Analysis of the mechanism of assembly of mouse keratin 1/keratin 10 intermediate filaments in vitro suggests that intermediate filaments are built from multiple oligomeric units rather than a unique tetrameric building block.

The question as to whether keratin intermediate filaments (KIF) are built from a unique "building block" consisting of a pair of coiled-coil molecules has been studied by examining the earliest stages of reassembly of mouse K1/K10 KIF in vitro. Particles formed in protein solutions of about 45 micrograms/ml (near or below the critical concentration for assembly) or 0.5-1.65 mg/ml were monitored by turbidity, visualized by electron microscopy, and their structures resolved biochemically using crosslinking, limited proteolysis, and amino acid sequencing. The rate of KIF reassembly in vitro is limited by an initial slow step involving the formation of a three- or four-molecule oligomer. At 2 min, the particles in solution are about 65 nm long and consist of two molecules aligned antiparallel and staggered. A few minutes later, a three- and/or four-molecule species appears that may be the rate-limiting particle(s). It is also 65 nm long, but contains one or two additional molecules aligned in register but antiparallel with respect to one of the molecules on the two-molecule particle. The present data cannot establish whether the rate-limiting particle contains three or four molecules, or in fact consists of a mixture of both. Below the critical concentration for KIF assembly, it exists in solution in rapid exchange with particles containing one and two molecules. In solutions above the critical concentration for assembly, once this oligomer has formed in sufficient quantity, further assembly into KIF occurs rapidly; 90, 110, and 130-nm particles soon appear by apparent addition of a single molecule or oligomers containing two, three, four, or even several molecules. Within about 20 min short KIF about 200-500 nm long appear which later elongate to long (greater than 1 micron) KIF. These data suggest that KIF assembly requires the initial correct alignment of three or four molecules which, once formed, provides a template for further rapid addition of molecules leading to KIF assembly. Furthermore, the data establish that KIF are built from alternating rows of in-register and staggered antiparallel molecules. The present data confirm independently the observations of the previous paper and do not support earlier notions that IF are built from a tetrameric building block consisting of a pair of in-register molecules. Finally, the data suggest that the mechanism of assembly in vitro and the dynamic in vivo assembly-disassembly characteristics of KIF in particular and IF in general are mediated through a variety of small oligomeric species ranging in size from one to several molecules.