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.

Regulation of postsynaptic structure and protein localization by the Rho-type guanine nucleotide exchange factor dPix.

Mutations in dpix were recovered from a large-scale screen in Drosophila for genes that control synaptic structure. dpix encodes dPix, a Rho-type guanine nucleotide exchange factor (RtGEF) homologous to mammalian Pix. Here we show that dPix plays a major role in regulating postsynaptic structure and protein localization at the Drosophila glutamatergic neuromuscular junction. dpix mutations lead to decreased synaptic levels of the PDZ protein Dlg, the cell adhesion molecule Fas II, and the glutamate receptor subunit GluRIIA, and to a complete reduction of the serine/threonine kinase Pak and the subsynaptic reticulum. The electrophysiology of these mutant synapses is nearly normal. Many, but not all, dpix defects are mediated through dPak, a member of the family of Cdc42/Rac1-activated kinases. Thus, a Rho-type GEF and Rho-type effector kinase regulate postsynaptic structure.

Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth.

The glutamatergic neuromuscular synapse in Drosophila forms and differentiates into distinct boutons in the embryo and grows by sprouting new boutons throughout larval life. We demonstrate that two axons form approximately 18 boutons on muscles 7 and 6 by hatching and grow to approximately 180 boutons by third instar. We further show that, after synapse formation, the homophilic cell adhesion molecule Fasciclin II (Fas II) is localized both pre- and postsynaptically where it controls synapse stabilization. In FasII null mutants, synapse formation is normal, but boutons then retract during larval development. Synapse elimination and resulting lethality are rescued by transgenes that drive Fas II expression both pre- and postsynaptically; driving Fas II expression on either side alone is insufficient. Fas II can also control synaptic growth; various FasII alleles lead to either an increase or decrease in sprouting, depending upon the level of Fas II.

Genetic dissection of structural and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity.

Increased neuronal activity (eag Shaker mutants) and cAMP concentration (dunce mutants) lead to increased synaptic structure and function at the Drosophila neuromuscular junction. Here, we show that the increase in synaptic growth is accompanied by an approximately 50% decrease in synaptic levels of the cell adhesion molecule Fasciclin II (Fas II). This decrease in Fas II is both necessary and sufficient for presynaptic sprouting; FasII mutants that decrease Fas II levels by approximately 50% lead to sprouting similar to eag Shaker and dunce, while transgenes that maintain synaptic Fas II levels suppress sprouting in eag Shaker and dunce. However, FasII mutants that cause a 50% increase in bouton number do not alter synaptic strength; rather, evoked release from single boutons has a reduced quantal content, suggesting that the wild-type amount of release machinery is distributed throughout more boutons.

Synaptic clustering of Fascilin II and Shaker: essential targeting sequences and role of Dlg.

Previous studies have shown that both the Fasciclin II (Fas II) cell adhesion molecule and the Shaker potassium channel are localized at the Drosophila neuromuscular junction, where they function in the growth and plasticity of the synapse. Here, we use the GAL4-UAS system to drive expression of the chimeric proteins CD8-Fas II and CD8-Shaker and show that the C-terminal sequences of both Fas II and Shaker are necessary and sufficient to drive the synaptic localization of a heterologous protein. Moreover, we show that the PDZ-containing protein Discs-Large (Dlg) controls the localization of these proteins, most likely through a direct interaction with their C-terminal amino acids. Finally, transient expression studies show that the pathway these proteins take to the synapse involves either an active clustering or a selective stabilization in the synaptic membrane.

Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release.

Postsynaptic sensitivity to glutamate was genetically manipulated at the Drosophila neuromuscular junction (NMJ) to test whether postsynaptic activity can regulate presynaptic function during development. We cloned the gene encoding a second muscle-specific glutamate receptor, DGluRIIB, which is closely related to the previously identified DGluRIIA and located adjacent to it in the genome. Mutations that eliminate DGluRIIA (but not DGluRIIB) or transgenic constructs that increase DGluRIIA expression were generated. When DGluRIIA is missing, the response of the muscle to a single vesicle of transmitter is substantially decreased. However, the response of the muscle to nerve stimulation is normal because quantal content is significantly increased. Thus, a decrease in postsynaptic receptors leads to an increase in presynaptic transmitter release, indicating that postsynaptic activity controls a retrograde signal that regulates presynaptic function.

Genetic analysis of Fasciclin II in Drosophila: defasciculation, refasciculation, and altered fasciculation.

