Relation of cytoplasmic alkalinization to microvillar elongation and microfilament formation in the sea urchin egg

Experiments have been carried out to test the proposal that the pH increase at fertilization in sea urchin eggs promotes microvillar elongation. Results presented herein show that microvillar elongation and microfilament formation occurred when sea urchin eggs were incubated in sodium-free seawater containing the calcium ionophore A23187, a treatment which initiates activation, i.e., induces a transient increase in intracellular free calcium, but prevents subsequent cytoplasmic alkalinization. Within elongated microvilli and cortices of these eggs, microfilaments were arranged in a loose meshwork. However, if the pH of the egg cytoplasm was increased experimentally, microfilament bundles appeared within individual microvilli. These findings suggest that: (1) microvillar elongation and microfilament formation in the sea urchin egg at fertilization may occur when cytoplasmic alkalinization is inhibited, and (2) formation of the microvillus bundle of microfilaments at egg activation is pH sensitive. Additionally, if the cytoplasmic pH of unfertilized eggs was experimentally elevated by NH₄Cl, microvilli failed to elongate. These data indicate that elevation of intracellular pH by this method is not sufficient to induce microvillar elongation.     

Purification of microvilli and an analysis of the protein components of the microfilament core bundle

A fast and convenient method for the purification of microvilli from chicken intestinal brush borders is described. The microvilli appear morphologically very similar to those found on intact brush borders. Removal of the microvillus membrane from the microvilli by Triton X-100 treatment reveals compact bundles of microfilaments with small regularly spaced projections along their length. SDS-polyacrylamide gel analysis of the protein components of the brush border, the microvilli and the microvillus core bundles shows that little or no tropomyosin, myosin or filamin is found in the microvillus, whereas polypeptide chains with mobilities characteristic for these proteins are present in the whole brush border. The majority of the microvillus core protein is actin, and the other major protein present has a polypeptide molecular weight of 95 000. Total actin from both brush borders and microvilli, characterized by isoelectric focussing analysis, contained about 40% β actin and 60% γ actin. The presence of both the β and γ cytoplasmic actins in the highly ordered parallel arrays of microfilaments of the microvilli is discussed in light of hypotheses for different functional roles of these two actin species.     

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

pH regulates the polymerization of actin in the sea urchin egg cortex

The state of actin in the isolated cortex of the unfertilized sea urchin egg can be controlled by experimentally manipulating the pH of the isolation medium. Cortices isolated at the pH of the unfertilized egg (6.5--6.7) do not contain filamentous actin, while those isolated at the pH of the fertilized egg (7.3--7.5) develop large numbers of microvilli which contain bundles of actin filaments. Cortices that are isolated at pH 6.5 and then transferred to isolation medium buffered at pH 7.5 also develop actin filaments. However, the filaments are not arranged in bundles and microvilli do not form. Although the cortical granules in cortices isolated at pH 6.5 discharge at a free Ca++ concentration of approximately 10 micrometer, actin polymerization is not induced by increasing the Ca++ concentration of the isolation medium. These results suggest that the increase in cytoplasmic pH which occurs following fertilization induces the polymerization of actin in the egg cortex.     

Microvillar elongation following parthenogenetic activation of sea urchin eggs

Parthenogenetic activation of unfertilized sea urchin eggs with ammonium chloride at pH 8.0 resulted in a slow, but dramatic, reorganization of surface microvilli in four species of sea urchin eggs. Following NH4Cl treatment, elongation of microvilli on the egg surface was observed concomitant with the formation of microfilament bundles within the microvillar cores. A minimum of 2 h of treatment was required for elongation and microfilament bundle formation to occur. The maintenance of elongated microvilli was pH-sensitive; removal of the activating agent resulted in the retraction of extended microvilli while readdition of NH4Cl caused microvilli to elongate again. Accompanying microvillar elongation in activated eggs, there was an increased calcium uptake as measured by 45Ca uptake. Blocking calcium uptake by incubation in lanthanum chloride or zero-calcium seawater containing 2 mM EGTA prevented microvillar elongation. These results suggested that elongation of microvilli following parthenogenetic activation by NH4Cl is pH- and calcium-dependent and is similar to that observed during normal fertilization.     

