Structural invariance of constitutively active and inactive mutants of Acanthamoeba myosin IC bound to F-actin in the rigor and ADP-bound states

The three single-headed monomeric myosin I isozymes of Acanthamoeba castellanii (AMIs)—AMIA, AMIB, and AMIC—are among the best-studied of all myosins. We have used AMIC to study structural correlates of myosin’s actin-activated ATPase. This activity is normally controlled by phosphorylation of Ser-329, but AMIC may be switched into constitutively active or inactive states by substituting this residue with Glu or Ala, respectively. To determine whether activation status is reflected in structural differences in the mode of attachment of myosin to actin, these mutant myosins were bound to actin filaments in the absence of nucleotide (rigor state) and visualized at 24-Å resolution by using cryoelectron microscopy and image reconstruction. No such difference was observed. Consequently, we suggest that regulation may be affected not by altering the static (time-averaged) structure of AMIC but by modulating its dynamic properties, i.e., molecular breathing. The tail domain of vertebrate intestinal brush-border myosin I has been observed to swing through 31° on binding of ADP. However, it was predicted on grounds of differing kinetics that any such effects with AMIC should be small [Jontes, J. D., Ostap, E. M., Pollard, T. D. & Milligan, R. A. (1998) J. Cell Biol. 141, 155–162]. We have confirmed this hypothesis by observing actin-associated AMIC in its ADP-bound state. Finally, we compared AMIC to brush-border myosin I and AMIB, which were previously studied under similar conditions. In each case, the shape and angle of attachment to F-actin of the catalytic domain is largely conserved, but the domain structure and disposition of the tail is distinctively different for each myosin.

Pollen selection: a transgenic reconstruction approach.

A transgenic reconstruction experiment has been performed to determine the feasibility of male gametophytic selection to enhance transmission of genes to the next sporophytic generation. For tobacco pollen from a transgenic plant containing a single hygromycin-resistance (hygromycin phosphotransferase, hpt-) gene under control of the dc3 promoter, which is active in both sporophytic and gametophytic tissues, 3 days of in vitro maturation in hygromycin-containing medium was sufficient to result in a 50% reduction of germinating pollen, as expected for meiotic segregation of a single locus insert. Pollination of wild-type plants with the selected pollen yielded 100% transgenic offspring, as determined by the activity of the linked kanamycin-resistance gene--present within the same transferred T-DNA borders--under control of the nos promoter. This is direct proof that selection acting on male gametophytes can be a means to alter the frequency of genes in the progeny.