Heterotypic interactions and filament assembly of type I and type II cytokeratins in vitro: viscometry and determinations of relative affinities.
We have determined relative affinities of type I and type II human non-epidermal cytokeratin (CK) polypeptides synthesized in Escherichia coli from cDNA, using the method of surface plasmon resonance, and have compared the influence of ionic strength and ion quality on the assembly to intermediate filaments (IFs) by viscometry and electron microscopy. By surface plasmon resonance (total internal reflection) we determined the real-time relative binding of various type I CKs to the type II CK8. Surprisingly, the various type I CKs examined (i.e., CKs 13, 18, 19 and 20) differed markedly in their relative binding rate: For example, CK18 and CK13 displayed much higher resonance signals than CK19 and CK20. In addition, soluble complexes of type II CK8 with various type I cytokeratins were reconstituted at slightly alkaline pH and low ionic strength. Subsequent IF assembly was induced by simultaneous shifting to neutral pH and increasing the ionic strength. Both mono- and divalent ions as well as tris (hydroxymethyl)-aminomethane (Tris) alone were able to induce IF assembly. When we compared the time course of viscosity increase of CK8 in combination with either CK13, CK18, CK19 or CK20, we found that the cytokeratin pairs 8:18 and 8:20 attained the highest viscosity values, whereas the cytokeratin pair 8:19 displayed a relatively low value. Significantly lower values of specific viscosity were also observed in competition experiments when CK18 was gradually replaced by CK19. The observed differences in relative affinities and assembly kinetics of certain CK combinations allow quantitations of the interactions between different CKs and are discussed in relation to IF stability and cell differentiation.
Hofmann
I
I
Division of Cell Biology, German Cancer Research Center, Heidelberg/Germany.
Franke
W W
WW
D001483:Base Sequence; D002413:Cations, Divalent; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007633:Keratins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D014783:Viscosity
D002413:Cations, Divalent; D018076:DNA, Complementary; D011994:Recombinant Proteins; D007633:Keratins