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.     

Dynamics of the contractile system in the pseudopodial tips of normally locomoting amoebae,demonstrated in vivo by video-enhancement

Summary Behaviour of the membrane and contractile system was directly recorded in the advancing and retracting frontal zones of spontaneously locomoting or stimulated amoebae. The advancing pseudopodial tips alternately slow down and accelerate. In the slowing phase the frontal hyaline caps are flat and compressed by countercontraction of the cortical actin network beneath the leading edge. At this stage the membrane-cytoskeleton complex splits: the detached contractile layer is retracted inwards, and the membrane lifted outwards. The fluid endoplasm fraction is filtered forward through the detached actin network. This results in a local hydrostatic pressure drop, immediately restores the forward flow of endoplasm and initiates the acceleration phase of the leading edge progression. The frontal membrane, temporarily disconnected from the cytoskeletal layer, is free to slide and extend forward, but the new submembrane contractile network is soon repolymerized. In this way, after making one step forward, the frontal zone recovers its former state, and the cycle is then repeated. The cortex disassembly-reassembly cycles at the leading edge are produced every 2 s, on average. Retraction of the frontal contractile layers is part of the general centripetal cortex flow observed during motor functions of amoebae and many other cells, and is therefore associated with various other backward movements observed within and on the surface of advancing frontal zones of amoebae. The backward movement of the contractile cortex is also responsible for the withdrawal of previously advancing pseudopodia, if the detachment of successive contractile sheets from the frontal membrane ceases. It was demonstrated that the action of attractants and repellents is based on the activation or inhibition, respectively, of rhythmic disassembly of the membrane-cytoskeleton complex at the leading edge.     

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.     

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.     

The morphology of multivesicular bodies in soybean protoplasts and their role in endocytosis

Summary Multivesicular bodies (MVBs) in soybean protoplasts are distinct organelles (generally 250–500 nm in diameter) consisting of a limiting membrane and a number of smaller internal vesicles (generally 40–100 nm in diameter). MVBs of soybean protoplasts are morphologically similar to MVBs of animal cell systems. They can have tubular protuberances which extend from the main body of the organelle and a lamellar plaque on the cytoplasmic surface of their limiting membrane. In addition, the internal vesicles can be labeled by a zinc iodide-osmium tetroxide postfixation and may form via invagination of the limiting membrane.The MVBs of soybean protoplasts are a major compartment in the endocytotic pathway. They accumulate, over time, exogenously applied cationized ferritin and may deliver it to the major lysosomal or lytic compartment of the plant cell, namely, the vacuoles.     

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.     

Dynamics of the cytoskeleton inAmoeba proteus: II. Influence of different agents on the spatial organization of microinjected fluorescein-labeled actin

Summary Iodoacetamido-fluorescein-(IAF)-labeled actin was microinjected into normal locomotingAmoeba proteus. Thereafter (30–60 minutes) changes in the cytoplasmic fluorescence distribution pattern and contractile activity were induced by internal and external chemical stimulation. Different agents such as phalloidin, procaine, 2.4-dinitrophenol (DNP), puromycin, ouabain and n-ethyl maleimide (NEM) interfere with the excitation-contraction mechanism involved in ordered pseudopodium formation during ameboid movement and cause various morphogenetic reactions based on actin polymerization-depolymerization cycles. Most frequent changes are (a) local condensation of IAF-actin and formation of a continuous IAF-actin layer at the cytoplasmic surface of the cell membrane and around the pulsating vacuole, (b) immobilization and hyalo-granuloplasm separation by combined contraction and detachment of the IAF-actin layer from the cell membrane, (c) organized and disorganized formation of pseudopodia by local contraction and disintegration of the IAF-actin layer, and (d) alterations in the rheological properties of the protoplasmic matrix by changes in the molecular state of soluble actin not incorporated into the cytoskeleton. The experimental approaches to the function of the actomyosin system in large amebas attainable by the method ofin vivo molecular cytochemistry are discussed in detail with respect to the participation of the cytoskeleton in motive force generation for cytoplasmic streaming and ameboid movement.     

Membrane-cytoskeletal interactions in the early mouse embryo

Differentiation in the early mouse embryo begins at the 8-cell stage when the blastomeres flatten against each other by active spreading movements and surface and cytoplasmic elements become concentrated in the apical (uncontacted) region of the cells. A ring of cortical myosin marks the demarcation between the contacted and the uncontacted cellular domains. The organization of the cortical contractile apparatus in the blastomeres bears a formal resemblance to that of other cells that are engaged in similar motile activities. It has been proposed that a flow of cortical filaments could provide the motor that powers these movements. The applicability of such a cortical flow model to the early embryo and the implications for cell flattening and cell polarization are discussed in this review.     

