The fin roundabout: Slit-Robo and S1P signaling coordinate fin morphogenesis.

Development of vertebrate limbs and fins requires that tissue growth is directed outwards, away from the body. How such directed growth is achieved is a fascinating biological problem. For limb/fin formation and outgrowth, signaling between mesenchymal cells and the overlying epithelium is essential. In particular, the epithelium at the distal margin of the growing limb/fin bud, termed the apical ectodermal ridge (AER), promotes directed outgrowth of the underlying mesenchyme, e.g., by providing polarization cues for mesenchymal cell migration. Several classical signaling pathways, such as fibroblast growth factor (Fgf), hedgehog, and Wnt signaling, are involved in the regulation of the cellular events that shape the limb/fin bud (Iovine, 2007). In this issue of EMBO Reports, Carney and colleagues surprisingly find that the Slit-Robo pathway, which is best known for its function in axon guidance, regulates the polarity of developing zebrafish fins (Mahabaleshwar et al, 2007). Intriguingly, they identify an intricate back and forth of signals between the mesenchyme and the AER. Slit ligands derived from mesenchyme act on Robo receptors in the AER to stimulate the production of sphingosine-1-phosphate, which then acts back on the mesenchyme to regulate cell polarity and orientation.

Recent advancements in understanding fin regeneration in zebrafish.

Zebrafish have the remarkable ability to fully regenerate a lost appendage, faithfully restoring its size, shape and tissue patterning. Studies over the past decades have identified mechanisms underlying the formation, spatial organization, and regenerative growth of the blastema, a pool of proliferative progenitor cells. The patterning of newly forming tissue is tightly regulated to ensure proper rebuilding of anatomy. Precise niche regulation of retinoic acid and sonic hedgehog signaling ensures adherence to ray-interray boundaries. The molecular underpinnings of systems underlying re-establishment of pre-amputation size and shape (positional information) are also slowly starting to emerge. Osteoblasts play an important role as a cellular source of regenerating skeletal elements, and in zebrafish both osteoblast dedifferentiation as well as de novo osteoblast formation occurs. Both dedifferentiation and proliferation are tightly controlled, which makes it interesting to compare it to tumorigenesis, and to identify potential players involved in these processes. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration.

Zebrafish fin and heart: what's special about regeneration?

Many organs regenerate well in adult zebrafish, but most research has been directed toward fin and heart regeneration. Cells have been found to remain generally lineage-restricted during regeneration, and proliferative regenerative progenitors can be formed by dedifferentiation from differentiated cells. Recent studies begin to shed light on the molecular underpinnings of differences between development and regeneration. Retinoic acid, BMP and NF-κB signaling are emerging as regulators of cellular dedifferentiation. Reactive oxygen species promote regeneration, and the dynamics of ROS signaling might help explain differences between wound healing and regeneration. Finally, the heart has been added to those organs that require a nerve supply to regenerate, and a trade-off between regeneration and tumor suppression has been proposed to help explain why mammals regenerate poorly.

Assembly and positioning of actomyosin rings by contractility and planar cell polarity.

The actomyosin cytoskeleton is a primary force-generating mechanism in morphogenesis, thus a robust spatial control of cytoskeletal positioning is essential. In this report, we demonstrate that actomyosin contractility and planar cell polarity (PCP) interact in post-mitotic Ciona notochord cells to self-assemble and reposition actomyosin rings, which play an essential role for cell elongation. Intriguingly, rings always form at the cells' anterior edge before migrating towards the center as contractility increases, reflecting a novel dynamical property of the cortex. Our drug and genetic manipulations uncover a tug-of-war between contractility, which localizes cortical flows toward the equator and PCP, which tries to reposition them. We develop a simple model of the physical forces underlying this tug-of-war, which quantitatively reproduces our results. We thus propose a quantitative framework for dissecting the relative contribution of contractility and PCP to the self-assembly and repositioning of cytoskeletal structures, which should be applicable to other morphogenetic events.

Regulation by a TGFß-ROCK-actomyosin axis secures a non-linear lumen expansion that is essential for tubulogenesis.

