Axonal neurofilaments differ in composition and morphology from those in the soma of the squid stellate ganglion.

Triton X-100 insoluble neurofilament (NF) fractions were obtained from two parts of the stellate ganglion and the main giant axon. These were analyzed by one- and two-dimensional gradient polyacrylamide gel electrophoresis, cyclic assembly and disassembly, and electron microscopy. The NF fractions from the ganglion cell bodies (GCB) and from the part of the ganglion mainly consisting of axon initial segments (GIS) were of similar composition; neither contained detectable amounts of the 220 kda and high molecular weight (greater than 400 kda) NF subunits that were prominent in the axonal NF fraction. However, the GCB and GIS did contain large quantities of a set of 65 kda polypeptides that were minor constituents of the axonal NF fraction. The 65 kda-containing NF fraction from the ganglion could be cyclically disassembled and reassembled, but only under low salt conditions, in contrast to the high salt conditions used to cycle axonal NFs. A comparison of the peptide map of the 65 kda polypeptides with that of the 60 kda axonal NF subunit showed them to be different. These biochemical differences between the ganglionic and axonal NF fractions correlated with morphologic distinctions: ganglionic NFs were relatively smooth surfaced, whereas axonal NFs had long sidearms. Such observations support the hypothesis that the NF cytoskeleton of the neuron soma is different from that of the axon. Furthermore, the change from the somal form to the axonal form of NFs appears to occur in the region where the axon initial segment increases in diameter to become the axon proper.

Isolation and characterization of keratin-like proteins from cultured cells with fibroblastic morphology.

Intermediate filaments (IF) isolated from a variety of cultured cells, conventionally described as fibroblasts, are composed predominantely of proteins of molecular weights of 54,000 and/or 55,000. Less than 15% of the protein found in native IF preparations from these cells is composed of three to four polypeptides of molecular weights 60,000-70,000. We have investigated some biochemical and immunological properties of these proteins isolated from BHK-21 and mouse 3T3 cells. They are capable of forming paracrystals that exhibit a light/dark banding pattern when negatively stained with uranyl acetate. The dark bands are composed of longitudinally aligned approximately 2-nm-diam filaments. The center-to-center spacing between either dark or light bands is 37-40 nm. These dimensions are consistent with the secondary structure of IF polypeptides and suggest that the dark bands represent lateral alignment of alpha-helical coiled-coil domains. Immunoblotting, secondary structure, as well as amino acid composition data indicate that the 60,000-70,000-mol-wt paracrystal polypeptides are similar to keratin. Thus, polypeptides with biochemical and immunological properties of epidermal keratin are present in cells normally considered to be fibroblasts.

In vitro reconstitution of intermediate filaments form mammalian neurofilament triplet polypeptides.

Intermediate filaments (IF) were reconstituted in vitro from bovine neurofilament triplet polypeptides. Neural IF, solubilized in either low salt or 8 M urea solution, assembled into IF when returned to near-physiological solution conditions. The 68,000-dalton component of the triplet, purified to homogeneity by preparative NaDodSO4 electrophoresis, was renatured and reassembled into short (approximatley 0.05-micrometer) approximatley 10 nm-diameter filaments. These results demonstrate that the triplet polypeptides are components of neural IF and that the 68,000-dalton polypeptide is an IF structural protein.

In vitro reassembly of squid brain intermediate filaments (neurofilaments): purification by assembly-disassembly.

Intermediate filaments from squid brain tissue were reassembled in vitro and purified by two cycles of assembly and disassembly. Purified squid brain filaments contained one major polypeptide (60,000 daltons), which constituted about 70 percent of the total protein, and three minor polypeptides (74,000, 100,000, and 220,000 daltons). Squid brain intermediate filaments were reconstituted from rod-shaped protofilamentous subunits. In addition to the intermediate filaments, dense bodies which may function in intermediate filament nucleation or organization were retained through two purification cycles.

In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells.

Intermediate filaments (IF) from baby hamster kidney (BHK-21) cells can be disassembled at low ionic strength and reassembled upon addition of salt. Turbidimetric analyses show that reassembled IF exhibit the light scattering properties of long rods under physiological conditions (5 mM Na+/K+ phosphate, pH 7.2/170 mM NaCl at 21 degrees C). IF weight concentration, determined by centrifugation, is directly proportional to the optical density at 3000 nm. Thus, turbidity can be used as a quantitative assay for IF assembly. Turbidimetric and centrifugation analyses both indicate that IF assembly exhibits a critical protein concentration of 0.05-0.15 mg/ml. Above the critical concentration, IF weight concentration at steady-state is a linear function of the total protein concentration. Negative stain observations at early stages of the assembly process suggest lateral association of protofilaments to form short IF. This lateral association is accompanied by a rapid turbidity increase which is then followed by IF elongation and a slower turbidity increase to plateau. Further purification of IF by low/high-NaCl-induced cycles of disassembly/reassembly results in retention of 54- and 55-kilodalton (decamin) polypeptides. These results constitute a quantitative description of in vitro reassembly of IF from homogeneous cultures of nonkeratinizing cells and establish conditions for further studies on the regulation of IF assembly.

Effects of RNase and RNA on in vitro aster assembly.

RNase alters the in vitro assembly of spindle asters in homogenates of meiotically dividing surf clam (Spisula solidissima) oocytes. Some effects of RNase, such as reduced astral fiber length, appear nonenzymatic and probably result from RNase binding to tubulin. However, RNase-induced changes in the microtubule organizing center are also observed. Since other polycations can mimic RNase effects, the existence of an RNA component of the spindle organizing center remains uncertain. Effects of RNase and other polycations on astral fiber length can be prevented and reversed by the RNase inhibitor, polyguanylic acid. Polyguanylic acid can also augment astral fiber length in the absence of added RNase or other polycations. Augmentation by polyguanylic acid is favored by high ionic strength, and can be duplicated by polyuridylic acid and, with less efficiency, by polyadenylic acid. Polycytidylic acid and unfractionated yeast RNA, however, are unable to augment aster assembly. Polyguanylic acid can also augment the length of astral fibers on complete spindles isolated under polymerizing conditions. These results demonstrate that specific polyribonucleotides can alter spindle assembly in vitro. The presence of an inhibitor of microtubule assembly in Spisula oocytes, which can be inactivated by specific RNAs, is suggested.