SUMMARYThe Hippo signaling pathway plays an important role in regulation of cell proliferation. Cell density regulates the Hippo pathway in cultured cells; however, the mechanism by which cells detect density remains unclear. In this study, we demonstrated that changes in cell morphology are a key factor. Morphological manipulation of single cells without cell-cell contact resulted in flat spread or round compact cells with nuclear or cytoplasmic Yap, respectively. Stress fibers increased in response to expanded cell areas, and F-actin regulated Yap downstream of cell morphology. Cell morphology-and F-actin-regulated phosphorylation of Yap, and the effects of F-actin were suppressed by modulation of Lats. Our results suggest that cell morphology is an important factor in the regulation of the Hippo pathway, which is mediated by stress fibers consisting of F-actin acting upstream of, or on Lats, and that cells can detect density through their resulting morphology. This cell morphology (stress-fiber)-mediated mechanism probably cooperates with a cell-cell contact (adhesion)-mediated mechanism involving the Hippo pathway to achieve density-dependent control of cell proliferation.

The Hippo tumour suppressor pathway is a conserved signalling pathway that controls organ size. The core of the Hpo pathway is a kinase cascade, which in Drosophila involves the Hpo and Warts kinases that negatively regulate the activity of the transcriptional coactivator Yorkie. Although several additional components of the Hippo pathway have been discovered, the inputs that regulate Hippo signalling are not fully understood. Here, we report that induction of extra F‐actin formation, by loss of Capping proteins A or B, or caused by overexpression of an activated version of the formin Diaphanous, induced strong overgrowth in Drosophila imaginal discs through modulating the activity of the Hippo pathway. Importantly, loss of Capping proteins and Diaphanous overexpression did not significantly affect cell polarity and other signalling pathways, including Hedgehog and Decapentaplegic signalling. The interaction between F‐actin and Hpo signalling is evolutionarily conserved, as the activity of the mammalian Yorkie‐orthologue Yap is modulated by changes in F‐actin. Thus, regulators of F‐actin, and in particular Capping proteins, are essential for proper growth control by affecting Hippo signalling.

Age-related but not artificially induced muscle fiber atrophy has been shown to occur without any decrease in myonuclear number, although these results remain controversial. The present study was carried out to clarify whether age difference affects the degree of decrease in myonuclear number occurring with denervation-induced fiber atrophy. After denervation of 3-wk-old (young) and 4-mo-old (mature) mice, single myofibers were isolated from the plantaris muscles by alkali maceration, and their fiber cross-sectional area (CSA), myonuclear number, and cytoplasm-to-myonucleus (C/N) ratios were analyzed. Fiber CSA in both young and mature mice decreased with denervation. Myonuclear number decreased in young mice 5 and 10 days after denervation but was unchanged in mature mice 10 and 120 days after denervation. C/N ratio decreased in mature mice but was unchanged in denervated young mice. These results suggest that age differences affect the degree of decrease of myonuclear number with denervation and that fiber cytoplasmic atrophy may occur without decrease in myonuclear number.

Two novel oxazolomycin isomers, oxazolomycins B (2) and C (3), were isolated from the fermentation broth of an oxazolomycin-producing strain, Streptomyces albus JA3453. Both compounds are geometrical isomers of oxazolomycin (1), the configurations of their triene moieties being (4'E, 6'E, 8'E) (2) and (4'Z, 6'E, 8'E) (3) while that of oxazolomycin (1) is (4'Z, 6'Z, 8'E). Compounds 2 and 3 exhibited potent inhibitory activity against crown gall formation with the same MIC (0.8 microgram/disk) as oxazolomycin. Compounds 2 and 3 showed no antibacterial activity against Agrobacterium tumefaciens, in contrast to oxazolomycin which has specific anti-A. tumefaciens activity.

Skeletal muscle can show change in its tissue size in response to functional demand, mediated by changes in myofiber size. Although the mechanism underlying this change is still unclear, the concept of DNA unit as proposed by Cheek [1] may help to illustrate the subcellular regulatory mechanism. According to this concept, the DNA unit (protein/DNA) is constant; therefore, increments in DNA owing to the fusion of myoblast or satellite cells into myofibers drive myofiber cytoplasmic enlargement. Indeed, many studies have shown that, in various situations, changes in myofiber size appear to be driven by changes in DNA content (i.e., changes in myonuclear number) within the myofiber.In overload-induced fiber hypertrophy [2] or during recovery from fiber atrophy [3], the myonuclear number increases proportionately. Further, X-ray irradiation of the muscle prevents fiber hypertrophy owing to the blockage of satellite cell incorporation into the myofiber [4,5]. These studies strongly suggest that the increase in myonuclear number is essential for fiber hypertrophy. Myofibers also undergo fiber atrophy accompanied by a decrease in myonuclear number under a range of conditions, including hind limb suspension [6], spinal cord isolation [2], and spaceflight [7,8]. In particular, long-term denervation induces both marked fiber atrophy and a decrease in myonuclear number [9,10]. However, we have clearly shown that mature mice undergo marked fiber atrophy without any decrease in myonuclear number, even after 120-d denervation [11], indicating that a reduction in myonuclear number is not always essential for fiber atrophy.Our new finding that no reduction in myonuclear number occurs after long-term denervation was made partly due to the methodological advantages of our myofiber isolation technique. In a previous study, we developed a novel fiber isolation method and observed Japanese Journal of Physiology, 53, 145-150, 2003 Key words: myofiber size, myonuclear number, lifespan, normal muscle. Abstract:It has been shown that changes in the nuclear number in myofibers are synchronized with myofiber size. Therefore, under some conditions, the myonuclear number is thought to be a determinant factor of myofiber size. However, we have clearly shown that denervation-induced fiber atrophy occurs without any decrease in myonuclear number, indicating that the myonuclear number is not always an important determinant factor of myofiber size. However, this was an event found under experimental conditions. In the present study, we examined the morphological features of single myofibers under normal conditions throughout the lifespan of normal mice. We discovered that the C/N ratio (cell volume/nucleus) greatly increases during the growth period and clearly decreases during the aging period. From 5 weeks to 6 months old, the myofibers undergo fiber hypertrophy accompanied by a decrease in myonuclear number. In muscle at 18 months, we found no correlation between myonuclear number and fiber cross-sectional area. These results suggest that,...