Nurit Kalderon

Myoblast fusion has been studied in cultures of chick embryonic muscle utilizing ultrastructural techniques. The multinucleated muscle cells (myotubes) are generated by the fusion of two plasma membranes from adjacent cells, apparently by forming a single bilayer that is particle-free in freeze-fracture replicas. This single bilayer subsequently collapses, and cytoplasmic continuity is established between the cells. The fusion between the two plasma membranes appears to take place primarily within particle-free domains (probably phospholipid enriched), and cytoplasmic unilamellar, particle-free vesicles are occasionally associated with these regions. These vesicles structurally resemble phospholipid vesicles (liposomes). They are present in normal myoblasts, but they are absent in certain fusion-arrested myoblast popluations, such as those treated with either 5-bromo-deoxyuridine (BUdR), cycloheximide (CHX), or pospholipase C (PLC). The unilamellar, particle-free vesicles are present in close proximity to the plasma membranes, and physical contact is observed frequently between the vesicle membrane and the plasma membrane. The regions of vesicle membrane-plasma membrane interaction are characteristically free of intramembrane particles. A model for myoblast fusion is presented that is based onan interpretation of these observations. This model suggests that the cytoplasmic vesicles initiate the generation of particle-depleted membrane domains, both being essential components in the fusion process.

Cell-to-cell communication was characterized in prefusion chick embryo myoblast cultures, and it was determined that the prefusion myoblasts can interact via gap junctions, ionic coupling, and metabolic coupling. The biological relevance of this communication was supported by the detection of gap junctions between myoblasts in embryonic muscle. Communication was also examined in fusion-arrested cultures to determine its potential relationship to fusion competency. In cultures that were fusion arrested by treatment with either 1.8 mM ethyleneglycolbis-(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), 3.3 X 10(-6) M 5-bromodeoxyuridine (BUdR), or 1 microgram/ml cycloheximide (CHX), both gap junctions and ionic coupling were present. Therefore, it is possible to conclude that cell communication is not a sufficient property by itself, to generate fusion between myob-asts. The potential role of communication in myogenesis is discusssed with respect to these observations.

The role of the serum proteolytic system plasminogen/plasminogen activator as a biochemical tool used by the glia or neurons, or both, for maintaining their temporary and flexible cellular interactions during histogenesis of the nervous system is under study. The present report identifies a glia cell type, the Schwann cell, as one of the cellular components of the nervous system that uses extracellular proteolysis at the time of the tissue construction. Purified dividing mouse Schwann cells in culture produce extracellular plasminogen activator. The levels of extracellular plasminogen-activator activity, as measured by the biochemical fibrinolytic assay, were directly related to the proliferation rates of the Schwann cells. The cellular plasminogen-activator specific activity at the maximal rate of cell proliferation was 3-4 times higher than that of the cells at low rate of mitosis. It is concluded that plasminogen-activator activity is expressed predominantly by the proliferating Schwann cell populations, suggesting that the extracellular proteolysis is used by the tissue at those stages when the cells divide.

Abstract Plasminogen activator (PA) is a key enzyme in control of the cascade of extracellular proteolytic activities, proteases that degrade the extracellular components. Mammalian cells produce two molecular forms of PA, the urokinase type (u‐PA) and the tissue type (t‐PA); the u‐PA type enzyme regulates cell migration/invasion and related tissue plasticity events. Thus, these plasticity properties of cells are defined by their PAs' biochemical profiles. The capacity of the differentiating glial cells of the central nervous system (CNS) to express and regulate the two types of PA activities has been examined as a function of cell age in culture. Results of the study suggest that only the immature astrocyte is endowed with these plasticity properties. Differentiating heterogeneous rat glial cells in culture express PA activity. Astroglia were identified as the primary source for the glial PA activity, as no PA activity was detected in the purified oligodendroglia. Cellular PA activity levels of differentiating rat and mouse astroglia are developmentally regulated. The specific activity of PA reached its highest level in rat astroglia at a cell age corresponding to 20–32 postnatal days (P20–P32) and in mouse astroglia at P8–P14; thereafter, this declined (three‐ to fourfold decrease) within 2 weeks to a low value. At comparable ages (P0–P35), the magnitudes of the PA specific activities of the differentiating rat astroglia and of the developing cerebrum, the tissue from which these cells were purified, were similar. Differentiating rat astroglia produce u‐PA and t‐PA, the cellular content of both is developmentally regulated, and the u‐PA form is only found in the immature cells. u‐PA is the predominant form in the immature astrocyte until age P13. Both forms are found in cells at ages P14–P30, and at later stages u‐PA disappears while the t‐PA type persists as the sole form. After 3 more weeks neither of the PA types was detected. Astroglia express also PA inhibitory activity; the rat astroglial PA inhibitor (PAI) seemed to be identical to PAI‐1, one of the known types of PAIs. Stimulation of astroglial proliferation by their subculturing in contrast to Schwann cells did not lead to an increase; rather, beyond a certain cell age (P13) it resulted in a threefold irreversible decline in the PA specific activity of the daughter cells. It has been established that various biochemical properties of CNS mature glia appear on schedule with cell age in culture, thus defining “mature” glia in vitro. According to our study, among differentiating and “mature” astroglial cells the immature astrocytes appear to be the sole source of the u‐PA molecular form and of high levels of PA activity. Astroglia are equipped with these plasticity tools in a limited developmental period, and upon maturation lose them altogether. Based on the known role of u‐PA in control of tissue plasticity processes, it is proposed that astroglia have a major role in regulating certain plasticity and/or remodeling processes in the developing and adult CNS.