Gender	Male
E-mail	dan.felsenfeld@mssm.edu

Department of Developmental & Regenerative Biology

Felsenfeld Laboratory

Intergrin, L1, cell migration, video microscopy, laser trap, adhesion receptors, extracellular matrix, cytoskeleton

The migration of adherent cells is central to variety of biological processes, including embryonic develpment, immune system function and the metastasis of tumor cells. During cell migration, cell-surface adhesion receptors play an essential role in both the recognition of molecular cues in the cellular enviroment and the transduction of cellular forces necessary for cell translocation. The ability of cell to generate traction forces depends critically on bonds between adhesion receptors and the cytoskeleton. However, for cell translocation to occur, force generation must be oriented with respect to the direction of cell movement. The organized regulation of adhesion receptor-cytoskeleton interactions across the surface is therefore, essential to the migration of adherent cells.

In the Felsenfeld Laboratory, we are interested in the spatial and temporal regulation of adhesion receptor-cytoskeleton interactions in migrating cells. To address this question, we employ a combination of video microscopy and molecular genetics to examine the function and dynamics of cell-surface glycoproteins in motile cells. To quantify cellular traction forces, we use an optical gradient laser trap or "laser tweezers" to place and manipulate microscopic beads on the cell surface. These beads, when coated with an antibody or a ligand for cell surface receptors, serve as a probe for the position of receptors in live cells. By following the movement of these beads in real time by video microscopy, we are able to detect and quantify the association between adhesion receptors and the underlying cytoskeleton with high spatial and temporal accuracy. By pulling on the bead with the laser trap, we can directly quantify the magnitude of the traction forces generated by the cell.

To understand better the regulation of adhesion receptor function in cell migration, we have focused on two classes of receptors: integrins and the neuronal immunoglobulin (Ig) family member L1. Each of these receptors has been implicated in cell migration. Integrins are a family of heterodimeric cell-surface adhesion receptors that mediate cellular interactions primarily with components of the extraellular matrix (ECM). A larger number of integrin alpha and beta isoforms have been identified; the heterodimer isoform permutation determines both integrin ligand binding specificity and function. Using microscopic particles coated with the integrin ligand fibronectin (FN), we have demonstrated that ligand activation causes the rapid associated between the fibronectin receptor(integrina5b1) and the actin cytoskeleton (Felsenfeld et al, 1996). Moreover, if FN-coated beads are held with the laser trap after binding to the cell surface, the strength of integrin-cytoskeleton linkages is strengthened in proportion to the strength of the trap (Choquet et al 1997). That is, the cell can respond to both the biochemical and mechanical (elastic) properties of the ECM substrate to which it is bound. Finally, we have shown that this process is modulated by the non-receptor tyrosine kinase Src which inhibits selectively the strengthening of vitronectin receptor(avb3/5)-cytoskeleton interactions(Felsenfeld et al, 1999). Therefore, integrin-cytoskeleton linkages appear to be regulated by ligand activation, substrate elasticity and specific second messenger pathways.

More recent work has focused on characterizing other classes of cell surface adhesion receptors, particularly the neuronal Ig-family member L1. Like intregrins, L1 has been implicated in cell migration: ligands for L1 (including L1 itself and the related Ig-family member TAG-1) promote axon outgrowth from cultured neurons. More significantly, L1 has been implicated in mediating axon guidance in the developing vertebrate central nervous system: mice deficient in L1 show defects in the guidance of spec ific cortico-spinal axon tracts. Mutations in the Human L1 gene lead to severe mental retardation, consistent with a role for L1 in nervous system function. L1 is a trans-membrane glycoprotein that is comprised of 6 amino-terminal Ig domains followed by 5 fibronectin type III repeats outside the cell. Inside the cell, the L1 binds to components of the actin cytoskeleton permitting the generation of organized traction forces at the front of the cell. Experiments are currently underway to examine the role of the L1-ankyrin interaction in the regulation of L1 cytoplasmic tail, the amino acid sequence FIGQY, which is regulated in its activity by tyrosine phosphorylation. By examining the dynamics of mutagenized L1 in cultured neurons, we hope to understand how L1-cytoskeleton interactions are regulated during neuronal growth and guidance.

For more information, please visit the Felsenfeld Laboratory website.

Whittard JD, Sakurai T, Cassella MR, Gazdoiu M, Felsenfeld DP. MAP Kinase Pathway-dependent Phosphorylation of the L1-CAM Ankyrin Binding Site Regulates Neuronal Growth. Mol Biol Cell 2006 Jun; 17(6): 2696-706.

Tombler E, Cabanilla NJ, Carman P, Permaul N, Hall JJ, Richman RW, Lee J, Rodriguez J, Felsenfeld DP, Hennigan RF, Diverse-Pierluissi MA. G protein-induced trafficking of voltage-dependent calcium channels. J Biol Chem 2006 Jan 20; 281(3): 1827-39.

Felsenfeld DP. Regulation of complexes by cytoskeletal elements: integrins serve as force transducers linking mechanical stimuli and biochemical signals. Sci STKE 2005 Dec 6; 2005(313): tr27.

Gil OD, Sakurai T, Bradley AE, Fink MY, Cassella MR, Kuo JA, Felsenfeld DP. Ankyrin binding mediates L1CAM interactions with static components of the cytoskeleton and inhibits retrograde movement of L1CAM on the cell surface. J Cell Biol 2003 Aug 18; 162(4): 719-30.

Okigaki M, Davis C, Falasca M, Harroch S, Felsenfeld DP, Sheetz MP, Schlessinger J. Pyk2 regulates multiple signaling events crucial for macrophage morphology and migration. Proc Natl Acad Sci USA 2003 Sep 16; 100(19): 10740-5.

Felsenfeld DP, Schwartzberg PL, Venegas A, Tse R, Sheetz MP. Selective regulation of integrin-cytoskeleton interactions by the tyrosine kinase Src. Nat Cell Biol 1999 Aug; 1(4): 200-6.

Sheetz MP, Felsenfeld DP, Galbraith CG. Cell migration: regulation of force on extracellular-matrix-integrin complexes. Trends Cell Biol 1998 Feb; 8(2): 51-4.

Choquet D, Felsenfeld DP, Sheetz MJ. Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages. Cell 1997 Jan 10; 88(1): 39-48.

Yauch RL, Felsenfeld DP, Kraeft SK, Chen LB, Sheetz MP, Hemler ME. Mutational evidence for control of cell adhesion through integrin diffusion/clustering, independent of ligand binding. J Exp Med 1997 Oct 20; 186(8): 1347-55.

Felsenfeld DP, Choquet D, Sheetz MJ. Ligand binding regulates the directed movement of beta1 integrins on fibroblasts. Nature 1996 Oct 3; 383(6599): 438-40.