In the past few years genetic, biochemical, and cytolocalization data have implicated members of the myosin superfamily of actin-based molecular motors in a variety of cellular functions including membrane trafficking, cell movements, and signal transduction. The importance of myosins is illustrated by the identification of myosin genes as targets for disease-causing mutations. The task at hand is to decipher how the multitude of myosins function at both the molecular and cellular level-a task facilitated by our understanding of myosin structure and function in muscle.

Formation and maintenance of specialized plasma membrane domains are crucial for many biological processes, such as cell polarization and signaling. During isotropic bud growth, the yeast cell periphery is divided into two domains: the bud surface, an active site of exocytosis and growth, and the relatively quiescent surface of the mother cell. We found that cells lacking septins at the bud neck failed to maintain the exocytosis and morphogenesis factors Spa2, Sec3, Sec5, and Myo2 in the bud during isotropic growth. Furthermore, we found that septins were required for proper regulation of actin patch stability; septin-defective cells permitted to enter isotropic growth lost actin and growth polarity. We propose that septins maintain cell polarity by specifying a boundary between cortical domains.

We have studied the interaction of CapZ, a barbed-end actin capping protein from the Z line of skeletal muscle, with actin. CapZ blocks actin polymerization and depolymerization (i.e., it "caps") at the barbed end with a Kd of approximately 0.5-1 nM or less, measured by three different assays. CapZ inhibits the polymerization of ATP-actin onto filament ends with ATP subunits slightly less than onto ends with ADP subunits, and onto ends with ADP-BeF3- subunits about as much as ends with ADP subunits. No effect of CapZ is seen at the pointed end by measurements either of polymerization from acrosomal processes or of the critical concentration for polymerization at steady state. CapZ has no measureable ability to sever actin filaments in a filament dilution assay. CapZ nucleates actin polymerization at a rate proportional to the first power of the CapZ concentration and the 2.5 power of the actin concentration. No significant binding is observed between CapZ and rhodamine-labeled actin monomers by fluorescence photobleaching recovery. These new experiments are consistent with but do not distinguish between three models for nucleation proposed previously (Cooper & Pollard, 1985). As a prelude to the functional studies, the purification protocol for CapZ was refined to yield 2 mg/kg of chicken breast muscle in 1 week. The activity is stable in solution and can be lyophilized. The native molecular weight is 59,600 +/- 2000 by equilibrium ultracentrifugation, and the extinction coefficient is 1.25 mL mg-1 cm-1 by interference optics. Polymorphism of the alpha and beta subunits has been detected by isoelectric focusing and reverse-phase chromatography. CapZ contains no phosphate (less than 0.1 mol/mol).

Myosins are actin-activated ATPases that are able to translocate along actin filaments using energy derived from ATP hydrolysis. Non-muscle cells contain conventional myosins, which are similar in sequence and structure to muscle myosin, and a number of unconventional myosins whose head sequences are similar but tail sequences are unrelated to conventional myosins. The myosin superfamily currently consists of nine classes; Drosophila 95F is an unconventional myosin and the original member of class VI, which includes a homologue found in pig kidney. Some unconventional myosins have been suggested as mediators of some types of intracellular transport, but there is little direct evidence for this function (but see ref. 6). We have observed transport of cytoplasmic particles in live Drosophila embryos in three dimensions using computational optical sectioning microscopy. We present here evidence that this transport is actin-based, ATP-dependent and catalysed by one such unconventional myosin, the 95F myosin. This is, to our knowledge, the first direct observation of transport catalysed by an unconventional myosin in living cells.

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