Tubular-vesicular transformation in the contractile vacuole system of Dictyostelium

The contractile vacuole complex of Dictyostelium is the paradigm of a membrane system that undergoes tubular-vesicular transitions during its regular cycle of activities. This system acts as an osmoregulatory organelle in freshwater amoebae and protozoa. It collects fluid in a network of tubules and cisternae, and pumps it out of the cell through transient pores in the plasma membrane. Tubules and vacuoles are interconvertible. The tubular channels are associated with the cortical actin network and are capable of moving and fusing. The contractile vacuole complex is separate from vesicles of the endosomal pathway and preserves its identity in a dispersed state during cell division. We outline techniques to visualize the contractile vacuole system by electron and light microscopy. Emphasis is placed on GFP-fusion proteins that allow visualization of the dynamics of the contractile vacuole network in living cells. Proteins that control activities of this specialized organelle in Dictyostelium have been conserved during evolution and also regulate membrane trafficking in man.     

Morphogenesis and Disassembly of the Circular Plasmalemma Invagination System in Physarum polycephalum

During the morphogenesis of small plasmodia developing from endoplasmic drops, an extended plasmalemma invagination system is formed. This system is a characteristic constituent of the ectoplasm. The invaginations have different cytophysiological functions.
The transition from the initial very irregular plasmalemma indentations in protoplasmic drops to the highly organized circular invagination ring of protoplasmic strands, i.e., the differentiation as well as the disassembly of this circular invagination system in retracting endings of strands was investigated with the aid of the semithin- and ultrathin-sectioning technique.
Live observation of protoplasmic drops revealed that simultaneously with the onset of initially irregular oscillating contractions, small endoplasmic streamlets are generated. Subsequently, an improvement of the coordination of contraction activities leads to an oriented mass transport of protoplasm and thereby to locomotion. The growing endoplasmic channels continuously develop into the well-known structure of protoplasmic strands. Differentiation and disassembly of circular plasmalemma invaginations are based on processes of membrane invagination in combination with intracytosis and exocytosis.
The importance and correlations of the following phenomena for morphogenesis and differentiation are discussed: 1) the formation and distribution of the contractile apparatus, i.e., the system of cytoplasmic actomyosin fibrils, 2) plasmalemma invaginations, 3) the generation of oscillating contractions, and 4) the endoplasmic streaming.     

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.     

Velocity distribution of the anterograde and retrograde transport of extracellular particles byAmoeba proteus

Summary The transverse velocity profiles of the anterograde flow of particles on the cell surface and around it are approximately parabolic. The peak velocity is recorded close to the membrane and the descendent arm of the profile is viscosity-dependent. It indicates that the extracellular forward flow is probably generated by a forward movement of the fluid fraction of the membrane itself. The retrograde component of extracellular movements is manifested by particles kept on the cell surface by adhesion, which behave exactly as the ectoplasmic layer on the opposite side of the membrane,i.e., they probably reflect the movement of that fraction of the surface material which is attached to the cortical microfilaments. In the longitudinal profile, the velocity of anterograde flow rises from the tail to the front of amoeba, but is generally related to the effective cell locomotion rate and not to the movements of any intracellular layer. Around the cells deprived of any attachment to the substratum, which cannot locomote but manifest vigorous intracellular movements, the anterograde flow ceases at least along 2/3 of their lenght. It persists, however, around the frontal fountain zone, where other particles still move backwards together with the retracted ectoplasmic layer. This indicates that the role of the forward flow of and on the cell surface is to compensate for: (1) the increase of the surface area in the frontal regions due to locomotion, (2) the withdrawal of a part of material which is hauled back by the retracting cortical layer. A comprehensive scheme of the velocity distribution within the different layers of a moving amoeba and around it has been constructed on the basis of present and earlier data.Study supported by the Research Project CPBP 04.01 of the Polish Academy of Science.I dedicate this paper to Professor K. E. Wohlfarth-Bottermann with the best wishes for his 65th birthday.     

