Mutant actin: A dead end?

Yeast actin with a subdomain 4 mutation (A204C) exhibits increased pointed-end critical concentration

“…Developing nonpolymerizing actin mutants is complicated because actin requires the activity of the eukaryotic chaperonin CCT to properly fold, and thus, only eukaryotic systems can be used for expression (Spiess et al 2004). Moreover, actin mutants that cannot polymerize produce dominant lethal effects (Hennessey et al 1992).…”

Molecular genetics of actin function

“…One study has investigated the effects observed in vivo using in vitro methods. A range of actin mutants, with different in vivo effects, all showed the same ability to polymerize [33] or bind to Drosophila myofibrils in vitro [36] as wild-type: the differences seen in vivo were not reproduced in vitro, though doubts have been expressed about what was actually being measured in these experiments [36]. However, electron microscopy (M. Stark and J. C. Sparrow, unpublished work) has shown that all the mutants used [33] make thin filaments in vivo although to very different degrees.…”

“…Although the interpretation of these experiments has been questioned [36], a number of mutants are able to co-polymerize as well as wild-type actin expressed in vitro [28,29,33,36] (Table 1). None of these mutants were in actin-actin contact sites.…”

“…Although it may not be fully processed [32,121], such actin behaves the same as the product of the same gene expressed in vivo [32]. Mutant actins have been expressed from the genes for human a-skeletal muscle actin [28][29][30], rat brain cytoplasmic actin [27] and Drosophila Act88F [31][32][33]35,36,148] and used in co-polymerization and protein binding studies. Although the costs are prohibitive for making large amounts of actin this way, expression is quickly and easily achieved and binding assays can be used to screen rapidly large numbers of mutations and pinpoint those of interest for more detailed study.…”

“…Actin mutations have been described in the yeast Saccharomyces cerevisiae [15,16], the fruitfly Drosophila melanogaster [17][18][19][20][21][22], the nematode Caenorhabditis elegans [23,24] and cultured human cells [25,26]. Mutations made in cloned actin genes have been studied after expression either by transcription and translation in vitro [27][28][29][30][31][32][33][34][35][36] or in vivo following transformation of suitable organisms such as S. cerevisiae [37][38][39][40][41][42][43], Dictyostelium discoideum [44], D. melanogaster [20,33,[45][46][47][48][49], or cultured cell lines [25,26,28,50].…”