Irradiations of rabbit myofibrils with an ultraviolet microbeam. II. Phalloidin protects actin in solution but not in myofibrils from depolymerization by ultraviolet light

We tested whether phalloidin protects actin in myofibrils from depolymerization by ultraviolet light (UV). I bands in glycerinated rabbit psoas myofibrils were irradiated with a UV microbeam in the presence and absence of phalloidin. We used the retention of contractility of the irradiated I band as the assay for protection of actin by phalloidin, since previous experiments indicated that UV blocks contraction of an irradiated I band by depolymerizing the thin filaments. The I bands of myofibrils incubated in phalloidin were as sensitive to UV as control I bands, indicating that phalloidin did not protect the thin filaments. However, phalloidin did protect F-actin in solution from depolymerization by UV. This apparent contradiction between F-actin in myofibrils and F-actin in solution was resolved by observing unirradiated myofibrils that were stained with rhodamine-phalloidin. It was found that phalloidin does not bind uniformly to the thin filaments, though as the fluorescence image is observed over time the staining pattern changes until it does appear to bind uniformly. We conclude that phalloidin does not protect F-actin in myofibrils from depolymerization by UV because it does not bind uniformly to the filaments.     

Aggregation of isolated myofibrils stimulated by their contraction. I. Origin of the second phase of optical changes during myofibril contraction

Aggregation of contracted myofibrils was shown to be responsible for spontaneous: slow reduction of optical density of myofibril suspension on the final stage of their contraction. This effect faded with an increase in light wavelength, with increased angle of view of the photocell and diminished sizes of myofibrils.     

Observations on the actin content of the rabbit myofibril

1. On extraction of whole muscle by the procedure of Hasselbach & Schneider (1951), the amount of actin that passes into solution seems to account for little more than 10% of the protein content of the myofibrils. 2. Extraction of isolated myofibrils with suitable media that allow identification and estimation of dissolved proteins seems to give about the same yield of actin (10-13% of the total). 3. A comparatively large residue of myofibrillar components remains after extraction. The amount of actin present in the residue can be only hypothetical.     

Effects of microbeam light on growth and phototropism ofPilobolus crystallinus sporangiophores

A small blue-light beam (50 μm in diam) was used to examine light-growth response and phototropism inPilobolus crystallinus sporangiophores. Continuous irradiation by microbeam of a region 100–150 μm from the apex promoted the growth of a dark-adapted sporangiophore for about 15 min after a lag period of 1–2 min. After the promotion, the growth rate fell below that before the irradiation. Irradiation of the apex of sporangiophore slightly promoted the growth but strongly inhibited the growth after the promotion. A smaller light beam (10 μm in diam) applied continuously at grazing incidence along one side of the sporangiophore caused bending toward the shaded side, implying that the irradiated side grew more rapidly than the shaded side and that the lens effect is involved in the phototropism of young sporangiophores ofP. crystallinus. The involvement of the lens effect was confirmed by the fact that a carotenoid-less mutant was 1.5–2 times more sensitive to unilateral blue light than the wild type, probably because of a smaller intracellular light attenuation during passage through the mutant cell.     

Rhodamine-phalloidin and anti-tubulin antibody staining of spindle fibres that were irradiated with an ultraviolet microbeam

Summary We irradiated chromosomal spindle fibres in crane-fly spermatocytes with an ultraviolet microbeam of 270 nm wavelength light with total energies near those that cause actin filaments in myofibrils to depolymerize; after irradiation we stained the cells with rhodamine-labelled phalloidin and with anti-tubulin antibodies. In some cells, the irradiation reduced both phalloidin and tubulin staining of the chromosomal spindle fibres; in other cells, the irradiations reduced phalloidin staining but not tubulin staining; in yet other cells, the irradiations reduced tubulin staining but not phalloidin staining. In all irradiated cells in which phalloidin staining was reduced in the irradiated areas phalloidin staining also was reduced poleward from the irradiated areas. These results show that phalloidin staining of chromosomal spindle fibres is not dependent on the presence of kinetochore microtubules, and, therefore, that actin filaments are present in the spindle fibres in vivo. We suggest that actin filaments present in spindle fibres in vivo may be involved in causing chromosome movements during anaphase.     

Effect of calcium ion on the interaction of aldolase with rabbit muscle myofibrils

A partition equilibrium study has shown calcium ion to be a noncompetitive inhibitor of aldolase adsorption by rabbit muscle myofibrils. This inhibition is interpreted quantitatively in terms of a 10-fold decrease in the intrinsic association constant for the aldolase-myofibril interaction upon Ca2+ binding to either or both of the low-affinity troponin sites associated with regulation of muscle contraction.     

Inactivation of transforming DNA by ultraviolet light. I. Near-UV action spectrum for marker inactivation