K+ transport properties of K+ channels in the plasma membrane of Vicia faba guard cells

Electrical properties of the plasma membrane of guard cell protoplasts isolated from stomates of Vicia faba leaves were studied by application of the whole-cell configuration of the patch-clamp technique. The two types of K+ currents that have recently been identified in guard cells may allow efflux of K+ during stomatal closing, and uptake of K+ during stomatal opening (Schroeder et al., 1987). A detailed characterization of ion transport properties of the inward-rectifying (IK+,in) and the outward-rectifying (IK+,out) K+ conductance is presented here. The permeability ratios of IK+,in and IK+,out currents for K+ over monovalent alkali metal ions were determined. The resulting permeability sequences (PK+ greater than PRb+ greater than PNa+ greater than PLi+ much greater than PCs+) corresponded closely to the ion specificity of guard cell movements in V. faba. Neither K+ currents exhibited significant inactivation when K+ channels were activated for prolonged periods (greater than 10 min). The absence of inactivation may permit long durations of K+ fluxes, which occur during guard cell movements. Activation potentials of inward K+ currents were not shifted when external K+ concentrations were changed. This differs strongly from the behavior of inward-rectifying K+ channels in animal tissue. Blue light and fusicoccin induce hyperpolarization by stimulation of an electrogenic pump. From slow-whole-cell recordings it was concluded that electrogenic pumps require cytoplasmic substrates for full activation and that the magnitude of the pump current is sufficient to drive K+ uptake through IK+,in channels. First, direct evidence was gained for the hypothesis that IK+,in channels are a molecular pathway for K+ accumulation by the finding that IK+,in was blocked by Al3+ ions, which are known to inhibit stomatal opening but not closing. The results presented in this study strongly support a prominent role for IK+,in and IK+,out channels in K+ transport across the plasma membrane of guard cells.     

<Emphasis Type="Italic">Ignicoccus hospitalis</Emphasis> and <Emphasis Type="Italic">Nanoarchaeum equitans</Emphasis>: ultrastructure,cell–cell interaction,and 3D reconstruction from serial sections of freeze-substituted cells and by electron cryotomography

Ultrastructure and intercellular interaction of Ignicoccus hospitalis and Nanoarchaeum equitans were investigated using two different electron microscopy approaches, by three-dimensional reconstructions from serial sections, and by electron cryotomography. Serial sections were assembled into 3D reconstructions, for visualizing the unusual complexity of I. hospitalis, its huge periplasmic space, the vesiculating cytoplasmic membrane, and the outer membrane. The cytoplasm contains fibres which are reminiscent to a cytoskeleton. Cell division in I. hospitalis is complex, and different to that in Euryarchaeota or Bacteria. An irregular invagination of the cytoplasmic membrane is followed by separation of the two cytoplasms. Simultaneous constriction of cytoplasmic plus outer membrane is not observed. Cells of N. equitans show a classical mode of cell division, by constriction in the mid-plane. Their cytoplasm exhibits two types of fibres, elongated and ring-shaped. Electron micrographs of contact sites between I. hospitalis and N. equitans exhibit two modes of interaction. One is indirect and mediated by thin fibres; in other cells the two cell surfaces are in direct contact. The two membranes of I. hospitalis cells are frequently seen in direct contact, possibly a prerequisite for transporting metabolites or substrates from the cytoplasm of one cell to the other. Rarely, a transport based on cargo vesicles is observed between I. hospitalis and N. equitans.     

The penetration of the spermatozoon through the sea urchin egg surface at fertilization : Observations from the outside on whole eggs and from the inside on isolated surfaces

The external and cytoplasmic surfaces of the sea urchin egg at fertilization have been examined with the scanning electron microscope (SEM). The outside events were documented by glueing eggs to polylysine coated glass plates, adding sperm and fixing rapidly. To reveal the inner aspects of the surface as the sperm travels through it to reach the egg cytoplasm, the fertilized egg surface was isolated in 0.3 M KC1, 0.35 M glycine, 2 mM MgCl₂, 2 mM EGTA, pH 7.5, glued onto a polylysine-coated plate and processed for the SEM. The events of spermatozoon attachment, membrane fusion, sperm entry, rotation and detachment into the egg cytoplasm as well as the associated cortical changes are described. The egg cortex is revealed to be a uniform network of fibrous bundles.The spermatozoon initially attaches to the egg surface by the acrosomal filament. As membrane fusion occurs between the gametes, the plasma membrane of the egg engulfs the sperm, the cortical granules start to discharge and a spreading surface deformation, possibly caused by a cortical contraction, is initiated. The perpendicularly entering spermatozoon is surrounded by a cluster of elongate microvilli which appear to have 235 nm vesicles associated with their bases. The sperm is prevented by the cortex from directly entering the egg cytoplasm and lies upon the egg surface between the plasma membrane and the matrix of cortical fibers. It is subsequently rotated additionally to enter the egg cytoplasm with the posterior end first. A scar is left in the cortex where the spermatozoon penetrated. The egg cortex is shown to consist of 50–200 nm uniformly arranged fibers, and its thickness ranges from 0.2 to 0.5 μm. It is speculated that this structure may be contractile.     

