The morphology of cultured melanophores from tadpoles of Xenopus laevis: Scanning electron microscopical observations

Summary Tail-fin melanophores of tadpoles of Xenopus laevis (Daudin) in primary culture were examined scanning electron microscopically in the aggregated and in the dispersed state. After isolation, the melanophores are spherical, but within 24 h they develop thin filopodia for attachment to the substratum. Subsequently, cylinder-like as well as flat sheet-like processes are formed, which adhere to the substratum with terminal pseudopodia and filopodia. The processes of adjacent melanophores contact each other, thus forming an interconnecting network between the melanophores.In the aggregated state the central part of the melanophore is spherical and voluminous. Both the central part and the processes bear microvilli. In melanophores with dispersed melanosomes the central part is much flatter; the distal parts have a thickness that equals a monolayer of melanosomes. The surface of the cell bears only scarce microvilli.These features indicate that melanophores do not have a fixed shape and that pigment migration is accompanied by reciprocal volume transformation between the cell body and its processes.     

Sexual dimorphism of motorneurons: Timbal muscle innervation in male periodical cicadas and homologous structures in females

Summary Treatment of cultured goldfish xanthophores by hormone (ACTH) or c-AMP induces not only pigment dispersion, but subsequent outgrowth of processes, and pigment translocation into these processes. These latter effects are shown to proceed as follows: First the edge of the cytoplasmic lamellae takes on a scalloped contour with numerous protrusions. These presumably serve as nucleation centers where short microfilament bundles are assembled, Later, the microfilament bundles elongate (grow), often resulting in an extension of the protrusions to become filopodia while the proximal end of the microfilaments penetrates into the thicker portion of the cellular process which now houses the pigment, i.e., the carotenoid droplets. Carotenoid droplets appear to migrate along the microfilament bundles, or cytoplasmic channels associated with them, into the filopodia. Finally, some of the filopodia become broader, thicker and laden with carotenoid droplets and are then recognized by light microscopy as pigmented cellular processes. The microfilaments have been shown to be actin filaments by their thickness, the size of their subunits, and decoration by heavy meromyosin. Evidence is presented which suggests that the growth of these actin filaments may come about by recruitment from short F-actin strands found in random orientation in adjacent areas.     

Myosin-X Induces Filopodia by Multiple Elongation Mechanism

Filopodia are actin-rich finger-like cytoplasmic projections extending from the leading edge of cells. Unconventional myosin-X is involved in the protrusion of filopodia. However, the underlying mechanism of myosin-X-induced filopodia formation is obscure. Here, we studied the movements of myosin-X during filopodia protrusion using a total internal reflection microscope to clarify the mechanism of myosin-X-induced filopodia formation. Myosin-X was recruited to the discrete site at the leading edge where it assembles with exponential kinetics before the filopodia extension. The myosin-X-induced filopodia showed repeated extension-retraction cycles with each extension of 2.4 μm, which was critical to produce long filopodia. Myosin-X, lacking the FERM domain, could move to the tip as does the wild type. However, it was transported toward the cell body during filopodia retraction, did not undergo multiple extension-retraction cycles, and failed to produce long filopodia. During the filopodia protrusion, the single molecules of full-length myosin-X moved within filopodia. The majority of the fluorescence spots showed two-step photobleaching, suggesting that the moving myosin-X is a dimer. Deletion of the FERM domain did not change the movement at the single molecule level with the same velocity of ∼600 nm/s as wild-type, suggesting that the myosin-X in filopodia moves without interaction with the attached membrane via the FERM domain. Based upon these results, we have proposed a model of myosin-X-induced filopodia protrusion.     

Filopodia and adhesion in cancer cell motility

Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PtdIns(3,4,5)P₃ and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia-inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas.Key words: filopodia, integrins, migration, cancer     

Filopodia and adhesion in cancer cell motility

Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PIP3 and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas.     