The Drosophila neural cell adhesion molecule Fasciclin II (Fas II) is expressed dynamically on a subset of embryonic CNS axons, many of which selectively fasciculate in the vMP2, MP1, and FN3 pathways. Here we show complementary fasII loss-of-function and gain-of-function phenotypes. Loss-of-function fasII mutations lead to the complete or partial defasciculation of all three pathways. Gain-of-function conditions, using a specific control element to direct increased levels of Fas II on the axons in these three pathways, rescue the loss-of-function phenotype. Moreover, the gain-of-function can alter fasciculation by abnormally fusing pathways together, in one case apparently by preventing normal defasciculation. These results define an in vivo function for Fas II as a neuronal recognition molecule that controls one mechanism of growth cone guidance-selective axon fasciculation--and genetically separates this function from other aspects of outgrowth and directional guidance.

Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.

The glutamatergic neuromuscular junction (NMJ) in Drosophila adds new boutons and branches during larval development. We generated transgenic fruit flies that express a novel green fluorescent membrane protein at the postsynaptic specialization, allowing for repeated noninvasive confocal imaging of synapses in live, developing larvae. As synapses grow, existing synaptic boutons stretch apart and new boutons insert between them; in addition, new boutons are added at the ends of existing strings of boutons. Some boutons are added de novo, while others bud from existing boutons. New branches form as multiple boutons bud from existing boutons. Nascent boutons contain active zones, T bars, and synaptic vesicles; we observe no specialized growth structures. Some new boutons exhibit a lower level of Fasciclin II, suggesting that the levels of this synaptic cell adhesion molecule vary locally during synaptic growth.

Wrapper, a novel member of the Ig superfamily, is expressed by midline glia and is required for them to ensheath commissural axons in Drosophila.

The midline glia are specialized, nonneuronal cells at the midline of the Drosophila central nervous system (CNS). During development, the midline glia provide guidance cues for extending axons. At the same time, they migrate and help separate the two axon commissures. They then wrap around and ensheath the commissural axons. In many segments, a few of the glia do not enwrap the axons, and these cells die. The wrapper gene encodes a novel member of the immunoglobulin (Ig) superfamily. Wrapper protein is expressed specifically on the surface of midline glia. In wrapper mutant embryos, the midline glia express their normal guidance cues and migrate normally. However, they do not ensheath the commissural axons, and as a result, the glia die. In the absence of Wrapper, the two axon commissures are not properly separated.

A genetic analysis of synaptic development: pre- and postsynaptic dCBP control transmitter release at the Drosophila NMJ.

Postsynaptic dCBP (Drosophila homolog of the CREB binding protein) is required for presynaptic functional development. Viable, hypomorphic dCBP mutations have a approximately 50% reduction in presynaptic transmitter release without altering the Ca2+ cooperativity of release or synaptic ultrastructure (total bouton number is increased by 25%-30%). Exogenous expression of dCBP in muscle rescues impaired presynaptic release in the dCBP mutant background, while presynaptic dCBP expression does not. In addition, overexpression experiments indicate that elevated dCBP can also inhibit presynaptic functional development in a manner distinct from the effects of dCBP loss of function. Pre- or postsynaptic overexpression of dCBP (in wild type) reduces presynaptic release. However, we do not observe an increase in bouton number, and presynaptic overexpression impairs short-term facilitation. These data suggest that dCBP participates in a postsynaptic regulatory system that controls functional synaptic development.

Highwire regulates synaptic growth in Drosophila.

The formation, stabilization, and growth of synaptic connections are dynamic and highly regulated processes. The glutamatergic neuromuscular junction (NMJ) in Drosophila grows new boutons and branches throughout larval development. A primary walking behavior screen followed by a secondary anatomical screen led to the identification of the highwire (hiw) gene. In hiw mutants, the specificity of motor axon pathfinding and synapse formation appears normal. However, NMJ synapses grow exuberantly and are greatly expanded in both the number of boutons and the extent and length of branches. These synapses appear normal ultrastructurally but have reduced quantal content physiologically. hiw encodes a large protein found at presynaptic terminals. Within presynaptic terminals, HIW is localized to the periactive zone surrounding active zones; Fasciclin II (Fas II), which also controls synaptic growth, is found at the same location.

Structural features of the terminal loop region of frog retinal rod outer segment disk membranes: III. Implications of the terminal loop complex for disk morphogenesis, membrane fusion, and cell surface interactions.