Regulation of lumen growth is crucial to ensure the correct morphology, dimensions and function of a tubular structure. How this is controlled is still poorly understood. During Ciona intestinalis notochord tubulogenesis, single extracellular lumen pockets grow between pairs of cells and eventually fuse into a continuous tube. Here, we show that lumen growth exhibits a lag phase, during which the luminal membranes continue to grow but the expansion of the apical/lateral junction pauses for ∼30 min. Inhibition of non-muscle myosin II activity abolishes this lag phase and accelerates expansion of the junction, resulting in the formation of narrower lumen pockets partially fusing into a tube of reduced size. Disruption of actin dynamics, conversely, causes a reversal of apical/lateral junction expansion, leading to a dramatic conversion of extracellular lumen pockets to intracellular vacuoles and a tubulogenesis arrest. The onset of the lag phase is correlated with a de novo accumulation of actin that forms a contractile ring at the apical/lateral junctions. This actin ring actively restricts the opening of the lumen in the transverse plane, allowing sufficient time for lumen growth via an osmotic process along the longitudinal dimension. The dynamics of lumen formation is controlled by the TGFß pathway and ROCK activity. Our findings reveal a TGFß-ROCK-actomyosin contractility axis that coordinates lumen growth, which is powered by the dynamics of luminal osmolarity. The regulatory system may function like a sensor/checkpoint that responds to the change of luminal pressure and fine-tunes actomyosin contractility to effect proper tubulogenesis.

An equatorial contractile mechanism drives cell elongation but not cell division.

Cell shape changes and proliferation are two fundamental strategies for morphogenesis in animal development. During embryogenesis of the simple chordate Ciona intestinalis, elongation of individual notochord cells constitutes a crucial stage of notochord growth, which contributes to the establishment of the larval body plan. The mechanism of cell elongation is elusive. Here we show that although notochord cells do not divide, they use a cytokinesis-like actomyosin mechanism to drive cell elongation. The actomyosin network forming at the equator of each notochord cell includes phosphorylated myosin regulatory light chain, α-actinin, cofilin, tropomyosin, and talin. We demonstrate that cofilin and α-actinin are two crucial components for cell elongation. Cortical flow contributes to the assembly of the actomyosin ring. Similar to cytokinetic cells, membrane blebs that cause local contractions form at the basal cortex next to the equator and participate in force generation. We present a model in which the cooperation of equatorial actomyosin ring-based constriction and bleb-associated contractions at the basal cortex promotes cell elongation. Our results demonstrate that a cytokinesis-like contractile mechanism is co-opted in a completely different developmental scenario to achieve cell shape change instead of cell division. We discuss the occurrences of actomyosin rings aside from cell division, suggesting that circumferential contraction is an evolutionally conserved mechanism to drive cell or tissue elongation.

A set of SNARE proteins in the contractile vacuole complex of Paramecium regulates cellular calcium tolerance and also contributes to organelle biogenesis.

The contractile vacuole complex (CVC) of freshwater protists serves the extrusion of water and ions, including Ca(2+). No vesicle trafficking based on SNAREs has been detected so far in any CVC. SNAREs (soluble NSF [N-ethylmaleimide sensitive factor] attachment protein receptors) are required for membrane-to-membrane interaction, i.e. docking and fusion also in Paramecium. We have identified three v-/R- and three t/Q-SNAREs selectively in the CVC. Posttranscriptional silencing of Syb2, Syb6 or Syx2 slows down the pumping cycle; silencing of the latter two also causes vacuole swelling. Increase in extracellular Ca(2+) after Syb2, Syb6 or Syx2 silencing causes further swelling of the contractile vacuole and deceleration of its pulsation. Silencing of Syx14 or Syx15 entails lethality in the Ca(2+) stress test. Thus, the effects of silencing strictly depend on the type of the silenced SNARE and on the concentration of Ca(2+) in the medium. This shows the importance of organelle-resident SNARE functions (which may encompass the vesicular delivery of other organelle-resident proteins) for Ca(2+) tolerance. A similar principle may be applicable also to the CVC in widely different unicellular organisms. In addition, in Paramecium, silencing particularly of Syx6 causes aberrant positioning of the CVC during de novo biogenesis before cytokinesis.

The actin subfamily PtAct4, out of many subfamilies, is differentially localized for specific local functions in Paramecium tetraurelia cells.

