Cell motility and the cytoskeleton最新文献

A procedure for preparing cytosol-free ram sperm models was developed. Sperm are introduced to a Triton X-100-containing demembranation medium layered on top of a discontinuous Percoll gradient. After brief exposure to the demembranating solution, the sperm are separated from the detergent-soluble components by centrifugation through a 55% Percoll layer, finally collecting on top of a 90% Percoll cushion from where they are recovered. Optimum conditions consisted of Triton X-100 at 0.20% and a demembranation time of 35 sec. Cross-sections of midpieces and principal pieces of the demembranated sperm were examined by electron microscopy. With 0.20% Triton X-100 in the demembranation medium, 86% of the cross-sections showed no plasma membranes and the rest had broken plasma membranes. The remaining tail structures appeared to be morphologically intact. Assay of phosphoglucose isomerase as a marker enzyme confirmed that at least 98% of the cytosolic protein was removed. Ram sperm models obtained by this procedure could be reactivated, and the percent motility and beat parameters were similar to those of the intact sperm. Reconstitution with the detergent-soluble components was neither required for, nor enhanced, reactivation. Therefore, demembranated ram sperm do not require a detergent-soluble protein factor for reactivation.

A "freeze-break" technique (Trombitás, K.: Acta Biochim. Biophys. Hung. 6:419-427, 1971) and immunoelectron microscopy were used to study the elastic properties of titin filaments. Small bundles of freshly prepared rabbit psoas muscle fibers were quickly frozen and broken under liquid nitrogen to fracture sarcomeres in planes perpendicular to the filament axis, in each of various regions along the sarcomere. The still-frozen specimens were thawed during fixation to allow elastic filaments to retract. The broken specimens were then labelled with monoclonal anti-titin antibodies against an unique epitope in the I-band. The titin epitopes were normally positioned symmetrically about the Z-line. However, in sarcomeres broken at the A-I junction, the epitopes no longer remained symmetrical: the titin filaments in the broken half-sarcomere retracted, independently of the thin filaments, forming a dense band just near the Z-line. The retracted density apparently did not reach the Z-line; retraction stopped at the level of the so-called N1-line. In sarcomeres broken at the Z-line level, the titin filaments retracted in the opposite direction. In this case the titin epitope retracted all the way to the ends of the thick filaments. It appears then that titin molecules form elastic filaments that are independent of thin filaments in most of the I-band. Near the Z-line, however, the titin filaments either have an inelastic domain or associate firmly with the thin filaments at the N1-line level.

NG108-15 cells extend "rapid-onset" neurites vigorously within the first hour after plating in minimal serum-free medium on Petri dishes coated with polylysine and laminin (1 ng/mm2). We recently reported that the initial rates of neurite formation and cell translocation are further accelerated in this system when non-specific substratum attachment sites are partially blocked by polyglutamate, bovine serum albumin, or polyethylene glycol polymers [Smalheiser, N. R. (1991): Dev. Brain Res. 62:81-89]. When cells were plated in the presence of the monovalent cation ionophore monensin (1-5 microM) or hypertonic sucrose (50-100 mM), the initial rate of outgrowth on laminin/polylysine-treated Petri dishes was not affected, yet the acceleration produced by polyglutamate was strongly inhibited. These data indicate that monensin-sensitive intracellular events can regulate neurite extension on laminin indirectly, through modulating the effects exerted on cells by nonspecific substratum sites. Although the critical events affected by monensin remain to be identified, movements of laminin receptors (their clustering, internalization, and recycling) are likely targets for further study.

We have constructed a chimeric beta-tubulin gene that places the structural gene for the tubC beta-tubulin of Aspergillus nidulans under the control of the benA beta-tubulin promoter. Introduction of either this chimeric gene or a second wild-type benA gene into a benomyl-resistant benA22 strain causes it to become benomyl sensitive, indicating that the introduced genes are functional. Analysis of the tubulin proteins synthesized in benA22 strains into which a second wild-type benA beta-tubulin gene was transformed showed that the total amount of beta-tubulin protein was the same as in the parental strain with a single benA gene. Thus the level of beta-tubulin must be regulated. This was also true of transformants carrying an extra copy of the chimeric beta-tubulin gene. The total amount of beta-tubulin was the same as in the parental strain. Two-dimensional gel analysis showed that the endogenous benA22 and the introduced chimeric tubC gene contributed equally to the total beta-tubulin pool. The fact that one-half of the benA beta-tubulin could be replaced by tubC beta-tubulin with no effect on the growth of the cells suggests that the benA and tubC beta-tubulins are functionally interchangeable.

Rabbit synovial fibroblasts respond to changes in cell shape and cytoskeletal architecture by altering specific gene expression. We have tested the ability of acrylamide, a neurotoxin that alters the distribution of intermediate filaments in cultured PtK1 cells, to induce metalloprotease expression in synovial fibroblasts. Cells treated with 2-20 mM acrylamide for 5 to 24 h underwent shape changes similar to cells treated with the tumor promoter phorbol myristate acetate. Intermediate filaments visualized with anti-vimentin antibodies did not collapse into a perinuclear cap in these rounded cells, but were still present in the extended cell processes. Unexpectedly, when actin was visualized in acrylamide-treated cells, extensive dissociation and clumping of microfilaments was observed. Concentrations of acrylamide greater than 10 mM were cytotoxic, but cells recovered completely after 24 h incubation with 5 mM acrylamide. Like other agents that alter cell shape and actin distribution in synovial fibroblasts, acrylamide also induced expression of the secreted metalloprotease collagenase. Although some recent evidence suggests that acrylamide may be able to exert its collagenase-inducing effects extracellularly, perhaps through transmembrane matrix receptors, our observation that this neurotoxin dramatically alters protein synthesis in synovial fibroblasts suggests that direct effects on cell metabolism may also play a role in acute acrylamide intoxication.

T-1 induces modifications in the shape of the centrosome at division in fertilized eggs of the North American sea urchin, Lytechinus pictus. Phase contrast microscopy observations of mitotic apparatus isolated from T-1-treated (1.7-8.5 microM) eggs at first division shows that the centrosomes already begin to spread or to separate by prophase and that the mitotic spindle is barrel-shaped. When eggs are fertilized with sperm that have been preteated with T-1, the centrosomes become flattened; the spindles are of normal length. Immunofluorescence microscopy using an anti-centrosomal monoclonal antibody reveals that T-1 modifies the structure of the centrosome so that barrel-shaped spindles with broad centrosomes are observed at metaphase, rather than the expected focused poles and fusiform spindle. Higher concentrations of T-1 induce fragmentation of centrosomes, causing abnormal accumulation of microtubules in polar regions. These results indicate that T-1 directly alters centrosomal configuration from a compact structure to a flattened or a spread structure. T-1 can be classified as a new category of mitotic drugs that may prove valuable in dissecting the molecular nature of centrosomes.