The cytoskeletal involvement in cellularization of theDrosophila melanogaster embryo

Summary The distribution and arrangement of cytoskeletal components in the early embryo ofDrosophila melanogaster were examined by thin-section electron microscopy to elucidate their involvement in the formation of the cellular blastoderm, a process called cellularization. During the final nuclear division in the cortex of the syncytial blastoderm bundles of astral microtubules were closely associated with the surface plasma membrane along the midline where a new gutter was initiated. Thus the new gutter together with the pre-formed ones compartmentalized the embryo surface to reflect underlying individual daughter nuclei. Subsequently such gutters became deeper by further invagination of the plasma membrane between adjacent nuclei to form so-called cleavage furrows. Nuclei simultaneously elongated in the direction perpendicular to the embryo surface and numerous microtubules from the centrosomes ran longitudinally between the nucleus and the cleavage furrow. Microtubules often appeared to be in close association with the nuclear envelope and the cleavage furrow membrane. The plasma membrane at the advancing tip of the furrow was always undercoated with an electron-dense layer, which could be shown to be mainly composed of 5–6 nm microfilaments. These microfilaments were decorated with H-meromyosin to be identified as actin filaments. As cleavage proceeded, each nucleus with its perikaryon became demarcated by the furrow membrane, which then extended laterally to constrict the cytoplasmic connection between each newly forming cell and the central yolk region. The cytoplasmic strand thus formed possessed a prominent circular bundle of microfilaments which were also decorated with H-meromyosin and bidirectionally arranged, similar in structure to the contractile ring in cytokinesis. These observations strongly suggest that both microtubules and actin filaments play a crucial role in cellularization ofDrosophila embryos.     

Plasmodial ultrastructure of the myxomycete Physarum polycephalum

When plasmodia of P. polyeephalutu are fixed simultaneously with osmium tetroxide and glutaraldchyde, the cytoplasm is so preserved that a system of microchannels, resembling pinocytosis channels, and numerous discrete vacuoles can be observed. With either fixative alone, the cytoplasm appears to contain large irregular vacuoles or a vacuolar continuum. The microehannels, approximately l µ in diameter, arising as invaginations of the plasma membrane are each surrounded by a sheath of thin filaments which in areas of invagination at the plasmodial surface seems to merge with a well-defined cortex underlying the plasma membrane. Coating the plasma membrane is a structured slime layer continuous with the microchannel contents. The location and structure of the microchannel-cortex system strongly suggests it as the site for localization of contractile function implicated in cyclosis and motility. Mitochondrial structures of special interest are also described.     

Studies on microplasmodia ofPhysarum polycephalum: Endocytotic activity,morphology of the vacuolar apparatus and defecation mechanism

Summary The investigation of endocytotic processes in axenically cultured microplasmodia ofPhysarum polycephalum is considerably complicated by the development of an extensive cell membrane invagination system. Cross-sections through single channels of this system are difficult to distinguish from vacuoles formed endocytotically. Therefore the whole system was labelled by staining the extracellular slime with ruthenium red or lanthanum hydroxide. In this way endosomes produced during the incubation period could be clearly identified. Aerosil andThorotrast are suitable markers for food vacuoles because they can easily be detected with the electron microscope. The application of these substances revealed that submerged cultured microplasmodia are able to form endosomes which contain material of extracellular origin. However, the endocytotic uptake of food material is of much less intensity than in normal macroplasmodia. Microplasmodia seem to cover most of their requirements for metabolic substances by active trans-membrane transport.The intracellular digestive system of microplasmodia corresponds to the vacuolar apparatus of other cells. Preexisting lysosomes originating by autophagic processes play a central role in this system: They coalesce with endosomes or secondary lysosomes thus forming digestion vacuoles. Indigestible food components are extruded together withCa-containing granules into the cell surface invagination system by defecation. The physiological significance of theCa-granules is unknown.     

