of the cell.

[27]  M. McNiven,et al.  Epithelial Growth Factor-induced Phosphorylation of Caveolin 1 at Tyrosine 14 Stimulates Caveolae Formation in Epithelial Cells* , 2006, Journal of Biological Chemistry.

[28]  T. Kirchhausen,et al.  Dynasore, a cell-permeable inhibitor of dynamin. , 2006, Developmental cell.

[29]  M. McNiven,et al.  Dynamin as a mover and pincher during cell migration and invasion , 2006, Journal of Cell Science.

[30]  M. Buhusi,et al.  Endocytosis of beta1 integrins is an early event in migration promoted by the cell adhesion molecule L1. , 2005, Experimental cell research.

[31]  M. McNiven,et al.  A novel endocytic mechanism of epidermal growth factor receptor sequestration and internalization. , 2006, Cancer research.

[32]  C. Parent,et al.  Chemotaxis: moving forward and holding on to the past , 2005, Thrombosis and Haemostasis.

[33]  L. E. Johannessen,et al.  Activation of the Epidermal Growth Factor (EGF) Receptor Induces Formation of EGF Receptor- and Grb2-Containing Clathrin-Coated Pits , 2006, Molecular and Cellular Biology.

[34]  Graydon B. Gonsalvez,et al.  Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins , 2007, The Journal of cell biology.

[35]  C. Waterman-Storer,et al.  Caveolin-1 regulates cell polarization and directional migration through Src kinase and Rho GTPases , 2007, The Journal of cell biology.

[36]  K. Kaibuchi,et al.  Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3. , 2007, Developmental cell.

[37]  C. Turner,et al.  Paxillin comes of age , 2008, Journal of Cell Science.

[38]  J. Norman,et al.  Endocytic transport of integrins during cell migration and invasion. , 2008, Trends in cell biology.

[39]  C. Parent,et al.  Eukaryotic chemotaxis at a glance , 2008, Journal of Cell Science.

[40]  J. Vilardaga,et al.  Cell Migration Is Regulated by Platelet-Derived Growth Factor Receptor Endocytosis , 2009, Molecular and Cellular Biology.

[41]  H. McMahon,et al.  Mechanisms of endocytosis. , 2009, Annual review of biochemistry.

[42]  C. Heisenberg,et al.  Trafficking and Cell Migration , 2009, Traffic.

[43]  J. Kunz,et al.  Focal adhesion disassembly requires clathrin‐dependent endocytosis of integrins , 2009, FEBS letters.

[44]  S. Simon,et al.  Endocytic trafficking of activated EGFR is AP-2 dependent and occurs through preformed clathrin spots , 2009, Journal of Cell Science.

[45]  G. Gundersen,et al.  Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells , 2009, The Journal of cell biology.

[46]  J. Norman,et al.  Integrins: masters and slaves of endocytic transport , 2009, Nature Reviews Molecular Cell Biology.

[47]  M. Gonzalez-Gaitan,et al.  Spatial restriction of receptor tyrosine kinase activity through a polarized endocytic cycle controls border cell migration , 2010, Proceedings of the National Academy of Sciences.

[48]  J. Rappoport,et al.  An agarose spot assay for chemotactic invasion. , 2010, BioTechniques.

[49]  F. Sotgia,et al.  CAV1 inhibits metastatic potential in melanomas through suppression of the integrin/Src/FAK signaling pathway. , 2010, Cancer research.

[50]  S. Simon,et al.  Imaging with total internal reflection fluorescence microscopy for the cell biologist , 2010, Journal of Cell Science.

[51]  J. Rappoport,et al.  Moving forward: polarised trafficking in cell migration. , 2010, Trends in cell biology.

[52]  James S. Duncan,et al.  3-D Reconstruction of Microtubules From Multi-Angle Total Internal Reflection Fluorescence Microscopy Using Bayesian Framework , 2011, IEEE Transactions on Image Processing.

[53]  P. Mancini,et al.  Polarized Endocytosis of the Keratinocyte Growth Factor Receptor in Migrating Cells: Role of Src-Signaling and Cortactin , 2011, PloS one.

[54]  V. Hsu,et al.  Integrins traffic rapidly via circular dorsal ruffles and macropinocytosis during stimulated cell migration , 2011, The Journal of cell biology.

[55]  N. Carragher,et al.  Spatial regulation of RhoA activity during pancreatic cancer cell invasion driven by mutant p53. , 2011, Cancer research.

[56]  Suyong Choi,et al.  Phosphoinositide signaling regulates the exocyst complex and polarized integrin trafficking in directionally migrating cells. , 2012, Developmental cell.

[57]  S. Shao,et al.  Caveolin-1 knockdown is associated with the metastasis and proliferation of human lung cancer cell line NCI-H460. , 2012, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[58]  Natalie S. Poulter,et al.  Clathrin‐mediated endocytosis regulates occludin, and not focal adhesion, distribution during epithelial wound healing , 2012, Biology of the cell.

[59]  J. Norman,et al.  Rab25 and CLIC3 Collaborate to Promote Integrin Recycling from Late Endosomes/Lysosomes and Drive Cancer Progression , 2012, Developmental cell.

[60]  M. McNiven,et al.  Increased Expression of the Large GTPase Dynamin 2 Potentiates Metastatic Migration and Invasion of Pancreatic Ductal Carcinoma , 2011, Oncogene.

[61]  T. Mak,et al.  The interaction between caveolin-1 and Rho-GTPases promotes metastasis by controlling the expression of alpha5-integrin and the activation of Src, Ras and Erk , 2011, Oncogene.

[62]  H. Urra,et al.  Caveolin-1-Enhanced Motility and Focal Adhesion Turnover Require Tyrosine-14 but Not Accumulation to the Rear in Metastatic Cancer Cells , 2012, PloS one.

[63]  K. Kaibuchi,et al.  Spatial regulation of VEGF receptor endocytosis in angiogenesis , 2013, Nature Cell Biology.

[64]  Y. Kalaidzidis,et al.  Dynamin Inhibitors Impair Endocytosis and Mitogenic Signaling of PDGF , 2013, Traffic.

[65]  J. Heath,et al.  Regulation of fibroblast growth factor receptor signalling and trafficking by Src and Eps8 , 2013, Journal of Cell Science.