Egg shells of mallophagans and anoplurans (Insecta: Phthiraptera): morphogenesis of specialized regions and the relation to F-actin cytoskeleton of follicular cells

The egg shells of investigated phthirapterans consist of three basic elements: an anterior operculum, a main egg shell and a posterior hydropyle. In some species these elements show further regional specializations: bristles and projections that facilitate attachment to feathers of the host, micropyles and aeropylar openings. All of the egg shell specializations are formed by distinct subpopulations of follicular cells. Staining with rhodamine-conjugated phalloidin has revealed that these subpopulations significantly differ in the distribution of microfilaments (F-actin). In this respect four morphological categories of the follicular cells have been distinguished: (1) cells devoid of processes and microvilli, with basal arrays of microfilaments, responsible for the secretion of a flat chorion; (2) cells devoid of processes and microvilli, separated by intercellular spaces, with basal arrays of microfilaments, responsible for the secretion of attachment structures; (3) cells equipped with actin-containing processes, responsible for the formation of micropyles or aeropyles, and (4) cells equipped with bundles of microvilli, responsible for the formation of hydropyles.     

Formation of bundles of microfilaments during spreading of fibroblasts on the substrate

Electron microscopy was used to study the sites of formation of bundles of parallel microfilaments in the early stages of spreading of normal mouse embryo fibroblasts on the substrate. Bundles of microfilaments were not found in suspended cells. Contact of the surface of spherical cells with the substrate was not sufficient for the formation of bundles: these bundles were not seen near the under surface of cells that were already attached to the substrate but had not yet developed cytoplasmic outgrowths at their periphery. Peripheral cytoplasmic outgrowths (microspikes and lamellar processes) attached to the substrate were found to be the only sites of localization of the first bundles of microfilaments seen in the spreading cells. It is suggested that surface and/or cytoplasm of the newly-formed peripheral cytoplasmic outgrowth may have some special properties necessary for the initiation of the development of microfilament bundles.     

Dynamic aspects of filopodial formation by reorganization of microfilaments

The coelomocytes of the sea urchin, Strongylocentrotus droebachiensis, may be prevented from clotting with 50 mM ethylene glycol-bis(beta-aminoethyl)-N,N,N',N'-tetraacetate, 50 mM Tris-HCl, pH 7.8 and subsequently separated into various cell types on sucrose gradients. One cell type, the petaloid coelomocyte, spontaneously undergoes a striking morphological transformation to a form exhibiting numerous, t-in cytoplasmic projections (filopodia). Moreover, the transformation is reversible. Ultrastructurally, the formation of the filopodia results from a progressive reorganization of actin-containing filaments into bundles that are radially oriented. The formation of the filament bundles is initiated at the cell's periphery and proceeds inward. Simultaneously, the cytoplasm in between the bundles is withdrawn, exposing finger-like filopodia. Ultimately, the filopodia can be extended by up to four times their original length. Biochemically, actin is the most abundant protein in while cell homogenates and is extractable in milligram quantities via acetone powders. An actomyosin complex may also be isolated from these cells and is presumed to be active in producing the various forms of motility observed.     

Different cytoskeletal organization in two maturation stages of Discoglossus pictus (Anura) oocytes: thickness and stability of actin microfilaments and tropomyosin immunolocalization

In Discoglossus pictus oocytes, the germinative area (GA) contains long and irregular microvilli where actin microfilaments are located. In the egg, the funnel-shaped dimple that originates by invagination of the GA is present. In the dimple both microvilli and microfilament bundles have a very orderly appearance. This report extends previous observations (Campanella and Gabbiani, Gamete Res 3:99-114, 1980) and shows that GA microfilaments are thinner (36 A average) than dimple microfilaments (60 A average), as measured in ultrathin section. Moreover, the interfilament distance is smaller in GA bundles than in the dimple bundles. To get an insight into actin organization in oocytes and eggs, we used an actin-depolymerizing factor (ADF) in which cryostat sections were incubated prior to immunofluorescent staining with antiactin antibodies. The microfilaments of the GA microvilli and partially of the oocyte cortex are resistant to ADF when compared to those in the dimple and the rest of the egg cortex. We also investigated immunocytochemically the presence of tropomyosin and found that this protein is localized in the dimple and in the cortex of oocytes and eggs but is absent in the GA.     