Paramecium tetraurelia possesses more actin isoforms than most other cells. With monospecific antibodies against actin subfamily 4 members, we could label cleavage furrow, nascent food vacuoles, oral apparatus, cilia, cell surface and macronucleus. Expression as green fluorescent protein- (GFP-) fusion protein now allowed us to localize more stringently actin4, e.g., in the macronucleus, particularly when enhanced with anti-GFP antibodies. Posttranscriptional gene silencing of actin4 resulted in disturbances at sites where actin4 has been localized. Cell division was impaired already early on, occasionally resulting in deformed cells. Both micro- and macronuclear development during vegetative cell fission were disturbed. Over longer periods, actin4 silencing entailed reduced phagocytotic activity, paralleled by accumulation of "acidosomes" (late endosomes) near the cytopharynx where they normally fuse with nascent phagosomes. In addition, near the cell surface, extensively misshapen "terminal cisternae" (early endosomes) occurred. In deformed cells, both constitutive endocytosis and stimulated trichocyst exocytosis were impaired. Thus, actin4 exerts pleiotropic effects at widely different sites of the Paramecium cell and disturbances generally coincide with sites where actin4 is normally enriched. Evidently the loss of actin4 cannot easily be compensated for by any other of the large number of actin isoforms occurring in a Paramecium cell.

Distinct subcellular localization of a group of synaptobrevin-like SNAREs in Paramecium tetraurelia and effects of silencing SNARE-specific chaperone NSF.

We have identified new synaptobrevin-like SNAREs and localized the corresponding gene products with green fluorescent protein (GFP)-fusion constructs and specific antibodies at the light and electron microscope (EM) levels. These SNAREs, named Paramecium tetraurelia synaptobrevins 8 to 12 (PtSyb8 to PtSyb12), showed mostly very restricted, specific localization, as they were found predominantly on structures involved in endo- or phagocytosis. In summary, we found PtSyb8 and PtSyb9 associated with the nascent food vacuole, PtSyb10 near the cell surface, at the cytostome, and in close association with ciliary basal bodies, and PtSyb11 on early endosomes and on one side of the cytostome, while PtSyb12 was found in the cytosol. PtSyb4 and PtSyb5 (identified previously) were localized on small vesicles, PtSyb5 probably being engaged in trichocyst (dense core secretory vesicle) processing. PtSyb4 and PtSyb5 are related to each other and are the furthest deviating of all SNAREs identified so far. Because they show no similarity with any other R-SNAREs outside ciliates, they may represent a ciliate-specific adaptation. PtSyb10 forms small domains near ciliary bases, and silencing slows down cell rotation during depolarization-induced ciliary reversal. NSF silencing supports a function of cell surface SNAREs by revealing vesicles along the cell membrane at sites normally devoid of vesicles. The distinct distributions of these SNAREs emphasize the considerable differentiation of membrane trafficking, particularly along the endo-/phagocytic pathway, in this protozoan.

Novel types of Ca2+ release channels participate in the secretory cycle of Paramecium cells.

A database search of the Paramecium genome reveals 34 genes related to Ca(2+)-release channels of the inositol-1,4,5-trisphosphate (IP(3)) or ryanodine receptor type (IP(3)R, RyR). Phylogenetic analyses show that these Ca(2+) release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I to CRC-VI), each one with features in part reminiscent of IP(3)Rs and RyRs. We characterize here the P. tetraurelia CRC-IV-1 gene family, whose relationship to IP(3)Rs and RyRs is restricted to their C-terminal channel domain. CRC-IV-1 channels localize to cortical Ca(2+) stores (alveolar sacs) and also to the endoplasmic reticulum. This is in contrast to a recently described true IP(3) channel, a group II member (P. tetraurelia IP(3)R(N)-1), found associated with the contractile vacuole system. Silencing of either one of these CRCs results in reduced exocytosis of dense core vesicles (trichocysts), although for different reasons. Knockdown of P. tetraurelia IP(3)R(N) affects trichocyst biogenesis, while CRC-IV-1 channels are involved in signal transduction since silenced cells show an impaired release of Ca(2+) from cortical stores in response to exocytotic stimuli. Our discovery of a range of CRCs in Paramecium indicates that protozoans already have evolved multiple ways for the use of Ca(2+) as signaling molecule.

Pharmacology of ciliated protozoa--drug (in)sensitivity and experimental drug (ab)use.

Most data on the effects of drugs as inhibitors, modulators, or stimulators have been collected with higher eukaryotic, mainly mammalian cells. Although in cell biological experiments with lower eukaryotes, including ciliates, the same drugs have frequently been applied, many results remained questionable for several reasons. Most drugs had to be used in unusually high concentrations. Moreover, drug effects have rarely been verified at the biochemical or molecular level. Data steadily emerging from genomics of ciliates, mainly Paramecium tetraurelia and Tetrahymena thermophila, show that drug-binding sites have only occasionally been conserved during evolution. They may vary or be totally absent in ciliate orthologs or specifically in certain paralogs. We here try to evaluate data available so far on the pharmacology of ciliates. In the future, domain analysis and drug screenings may detect compounds specifically effective in specific ciliated protozoa, including pathogenic forms, and, thus, yield an important basis not only for cell biology but also for ecotoxicology.

