Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-03-30
2004-04-06
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
Reexamination Certificate
active
06716597
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to methods for regulating cell motility and related products. In particular methods for promoting and preventing cell migration are described herein.
BACKGROUND OF THE INVENTION
How a cell moves is one of the most compelling mysteries of cell biology. Cell migration forms the basis for higher order processes such as immune cell homing, wound healing, and axonal pathfinding. Migration depends on the coordinated execution and integration of complex individual processes. Although different cell types have unique approaches to cell movement, it is useful to consider animal cell migration in a generalized way. In its simplest form, movement requires that a cell generates and maintains a state of asymmetry or polarity.
Once polarized, a cell must execute a four-step cycle to migrate or translocate (reviewed in Lauffenburger, D. A., and Horwitz, A. F. (1996). Cell migration: a physically integrated molecular process.
Cell
84, 359-69). First, a cell must extend a process, known as the leading edge, in the direction of movement. During this step, increased actin polymerization is seen in the area of the leading edge. This increased polymerization arises from the creation of new barbed ends that are oriented towards the membrane, either by nucleation of new filaments from pools of G-actin or by severing or uncapping of existing filaments. Actin monomers are added onto barbed ends until they are capped (Schafer, D. A., and Cooper, J. A. (1995). Control of actin assembly at filament ends.
Annu Rev Cell Dev Biol
11, 497-518). The combination of actin nucleation and filament elongation is thought to play a critical role in the protrusion of the leading edge (Eddy, R. J., Han, J., and Condeelis, J. S. (1997). Capping protein terminates but does not initiate chemoattractant—induced actin assembly in Dictyostelium.
J Cell Biol
139, 1243-53). Second, once a cell has extended a process, it must form semi-stable points of attachment with the underlying substratum to serve as anchor points. One class of attachment points, focal adhesions, contain aggregates of integrin receptors and a variety of cytosolic signaling and cytoskeletal proteins and serve as sites of bidirectional signaling between the extracellular matrix and the actin cytoskeleton (Schoenwaelder, S. M., and Burridge, K. (1999). Bidirectional signaling between the cytoskeleton and integrins.
Curr Opin Cell Biol
11, 274-86). Although attachment of newly extended processes may be critical for cell translocation, process extension itself does not require adhesion (Bailly, M., Yan, L., Whitesides, G. M., Condeelis, J. S., and Segall, J. E. (1998). Regulation of protrusion shape and adhesion to the substratum during chemotactic responses of mammalian carcinoma cells.
Exp Cell Res
241, 285-99). Third, once a cell has extended and anchored a new process, it must slide the cell body forward by traction. The fourth step is release of points of substratum attachment at the rear of the cell.
The evolutionarily-conserved Ena/VASP protein family has been implicated in the regulation of cell migration (Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J., and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics.
Cell
87, 227-39). Enabled (Ena; SEQ ID NO: 9) was identified as a genetic suppressor of loss-of-function mutations in Drosophila Ableson tyrosine kinase (D-Ab1) (Gertler, F. B., Doctor, J. S., and Hoffinann, F. M. (1990). Genetic suppression of mutations in the Drosophila abl proto-oncogene homolog.
Science
248, 857-60). Loss-of-function mutations in Ena ameliorated the embryonic central nervous system defects associated with loss of D-Ab1 in combination with mutations in any of several known D-Ab1 modifier genes (Gertler, F. B., Corner, A. R., Juang, J L., Ahern, S. M., Clark, M. J., Liebl, E. C., and Hoffmann, F. M. (1995).
enabled, a dosage-sensitive suppressor of mutations in the Drosophila Abl tyrosine kinase, encodes an Abl substrate with SH
3 domain-binding properties. Genes Dev 9, 521-33). VASP was identified biochemically as an abundant substrate for cyclic-nucleotide dependent kinases in mammalian platelets (SEQ ID NO: 10); (Halbrugge, M., and Walter, U. (1990).
Analysis, purification and properties of a
50,000-dalton membrane- associated phosphoprotein from human platelets. J Chromatogr 521, 335-43). Two other mammalian members of this protein family, Mena (mammalian Enabled; SEQ ID NO: 2 and EVL (Ena/VASP like; SEQ ID NO: 11), were identified by sequence similarity (Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J., and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics.
Cell
87, 227-39).
All Ena/VASP family members share a conserved domain structure. The N-terminal third of the protein, the EVH1 (Ena VASP Homology) domain (Gertler, F. B., Niebuhr, K., Reinhard, M, Wehland, J, and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics.
Cell
87, 227-39), mediates subcellular targeting of Ena/VASP proteins to focal adhesions by binding to proteins containing a motif whose consensus is D/E FPPPPX D/E (SEQ ID NO: 1) (Niebuhr, K., Ebel, F., Frank, R., Reinhard, M., Domann, E., Carl, U. D., Walter, U., Gertler, F. B., Wehland, J., and Chakraborty, T. (1997).
A novelproline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligandfor the EVH
1 domain, a protein module present in the Ena/VASP family.
Embo J
16, 5433-44). Mutational analysis indicated that the phenylalanine residue, along with flanking acidic residues on either side, are critical for optimal binding (Carl, U. D., Pollmann, M., Orr, E., Gertler, F. B., Chakraborty, T., and Wehland, J. (1999). Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with proline-rich ligands.
Curr Biol
9, 715-8). The EVH1 ligand motif is found in a number of cellular proteins, including the focal adhesion proteins zyxin and vinculin. The central portion of Ena/VASP proteins contains proline-rich stretches, which have been reported to be binding sites for three types of proteins: the G-actin binding protein profilin, SH3 domain-containing proteins, and WW domain-containing proteins (Ermekova, K. S., Zambrano, N., Linn, H., Minopoli, G., Gertler, F., Russo, T., and Sudol, M. (1997). The WW domain of neural protein FE65 interacts with proline-rich motifs in Mena, the mammalian homolog of Drosophila enabled.
J Biol Chem
272, 32869-77; Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J., and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics.
Cell
87, 227-39). The C-terminal third of Ena/VASP proteins contains the EVH2 domain that binds in vitro to F-actin and has a putative coiled-coil region reported to be important for multimerization (Bachmann, C., Fischer, L., Walter, U., and Reinhard, M. (1999). The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin bundle formation.
J Biol Chem
274, 23549-57,; Huttelmaier, S., Harbeck, B., Steffens, O., Messerschmidt, T., Illenberger, S., and Jockusch, B. M. (1999). Characterization of the actin binding properties of the vasodilator-stimulatedphosphoprotein VASP.
FEBS Lett
451, 68-74).
In addition to their capacity to bind profilin and actin, the localization of Ena/VASP proteins suggests that they may be involved in regulating actin dynamics and/or adhesion. In fibroblasts, Ena/VASP proteins are localized to focal adhesions, in a weak punctuate pattern along stress fibers and to the leading edge, while in neuronal growth cones, they are concentrated at the distal tips of filopodia (Reinhard, M., Halbrugge, M., Scheer, U., Wiegand, C., Jockusch, B. M., and Walter, U. (1992). The 46/50 kDa phosphoprotein VASP purifiedfrom human platelets is a novel protein associated w
Bear James E.
Gertler Frank B.
Loureiro Joseph J.
Wehland Jurgen
Massachusetts Institute of Technology
McKelvey Terry
Wolf Greenfield & Sacks P.C.
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