Isolation and characterization of two forms of a cytoskeleton

Isolated petaloid coelomocytes from the sea urchin Strongylocentrotus droebachiensis transform to a filopodial morphology in hypotonic media. Electron micrographs of negatively stained Triton-insoluble cytoskeletons show that the petaloid form consists of a loose net of microfilaments while the filopodial form consists of paracrystalline bundles of microfilaments. Actin is the major protein of both forms of the cytoskeleton. Additional polypeptides have molecular weights of approximately 220,000, 64,000, 57,000, and 27,000 daltons. Relative to actin the filopodial cytoskeletons have an average of 2.5 times as much 57k polypeptide as the petaloid cytoskeletons. Treatment with 0.25 M NaCl dissociates the filament bundles into individual actin filaments free of the actin-associated polypeptides. Thus, one or more of these actin-associated polypeptides may be responsible for crosslinking the actin filaments into bundles and maintaining the three-dimensional nature of the cytoskeletons.     

Reorganization of the apical cytoskeleton of uterine epithelial cells during early pregnancy in the rat: a study with myosin subfragment 1.

Actin filaments were identified in the epithelial cells of rat uterus following detergent extraction and decoration of microfilaments (MF) with myosin subfragment 1 (S1). MF connections with cytoplasmic organelles and the apical plasma membrane are also described. Transmission electron microscopy revealed that the regular microvilli of non-pregnant, oestrous animals contain several decorated MF with rootlets descending into a densely filamentous terminal web. Following mating, the actin cytoskeleton was examined on days 1, 3 and 6 of pregnancy. In this period, the irregular projections that replace MV assumed an underlying, dense network of decorated MF, whilst smoother surfaces displayed few cytoplasmic filaments. At the time of blastocyst implantation, a structured terminal web was no longer present. Structural details were revealed concerning the contents of large, bleb-like projections found on the apical surface.     

Latrunculin inhibits the microfilament-mediated processes during fertilization, cleavage and early development in sea urchins and mice

Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.     

Immunofluorescent and ultrastructural studies of polygonal microfilament networks in respreading non-muscle cells

Respreading gerbil fibroma cells (CCL146) have been found to display cytoplasmic actin-based polygonal fiber networks 10 h after replating (stage III of respreading according to Vasiliev & Gelfand, [1]). The networks have been analyzed by immunofluorescence and electron microscopy. The foci, sites of actin, α-actinin and filamin distribution, are condensed meshworks of microfilaments attached to the inner surface of the plasma membrane. The interconnecting fibers, sites of uniform actin distribution and complementary periodicities of α-actinin and myosin, are bundles of parallel microfilaments with periodic dense bodies. Heavy meromyosin (HMM) labelling of the microfilaments in the foci and interconnecting bundles confirm that they contain actin. In addition, approx. 70% of the microfilaments associated with an individual focus have a uniform polarity relative to it (arrowheads pointing away) suggesting that they have their origin there. Our results support earlier conclusions [2] that polygonal networks are structural intermediates responsible for organizing contractile proteins of the cortical microfilament layer into stress fibers.     

Propranolol, a beta-adrenergic receptor blocker, affects microfilament organization, but not microtubules, during the first division in sea urchin eggs

Propranolol, a beta-adrenergic receptor blocker, blocks the formation of the cleavage furrow, while karyokinesis is unaffected during first division in the sea urchins Paracentrotus lividus or Lytechinus pictus. This effect is reversed by adrenalin, indicating that it is mediated by an adrenergic mechanism. The staining of F-actin microfilaments by rhodamine phalloidin in eggs in which the cleavage is blocked by the drug has revealed that propranolol affects both the distribution and the organization of actin microfilaments. A low-voltage scanning electron microscopy (LVSEM) study of microvilli in these eggs shows an extensive rearrangement of the egg surface. Anti-tubulin immunofluorescence microscopy of eggs treated with propranolol shows that they form normal mitotic asters. This indicates that while cleavage is affected, mitotic spindle formation is not. These results suggest that neurotransmitter monoamines known to be present in the sea urchin egg might be involved in the reorganization of the actin cytoskeleton underlying the formation of the cleavage furrow.     