The storage effect is defined as an increase of mutational damage after cessation of treatment. It differs from other kinds of delayed effect (replication errors, replicating instabilities) in not requiring replication of DNA. In Neurospora ad3A 38701 inos 37401 diepoxybutane (DEB) yields a storage effect for adenine reversions, but none for inositol reversions. The storage effect takes place in treated washed spores that are sedimented in a centrifuge tube, but not in spores that are agitated in water. Under the latter conditions, response to storage is gradually lost. The storage effect can be imitated by administering very small amounts of DEB to cells that had been previously treated with a moderately high dose (booster effect). During post-treatment shaking in water, responses to booster and to storage conditions disappear together; response to booster disappears at the same rate in spores that are sedimented in centrifuge tubes. No mutagenic action could be detected in eluates from heavily treated cells. We have concluded that treatment with DEB sensitizes the conidia to further small doses of DEB whether these are administered extraneously as booster or present intracellularly during storage. Sensitization is lost in the course of a few hours in shaken as well as in sedimented spores. Thus, while the storage effect is due to traces of mutagen, its gradual disappearance after treatment is not due to loss of these traces.Correlated with the ability to yield a storage effect, and probably part of the storage effect, is the response to temperature between treatment and plating. Conidia that can give a storage effect yield fewer mutations when spread on cold agar than when inplated into warm agar or heat-shocked before spreading; the excess of mutation under the latter two conditions forms part of the final storage effect. The true base line for calculation of the storage effect is therefore mutation frequency among spread spores.For DEB as mutagen, response to storage by the adenine locus and lack of response by the inositol locus are correlated with the responses of these two loci to dose of DEB and to combination treatment of DEB with UV, or DEB with nitrous acid (NA). This makes it possible to fit all observations into the picture of a general hypothesis on the cellular effects of DEB. Because of the differential response of the two loci, storage and plating procedures offer two additional means for manipulating specificity in this system.     

Effects of ultraviolet light on the in vitro assembly of microtubules

Exposure of microtubular protein to ultraviolet light inhibits its assembly into morphologically normal microtubules. This effect appeared to result primarily from damage to the tubulin dimers. The damage consisted of a conformational change, a loss of two free sulfhydryl groups, a production of higher molecular weight cross-linked species, and the formation of aggregated amorphous material upon polymerization.     

Mechanisms of chromosomal aberration production I. Aberration induction by ultraviolet light

We have studied chromosomal aberration production in V-79 Chinese hamster tissue culture cells by UV light administered during the post-DNA-synthetic G₂ phase of the cell cycle. The treatment produced achromatic lesions and some chromatid deletions in the first post-irradiation mitosis, but no isochromatid deletions or chromatid exchange aberrations. In contrast, when G₂ UV-irradiated cells were examined in their second post-irradiation mitosis, there were significant yields of chromatid-type aberrations of all types, including isochromatid deletions and chromatid exchanges.

We have earlier reported²¹ that UV-irradiation during the pre-DNA-synthetic G₁ phase of the cell cycle induces only chromatid aberrations and also that most chromosomal aberration production by UV in G₁ can be photoreactivated in cells possessing the photoreactivating enzyme. We present here a model for chromosomal aberration production by UV. In the model all aberration production is enzymatically mediated, a consequence of the functioning of known molecular repair mechanisms. The important elements in the model are the following:

1. (1) The vertebrate chromosome is mononeme; i.e., contains but a single DNA double helix during the prereplication G₁ phase of the cell cycle.

2. (2) The UV-induced DNA lesion leading to the production of most aberrations is the cyclobutane dimer between adjacent pyrimidines in one polynucleotide strand.

3. (3) Single chain breaks appear at metaphase as achromatic lesions.

4. (4) Dimer removal sometimes leaves unrepaired single chain gaps, possibly as a result of incomplete excision repair.

5. (5) The single-stranded DNA opposite a single chain gap can be cleaved by a single-strand DNAase.

6. (6) Gaps are left in newly synthesized DNA polynucleotide chains opposite defective template chains (i.e., opposite dimers and chain breaks).

7. (7) Double-strand breaks present following local DNA replication may “spread” to the other chromatid by a recombinational process between template and new polynucleotide chains, one from each of the homologous double helices.

The model predicts the occurrence of isoachromatic lesions and of chromatid deletions paired (isolocus) with achromatic lesions. Though often not reported, both do, in fact, occur. In addition, the model accounts for the phenomenon of sister-chromatid exchange as a manifestation of a recombinational, or post-replication, repair mechanism. Finally, the model offers a simple interpretation of chromosomal aberration production by a variety of chemical agents.     

Induction of mutations in the zebrafish with ultraviolet light.

Recessive lethal germline and specific locus somatic mutations were induced efficiently in the zebrafish by exposure of mature sperm to UV light. Mutagenesis of sperm yielded mosaic individuals: clones bearing novel mutations represented approximately 12-25% of the haploid germ cells and 25-50% of the somatic tissue. Simple methods are described for the reliable identification and propagation of newly arising developmental mutations in zebrafish.     

Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers.

We have investigated (a) effects of varying proton concentration on force and shortening velocity of glycerinated muscle fibers, (b) differences between these effects on fibers from psoas (fast) and soleus (slow) muscles, possibly due to differences in the actomyosin ATPase kinetic cycles, and (c) whether changes in intracellular pH explain altered contractility typically associated with prolonged excitation of fast, glycolytic muscle. The pH range was chosen to cover the physiological pH range (6.0-7.5) as well as pH 8.0, which has often been used for in vitro measurements of myosin ATPase activity. Steady-state isometric force increased monotonically (by about threefold) as pH was increased from pH 6.0; force in soleus (slow) fibers was less affected by pH than in psoas (fast) fibers. For both fiber types, the velocity of unloaded shortening was maximum near resting intracellular pH in vivo and was decreased at acid pH (by about one-half). At pH 6.0, force increased when the pH buffer concentration was decreased from 100 mM, as predicted by inadequate pH buffering and pH heterogeneity in the fiber. This heterogeneity was modeled by net proton consumption within the fiber, due to production by the actomyosin ATPase coupled to consumption by the creatine kinase reaction, with replenishment by diffusion of protons in equilibrium with a mobile buffer. Lactate anion had little mechanical effect. Inorganic phosphate (15 mM total) had an additive effect of depressing force that was similar at pH 7.1 and 6.0. By directly affecting the actomyosin interaction, decreased pH is at least partly responsible for the observed decreases in force and velocity in stimulated muscle with sufficient glycolytic capacity to decrease pH.