Overview
Our research is divided along three themes: the dynamics of assembly of a macrophage adhesion, the energetics, mechanics, and chemistry of cell movement, and development of MEMS-based devices to study molecules and cells.

Research Summary
Assembly of a macrophage adhesion: A punctate set of adhesions called podosomes assembles at the leading lamellum of a macrophage. The adhesions lie in the midst of the dynamic actin network that powers the protrusion of the cell membrane. Each adhesion is a vertical bundle of actin filaments crosslinked by fimbrin and is associated with integrin receptors, vimentin, and microtubules. Our analysis of 4D multispectral images shows that podosomes assemble de novo at the leading edge of the cell. Once assembled, the podosomes have four fates, disassembly, fusion with other podosomes, fission into two podosomes, or growth and fission into a cluster of podosomes. Assembly and disassembly is microtubule-independent while fusion and fission are microtubule-dependent. The structure of an adhesion and the dynamics of its assembly are important steps in understanding how the complex process of cell movement is controlled by signaling by mechanical and chemical receptors. [J. Evans, A. Goodman, W. Timp, and P. Matsudaira in collaboration with D. Hanein, N. Volkmann, Burnham Institute].

A new cellular engine; an actin spring: Cell movements are powered by engines that operate as rotors, ratchets, and springs. We study one of the most powerful cellular engines, an actin spring. Within a few seconds a bundle of actin filaments uncoils and extends 50 µm from the sperm head. The finger of membrane supported by the bundle bridges between sperm and egg to initiate fertilization of the horseshoe crab, Limulus polyphemus. Our lab studies the structure of the actin bundle, the energetics of converting the potential energy of a macromolecular spring into the extension of a crystalline bundle, and the mechanism by which the crosslinking protein, scruin, controls these changes in actin structure. Energy is stored in “tight” conformation of scruin, calcium binding unlatches scruin and permits the actin filament to relax. Change in actin twist is coupled to uncoiling and the bundle extends this process. [P. Matsudaira, G. Waller, J. Shin, B. Tam and L. Mahadevan].

Structure of an actin spring: Actin filaments are crosslinked into a bundle. A reconstruction of the actin bundles shows an actin filament decorated by bi-lobed scruin molecules. One domain of scruin is bound to one actin subunit while the other domain is bound to a neighboring subunit. A single filament appears to contact two adjacent filaments through the outer scruin molecules. The crystalline state appears to permit sliding and conformation changes through velcro-like interactions between scruin molecules on several filaments. Independent EM, synchrotron x-ray solution studies, analytical ultracentrifugation, and calorimetric studies document a calcium-dependent conformation that involves a relaxation of the scruin structure and release of energy. [Matsudaira, Waller in collaboration with M. Sherman, M. Schmid, and W. Chiu, Baylor College of Medicine, W. Stafford and K. Langsetmo, BBRI, and H. Tsuruta, Stanford].

BioMEMS: High speed, high throughput bioanalytic tools are required to study complex problems in biology. Our project is focused on technology development in two areas: ultra-fast and automatic cell, protein, and DNA analysis using small microfabricated BioMEMS systems, and novel photon-emitting protein interaction sensors. To date we have developed microelectrophoresis chips for DNA genotyping and sequencing that are capable of resolving simple tandem repeats in a minute time frame and reading 400 bases in ten minutes and 800 bases in an hour. These chips are being incorporated into automated systems for high speed, high through-put analysis. [Ehrlich, Aborn, Ait-Ghezala, Carey, Chiou, Desmarais, El-Difrawy, Lam, McKenna, Mitnik-Gankin, Novotny, O’Neil, Srivastava, Streecham, Freyzon, and Matsudaira].

Selected Publications
Schmid, M.F., M.B. Sherman, P. Matsudaira and W. Chiu, Structure of the Acrosomal Bundle. Nature (in press.)

Gardel, M.L., J.H. Shin, F.C. MacKintosh, L. Mahadevan, P. Matsudaira and D.A. Weitz, Elastic behavior of Cross-Linked and Bundled Actin Networks. Science 304: 1301-1305 (2004).

Evans, J.G., Correia, I., Krasavina. O., Watson, N. and P. Matsudaira. Macrophage podosomes assemble at the leading lamella by growth and fragmentation. J. Cell Biol 161,697-705 (2003).

Lodish, Berk, Matsudaira, Kaiser, Krieger, Scott, Zipursky, and Darnell. Molecular Biology of the Cell, 5th edition, W.H. Freeman (2003)