Actin Typing on Total Cellular Extracts

Abstract

Based on the finding that the amino-terminal tryptic peptide of actin is a reliable marker for actin divergence, we describe in detail a highly sensitive protein-chemical procedure for actin typing. The method is performed on non-radioactively labelled cells and tissues and six actins can be identified unambiguously in warm-blooded vertebrates. The method is quantitative and gives directly the ratio of the different actins present in the specimens. It does not require previous purification of actin and can be used on total cellular extracts without any prior fractionation. The procedure can be extended to actins not previously characterized by amino acid sequence analysis and makes certain predictions possible about the partial amino acid sequences of the amino-terminal tryptic peptides, mostly sufficient for a correlation with DNA sequences derived from cloned actin genes. This is done as an example for the cytoplasmic actin present in Schneider L-2 Drosophilu melunoguster cells. Although the method is currently used routinely on lo5 cells, modifications are discussed, which should allow the analysis to be performed with even higher sensitivity.Actin is a major protein expressed probably in all different eukaryotic cell types (for a review see [1,2]). Mammals and most likely also birds express at least six different actin genes in a tissue-specific and species-independent manner : two nonmuscle actins, two smooth-muscle actins, cardiac-muscle actin and skeletal-muscle actin [3,4] (and our unpublished results). The complete amino acid sequences now available show that these six actin polypeptides are extremely closely related. Their polypeptide length differs by maximally one amino acid residue and the highest number of amino acid exchanges observed is 25 in a comparison of non-muscle y-actin and skeletal-muscle actin [4-101. The four muscle actins are even more similar and in the most extreme case, i.e. skeletal-muscle actin and one type of smooth-muscle actin, differ by only eight residues [4].Actin differentiation is routinely assessed by one-dimensional or two-dimensional polyacrylamide gel electrophoresis using isoelectric focussing analysis in the first dimension [11 -131. This system resolves three actin types easily: fi and y nonmuscle actins and the so-called a-actins typical of sarcomeric actins [12]. The two smooth-muscle actins are not easily distinguished from a and y-actin, i.e. the a-type smooth-muscle actin is easily confused with sarcomeric actins [3,4] and the y-type smooth-muscle actin with non-muscle y-actin [14], potentially giving rise to erroneous assignments. The latter results, however, explain the different isoelectric points of various actins. Thus for instance the a and y-like smooth-muscle actins, which differ by as much as a full charge, show only three amino acid differences [4], whereas non-muscle y-actin and y-like smooth-muscle actin which are only marginally separated by isoelectric focussing analysis differ by up to 17 residues [10,15]. Similar difficulties can be expected in ...

The ‘CC.Ar.GG’ box

“…Transient CAT expression in the muscle C2C12 cell line from the HCA1/85 promoter was 50-75% that from the HCAl18 promoter (Fig. 6), showing that the deletion of nucleotides -11 8 to -11 6 and the linker substitution for nucleotides -93 to -86 did not drastically affect promoter function. The CC/AA and GG/TT substi- In other experiments using, in each case, DNA preparations from independent clones, we obtained values for CAT expression in C2C12 cells (normalised to that from pHCA118CAT at 100%) of 51%, 58% and 74% for pHCAlj85CAT and 15%, 18% and 26% for pMlj85 CAT, compared to values of 15% and 20% for pHCA47CAT.…”

“…The mouse C2C12 myogenic cell line and the mouse L fibroblastic cell line were obtained and cultured as previously described [9, 33, 341. Nuclear extracts C2C12 cells were harvested at an early stage of myotube differentiation, 1 day after switching to low-serum (2%) medium, the stage at which cardiac actin expression is maximum [6]. C2C12 and L cell nuclei were isolated according to the method of Mellon and Bhorjee [35] except that cells were harvested by swelling in a 10 mM Tris/HCl pH 7.8, 1 mM EDTA, 3 mM CaCl,, 10 mM KCl buffer for 20 min, and scraping with a rubber policeman.…”

“…Cell Biol. 6,2125-21361. Here we show that this sequence binds a nuclear protein, and that binding is abolished by mutating either the CC and G G dinucleotides or the (A + T)-rich centre.…”

Polarized distribution of actin isoforms in gastric parietal cells.

“…Such structural comparisons might provide insights into the physiological significance of the isoforms caused by the small difference in primary sequences. Cytoplasmic 1-and y-actin isoforms in nonmuscle cells are present in amounts that vary among different cell types (Firtel 1981;Vandekerckhove and Weber, 1981). Although there have been speculations on differential functions and/or subcellular localizations (Pardo et al, 1983), differences in isoform distribution and function have been minimally explored.…”

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle α-actin regulatory elements

“…20 Although the gene is transiently expressed in developing cardiac and skeletal muscle, and in myofibroblasts within tumours and healing wounds, 21,22 in adults it is constitutively expressed solely in VSMCs and smooth musclerelated cells. 23 A number of elements controlling the transcription of VSMC ␣-actin have been defined in the chicken, 24,25 rat, 26,27 mouse [28][29][30] and human [31][32][33] (schematic in Figure 1a). The sequences immediately flanking the VSMC ␣-actin TATA box are highly conserved in human (89% Vs rat), mouse (98% Vs rat), rat and chicken (73% Vs rat) between approximately −255 and +12.…”