A PRIMER ON ACTIN CYTOSKELETON PROTEINS

Some basic knowledge of the dynamics of actin assembly and disassembly helps one to understand how Listeria spread from cell to cell. In nonmuscle cells, particularly neutrophils and macrophages, regulation of actin concentration and filament length underlie the changes in the consistency and structure of the cytoplasm required for amoeboid movement and phagocytosis. Actin is the most abundant protein in the cytoplasm of mammalian cells, often amounting 10 to 20 percent of the total cytoplasmic protein. It exists either as a globular monomer (called G-actin) or as a filament (designated F-actin), the latter formed by head-to-tail polymerization of asymmetric monomers. In fact, the two filament ends (designated the plus and minus ends) differ in geometry, stability, and growth rate, and this polarity is fundamentally important in understanding how net assembly at the plus ends (situated nearest the peripheral membrane) and net disassembly (deeper within the cytoplasm) results in the steady-state flux of subunits through F-actin.

The first step in actin filament formation is called nucleation. In this low probability event, three actin monomers are thought to combine simultaneously, forming a thermodynamically unstable trimeric nucleus. Once a trimer is formed, the nucleus most frequently dissociates back into monomers; however, the nucleus occasionally survives long enough to permit subsequent binding of additional actin molecules. As this elongation reaction proceeds in vitro, monomeric actin molecules (each containing ATP) add one by one to both ends of the elongating nucleus to form filaments. Monomer addition is more rapid at the plus end, and in the cell little or no monomer addition takes place at the minus end. After monomer incorporation into growing filaments, ATP-actin undergoes nucleotide hydrolysis to form ADP-actin subunits within the filament's helical lattice. Once actin filaments reach a steady-state length, ADP-actin monomers are released from the minus ends of the filaments at the same rate as new ATP-actin monomers are added to the plus ends. In the cell, two additional issues are important: (a) actin monomer addition occurs mostly, if not exclusively, at the plus-end; and (b) this process probably produces a pool of ADP-actin from which actin-ATP must be regenerated by exchange (not direct phosphoryl transfer) with ATP in the cytoplasm. ATP-actin has a much higher affinity for the ends of actin filaments than does ADP-actin, and ATP-actin is the primary monomeric species of actin that adds to filament ends in the cell.

Within the host cell, actin filament assembly is exquisitely well regulated through the action of a number of actin-regulatory proteins, including actin filament capping and severing proteins, actin monomer sequestering proteins, actin bundling proteins and actin cross-linking proteins. One host cell component likely to play a central role in Listeria actin-based motility is profilin. This protein binds to actin monomers in a one to one complex, alters the conformation of the actin monomer, and accelerates the exchange of ATP with actin-bound ADP. Because ATP-actin has a higher affinity for the ends of actin filaments, catalysis of nucleotide exchange should enhance actin-filament assembly. Profilin is likely to be most highly concentrated wherever new actin filaments assemble. Profilin is the only actin regulatory protein that binds polyproline, and the host cell protein vasodilator-stimulated phosphoprotein (VASP) has taken advantage of this unique characteristic to concentrate profilin in specific regions of the cell where new actin filaments assemble. The VASP monomer contains four potential profilin binding sites each containing a series of proline residues. VASP exists as a tetramer in the host cell; therefore each VASP molecule could attract up to 16 profilin molecules. Finally, a third actin-regulatory protein of importance in Listeria intracellular movement is called alpha-actinin. This host cell cytoskeletal protein binds to the sides of actin filaments, linking them into bundles. Bundling of actin filaments creates the more rigid co-linear filament network required to form structures such as actin stress fibers and Listeria actin tails.