Fine structure and distribution of contractile layers inAmoeba proteus preincubated at high temperature

Summary Ultrastructural and immunocytochemical studies allow the localization and identification of a microfilament cortex in heat-shockedAmoeba proteus at different stages of recovery to room temperature. Immediately after heating the cortex is in close contact with the cytoplasmic face of the plasma membrane; however, during cooling it detaches from the membrane and shifts toward the cell centre thus separating a region of peripheral hyaloplasm from central granuloplasm. After polymerization of a new submembrane cortex several detachment and reformation cycles rhythmically repeated for 2–3 hours until a multitude of stratified layers has been formed in the hyaloplasm.Electron micrographs reveal that the cortical layer at the plasma membrane is merely composed of a network of actin filaments, whereas the retracted contractile layers in the hyaloplasm and at the granuloplasmic border contain both, thick and thin filaments often arranged in bundles. The heat-shock induced activities of the microfilament cortex are based on the highly contractile properties of this system in conjunction with controlled displacements in the equilibrium between F- and G-actin.     

Interrelationships among the plasma membrane,the membrane skeleton and surface form in a unicellular flagellate

Summary Surface isolates or membrane skeletons from surface isolates can maintain the cell and surface form characteristic of euglenoids. We now report that the plasma membrane alone obtained by trypsin or urea digestion of surface isolates can also maintain surface form, but the membrane skeleton is able to produce striking changes in membrane organization. Trypsin digests microtubules, the membrane skeleton and partially digests the major integral membrane protein from surface isolates but does not alter the paracrystalline plasma membrane interior. Extraction of surface isolates with 4M urea leaves an insoluble plasma membrane and a subset of proteins arranged perpendicularly to the membrane surface. To resolve further the relationship between the plasma membrane and the membrane skeleton we have perturbed membrane organization by extraction of surface isolates with NaOH and find that readdition of the extract followed by neutralization restored important features of the membrane skeleton and caused patching of the membrane interior. Biochemically, the reassembled membrane skeleton consisted of 80 and 86 kD polypeptides and other less abundant proteins, and structurally the reassembled membrane skeleton was about the same thickness as the native membrane skeleton. Reassembly of the membrane skeleton appeared to be saturatable in that addition of an excess of extract had no effect on the thickness of the membrane skeletal layer. When the 80 kD protein was depleted from the reassembly mixture by affinity chromatography using Sepharose-bound monoclonal antibodies, the amount of 86 kD protein bound was significantly reduced, suggesting a dependance of 86 kD protein on 80 kD binding. A urea soluble fraction enriched in the 80 and 86 kD proteins was added to alkali-stripped membranes and 170 Å filaments were formed perpendicularly to the membrane surface. From the sum of these experiments we suggest that a) the native amorphous membrane skeleton ofEuglena may consist of a framework of 80 and 86 kD filaments arranged in a brush-like layer, b) the framework can direct plasma membrane organization, but once determined, membrane form remains stable to urea and trypsin but not to alkali, and c) new surface growth can in theory occur as an expansion of the brush-like layer by direct intercalation of filaments enriched in or consisting wholly of 80 and 86 kD proteins.Abbreviations BSA bovine serum albumin - ELISA enzyme linked immunosorbant assay - EF ectoplasmic fracture face - IMPs intramembrane particles - PF protoplasmic fracture face This work was supported by a University of Illinois Fellowship to RRD and NSF grant DCB-8602793 to GBB.     

Spatial organization and fine structure of the cortical filament layer in normal locomoting Amoeba proteus

Summary The fine structural organization of a cortical filament layer in normal locomoting Amoeba proteus was demonstrated using improved fixation and embedding techniques. Best results were obtained after application of PIPES-buffered glutaraldehyde in connection with substances known to prevent the depolymerization of F-actin, followed by careful dehydration and freeze-substitution.The filament layer is continuous along the entire surface; it exhibits a varying thickness depending on the cell polarity, measuring several nm in advancing regions and 0.5–1 m in retracting ones. Two different types of filaments are responsible for the construction of the layer: randomly distributed thin (actin) filaments forming an unordered meshwork beneath the plasma membrane, and thick (myosin) filaments mostly restricted to the uroid region in close association with F-actin.The cortical filament layer generates the motive force for amoeboid movement by contraction at posterior cell regions and induces a pressure flow that continues between the uroid with a high hydrostatic pressure and advancing pseudopodia with a low one. The local destabilization of the cell surface as a precondition for the formation of pseudopodia is enabled by the detachment of the cortical filament layer from the plasma membrane. This results in morphological changes by the active separation of peripheral hyaloplasmic and central granuloplasmic regions.     

