let-7 miRNA controls CED-7 homotypic adhesion and EFF-1–mediated axonal self-fusion to restore touch sensation following injury

Neuronal injury often leads to devastating consequences such as loss of senses or locomotion. Restoration of function after injury relies on whether the injured axons can find their target cells. Although fusion between injured proximal axon and distal fragment has been observed in many organisms, its functional significance is not clear. Here, using Caenorhabditis elegans mechanosensory neurons, we address this question. Using two femtosecond lasers simultaneously, we could scan and sever posterior lateral microtubule neurons [posterior lateral microtubules (PLMs)] on both sides of the worm. We showed that axotomy of both PLMs leads to a dramatic loss of posterior touch sensation. During the regenerative phase, only axons that fuse to their distal counterparts contribute to functional recovery. Loss of let-7 miRNA promotes functional restoration in both larval and adult stages. In the L4 stage, loss of let-7 increases fusion events by increasing the mRNA level of one of the cell-recognition molecules, CED-7. The ability to establish cytoplasmic continuity between the proximal and distal ends declines with age. Loss of let-7 overcomes this barrier by promoting axonal transport and enrichment of the EFF-1 fusogen at the growing tip of cut processes. Our data reveal the functional property of a regenerating neuron.

Physiological Significance of Network Organization in Fungi

ABSTRACTThe evolution of multicellularity has occurred in diverse lineages and in multiple ways among eukaryotic species. For plants and fungi, multicellular forms are derived from ancestors that failed to separate following cell division, thus retaining cytoplasmic continuity between the daughter cells. In networked organisms, such as filamentous fungi, cytoplasmic continuity facilitates the long-distance transport of resources without the elaboration of a separate vascular system. Nutrient translocation in fungi is essential for nutrient cycling in ecosystems, mycorrhizal symbioses, virulence, and substrate utilization. It has been proposed that an interconnected mycelial network influences resource translocation, but the theory has not been empirically tested. Here we show, by using mutants that disrupt network formation inNeurospora crassa(Δsomutant, no fusion; ΔPrm-1mutant, ∼50% fusion), that the translocation of labeled nutrients is adversely affected in homogeneous environments and is even more severely impacted in heterogeneous environments. We also show that the ability to share resources and genetic exchange between colonies (via hyphal fusion) is very limited in mature colonies, in contrast to in young colonies and germlings that readily share nutrients and genetic resources. The differences in genetic/resource sharing between young and mature colonies were associated with variations in colony architecture (hyphal differentiation/diameters, branching patterns, and angles). Thus, the ability to share resources and genetic material between colonies is developmentally regulated and is a function of the age of a colony. This study highlights the necessity of hyphal fusion for efficient nutrient translocation within anN. crassacolony but also shows that establishedN. crassacolonies do not share resources in a significant manner.

Genetic Analysis of Myoblast Fusion: blown fuse Is Required for Progression Beyond the Prefusion Complex

The events of myoblast fusion in Drosophila are dissected here by combining genetic analysis with light and electron microscopy. We describe a new and essential intermediate step in the process, the formation of a prefusion complex consisting of “paired vesicles.” These pairs of vesicles from different cells align with each other across apposed plasma membranes. This prefusion complex resolves into dense membrane plaques between apposed cells; these cells then establish cytoplasmic continuity by fusion of small areas of plasma membrane followed by vesiculation of apposed membranes. Different steps in this process are specifically blocked by mutations in four genes required for myoblast fusion. One of these genes, blown fuse, encodes a novel cytoplasmic protein expressed in unfused myoblasts that is essential for progression beyond the prefusion complex stage.

Sperm-egg fusion is the prelude to the initial Ca2+ increase at fertilization in the mouse

Fusion of sperm and egg plasma membranes is an early and essential event at fertilization but it is not known if it plays a part in the signal transduction mechanism that leads to the oscillations in the cytoplasmic free Ca2+ concentration ([Ca2+]i) that accompany mammalian egg activation. We have used two independent fluorescence methods and confocal microscopy to show that cytoplasmic continuity of egg and sperm precedes the onset of the first [Ca2+]i increase in mouse eggs. The Ca2+ indicator dye Ca2+-green dextran was microinjected and its transfer from egg to sperm was monitored. We found that it occurred before, and without a requirement for, any detectable [Ca2+]i increase in the egg. In separate experiments [Ca2+]i changes were recorded in populations of eggs, using fura red, and the eggs fixed at various times after some of the eggs had shown a [Ca2+]i transient. Fusion of the sperm and egg was then assessed by Hoechst dye transfer. All eggs that showed a [Ca2+]i increase had a fused sperm but more than half of the eggs contained a sperm but had not undergone a [Ca2+]i increase. These data indicate that sperm-egg fusion precedes [Ca2+]i changes and we estimate that the elapsed time between sperm-egg fusion and the onset of the [Ca2+li oscillations is 1–3 minutes. Finally, sperm-egg fusion was prevented by using low pH medium which reversibly prevented [Ca2+]i oscillations in eggs that had been inseminated. This was not due to disruption of signalling mechanisms, since [Ca2+]i changes still occurred if low pH was applied after the onset of oscillations at fertilization. [Ca2+]i changes also occurred in eggs in low pH in response to the muscarinic agonist carbachol. These data are consistent with the idea that the [Ca2+]i signals that occur in mammalian eggs at fertilization are initiated by events that are closely coupled to the fusion of the sperm and egg membranes.