A Combination of Diffusion and Active Translocation Localizes Myosin 10 to the Filopodial Tip

Myosin 10 is an actin-based molecular motor that localizes to the tips of filopodia in mammalian cells. To understand how it is targeted to this distinct region of the cell, we have used total internal reflection fluorescence microscopy to study the movement of individual full-length and truncated GFP-tagged molecules. Truncation mutants lacking the motor region failed to localize to filopodial tips but still bound transiently at the plasma membrane. Deletion of the single α-helical and anti-parallel coiled-coil forming regions, which lie between the motor and pleckstrin homology domains, reduced the instantaneous velocity of intrafilopodial movement but did not affect the number of substrate adherent filopodia. Deletion of the anti-parallel coiled-coil forming region, but not the EKR-rich region of the single α-helical domain, restored intrafilopodial trafficking, suggesting this region is important in determining myosin 10 motility. We propose a model by which myosin 10 rapidly targets to the filopodial tip via a sequential reduction in dimensionality. Molecules first undergo rapid diffusion within the three-dimensional volume of the cell body. They then exhibit periods of slower two-dimensional diffusion in the plane of the plasma membrane. Finally, they move in a unidimensional, highly directed manner along the polarized actin filament bundle within the filopodium becoming confined to a single point at the tip. Here we have observed directly each phase of the trafficking process using single molecule fluorescence imaging of live cells and have quantified our observations using single particle tracking, autocorrelation analysis, and kymographs.     

Microtubules and pigment migration in the melanophores ofFundulus heteroclitus L.

Summary The dermal melanophores ofFundulus heteroclitus L. have been investigated by light and electron microscopy with the purpose of revealing the mechanisms controlling pigment migration. As predicted by earlier studies, the nerve endings of a double innervation were found adjacent to and in synaptic relation to the melanophore surface. Not expected were the large number of small pits or invaginations present in the cell surface. These appear identical to the so-called micropinocytotic vesicles found generally in cells of the vascular endothalium and smooth muscle. In chromatophores they are more reasonably interpreted as receptor sites for neurohormones than as uptake and transport mechanisms.Observations made on the kinetics of pigment migration within the processes of these melanophores indicate that the granules move along relatively fixed channels arranged parallel to the long axes of the processes. Examined at fine structure levels, the zones of cytoplasm around these channels are found to be populated by microtubules about 225 Å in diameter aligned parallel to the direction of pigment movement. These long slender elements are present in the processes regardless of whether the melanin is concentrated in the cell center or dispersed. It is reasoned from these and other observations that the microtubules function as cytoskeletal elements which help maintain the extended form of the melanophore arms and at the same time define the channels in which the pigment moves. The possible role of the tubule in generating the motive force for pigment migration is discussed.Supported by US Public Health Service Training Grant, 5 TIGM-707.     

Measurements of Light Transmission Through Single Melanophores

A photometrical method has been developed that allows assessment of subcellular pigment migration in melanophores of the fish cockoo wrasse (Labrus ossifagus L.). The pigment migration was studied with local light spot transmission measurements. Depending on where the light beam is placed on the melanophores it is possible to study events within an area of approximately 75 μm². Measuring pigment translocation in different parts of a melanophore gives new possibilities to study how cell membrane receptor-mediated signals are spread within a single cell, which will increase our understanding of how receptor activating drugs exert their cellular effect. The technique can be used in pharmacological and biophysical studies and in biosensors, pharmaceutical screens, environmental detectors, etc. The method clearly has the ability to study local and small changes in light transmission due to displacement of melanophore pigment granules. Since one melanophore on the tip of an optical fibre would be enough to obtain a measurable effect, the presented technique provides the basis for future development of biosensors small enough for in vivo applications, e.g., to monitor the catecholamine levels of circulating blood.     

Pigmentation and tunic cells in Cystodytes dellechiajei (Urochordata, Ascidiacea)

The tunic of Cystodytes dellechiajei (Poly- citoridae), a colony-forming species of the Ascidiacea that contains biologically active alkaloids, was investigated using light microscopy, laser-scanning microscopy and nuclear magnetic resonance techniques. The colonies contain numerous individual zooids, which are embedded in a common tunic. Each zooid is protected by a firm capsule of overlapping calcareous spicules. The colonies lack blood vessels in the tunic, but six morphologically different types of tunic cells were found: pigment cells, bladder cells, vacuolated filopodial cells, granular filopodial cells, morula cells and granular cells. Rod-like bacteria were found in the tunic matrix. Bladder cells and pigment cells could be identified as storage units for acid and pyridoacridine alkaloids, making the tunic inedible and repelling predators. Filopodial cells have long filopodia, which probably are connected to each other. They may be involved in transportation processes within the tunic tissue. The functions of the morula cells and the granular cells are unknown as yet. With its several specialised cells, the tunic of C. dellechiajei represents a dynamic living tissue containing biologically active compounds. Accepted: 20 September 2000     