Ultrastructure of the wax-gland system in subterranean scale insects (homoptera,coccoidea, margarodidae)

The ultrastructure of wax glands (integumentary, stigmatic, and peristigmatic glands) was investigated in larvae, cysts, and adult females and males of species belonging to the genera Porphyrophora, Sphaeraspis, and Eurhizococcus. The general organization and cytological characteristics are similar for all glands studied. Each gland is composed of a single layer of 8 to 40 cells. The glandular cells are characterized by a very large quantity of smooth endoplasmic reticulum which forms dense zones throughout the cytoplasm, but is always placed near the collecting canals in the presence of mitochondria. Each cell has a central canal reservoir which penetrates it deeply and gives rise to a large number of lateral collecting canals, formed by the invagination of the apical plasma membrane. The canals open into a subcuticular cavity forming a common reservoir in which the secretion is accumulated. This reservoir is covered by a modified cuticle formed from the endocuticle and the epicuticle. The endocuticle is composed of a network of fine tubular structures and has many filaments on its surface. The epicuticle is perforated by numerous pores. There is no cuticular duct. The secretion crosses the cuticle in three successive steps. First, it passes through the filaments, then through fine tubular structures of the endocuticle, and finally through the epicuticular pores.     

Fine structure of the intersegmental membrane glands of the sixth abdominal sternum of female Nomia melanderi (Hymenoptera,Apoidea)

The fine structure of the intersegmental glands of the sixth abdominal sternum in 1-week old females of Nomia melanderi is presented. The plasma membrane of the secretory cell is unfolded in many places and is covered by a basement membrane. The microvillous surface is invaginated to form a rather long sinuous cavity. The endoplasm is almost entirely filled by secretory granules. Many secretory granules are located close to the inner surface of the invaginated plasma membrane. The invagination contains a porous ductule, apparently of cuticulin origin, that is connected directly with the inner layer of the transport duct of the duct-forming cell. This type of arrangement allows the direct flow of the secretory substance to the outside in a continuous way. The cylindrical duct-forming cell, besides having typical cell organelles, contains a cuticular transport duct. This duct is composed of a thin cuticulin layer surrounded by a rather thick epicuticular one. The results suggest that the secretory cell has two secretory cycles. The first occurs while the gland is differentiating (at the pupal stage) and is involved in secretion of the cuticulin that forms the porous ductule. The second cycle, which starts by the beginning of nesting, is involved in the secretion of a substance that is carried to the outside via the transport duct of the duct-forming cell.     

Movement of surface markers along the pinocytotic pseudopodia ofAmoeba proteus

Summary The movement of latex beads over pinocytotic pseudopodia produced byAmoeba proteus was recorded in the presence of 117.65 mM EGTA as an inducer of pinocytosis. The results show that all particles flow in the direction of pseudopodial growth, with a slightly higher velocity than the advancing frontal edge. This means that markers are removed from the base of a pinocytotic pseudopodium and gradually approach the pseudopodium tip. Two particles on the surface of the same pseudopodium can move at the same rate or differ slightly in the velocity of their forward flow. A bead can move even if another blocks the channel orifice. Retrograde particle movement has never been observed. Whether all latex spheres bound to pinocytotic pseudopodia flow with the laterally mobile plasma membrane fraction, which slides over submembranous contractile layer, or whether the whole cortical complex, the actin network and the plasma membrane, move together towards the invagination site is discussed.     

Cytoskeletal organization and cell organelle transport in basal epithelial cells of the freshwater sponge Spongilla lacustris

Summary Different antibodies against actin, tubulin and cytokeratin were utilized to demonstrate the spatial organization of the cytoskeleton in basal epithelial cells of the freshwater sponge Spongilla lacustris. Accordingly, actin is localized in a cortical layer beneath the plasma membrane and in distinct fibers within the cytoplasmic matrix. Microtubules exhibit a different distributional pattern by radiating from a perinuclear sheath and terminating at, the cell periphery; in contrast, intermediate filaments are lacking. Cytoplasmic streaming activity was studied by in-vivo staining of mitochondria and endoplasmic reticulum by means of fluorescent dyes. Single-frame analysis of such specimens revealed a regular shuttle movement of mitochondria and other small particles between the cell nucleus and the plasma membrane, which can be stopped in a reversible manner with the use of colcemid or colchicine but not with cytochalasin D. The results point to the microtubular system as a candidate for cell organelle transport, whereas the actomyosin system rather serves for changes in cellular shape and motility.     

Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane

We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton- plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.     

Dense bodies of the contractile system of cardiac muscle in Venus mercenaria

Dense bodies in the heart muscle of Venus mercenaria exist in two forms, free and attached. Free dense bodies morphologically consist of fascicles of thin filaments in parallel array and bound together by a dense, amorphous proteinaceous material. The binding of dense bodies to the cell membrane is effected via connecting filaments of the amorphous material of the dense body which join a condensation of morphologically similar material attached to the inner osmiophilic layer of the unit membrane. This composite of dense body, connecting filaments, membrane condensation and unit cell membrane has been termed collectively the attachment plaque. The attachment plaque is part of an extensive network on the cell surface which obligates that surface to a role in the contractile process. Moreover, this set of attachment plaques imposes an organization and an orientation to most thin filaments of the cell and preserves the contractile axis of the cell.     

Participation of the contractile system in endocytosis demonstrated in vivo by video-enhancement in heat-pretreated amoebae

Summary The heat-pretreated amoebae (hyalospheres) are well suited cell models to study several manifestations of endocytosis: invagination of initial funnels, formation of pinocytotic channels, their activity and disintegration, production of microand macroendosomes directly from the surface membrane. All these phenomena are rhythmically reproduced (with periods ranging from 9 to 27 s) at the same active spots on the cell surface and accompanied by pulsation of the adjacent peripheral cytoplasmic layers. Successive portions of the contractile cortical network are serially detached from the plasma membrane and retracted inwards (on average 1 detachment per 15 s). They are suggested to be responsible for the traction component of endocytotic movements, i.e., for pulling the initial invagination funnels, elongation of channels, and inward transport of macroendosomes which are embedded in them. On the other hand, retraction of the cortical network squeezes the hyaloplasm outwards and thus the pressure component of endocytosic is produced. This results in cell surface expansion around the orifice of endocytotic channels or formation of macroendosomes by constriction at the mouth of large surface invaginations. Moreover, the retracting cortical network produces various radial transhyaline strands which seem to play a, not fully understood, role in membrane invagination and inward transport of microendosomes, and to accompany cytoplasmic pulsation around channels. The contractile network lining the walls of the channels may be detected in vivo, when some old channels are destroyed and their membrane dissociates from the cytoskeletal sleeve. The central role of the rhythmic detachment of the contractile network from the plasma membrane is common to the locomotory and endocytotic movements.     

The effect of salinity acclimation on the ultrastructure of the gills of Gammarus oceanicus (Segerstråle, 1947) (Crustacea: Amphipoda)

Summary Acclimation to low salinity induces changes in the ultrastructure of the gill cells of the marine euryhaline amphipod, Gammarus oceanicus. The gills are composed of a single cell type. In 100% artificial sea water, these cells contain moderate numbers of mitochondria which are randomly distributed in the cytoplasm. The plasma membrane is extensively invaginated at the apical, lateral, and basal surfaces. Acclimation to 20% artificial sea water induces a further invagination at the apical cell membrane to form an elaborate apical labyrinth. The extracellular spaces between the folds in the basal cell membrane dilate to 1500 Å or more. Mitochondria are more abundant and in many cells they undergo a change in conformation. The mitochondria are crowded into thin leaflets of cytoplasm between the dilated basal invaginations or into the narrow space between apical and basal cell membranes. Consequently, they lie in close contact with the plasma membrane over much of their surface.Supported in part by grants from the United States Public Health Service, 5 RO1 AM13455-03 and PHS FR-07085-04, and by a grant from the National Research Council of Canada administered by Dr. G. P. Morris.     

