February 18, 2025

Accordingly, wild-type K5-K14 assemblies behave like weak-viscoleastic solids under standard assembly conditions at pH 7

Accordingly, wild-type K5-K14 assemblies behave like weak-viscoleastic solids under standard assembly conditions at pH 7.4. properties when placed under strain. Loss of the C-terminal 41 residues contributes to these alterations. When transfected in cultured epithelial cells, K5-1649delG incorporates into preexisting keratin IFs and also forms multiple small aggregates that often colocalize with hsp70 in the cytoplasm. Aggregation is usually purely a function of the K5-1649delG tail domain name; in contrast, the cloned 109 residue-long tail domain name from wild type K5 is usually distributed throughout the cytoplasm and colocalizes partly with keratin IFs. These data provide a mechanistic basis for the cell fragility seen in individuals bearing the K5-1649delG allele, and point to the role of the C-terminal 41 residues in determining K5’s assembly properties. INTRODUCTION Intermediate filaments (IFs) are encoded by a large family of genes comprising 67 members in the human and mouse genomes (Coulombe strain BL21 (DE3) followed by fusion protein purification on a nickel column (Novagen, Madison, WI) before cosedimentation assays. Protein purity ( 99%) was assessed by SDS-PAGE electrophoresis and Coomassie Blue staining, and protein identity was assessed by Western immunoblotting by using monoclonal antibody (mAb) PCK-26 against K5 (Sigma-Aldrich, St. Louis, MO) and mAb 9E10 against Myc antibody (American Type Culture Collection, Manassas, VA). Construction of Fusion cDNAs The human K5 tail domain name coding sequence (residues 482C590; Wilson The coding sequence of wild-type human K5 (Wilson The full-length wtK5 and K5-1649delG cDNAs were subcloned from pcDNA3-Nmyc into the Bam H1 site of pEGFP-C3 (BD Biosciences Clontech, Palo Alto, CA). In both cases, the keratin open reading frame is usually fused in frame at the 3 Rabbit Polyclonal to TBX3 end (C terminus) Harringtonin of the EGFP coding sequences. All newly generated DNA constructs were validated by sequencing before use. Filament Assembly and Characterization Starting from type I-type II heterotypic complexes at 0.5 mg/ml (10 mM), keratin IFs were reconstituted by serial dialysis by using the following three buffers at room temperature (Ma and validated using Harringtonin electrophoretic assays (our unpublished data). K5C41 is usually a C-terminal deletion mutant in which the open reading frame has been interrupted precisely at the location of the frameshift in K5-1649delG (Physique 1A). Purified type ICtype II heterotypic keratin complexes were used for dialysis-based assembly. Polymerization efficiency was assessed by centrifugation, and structural features of individual polymers were visualized by unfavorable staining and electron microscopy. Mechanical properties of the polymers were assessed by rheological assays. None of the mutant tested was impaired in its ability to form ureastable heterotypic complexes (our unpublished data), as determined by chromatography and chemical cross-linking assays (Wawersik Sample pHaFilament width (nm)bElastic modulus G (dynes/cm2)cPhase angle ()cWtK5/K14 7.4 10.1 0.3 510 48 K5-1649 del G/K14 7.4 9.6 0.3 UNd UN K5-1649 del G/K14e 7.0 10.3 0.2 69 511 Open in a separate window apH refers to the pH of the assembly buffer: pH 7.4 corresponds to the standard condition for polymerizing purified keratins in vitro, whereas pH 7.0 promotes the bundling of K5-K14 filaments (Bousquet 2001 ; Ma Harringtonin 2001 ; Yamada 2002 , 2003 ) bFilament width (mean SEM) was decided from five filament profiles from each of three micrographs per sample analyzed cElastic modulus G and phase angle were determined by rheology using conditions of minute deformation (1% strain, and 1 rad/s frequency). The elastic modulus G defines the elastic response of a material to a deformation. G Harringtonin is usually directly related to the fraction of the input energy (deformation) that is stored within the material. The phase angle , expressed in degrees, corresponds to the delay in the material response due to energy dissipation. The stress response of elastic solids is usually perfectly in-phase with the imposed strain (deformation), and thus = 0. The stress response of viscous liquids is out of phase with.