Cellular Aspects of the Control of Physiological Color Changes in Fishes

SYNOPSIS. Recent advances in the cellular aspects of chromatophoricactivities in fishes are reviewed, special emphasis being laidon the black pigment-containing cells, the melanophores. A fewrecent electron-microscopic studies have disclosed the finestructure of melanophores. They are enclosed with a single cellmembrane, within which melanosomes and other cell organellesare found. All observations favor the view that melanosomesare selectively moved through the cellular processes, leavingthe cell contour rather fixed. In regard to these findings,current ideas about the mechanisms of pigment movements arediscussed. Particular attention is directed to the possibleintervention of microtubules and the theory of migration ofpigment by intracellular electrophoresis. The regulatory mechanismsof pigment cells are then dealt with. The adrenergic natureof transmission is affirmed in the peripheral melanin-aggregatingnervous system. The mode of nervous supply to a melanophoreis also analyzed. Investigations of the antagonistic, melanin—dispersing,nervous system are also considered, with special reference torecent physiological studies and to the finding of synapticvesicles by electron microscopy. On the basis of these results,a new interpretation of the so-called Parker effect is proposed.     

Amöboid bewegliche Pigmentzellen im Epithel des Seeigels Centrostephanus longispinus

Zusammenfassung Für den physiologischen Farbwechsel bei Vertebraten und Evertebraten gilt die Vorstellung, daß eine Pigmentbewegung innerhalb einer formkonstanten Zelle stattfindet. Am Seeigel Centrostephanus longispinus wird nun der Nachweis einer amoeboiden Bewegung von Pigmentzellen geführt: Die Epidermis von Centrostephanus enthält große braune Chromatophoren, die bei Belichtung eine Pigmentdispersion, bei Verdunkelung eine Konzentration des Pigments zeigen. Die Chromatophoren sind außerordentlich stark verzweigte Zellen, deren Arme dicht mit Pigmentgrana erfüllt sind. Im geballten Zustand ist die allgemeine Zellform mehr oder weniger ovoid, wobei die Zellarme eingezogen und dicht um die Zellmitte angeordnet sind. Dispersion des Pigments wird hervorgerufen durch Ausstrecken der pigmentierten Zellarme in den Interzellularraum des umgebenden Gewebes. Innerhalb der Zelle werden filamentöse Elemente nachgewiesen, die vermutlich für die Zellbeweglichkeit verantwortlich sind. — Ferner wird der zelluläre Aufbau des Integuments beschrieben.

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Amoeboid pigment cells in the epithelium of the sea urchin Centrostephanus longispinus A novel colour change mechanism

Summary Rapid colour changes in vertebrate and invertebrate species are considered to be due to movement of pigment granules within pigment cells of constant shape. Evidence is presented in this study to show that an amoeboid movement of chromatophores occurs in the epidermis of the Echinoderm Centrostephanus longispinus. The epidermis in this species contains large brown chromatophores, which display a dispersion of pigment on illumination and its concentration on darkening. The chromatophores are extensively branched cells, and their branches are densely packed with pigment granules. In the state of pigment concentration, the shape of the cell is more or less ovoid, and the cell branches are drawn in and closely arranged around the cell centre. Dispersion is attained by a stretching out of the pigmented cell branches into the intercellular spaces of the surrounding tissue. Within the cell, filamentous elements, which may be functional in the motility of the pigment cell, can be demonstrated.—Additionally the cellular composition of the integument is described.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft. Frl. A. Mikolaczick danken wir für sorgfältige technische Assistenz.     

Morphometry of cellular protrusions of mesodermal cells and fibrous extracellular matrix in the primitive streak stage chick embryo

Summary Chick mesodermal cells, having become invaginated and beginning to locomote prior to the formation of the mesodermal cell layer at an early primitive streak stage, extend many filopodia and flatten themselves against the basal surface of the epiblast. Morphometry on scanning electron micrographs of chick mesodermal cells revealed two statistically significant tendencies. Each cell took an extended form and protruded filopodia, preferably along its major axis, suggesting that the force extending the cell body was generated by both ends rich in filopodia. The cells also tended to protrude filopodia most frequently in a direction away from Hensen's node. The orientation of the fibrous extracellular matrix (fECM), running on the basal surface of the epiblast, was assessed quantitatively, and it was proved statistically that the orientation of the fECM was radial around the primitive streak: With an immunogold staining technique, fECM, to which the filopodia of the mesodermal cells attached frequently and closely, was confirmed to be rich in fibronectin (FN). These results lead us to conclude that the mesodermal cells in chick gastrula were guided to locomote towards the periphery of the area pellucida by FN-rich fECM laid on the basal surface of the epiblast, and that this movement was due to an in vivo locomotive mechanism using filopodia. Offprint requests to: R. Toyoizumi     