The V-ATPase in Paramecium: functional specialization by multiple gene isoforms.

The vacuolar H(+)-ATPase (V-ATPase), a multisubunit, adenosine triphosphate (ATP)-driven proton pump, is essential for numerous cellular processes in all eukaryotes investigated so far. While structure and catalytic mechanism are similar to the evolutionarily related F-type ATPases, the V-ATPase's main function is to establish an electrochemical proton potential across membranes using ATP hydrolysis. The holoenzyme is formed by two subcomplexes, the transmembraneous V(0) and the cytoplasmic V(1) complexes. Sequencing of the whole genome of the ciliate Paramecium tetraurelia enabled the identification of virtually all the genes encoding V-ATPase subunits in this organism and the studying of the localization of the enzyme and roles in membrane trafficking and osmoregulation. Surprisingly, the number of V-ATPase genes in this free-living protozoan is strikingly higher than in any other species previously studied. Especially abundant are V(0)-a-subunits with as many as 17 encoding genes. This abundance creates the possibility of forming a large number of different V-ATPase holoenzymes by combination and has functional consequences by differential targeting to various organelles.

Rapid downregulation of the Ca2+-signal after exocytosis stimulation in Paramecium cells: essential role of a centrin-rich filamentous cortical network, the infraciliary lattice.

We analysed in Paramecium tetraurelia cells the role of the infraciliary lattice, a cytoskeletal network containing numerous centrin isoforms tightly bound to large binding proteins, in the re-establishment of Ca2+ homeostasis following exocytosis stimulation. The wild type strain d4-2 has been compared with the mutant cell line Delta-PtCenBP1 which is devoid of the infraciliary lattice ("Delta-PtCenBP1" cells). Exocytosis is known to involve the mobilization of cortical Ca2+-stores and a superimposed Ca2+-influx and was analysed using Fura Red ratio imaging. No difference in the initial signal generation was found between wild type and Delta-PtCenBP1 cells. In contrast, decay time was greatly increased in Delta-PtCenBP1 cells particularly when stimulated, e.g., in presence of 1mM extracellular Ca2+, [Ca2+]o. Apparent halftimes of f/f0 decrease were 8.5 s in wild type and approximately 125 s in Delta-PtCenBP1 cells, requiring approximately 30 s and approximately 180 s, respectively, to re-establish intracellular [Ca2+] homeostasis. Lowering [Ca2+]o to 0.1 and 0.01 mM caused an acceleration of intracellular [Ca2+] decay to t(1/2)=33 s and 28 s, respectively, in Delta-PtCenBP1 cells as compared to 8.1 and 5.6, respectively, for wild type cells. We conclude that, in Paramecium cells, the infraciliary lattice is the most efficient endogenous Ca2+ buffering system allowing the rapid downregulation of Ca2+ signals after exocytosis stimulation.

The actin multigene family of Paramecium tetraurelia.

BACKGROUND: A Paramecium tetraurelia pilot genome project, the subsequent sequencing of a Megabase chromosome as well as the Paramecium genome project aimed at gaining insight into the genome of Paramecium. These cells display a most elaborate membrane trafficking system, with distinct, predictable pathways in which actin could participate. Previously we had localized actin in Paramecium; however, none of the efforts so far could proof the occurrence of actin in the cleavage furrow of a dividing cell, despite the fact that actin is unequivocally involved in cell division. This gave a first hint that Paramecium may possess actin isoforms with unusual characteristics. The genome project gave us the chance to search the whole Paramecium genome, and, thus, to identify and characterize probably all actin isoforms in Paramecium. RESULTS: The ciliated protozoan, P. tetraurelia, contains an actin multigene family with at least 30 members encoding actin, actin-related and actin-like proteins. They group into twelve subfamilies; a large subfamily with 10 genes, seven pairs and one trio with > 82% amino acid identity, as well as three single genes. The different subfamilies are very distinct from each other. In comparison to actins in other organisms, P. tetraurelia actins are highly divergent, with identities topping 80% and falling to 30%. We analyzed their structure on nucleotide level regarding the number and position of introns. On amino acid level, we scanned the sequences for the presence of actin consensus regions, for amino acids of the intermonomer interface in filaments, for residues contributing to ATP binding, and for known binding sites for myosin and actin-specific drugs. Several of those characteristics are lacking in several subfamilies. The divergence of P. tetraurelia actins and actin-related proteins between different P. tetraurelia subfamilies as well as with sequences of other organisms is well represented in a phylogenetic tree, where P. tetraurelia sequences only partially cluster. CONCLUSION: Analysis of different features on nucleotide and amino acid level revealed striking differences in isoforms of actin and actin-related proteins in P. tetraurelia, both within the organism and in comparison to other organisms. This diversification suggests unprecedented specification in localization and function within a unicellular eukaryote.

