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The effects of the Ca(2+)/H(+) exchanger A23187 and the K(+)/H(+) exchanger nigericin, the electrogenic membrane-potential depleters valinomycin and CCCP, and the calcium channel blockers ruthenium red, nifedipine, and nitrendipine on the apical growth of Phycomyces blakesleeanus were analyzed. While all of the compounds inhibited the growth of germlings in liquid medium, the Ca(2+) channel blockers were the least effective. Chitin synthesis in vivo was also sensitive to the inhibitors; here again, the calcium channel blockers were less efficient, and their effect occurred after a lag phase, in contrast to the electroneutral ionophores whose effects were immediate. The ionophores rapidly inhibited protein secretion, and reduced the number of secretory vesicles and chitosomes in the hyphal apex of P. blakesleeanus. The results suggest that not only tip-to-base calcium gradients but also transmembrane ionic gradients and membrane potential have a role in the apical growth of P. blakesleeanus. They are probably involved in the formation, migration, and/or fusion with the plasmalemma of secretory vesicles and chitosomes.

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Addition of cycloheximide rapidly inhibited protein synthesis in Phycomyces blakesleeanus. In contrast, chitin biosynthesis decreased with biphasic kinetics displaying a slow and a rapid decay phases. Electron microscopic studies revealed a decrease in the number of apical vesicles and chitosomes after cycloheximide addition; and no change in wall thickness. It is proposed that the slow phase of decay in chitin biosynthesis represents the exhaustion of the pool of chitosomes which transport the chitin synthase necessary to maintain apical wall growth; whereas the second one corresponds to inactivation of the enzyme, which is short lived in vivo. Data also rule out a change in the polarization of wall synthesis induced by cycloheximide, as suggested in other systems.

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The sporangiophores of the zygomycete fungus Phycomyces blakesleeanus contain octahedral crystals with diameters of up to 5 micrometers in their vacuole. The crystals are associated with the intracellular membrane system. In tilted or horizontally placed sporangiophores, the crystals sediment to the respective lower face of the vacuole with a velocity of up to 100 micrometers per minute. The sedimentation is completed within about 2 minutes, well within the latency period for the negative gravitropic response of Phycomyces. Crystal-lacking mutant strains display a smaller maximal bending angle and a reduced gravitropic bending rate in comparison to the wild type. We therefore conclude that the crystals serve as statoliths for gravitropism in Phycomyces.

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An increasing number of studies on tropic responses includes the genetic analysis of mutants defective in these morphogenetic processes. This review collates the information and discusses the implications of this approach to such studies. The review is organized on a systematic basis because most genetic analyses are insufficiently complete for general principles to have emerged. The most advanced analyses are those of lower eukaryotes because of their haploidy and the ease with which they can be manipulated in vitro. The extensive studies of phototropism, gravitropism and autochemotropism in the fungus Phycomyces blakesleeanus and of phototropism, polarotropism and gravitropism in the moss Physcomitrella patens are reviewed. In comparison with these studies, the genetic analysis of tropic responses in particular species of flowering plants is more limited. However, comparative physiological and ultra-structural studies of individual mutant and wild-type strains have been performed for a number of species. These results are discussed with particular regard to their support for established hypotheses.

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Stability of chitin synthetase in cell-free extracts from mycelial fungi was markedly improved by the presence of sucrose in the homogenization media. Breakage of mycelium in sucrose-containing buffer yielded enzyme preparations from which chitosomal chitin synthetase could be purified by a procedure involving ammonium sulfate precipitation, gel filtration and centrifugation in sucrose density gradients. Purified chitosomes catalyzed the synthesis of chitin microfibrils in vitro upon incubation with substrate and activators. Chitosomal chitin synthetase from the filamentous form of M. rouxii was similar to the enzyme from yeast cells, except for the poorer stability and diminished sensitivity to GlcNAc activation of the former.

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The gravitropism of a mature stage IV Phycomyces sporangiophore has a shorter and more uniform latency if the sporangiophore is exposed horizontally to gravity during its earlier development (stage II and stage III). This early exposure to an altered gravitational orientation causes the sporangiophore to develop a gravireceptor as it matures to stage IV and resumes elongation. A technique has been developed to observe the spatial relationship between the vacuole and the protoplasm of a living sporangiophore and to show the reorganization caused by this exposure to altered gravity. Possible gravireceptor mechanisms are discussed.