Mitochondria-associated myosin 19 processively transports mitochondria on actin tracks in living cells

Mitochondria are fundamentally important in cell function, and their malfunction can cause the development of cancer, cardiovascular disease, and neuronal disorders. Myosin 19 (Myo19) shows discrete localization with mitochondria and is thought to play an important role in mitochondrial dynamics and function; however, the function of Myo19 in mitochondrial dynamics at the cellular and molecular levels is poorly understood. Critical missing information is whether Myo19 is a processive motor that is suitable for transportation of mitochondria. Here, we show for the first time that single Myo19 molecules processively move on actin filaments and can transport mitochondria in cells. We demonstrate that Myo19 dimers having a leucine zipper processively moved on cellular actin tracks in demembraned cells with a velocity of 50 to 60 nm/s and a run length of ∼0.4 μm, similar to the movement of isolated mitochondria from Myo19 dimer-transfected cells on actin tracks, suggesting that the Myo19 dimer can transport mitochondria. Furthermore, we show single molecules of Myo19 dimers processively moved on single actin filaments with a large step size of ∼34 nm. Importantly, WT Myo19 single molecules without the leucine zipper processively move in filopodia in living cells similar to Myo19 dimers, whereas deletion of the tail domain abolished such active movement. These results suggest that Myo19 can processively move on actin filaments when two Myo19 monomers form a dimer, presumably as a result of tail–tail association. In conclusion, Myo19 molecules can directly transport mitochondria on actin tracks within living cells.     

Fascin promotes filopodia formation independent of its role in actin bundling

Fascin is an evolutionarily conserved actin-binding protein that plays a key role in forming filopodia. It is widely thought that this function involves fascin directly bundling actin filaments, which is controlled by an N-terminal regulatory serine residue. In this paper, by studying cellular processes in Drosophila melanogaster that require fascin activity, we identify a regulatory residue within the C-terminal region of the protein (S289). Unexpectedly, although mutation (S289A) of this residue disrupted the actin-bundling capacity of fascin, fascin S289A fully rescued filopodia formation in fascin mutant flies. Live imaging of migrating macrophages in vivo revealed that this mutation restricted the localization of fascin to the distal ends of filopodia. The corresponding mutation of human fascin (S274) similarly affected its interaction with actin and altered filopodia dynamics within carcinoma cells. These data reveal an evolutionarily conserved role for this regulatory region and unveil a function for fascin, uncoupled from actin bundling, at the distal end of filopodia.     

Myosin X transports Mena/VASP to the tip of filopodia

Myosin X (M10) is a two-headed actin based motor expressed in a variety of cell types, that is thought to play a role in cargo movement in mammalian cells, but its cellular function is unknown. Here we found that M10 binds to Mena/VASP, which facilitates actin polymerization by competing with actin capping proteins. Immunocytochemistry revealed that endogenous M10 co-localized with Mena/VASP at the tip of filopodia. Consistently, both EGFP-M10 and RFP-VASP were found at the tip of filopodia. The result raises a hypothesis that M10 transports Mena/VASP towards the tip of filopodia. Supporting this idea, the amount of VASP at the tip of filopodia was proportional to that of M10. Furthermore, we directly visualized the movement of M10 and VASP in living HeLa cells under fluorescence microscope. EGFP-M10 and RFP-VASP move together from the root to the tip of the filopodia. Interestingly, the amount of M10 at the tip of filopodia was linearly related to the length of filopodia, consistent with the actin filament extending function of VASP. These results show that M10 is a specific motor carrying Mena/VASP from the root to the tip of the filopodia where extension of actin filament takes place.     

