Cell Signaling and Migration Erich Lidstone April 29, 2009

Cell Signaling and Migration Importance of cell migration Major steps of migration Signaling pathways Lipid rafts Adhesion-dependent trafficking

Importance of Cell Migration Embryonic development Chemotaxis Tissue turnover Wound healing Immunity

Cell Migration and the Immune Response _S-w _S-w

Importance of Cell Migration Pathological processes – Vascular disease – Osteoporosis Chronic inflammatory diseases – Rheumatoid arthritis – Multiple sclerosis – Cancer – Mental retardation

Cell Migration Cycle Polarize, extend protrusion Migration-promoting event Adhere to ECM or TM receptor proteins Move forward over proteins Disassemble adhesions Transport necessary proteins to movement front Microtubule dynamics promote efficient exploration of the peripheral cytoplasmic domain of a neuronal growth cone. An example of a single dynamically unstable microtubule undergoing assembly followed by disassembly (bottom). Microtubule behavior integrated over 10 minutes in the peripheral growth cone lamellipodium (top). Forscher Lab, Yale, Department of Molecular, Cellular, and Developmental Biology

Actin Polymerization FAST-growing “barbed” end SLOW-growing “pointed” end Lamellipodia – branching dendritic network configuration Filipodia – long parallel bundles of filaments Mediated by Arp2/3 complex Causes branching of existing filaments

Movement Leading edge moves by “Brownian ratchet” Uses thermal energy built up by deforming existing filaments Does not derive energy directly from actin polymerization

Cell migration detection Akt/PKB-GFP fusion  4-integrin staining PTEN-GFP fusion FRET of GFP-V12Rac with an effector molecule

Cell Migration Effectors Profilin Thymosin Cofilin Capping proteins Cortactin stabilizes branches Cross-linkers filamin A  -actinin

Cell Polarization Polarity intrinsic to migrating cell Vesicle trafficking toward the leading edge Localization of the MTOC and golgi apparatus PTEN resides at cell margins PI3K resides at the leading edge PIP3 production at the leading edge

Protrusion and Adhesion Formation

Integrins and Adhesion in Migration Integrins support adhesion to ECM or neighboring cells Activate migration-related signaling molecules  - and  - subunits interact with signaling proteins, undergo conformational changes Integrin clustering Downstream intracellular signaling – Tyrosine phosphorylation – GTPase activation – Phospholipid biosynthesis – Adhesion complex integrity

Tractional Forces Integrins also function as sites of traction Cell uses them as a substrate over which it moves during migration Balance between adhesion at the leading edge and disassembly at the trailing edge Junction density Rate of disassembly

Adhesion Disassembly Important at both the front and the rear of the cell Front – junctions disassembled as new ones are assembled in the formation of the protrusion Rear – junctions must be disassembled to provide materials and release cell from rear direction

Cell Migration Summary Migration is promoted Cell undergoes polarization Leading edge reconfigures junctions, actin filaments, and microtubules Trailing edge undergoes junction disassembly Cell uses integrins for a combination of traction and intracellular signaling Cell reevaluates direction of migration

Arf6 and Microtubules in Adhesion- Dependent Trafficking of Lipid Rafts Detailed analysis of adhesion modulation Lipid raft movement Cell movement

Cytoskeletal Regulation of Raft Endocytosis

Identification of the Intracellular Compartment

Role of Arf6 in Raft Trafficking

Inhibition of Arf6 Function

Adhesion-dependent Activation of Arf6 Promotes Raft Exocytosis

Microtubules in Raft Exocytosis

Lipid Raft Trafficking Summary

References