Elucidation of the separate roles of myosins IIA and IIB during neurite outgrowth, adhesion and retraction.

The functions of nonmuscle myosin isoforms are key to an understanding of process outgrowth from nerve cells during animal development. Despite considerable structural similarity, myosin IIA and myosin IIB play distinct and complementary roles during the actin-based mechanisms of nerve process extension. An overview is given of evidence that implicates myosin IIB as the motor essential for nerve process outgrowth and myosin IIA both as the motor required to maintain cell adhesion to the substrate as well as the motor required to power retraction of the nerve cell process. These actions are placed in context within a model for nerve process extension that is consistent with many observations in the literature and provides testable hypotheses regarding possible roles for these nonmuscle myosin motors. The relevance of a fundamental understanding of the mechanisms underpinning nerve cell process extension to the application of nanotechnology in this area is also discussed.

Separate but linked functions of conventional myosins modulate adhesion and neurite outgrowth.

The potential functional diversity of closely related myosin isoforms found in eukaryotic cells is not yet understood in detail. We have previously provided evidence from functional knockouts of Neuro-2A neuroblastoma cells that myosin IIB is essential for neurite outgrowth. Here we investigate the role of non-muscle myosin IIA in the same cell line. We show that suppression of myosin IIA transcript and protein expression, brought about through exposure to isoform-specific antisense oligonucleotides, caused a rearrangement of the actin cytoskeleton and loss of cell adhesion. This also led to disruption of focal contacts, as evidenced by coincident reduction in paxillin and vinculin immunofluorescence, but did not diminish transcript expression. All effects were fully reversible. Before myosin IIA antisense-induced detachment, neurite outgrowth remained unaffected. By contrast, antisense oligonucleotides directed against myosin IIB transcripts had no effect on adhesion but severely attenuated neurite outgrowth. We infer that the two main isoforms of neuronal conventional myosin, myosins IIA and IIB, have separate but linked functions during neuronal adhesion and neurite outgrowth.

A conventional myosin motor drives neurite outgrowth.

Neuritic outgrowth is a striking example of directed motility, powered through the actions of molecular motors. Members of the myosin superfamily of actin-associated motors have been implicated in this complex process. Although conventional myosin II is known to be present in neurons, where it is localized at the leading edge of growth cones and in the cell cortex close to the plasma membrane, its functional involvement in growth cone motility has remained unproven. Here, we show that antisense oligodeoxyribonucleotides, complementary to a specific isoform of conventional myosin (myosin IIB), attenuate filopodial extension whereas sense and scrambled control oligodeoxyribonucleotides have no effect. Attenuation is shown to be reversible, neurite outgrowth being restored after cessation of the antisense regimen. Myosin IIB mRNA was present during active neurite extension, but levels were minimal in phenotypically rounded cells before neurite outgrowth and message levels decreased during antisense treatment. By contrast, the myosin IIA isoform is shown to be expressed constitutively both before and during neurite outgrowth and throughout exposure to myosin IIB antisense oligodeoxyribonucleotides. These results provide direct evidence that a conventional two-headed myosin is required for growth cone motility and is responsible, at least in part, for driving neuritic process outgrowth.

Temperature effects on mammalian muscle contraction.

Effects of temperature (range 37 degrees C to 8 degrees C) on isometric twitch and tetanic tensions of mammalian (rat) fast and slow muscles are re-examined. The twitch tension of a fast muscle increased in cooling it 20 degrees C but decreased on further cooling, whereas that of a slow muscle decreased monotonically with cooling. The cooling-potentiation in the fast muscle was evident within a week after birth but the cooling-depression in the slow muscle was not established until about the fourth week. The maximal tetanic tension in either muscle, and at all ages, decreased 10-20% in cooling to 25 degrees C but decreased more markedly in cooling below 20 degrees C; the tension at 10 degrees C was 40-50% of that at 35 degrees C. Preliminary observations made on fast muscle showed that the apparent stiffness/tension ratio was higher at low temperatures. It is postulated that the cooling depression of tetanic tension in mammalian muscle may be due to a direct effect on cross-bridges, whereas twitch tension variation may represent a net outcome of the same effect and the cooling-increase in the calcium sensitivity of myofibrillar activation.

Temperature dependence of contraction characteristics in developing rat muscles.

Contractions of rat extensor digitorum longus (EDL, a fast muscle) and soleus (SOL, a slow muscle) muscles of different ages (1-4 weeks) were recorded in vitro with direct stimulation and at different temperatures (range 35-10 degrees C). Twitch tension in 4-week-old EDL muscle increased in cooling from 35 to 20 degrees C (cooling potentiation); the tension decreased in further cooling below 20 degrees C. This pattern of temperature dependence of twitch tension was seen in fast muscles of all ages (1-4 weeks). Twitch tension in 4-week-old SOL muscle decreased monotonically in cooling from 35 to 10 degrees C (cooling depression). This pattern of cooling depression was not clearly evident in younger SOL muscles. There was a marked hysteresis in the temperature dependence of twitch tension in the 1-week-old SOL muscles. Tetanic tension was depressed by low temperature in both EDL and SOL muscles at 1 week and at 4 weeks of age. Results show that the processes concerned with contractile activation are nearly fully developed in the fast muscle fibers at an early age (1 week), whereas they develop later in the slow muscle fibers.

Temperature-dependent transitions in isometric contractions of rat muscle.

The effect of temperature on tetanic tension development was examined in extensor digitorum longus (fast-twitch) and soleus (slow-twitch) muscles of the rat, in vitro and with direct stimulation. The temperature range was from 35 to 10 degrees C. 2. The maximum tetanic tension decreased slightly on cooling from 35 to 25 degrees C. Cooling below 20 degrees C resulted in a marked depression of tetanic tension. The results were similar in the two muscles. 3. Analysis (in the form of Arrhenius plots) of the rate of tetanic tension development and relaxation clearly showed the occurrence of two phases in their temperature dependence, due to an increased temperature sensitivity below about 25 degrees C. Arrhenius activation energy estimates for temperatures lower than 21 degrees C were around twice as high as those for temperatures higher than 24 degrees C in both muscles.