The Mesenchyme-Like Layer of the Fiber Cells of Trichoplax adhaerens (Placozoa), a Syncytium

The fiber cells of the middle layer of Trichoplax adhaerens are interconnected by slender extensions. Newly formed connections after mechanical disruption of the tissue studied in ultrathin sections revealed cytoplasmic continuity between the cell bodies, suggesting a syncytial organisation of the fiber cell layer. The slender extensions connecting the cell bodies are traversed by microtubules and microfilaments. The structure of rare osmiophilic cell contacts suggests a stage in the fusion of adjacent cell membranes.

The ultrastructure of hyphal anastomoses between vegetatively compatible and incompatible virulent and hypovirulent strains of Cryphonectria parasitica

European hypovirulent (dsRNA-containing) Cryphonectria parasitica strain Ep-50 was paired individually with West Virginia virulent (dsRNA-free) strains Ep 15-7-7 (vegetatively compatible) and EP 7-5-1 (vegetatively incompatible) on cellophane membranes. Four to six hours after anastomoses formed, the strains were preserved using freeze-substitution and observed using transmission electron microscopy. Hyphal anastomoses between Ep-50 and Ep 15-7-7 showed complete cytoplasmic continuity, with microtubules and mitochondria extending through the fusion aperture. Spherical, membrane-bound virus-like particles, measuring 50–90 nm in diameter, were located in the Ep-50 hypha, the Ep 15-7-7 hypha, and the short anastomosis bridge between them. All anastomoses between the compatible strains involved a hyphal peg that grew toward a swelling that developed on the receiving hypha. Fusion took place between the swelling and the lateral wall of the peg. Anastomoses between the incompatible strains showed cellular collapse and cytoplasmic degeneration that extended away from the anastomosis area in hyphae of both strains. Because of this, vegetative incompatibility would seem to be a formidable barrier to hypovirulence conversion and biocontrol of C. parasitica. Key words: Endothia parasitica, hyphal fusion, virus-like particles, hypovirulence conversion.

The stomatal complex of Persea borbonia

The ultrastructure of the guard cell complex of Persea borbonia is described. The guard cells are embedded in the midregion of the ventral wall of subsidiary cells. The outer portions of the subsidiary cell wall is extensively thickened forming a domelike structure with a large slot opening projecting above the stomatal aperture. The outer walls of epidermal cells are also extensively thickened. The fused ventral cell walls have perforations resulting in cytoplasmic continuity between guard cells. The guard cells and subsidiary cells contain normal cytoplasmic constituents, including chloroplasts. Preliminary studies of three other Lauraceae species, Persea americana, Sassafras albidum, and Laurus nobilis, indicate a similarly structured stomatal complex.

Ultrastructure and cytochemistry of the tegument ofOrthocoelium scoliocoeliumandParamphistomum cervi(Trematoda: Digenea)

The tegument ofOrthocoelium scoliocoeliumandParamphistomum cerviwas examined using histochemical techniques and electron microscopy. On the basis of the distribution of acid and alkaline phosphatase (E.C. 3.1.3.2, E.C. 3.1.3.1), non-specific esterase (E.C. 3.1.1.1), cholinesterase (E.C. 3.1.1.7) and succinate dehydrogenase (E.C. 1.3.99.1) at light microscope level two distinct regions were recognized, an outer and an inner zone. Electron microscopy revealed that the tegument comprises an outer surface syncytium underlain by a thick subsyncytial zone and musculature. Deeper still occur the nucleated “tegumental cells”. The latter are in cytoplasmic continuity with the surface syncytium via vacuolated cytoplasmic trabeculae which traverse the muscle layers and the subsyncytial zone. Three types of tegumental cells each lacking mitochondria were observed. The T1cells synthesize discoid and electron dense T1bodies while T2cells produce oval and electron lucent T2bodies. The third type of tegumental cells apparently produce no secretory bodies and may represent an embryonic cell type. The surface syncytium contains T1and T2secretory bodies and is bounded apically by a plasma membrane invested externally by a fuzzy and filamentous glycocalyx. The surface syncytium lacks mitochondria and is traversed by infoldings of the basal plasma membrane. Beneath the surface syncytium the subsyncytial zone is largely comprised of fibrous interstitial material. This zone, which is particularly thick in the amphistomes, is traversed by trabeculae and extensions of underlying parenchymal cells which usually contain mitochondria and lysosomes. The subsyncytial zone overlies numerous circular and longitudinal muscle fibres. The absence of mitochondria and enzymes associated with active transport suggests that the amphistome tegument may be mainly specialized for protection of the worm against mechanical and chemical conditions prevailing in the rumen. Active uptake of nutrients is probably not a primary function.

Evidence for involvement of metalloendoproteases in a step in sea urchin gamete fusion.

Membrane fusion events are required in three steps in sea urchin fertilization: the acrosome reaction in sperm, fusion of the plasma membrane of acrosome-reacted sperm with the plasma membrane of the egg, and exocytosis of the contents of the egg cortical granules. We recently reported the involvement of a Zn2+-dependent metalloendoprotease in the acrosome reaction (Farach, H. C., D. I. Mundy, W. J. Strittmatter, and W. J. Lennarz. 1987. J. Biol. Chem. 262:5483-5487). In the current study, we investigated the possible involvement of metalloendoproteases in the two other fusion events of fertilization. The use of inhibitors of metalloendoproteases provided evidence that at least one of the fusion events subsequent to the acrosome reaction requires such enzymes. These inhibitors did not block the binding of sperm to egg or the process of cortical granule exocytosis. However, sperm-egg fusion, assayed by the ability of the bound sperm to establish cytoplasmic continuity with the egg, was inhibited by metalloendoprotease substrate. Thus, in addition to the acrosome reaction, an event in the gamete fusion process requires a metalloendoprotease.