The perimeter of rod outer segment (ROS) disks displays a two-dimensional lattice of components referred to as the terminal loop complex (Corless, Fetter, Zampighi, Costello, and Wall-Buford: J. Comp. Neurol. 257:9-23, '87b). We take the view that this pattern of structural organization reflects the mechanism(s) whereby the disk perimeter is defined and constructed. Herein we develop and partially evaluate a generalized template mechanism of disk perimeter development, to account for the structure and the axial alignment of both marginal and incisural domains. Components of the terminal loop complex are conceived as the morphogens that determine the location and guide the differentiation of the disk perimeter. Briefly, we postulate that transmembranous components of the terminal loop complex are present within the reflection of plasmalemma that forms the base of the rod outer segment. These components interact with the cytoplasmic template provided by the perimeter lattice present along the lower surface of the most basal disk, thereby propagating the lattice and creating an extracellular template. The latter is then available to interact with corresponding elements distributed within the apical surface of the adjacent disk precursor evagination. The progressive interaction and alignment of these extracellular domains form the scaffolding that guides the restructuring of the plasmalemma, to form the mature disk margin topology. Successive repetitions of this process are seen to produce an axial stacking of disks whose perimeters are aligned and ensheathed by a two-dimensional net.

Modulation of disk margin structure during renewal of cone outer segments in the vertebrate retina.

In the process of disk renewal in retinal cone outer segments (COSs), apical displacement of disks must be coupled to systematic reductions in disk area and perimeter in order to retain overall conical geometry. We have quantified these changes in disk area and perimeter segment lengths by morphometric analyses of cross sections of fully formed disks taken from basal to apical ends of COSs. Specifically excluded from these analyses are data arising from partial or incomplete disks within the COS, which do not conform to the conical geometry and which constitute a minor fraction of the COS disk population. Thus, our results address the long-range pattern of structural changes affecting the major population of disks along the length of the COS. Our data indicate that decreases in total disk margin length associated with apical displacement of fully formed disks are due to decreases in the length of the margin opposite the cilium, i.e., the open margin segment. In contrast, the average length of the closed margin segment remains constant or increases slightly in the apical direction. The open margins of frog COS disks have recently been shown to possess a distinctive lattice of membrane-associated components (Fetter and Corless: Invest. Ophthalmol. Vis. Sci. 28:646-657, '87). We have also examined COSs by the freeze-fracture, deep-etch technique for evidence of a mechanism whereby measured changes in open margin length may be accommodated while maintaining the overall organization of the open margin segments. In regions of membrane continuity between open margins and the COS plasma membrane, we have observed elevated ridges on the plasma membrane that 1) tend to lie parallel to the open margin segments, 2) have a similar axial spacing, 3) occasionally demonstrate interconnecting filaments similar to those of the open margin lattice, and 4) appear to have a particulate substructure. The mechanism proposed for reducing open margin length involves tangential displacement of the lateral edges of the open margin lattice to the adjacent plasma membrane. These shifted lattice domains initially give rise to the plasmalemmal ridges, which subsequently disassemble, and whose components become redistributed in the COS plasma membrane. These structural features of COS open margins suggest several revisions of our earlier model of disk morphogenesis (Corless and Fetter: J. Comp. Neurol. 257:24-38, '87), which was based on the margin structure of ROS disks alone. Eckmiller (J. Cell Biol. 105:2267-2277, '87) has recently proposed that partial-disks observed within the COS represent sites of new disk formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural features of the terminal loop region of frog retinal rod outer segment disk membranes: I. Organization of lipid components.

We have applied thin sectioning and freeze-fracture techniques to investigate the terminal loop structure of photoreceptive disks in frog retinal rod outer segments. Our studies of this region demonstrate a highly curved terminal loop bilayer that is continuous with both lamellar bilayers of the disk, and equivalent to them in dimensions and staining properties. Rhodopsin, however, appears to be excluded from this region of high curvature.

Structural features of the terminal loop region of frog retinal rod outer segment disk membranes: II. Organization of the terminal loop complex.