Structure and Operation of the Feeding Apparatus in a Colorless Euglenoid,Entosiphon sulcatum1

Entosiphon sulcatum is a phagotrophic euglenoid. The tubular ingestion apparatus, called a siphon, is composed of three microtubular rods extending the length of the cell. Within the tube are four large striated vanes arranged much like the blades in a pinwheel. The vanes arise from the microtubular rods and curve towards the center of the feeding apparatus. Sheets of endoplasmic reticulum are positioned adjacent to each of the vanes and surround the perimeter of the apparatus. A cap, supported by a scaffold and anchored into the cytoplasm, covers the opening of the siphon. An elongate invagination of the plasma membrane is positioned adjacent to the edge of the cap and extends downward into the siphon forming the opening. The vanes converge at the anterior end of the siphon and surround the invagination. During feeding, the siphon protrudes from the cell. As the apparatus protrudes the cap is withdrawn to the side, opening the siphon. The vanes spread apart expanding the invagination of the plasma membrane into a large cavity into which ingested food particles are taken.     

A freeze-fracture study of the surface of the infective-stage larva of the nematode Trichinella

The surface layers of the cuticle of the infective, first-stage larva of the nematodes Trichinella spiralis and T. spiralis var. pseudospiralis have been studied by means of the freeze-fracturing technique. No obvious differences between the two nematodes were found. A double-layered structure covers the cuticle. Its outermost layer consists of particles embedded in an amorphous matrix; its inner layer is composed of a sheet of fine filaments which may be composed of globular subunits. This unique double layered structure is not like a normal cell membrane in structure. The surface of the cuticle beneath it is relatively smooth except for impressions from the inner surface of the double-layered structure. The cuticle surface did not fracture in the manner of a cell membrane.     

Effects of the actin-binding protein DNAase I on cytoplasmic streaming and ultrastructure of Amoeba proteus

Microinjection of DNAase I, which is known to form a specific complex with G-actin, induces characteristic changes in cytoplasmic streaming, locomotion and morphology of the contractile apparatus of A. proteus. Light microscopical studies show pronounced streaming originating from the uroid and/or the retracting pseudopods, which ceases 10--15 min after injection of DNAase I, at a time when ultrasctructural studies show that the actin filament system is very much reduced. These results suggest that a controlled reversible equilibrium between soluble and polymerized forms of actin is a necessary requirement for amoeboid movement. The topographic distribution of contractile filaments beneath the plasma membrane visualized by correlated light- and electron microscopy of DNAase I-injected cells establishes the importance of the membrane-bound filamentous layer for three major aspects of streaming: (1) Streaming originates by local contractions of a cell membrane-associated filament layer at the uroid and/or retracting pseudopods, creating a pressure flow. (2) This flow continues beneath the membrane, which is stabilized by filaments in the lateral regions between the posterior end, with a high hydrostatic pressure, and the anterior end, with a low hydrostatic pressure. (3) Pseudopods or extending areas are created by a local destabilization of the cell periphery caused by the separation of the filamentous layer from the plasma membrane.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.     

Actin filament array during side branch initiation in protonema cells of the mossFunaria hygrometrica: an actin organizing center at the plasma membrane

Summary With an improved method to visualize the actin filament system it is possible to detect a small, peculiar accumulation of actin filaments under the prospective area of side branch formation inFunaria protonema cells. It consists of a ring-like configuration of actin filaments from which filaments radiate, preferentially along the plasma membrane. During the transition to tip growth the arrangement becomes loosened and eventually disappears whereas the filaments are concentrated in inner regions of the cytoplasm with a maximum in the apical dome.     

Insights into eisosome assembly and organization

Eisosomes, large protein complexes that are predominantly composed of BAR-domain-containing proteins Pil1 and its homologs, are situated under the plasma membrane of ascomycetes. A successful targeting of Pil1 onto the future site of eisosome accompanies maturation of eisosome. During or after recruitment, Pil1 undergoes self-assembly into filaments that can serve as scaffolds to induce membrane furrows or invaginations. Although a consequence of the invagination is likely to redistribute particular proteins and lipids to a different location, the precise physiological role of membrane invagination and eisosome assembly awaits further investigation. The present review summarizes recent research findings within the field regarding the detailed structural and functional significance of Pil1 on eisosome organization.