A broad spectrum of actin paralogs in Paramecium tetraurelia cells display differential localization and function.

To localize the different actin paralogs found in Paramecium and to disclose functional implications, we used overexpression of GFP-fusion proteins and antibody labeling, as well as gene silencing. Several isoforms are associated with food vacuoles of different stages. GFP-actin either forms a tail at the lee side of the organelle, or it is vesicle bound in a homogenous or in a speckled arrangement, thus reflecting an actin-based mosaic of the phagosome surface appropriate for association and/or dissociation of other vesicles upon travel through the cell. Several paralogs occur in cilia. A set of actins is found in the cell cortex where actin outlines the regular surface pattern. Labeling of defined structures of the oral cavity is due to other types of actin, whereas yet more types are distributed in a pattern suggesting association with the numerous Golgi fields. A substantial fraction of actins is associated with cytoskeletal elements that are known to be composed of other proteins. Silencing of the respective actin genes or gene subfamilies entails inhibitory effects on organelles compatible with localization studies. Knock down of the actin found in the cleavage furrow abolishes cell division, whereas silencing of other actin genes alters vitality, cell shape and swimming behavior.

Immunolocalization of actin in Paramecium cells.

We have selected a conserved immunogenic region from several actin genes of Paramecium, recently cloned in our laboratory, to prepare antibodies for Western blots and immunolocalization. According to cell fractionation analysis, most actin is structurebound. Immunofluorescence shows signal enriched in the cell cortex, notably around ciliary basal bodies (identified by anti-centrin antibodies), as well as around the oral cavity, at the cytoproct and in association with vacuoles (phagosomes) up to several mum in size. Subtle strands run throughout the cell body. Postembedding immunogold labeling/EM analysis shows that actin in the cell cortex emanates, together with the infraciliary lattice, from basal bodies to around trichocyst tips. Label was also enriched around vacuoles and vesicles of different size including "discoidal" vesicles that serve the formation of new phagosomes. By all methods used, we show actin in cilia. Although none of the structurally well-defined filament systems in Paramecium are exclusively formed by actin, actin does display some ordered, though not very conspicuous, arrays throughout the cell. F-actin may somehow serve vesicle trafficking and as a cytoplasmic scaffold. This is particularly supported by the postembedding/EM labeling analysis we used, which would hardly allow for any large-scale redistribution during preparation.

Ca2+ oscillations mediated by exogenous GTP in Paramecium cells: assessment of possible Ca2+ sources.

We applied exogenous guanosine trisphosphate, GTP, to Paramecium tetraurelia cells injected with Fura Red for analysing changes of free intracellular Ca(2+) concentrations, [Ca(2+)](i), during periodic back-/forward swimming thus induced. Strain ginA (non-responsive to GTP) shows no Ca(2+) signal upon GTP application. In strain nd6 (normal Ca(2+) signalling) an oscillating [Ca(2+)](i) response with a prominent first peak occurs upon GTP stimulation, but none after mock-stimulation or after 15 min adaptation to GTP. While this is in agreement with previous electrophysiological analyses, we now try to identify more clearly the source(s) of Ca(2+). Stimulation of nd6 cells, after depletion of Ca(2+) from their cortical stores (alveolar sacs), shows the same Ca(2+) oscillation pattern but with reduced amplitudes, and a normal behavioural response is observed. Stimulation with GTP, supplemented with the Ca(2+) chelator BAPTA, results in loss of the first prominent Ca(2+) peak, in reduction of the following Ca(2+) amplitudes, and in the absence of any behavioural response. Both these observations strongly suggest that for the initiation of GTP-mediated back-/forward swimming Ca(2+) from the extracellular medium is needed. For the maintenance of the Ca(2+) oscillations a considerable fraction must come from internal stores, probably other than alveolar sacs, rather likely from the endoplasmic reticulum.