The organization of myosin and actin in rapid frozen nerve growth cones

Rapid freezing and freeze substitution were used in conjunction with immunofluorescence, whole mount EM, and immunoelectron microscopy to study the organization of myosin and actin in growth cones of cultured rat superior cervical ganglion neurons. The general cytoplasmic organization was determined by whole mount EM; tight microfilament bundles formed the core of filopodia while a dense meshwork formed the underlying structure of lamellipodia. Although the central microtubule and organelle-rich region of the growth cone had fewer microfilaments, dense foci and bundles of microfilaments were usually observed. Anti-actin immunofluorescence and rhodamine phalloidin staining of f-actin both showed intense staining of filopodia and lamellipodia. In addition, staining of bundles and foci were observed in central regions suggesting that the majority of the microfilaments seen by whole mount EM are actin filaments. Anti-myosin immunofluorescence was brightest in the central region and usually had a punctate pattern. Although less intense, anti-myosin staining was also seen in peripheral regions; it was most prominent at the border with the central region, in portions of lamellipodia undergoing ruffling, and in spots along the shaft and at the base of filopodia. Immunoelectron microscopy of myosin using postembedment labeling with colloidal gold showed a similar distribution to that seen by immunofluorescence. Label was scattered throughout the growth cone, but present as distinct aggregates in the peripheral region mainly along the border with the central region. Less frequently, aggregates were also seen centrally and along the shaft and at the base of filopodia. This distribution is consistent with myosins involvement in the production of tension and movements of growth cone filopodia and lamellipodia that occur during active neurite elongation.     

Microvilli on sea urchin eggs: a second burst of elongation

A scanning EM study reveals about 300,000 microvilli on each egg of the sea urchin Strongylocentrotus droebachiensis. The microvilli are about 0.2 μm long before fertilization, elongate to about 0.5 μm soon after fertilization (the “first burst” of microvillus elongation), and subsequently elongate again about midway between fertilization and first cell division (the “second burst” of elongation). The second burst occurs during a discrete 30-min period and results in some microvilli being as long as 10 μm, although the average length is about 1.8 μm. The surface area of the egg following the second burst is about 2.7 times the area of the unfertilized egg.     

Organization of actin microfilaments in the apical border of oviduct ciliated cells

Actin microfilaments were localized in quail oviduct ciliated cells using decoration with myosin subfragment S1 and immunogold labeling. These polarized epithelial cells show a well developed cytoskeleton due to the presence of numerous cilia and microvilli at their apical pole. Most S1-decorated microfilaments extend from the microvilli downward towards the upper part of the ciliary striated rootlets with which they are connected. From the microvillous roots, a few microfilaments connect the proximal part of the basal body or the basal foot associated with the basal body. Microfilament polarity is shown by S1 arrowheads pointing away from the microvillous tip to the cell body. Furthermore, short microfilaments are attached to the plasma membrane at the anchoring sites of basal bodies and run along the basal body. The polarity of these short microfilaments is directed from the basal body anchoring fibers downward to the cytoplasm. At the cell periphery, microfilaments from microvillous roots and ciliary apparatus are connected with those of the circumferential actin belt which is associated with the apical zonula adhaerens. Together with the other cytoskeletal elements, the microfilaments increase ciliary anchorage and could be involved in the coordination of ciliary beating. Moreover, microvilli surrounding the cilia probably modify ciliary beating by offering resistance to cilium bending. The presence of microvilli could explain the fact that mainly the upper part of the cilia appanars to be involved in the axonemal bending in metazoan ciliated cells.     