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Using freeze-fracture method it is shown that the direction of the fracture plane is a sensitive indicator of the membrane structural organization. A decrease in the proportion of longitudinal fractures is noted upon the membrane rearrangement presumably associated with changes in the lipid phase, and, on the contrary, an increase in the proportion of these fractures is observed upon some actions resulting, presumably, in changes of the protein phase of membranes.

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The surface layer of the cell wall of the sporangia of Albugo candida and of the sporangiophores of Phycomyces blakesleeanus was composed of a series of lamellae. The evidence from freeze-fracture, freeze-etch, and single-stage replicas indicated that the lamellae are organized as bilayers, an organization associated with the presence of lipids. The role of these lamellae in dispersibility and resistance is discussed.

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The plasma membrane of sporangiospores (spores) of Phycomyces blakeleeanus has distinct structural features that distinguish it from the plasma membrane of the sporangiophores. The plasma membrane of the spores is, however, derived from that of the sporangiophore. A study of sequential development of spores shows that the mass of cytoplasm in a sporangial initial is cleavaged by proliferation and subsequent anastomosis and membrane fusion of the clevage vesicles. The plasma membrane of the cleavage vesicles has a reversed curvature (inside out) to that of the sporangial plasma membrane. Otherwise, the distribution of intercalated membranous particles (Imp) on the fractured faces is similar to that of the plasma membrane of the sporangium. Lather, as a result of fusion of cleavage vesicles, curvature of the plasma membrane in the spore initials returns to their original state. In the processes of spore maturation, a new type of structural feature appears in the plasma membrane, which is not present either in the plasma membrane of spore initials or in the plasma membrane of sporangiophores or mycelia. This feature often is seen as large particles (30--35 nm) which are randomly distributed on the exoplasmic face (EF) with corresponding depressions on the plasmic half (PF) in freeze-fractured replicas. They increase in number with increasing spore maturity. These particles are, in fact, the inward protrusions of the plasma membrane of the spore. A change in the number of 5--8 nm intramembranous particles on the fractured faces is also noted during spor maturation, with an increase on the EF face and a corresponding decrease on the PF face. This alteration may result from vertical movement of Imp or from a change in the fracture plane as a consequence of alteration in the lipid content during maturation.

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Effect of cyclic AMP (cAMP) on Phycomyces blakesleeanus was studied by growing sporangiospores on glucose-asparagine agar or liquid medium containing three different levels of cAMP (10, 20 and 40 micronM) in addition to the control (no cAMP added). The response of Phycomyces to the exogenous cAMP concentration in the medium is as follows: (1) the time required for germ tube emergence is reduced; (2) the diameter of the mycelium is increased (sometimes more than 10 times) and frequency of branching is also increased; (3) the cell wall of the mycelium is thickened (in some cases more than 5 times); (4) the glycogen in the cytoplasm is decreased as visualized in thin sections and also demonstrated in biochemical quantitation; and (5) the distribution of intercalated membranous particles (Imp) on plasma membrane is altered and this can be easily detected in freeze-fractured replica. Such a change in Imp is seen in the formation of small clusters of aggregated particles on the plasmic half (PF) and craters on the complementary exoplasmic half (EF) of the plasma membrane. Although the mechanism of cAMP action requires further exploration, it is possible that the addition of cAMP to the culture medium leads to degradation of glycogen and enhancement of chitin synthesis since the cell wall is largely composed of chitin. The alteration in Imp may be related to a change in the activity of chitin synthetase which is a plasma membrane-bound enzyme.

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The multinucleate state of the vegetative spores of Phycomyces blakesleeanus arises as a consequence of cleavage of cytoplasm containing a variable number (1-6) of pre-existing nuclei. No nuclear division or incorporation of 3H-thymidine was detected during maturation of the spores.

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Part I. Migration and Diffusion of Graviceptors: The physical action of gravitational and inertial forces on graviceptors is considered. The motion of graviceptors as influenced by physical dimensions, density, electric charge, composition of the suspending medium and flow variables is demonstrated. Part II. Observations on Geotropism in Phycomyces blakesleeanus: Mutants of Phycomyces blakesleeanus exhibit strikingly different rates of geotropic response. It is shown that Phycomyces grown in the dark lack normal geotropic responses: pre-exposure to light is necessary for the synthesis of structures responsible for geotropism. A physical model is presented that may account for some of the geotropic phenomena observed in Phycomyces.

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