In addition to a lipid bilayer component (Corless, Fetter, and Costello: J. Comp. Neurol. 257:1-8, '87), the terminal loop region of frog rod outer segment (ROS) disks displays a clustering of discrete elements referred to as the terminal loop complex. It consists of (1) semicircular or crescentic densities within the terminal loop, (2) linear interdisk densities spanning the cytoplasm near terminal loops, and (3) distinctive freeze-fracture particles associated with the terminal loop, located between 1 and 2. The linear interdisk densities are organized on a two-dimensional lattice that appears to ensheath completely the lamellar domains of all ROS disks. Indirect evidence is presented for a net axial alignment of intraloop densities. We interpret the large freeze-fracture particles of the terminal loop region to reflect transmembrane components that connect the interdisk and intraloop densities. Thus, we propose that the entire terminal loop (TL) complex is organized on a two-dimensional net. We further infer that each TL complex is organized as a dimeric unit and that such dimers interact axially and laterally to generate the observed lattice structure. It is suggested that one component of the terminal loop complex is the high molecular weight protein localized along the disk perimeter by Papermaster, Schneider, Zorn, and Kraehenbuhl (J. Cell. Biol. 78:415-425, '78).

Freeze-fracture methods: preparation of complementary replicas for evaluating intracellular ice damage in ultrarapidly cooled specimens.

Thin biological specimen sandwiched between conductive metal foils and ultrarapidly frozen in the absence of chemical pretreatments are well suited for ultrastructural studies by the freeze-fracture technique. However, the roughness of the metal surfaces, together with the thinness of the specimen, produce highly irregular surfaces upon fracturing, and thus yield fragile metal replicas. The techniques described here for stabilizing the replicas, initially with an open mesh grid and finally with a Formvar film, provide a means for obtaining a high percentage of intact replicas from which complementary images can be prepared. With the aid of the montages, complementary regions on the replicas can be located at some later time, usually in less than 1 hr. The complementary images obtained are excellent for evaluating the factors which influence the quality and resolution of the replicas--such as heating and plastic deformation--and for describing the preservation of biological structures. Regions within the specimen which display unacceptable ice crystal growth or damage can be easily and confidently identified in complementary images.

Morphological components associated with frog cone outer segment disc margins.

The two margin morphologies of frog retinal cone outer segment (COS) discs were examined by thin sectioning, freeze-fracture, and deep-etch, rotary shadowing techniques. The disc margin adjacent to the connecting cilium, which morphologically resembles the terminal loop of rod outer segment (ROS) discs, exhibited a distinctive staining density tightly apposed to the membrane surface facing the lumen. This density was crescent-shaped in longitudinal sections, and a continuous band in cross-sections. Associated with the disc margin opposite the cilium, two additional extracellular structures were observed: a globular staining density located at the outer edge of the membrane loop forming the margin, and filaments axially interconnecting adjacent margins. The globular densities and filaments were spaced at regular intervals along the margin. Where these margins were adjacent to calycal processes, the globular densities appeared to span the extracellular gap and interconnect the membranes of the COS discs and the calycal process. Distinctive intramembrane particles were observed along both margins by freeze-fracture. The distribution of the globular and filamentous elements suggests that they may have a role in maintaining the radial dimensions and axial spacing of the associated disc margin by forming an extracellular framework.

Planning, budgeting, and controlling--one look at the future: case-mix cost accounting.

This paper outlines the system for cost accounting and managerial control which is an extension of the usually accepted departmental costing systems and takes as its units the 383 Diagnosis Related Groups (DRGs) considered to be the hospital's products. It is held that such an approach offers hospital managers a more powerful, analytic, budgeting, and cost-finding tool and offers the opportunity to involve the medical staff in the issues of how their practice patterns are affecting hospital costs.

Ambulatory visit groups: a framework for measuring productivity in ambulatory care.

This article describes Ambulatory Visit Groups (AVGs) and the process by which they were defined. An approach to the analysis of physician productivity in the ambulatory setting is then demonstrated, with data derived from the National Ambulatory Medical Care Survey [1]. Finally, recommendations for future work are presented to make this approach more effective in designing and managing ambulatory care delivery organizations.

Nursing home reimbursement and the allocation of rehabilitation therapy resources.

Most public funding methods for long-term care do not adequately match payment rates with patient need for services. Case-mix payment systems are designed to encourage a more efficient and equitable allocation of limited health care resources. Even nursing home case-mix payment systems, however, do not currently provide the proper incentives to match rehabilitation therapy resources to a patient's needs. We were able to determine by a review of over 8,500 patients in 65 nursing homes that certain diagnoses, partial dependence in activities of daily living (ADLs), clear mental status, and improving medical status are associated with the provision of rehabilitation services to nursing home residents. These patient characteristics are clinically reasonable predictors of the need for therapy and should be considered for use in nursing home case-mix reimbursement systems. Primary payment source also was associated with the provision of rehabilitation services even after taking into account significant patient characteristics. It is unclear how much of the variation in service use across payers is due to differences in patient need as opposed to differences in the financial incentives associated with current payment methods.