Cellular Events of Wrinkled Blastula Formation and the Influence of the Fertilization Envelope on Wrinkling in the Seastar Patiriella exigua

Like many echinoderms, the seastar, Patiriella exigua has a wrinkled blastula rather than the smooth-walled blastula typical of most phyla. The cellular events of wrinkled blastula formation in P. exigua were documented using light, confocal and electron microscopy. Wrinkled blastulae have a highly infolded epithelium. Prior to wrinkling, the blastomeres are cuboidal with lipid droplets and yolk granules distributed throughout their cytoplasm. During wrinkling, the cells become columnar and the lipid and yolk reserves become redistributed to the basal and apical ends of the cells, respectively. Gastrulae have a tall columnar epithelium, with a basal accumulation of lipid. Interdigitation of numerous cell projections, including short lateral processes, basal lamellipodia and apical filopodia, assists in maintaining epithelial integrity during wrinkling. Apical filopodia have not been observed in other echinoderm embryos. Although 1 M urea caused elevation of the fertilization envelope, the embryos did not expand into the newly-created space. This is suggested to be due to the adhesive properties of the hyaline layer. Embryos removed from their envelope were enlarged with shallower and fewer wrinkles compared with controls. It appears that the integrity of the hyaline layer and fertilization envelope both influence the compact wrinkled profile of P. exigua blastulae.     

Regulated actin cytoskeleton assembly at filopodium tips controls their extension and retraction.

The extension and retraction of filopodia in response to extracellular cues is thought to be an important initial step that determines the direction of growth cone advance. We sought to understand how the dynamic behavior of the actin cytoskeleton is regulated to produce extension or retraction. By observing the movement of fiduciary marks on actin filaments in growth cones of a neuroblastoma cell line, we found that filopodium extension and retraction are governed by a balance between the rate of actin cytoskeleton assembly at the tip and retrograde flow. Both assembly and flow rate can vary with time in a single filopodium and between filopodia in a single growth cone. Regulation of assembly rate is the dominant factor in controlling filopodia behavior in our system.     

Epithelial morphogenesis: filopodia at work

Spreading and fusion of epithelial sheets are conserved morphogenetic mechanisms that help shape embryos and tissues. Recent findings suggest that the formation of dynamic filopodia at the leading front of the epithelia plays a critical role in regulating cell movement and recognition during these processes.     

Development and differentiation of the eye in Platynereis dumerilii (Annelida,Polychaeta)

The nereid polychaete, Platynereis dumerilii, possess two pairs of post-trochophoral eyes with one vitreous body each. The development of these eyes has first been observed in 2-day-old larvae. Whether the eye anlagen arise from stem cells or from undifferentiated ectodermal tissue was not determined. At first, the anlagen of the anterior and the posterior eyes adjoin each other. They separate in late 3-day-old larvae. The first separated eye complexes consist each of two supporting and two sensory cells. The supporting cells synthesize two different kinds of granules, the pigment granules of the pigment cup and the prospective tubules of the vitreous body. These tubules accumulate in the distal process of the supporting cell. The vitreous body is formed by compartments of the supporting cells filled with the osmiophilic vitreous body tubules. The short, bulbar photosensory processes bear microvilli that emerge into the ocular cavity. At the apex of each sensory cell process, a single cilium (or occasionally two) arises. The sensory cells contain a different kind of pigment granule within their necks at the level of the pigment cup. The rate of eye development and differentiation varies. New supporting cells are added to the rim of the eye cup. They contribute to the periphery of the vitreous body like onion skins, and sensory cells move between supporting cells. The older the individual compartments of the vitreous body are, the more densely packed is their content of vitreous body tubules. Elongation of the sensory and supporting cell processes of the older cells increases the volume of the eye. The eyespots of the trochophore are briefly described as of the two-celled rhabdomeric type with a single basal body with ciliary rootlet.     

The Effect of Temperature on the Light-Induced Pigment Movement in Fish Corneal Chromatophores

Corneal chromatophores of unusual morphology were used for studies on the influence of temperature on the intracellular pigment movement in two species of marine fish from different temperature zones: the tropical puffer, Canthigaster cinctus, and boreal whitespotted greenling, Hexagrammos stelleri. It was shown that both dispersion under bright illumination and aggregation at darkening are slower or decrease at lower temperatures when examined in the range of 12–27°C. The mean speed of the pigment translocations in the individual cell process was 0.38 μm/s at the highest temperature examined, with a range of 0.17–1.0 μm/s. Near the middle of the temperature range, the dynamic characteristics of cell pigment movement in tropical and boreal species were rather close, suggesting that there would be little divergent adaptations with respect to the mechanisms of the pigment transport. Corneal chromatophores are considered as a new promising model for cell motility studies.