Studies on microplasmodia ofPhysarum polycephalum

Summary The new technique of fluorescent analog cytochemistry was used to investigate the cell surface morphology (RITC-WGA staining), the organization of the microfilament system (Rh-phalloidin staining) and the spatial distribution of mitochondria (Rh-123 staining) in the various growth stages of axenically cultured living and fixed microplasmodia ofPhysarum polycephalum. The differentiation degree of the cell surface is generally size- and age-dependent: the invagination system develops by degrees from small spherical stages (50–100 m) without invaginations to large vein-like or dumbbell-shaped specimens (300–1,000 (m long) with extensive invagination systems. The microfilaments are always organized in a cortical system along the entire cell surface and sometimes in a fibrillar system as well, extending throughout the cytoplasmic matrix. Results on living microplasmodia demonstrate that the cortical microfilament system is mainly involved in motive force generation for changes of cell surface morphology and protoplasmic streaming activity, whereas the fibrillar system rather serves a stabilizing and adhering function. Moreover, the functional differences of the two microfilament systems are indicated by the position of a large population of stationary mitochondria in close vicinity to the cell surface, thus pointing to a reasonable arrangement of the energy-supplying and energy-transforming system.     

Involvement of Golgin-160 in cell surface transport of renal ROMK channel: co-expression of Golgin-160 increases ROMK currents.

The weak inward rectifier potassium channel ROMK is important for water and salt reabsorption in the kidney. Here we identified Golgin-160 as a novel interacting partner of the ROMK channel. By using yeast two-hybrid assays and co-immunoprecipitations from transfected cells, we demonstrate that Golgin-160 associates with the ROMK C-terminus. Immunofluorescence microscopy confirmed that both proteins are co-localized in the Golgi region. The interaction was further confirmed by the enhancement of ROMK currents by the co-expressed Golgin-160 in Xenopus oocytes. The increase in ROMK current amplitude was due to an increase in cell surface density of ROMK protein. Golgin-160 also stimulated current amplitudes of the related Kir2.1, and of voltage-gated Kv1.5 and Kv4.3 channels, but not the current amplitude of co-expressed HERG channel. These results demonstrate that the Golgi-associated Golgin-160 recognizes the cytoplasmic C-terminus of ROMK, thereby facilitating the transport of ROMK to the cell surface. However, the stimulatory effect on the activity of more distantly-related potassium channels suggests a more general role of Golgin-160 in the trafficking of plasma membrane proteins.     

Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers

Spontaneous oscillatory fluctuations in membrane potential are often observed in heart cells, but their basis remains controversial. Such activity is enhanced in cardiac Purkinje fibers by exposure to digitalis or K-free solutions. Under these conditions, we find that voltage noise is generated by current fluctuations that persist when membrane potential is voltage clamped. Power spectra of current signals are not made up of single time-constant components, as expected from gating of independent channels, but are dominated by resonant characteristics between 0.5 and 2 HZ. Our evidence suggests that the periodicity arises from oscillatory variations in intracellular free Ca that control ion movements across the surface membrane. The current fluctuations are strongly cross-correlated with oscillatory fluctuations in contractile force, and are inhibited by removing extracellular Ca or exposure to D600. Chelating intracellular Ca with injected EGTA also abolishes the current fluctuations. The oscillatory mechanism may involve cycles of Ca (or Sr) movement between sarcoplasmic reticulum and myoplasm, as previously suggested for skinned cardiac preparations. Our experiments in intact cells indicate that changes in surface membrane potential can modulate cytoplasmic Ca oscillations in frequency and perhaps amplitude as well. A two-way interaction between surface membrane potential and intracellular Ca stores may be a common feature of heart, neuron, and other cell types.     

Surface contraction waves (SCWs) in the Xenopus egg are required for the localization of the germ plasm and are dependent upon maternal stores of the kinesin-like protein Xklp1

During the first four cell cycles in Xenopus, islands of germ plasm, initially distributed throughout the vegetal half of the egg cortex, move to the vegetal pole of the egg, fusing with each other as they do so, and form four large cytoplasmic masses. These are inherited by the vegetal cells that will enter the germ line. It has previously been shown that germ plasm islands are embedded in a cortical network of microtubules and that the microtubule motor protein Xklp1 is required for their localization to the vegetal pole [Robb, D., Heasman, J., Raats, J., and Wylie, C. (1996). Cell 87, 823-831]. Here, we show that germ plasm islands fail to localize and fuse in Xklp1-depleted eggs due to the abrogation of the global cytoplasmic movements known as surface contraction waves (SCWs). Thus, SCWs are shown to require a microtubule-based transport system for which Xklp1 is absolutely required, and the SCWs themselves represent a cortical transport system in the egg required for the correct distribution of at least one cytoplasmic determinant of future pattern.