Development of the Nervous System of Sea Urchin Embryos: Formation of Ciliary Bands and the Appearance of Two Types of Ectoneural Cells in the Pluteus

An ultrastructural study of the larval integument of the sea urchin, Hemicentrotus pulcherrimus , was conducted with special emphasis on the development of the nervous system in relation to the formation of ciliary bands. In the integument of 4-armed pluteus larvae, cells associated with the ciliary band, which have 200 nm-thick projections at their apices, and cells in the squamous epithelium, which have a cilium and long, fine radiating processes in the apical region, were observed. Both cell types have axons at their basal ends that form nerve bundles beneath the ciliary bands, where the axons make contact with ectodermal effector cells with motile cilia. The cilia and other apical projections of these ectoneural cells run parallel to the surface of the cells, and are under the hyaline layer. The axoneme of the cilium has a typical "9 + 2" microtubular arrangement, but generally has no dynein arms. These ectoneural cells are more frequent on the oral surface than on the antioral surface.     

The changes in structural organization of actin in the sea urchin egg cortex in response to hydrostatic pressure

We have used hydrostatic pressure to study the structural organization of actin in the sea urchin egg cortex and the role of cortical actin in early development. Pressurization of Arbacia punctulata eggs to 6,000 psi at the first cleavage division caused the regression of the cleavage furrow and the disappearance of actin filament bundles from the microvilli. Within 30 s to 1 min of decompression these bundles reformed and furrowing resumed. Pressurization of dividing eggs to 7,500 psi caused both the regression of the cleavage furrow and the complete loss of microvilli from the egg surface. Following release from this higher pressure, the eggs underwent extensive, uncoordinated surface contractions, but failed to cleave. The eggs gradually regained their spherical shape and cleaved directly into four cells at the second cleavage division. Microvilli reformed on the egg surface over a period of time corresponding to that required for the recovery of normal egg shape and stability. During the initial stages of their regrowth the microvilli contained a network of actin filaments that began to transform into bundles when the microvilli had reached approximately 2/3 of their final length. These results demonstrate that moderate levels of hydrostatic pressure cause the reversible disruption of cortical actin organization, and suggest that this network of actin stabilizes the egg surface and participates in the formation of the contractile ring during cytokinesis. The results also demonstrate that actin filament bundles are not required for the regrowth of microvilli after their removal by pressurization. Preliminary experiments demonstrate that F-actin is not depolymerized in vitro by pressures up to 10,000 psi and suggest that pressure may act indirectly in vivo, either by changing the intracellular ionic environment or by altering the interaction of actin binding proteins with actin.     

A thin-section and freeze-fracture study of microfilament-membrane attachments in choroid plexus and intestinal microvilli

Choroid plexus and intestinal microvilli in thin sections have microfilaments in the cytoplasm adjacent to the membranes, and in replicas have broken strands of filaments in both cytoplasm and on E faces of plasm membranes. The microfilaments contain actin as indicated by their binding of heavy meromyosin (HMM). In sections of choroid plexus, the microfilaments are 7-8 nm in diameter and form a loose meshwork which lies parallel to the membrane and which is connected to the membranes both by short, connecting filaments (8 times 30 nm) and dense globules (approximately 15-20 nm). The filamentous strands seen in replicas are approximately 8 nm in diameter. Because they are similar in diameter and are connected to the membrane, these filamentous strands seen in replicas apparently represent the connecting structures, portions of the microfilaments, or both. The filamentous strands attached to the membrane are usually associated with the E face and appear to be pulled through the P half-membrane. In replicas of intestinal brush border microvilli, the connecting strands attaching core microfilaments to the membrane are readily visualized. In contrast, regions of attachment of core microfilaments to dense material at the tips of microvilli are associated with few particles on P faces and with few filamentous strands on the E faces of the membranes. Freeze-fracture replicas suggest a morphologically similar type of connecting strand attachment for microfilament-membrane binding in both choroid plexus and intestinal microvilli, despite the lack of a prominent core bundle of microfilaments in choroid plexus microvilli.