ABSTRACT
The Rho GTPases - RHOA, RAC1 and CDC42 - are small GTP binding proteins that regulate basic biological processes such as cell locomotion, cell division and morphogenesis by promoting cytoskeleton-based changes in the cell cortex. This regulation results from active (GTP-bound) Rho GTPases stimulating target proteins that, in turn, promote actin assembly and myosin 2-based contraction to organize the cortex. This basic regulatory scheme, well supported by in vitro studies, led to the natural assumption that Rho GTPases function in vivo in an essentially linear matter, with a given process being initiated by GTPase activation and terminated by GTPase inactivation. However, a growing body of evidence based on live cell imaging, modelling and experimental manipulation indicates that Rho GTPase activation and inactivation are often tightly coupled in space and time via signalling circuits and networks based on positive and negative feedback. In this Review, we present and discuss this evidence, and we address one of the fundamental consequences of coupled activation and inactivation: the ability of the Rho GTPases to self-organize, that is, direct their own transition from states of low order to states of high order. We discuss how Rho GTPase self-organization results in the formation of diverse spatiotemporal cortical patterns such as static clusters, oscillatory pulses, travelling wave trains and ring-like waves. Finally, we discuss the advantages of Rho GTPase self-organization and pattern formation for cell function.
Subject(s)
Cytoskeleton , rho GTP-Binding Proteins , rho GTP-Binding Proteins/metabolism , Cytoskeleton/metabolism , Actins/metabolism , Signal Transduction , Cell Movement , rac1 GTP-Binding Protein/metabolismABSTRACT
Endocycles are variant cell cycles comprised of DNA synthesis (S)- and gap (G)-phases but lacking mitosis. Such cycles facilitate post-mitotic growth in many invertebrate and plant cells, and are so ubiquitous that they may account for up to half the world's biomass. DNA replication in endocycling Drosophila cells is triggered by cyclin E/cyclin dependent kinase 2 (CYCE/CDK2), but this kinase must be inactivated during each G-phase to allow the assembly of pre-Replication Complexes (preRCs) for the next S-phase. How CYCE/CDK2 is periodically silenced to allow re-replication has not been established. Here, using genetic tests in parallel with computational modelling, we show that the endocycles of Drosophila are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the CRL4(CDT2) ubiquitin ligase, and this in turn mediates the destruction of E2F1 (ref. 7). We propose that it is the transient loss of E2F1 during S phases that creates the window of low Cdk activity required for preRC formation. In support of this model overexpressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by deregulating target genes, including CycE, as well as Cdk1 and mitotic cyclins. Moreover, we find that altering cell growth by changing nutrition or target of rapamycin (TOR) signalling impacts E2F1 translation, thereby making endocycle progression growth-dependent. Many of the regulatory interactions essential to this novel cell cycle oscillator are conserved in animals and plants, indicating that elements of this mechanism act in most growth-dependent cell cycles.
Subject(s)
Cell Cycle/physiology , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/enzymology , E2F Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Female , Male , S Phase/physiology , Salivary Glands/cytology , Transcription Factors , Ubiquitin-Protein Ligase ComplexesABSTRACT
The Rho GTPases pattern the cell cortex in a variety of fundamental cell-morphogenetic processes including division, wound repair, and locomotion. It has recently become apparent that this patterning arises from the ability of the Rho GTPases to self-organize into static and migrating spots, contractile pulses, and propagating waves in cells from yeasts to mammals 1 . These self-organizing Rho GTPase patterns have been explained by a variety of theoretical models which require multiple interacting positive and negative feedback loops. However, it is often difficult, if not impossible, to discriminate between different models simply because the available experimental data do not simultaneously capture the dynamics of multiple molecular concentrations and biomechanical variables at fine spatial and temporal resolution. Specifically, most studies typically provide either the total Rho GTPase signal or the Rho GTPase activity as reported by various sensors, but not both. Therefore, it remains largely unknown how membrane accumulation of Rho GTPases (i.e., Rho membrane enrichment) is related to Rho activity. Here we dissect the dynamics of RhoA by simultaneously imaging both total RhoA and active RhoA in the regime of acute cortical excitability 2 , characterized by pronounced waves of Rho activity and F-actin polymerization 3-5 . We find that within nascent waves, accumulation of active RhoA precedes that of total RhoA, and we exploit this finding to distinguish between two popular theoretical models previously used to explain propagating cortical Rho waves.
ABSTRACT
During vertebrate cell division, chromosomes oscillate with periods of smooth motion interrupted by abrupt reversals in direction. These oscillations must be spatially constrained in order to align and segregate chromosomes with high fidelity, but the molecular mechanism for this activity is uncertain. We report here that the human kinesin-8 Kif18A has a primary role in the control of chromosome oscillations. Kif18A accumulates as a gradient on kinetochore microtubules in a manner dependent on its motor activity. Quantitative analyses of kinetochore movements reveal that Kif18A reduces the amplitude of preanaphase oscillations and slows poleward movement during anaphase. Thus, the microtubule-depolymerizing kinesin Kif18A has the unexpected function of suppressing chromosome movements. Based on these findings, we propose a molecular model in which Kif18A regulates kinetochore microtubule dynamics to control mitotic chromosome positioning.
Subject(s)
Chromosome Positioning , Chromosomes, Human/metabolism , Kinesins/metabolism , Kinetochores/metabolism , Mitosis , Anaphase , Cell Polarity , HeLa Cells , Humans , Microtubules/metabolism , Models, Biological , Protein Transport , Spindle Apparatus/metabolismABSTRACT
INTRODUCTION: The nemertean pilidium is a long-lived feeding larva unique to the life cycle of a single monophyletic group, the Pilidiophora, which is characterized by this innovation. That the pilidium feeds on small planktonic unicells seems clear; how it does so is unknown and not readily inferred, because it shares little morphological similarity with other planktotrophic larvae. RESULTS: Using high-speed video of trapped lab-reared pilidia of Micrura alaskensis, we documented a multi-stage feeding mechanism. First, the external ciliation of the pilidium creates a swimming and feeding current which carries suspended prey past the primary ciliated band spanning the posterior margins of the larval body. Next, the larva detects prey that pass within reach, then conducts rapid and coordinated deformations of the larval body to re-direct passing cells and surrounding water into a vestibular space between the lappets, isolated from external currents but not quite inside the larva. Once a prey cell is thus captured, internal ciliary bands arranged within this vestibule prevent prey escape. Finally, captured cells are transported by currents within a buccal funnel toward the stomach entrance. Remarkably, we observed that the prey of choice - various cultured cryptomonads - attempt to escape their fate. CONCLUSIONS: The feeding mechanism deployed by the pilidium larva coordinates local control of cilia-driven water transport with sensorimotor behavior, in a manner clearly distinct from any other well-studied larval feeding mechanisms. We hypothesize that the pilidium's feeding strategy may be adapted to counter escape responses such as those deployed by cryptomonads, and speculate that similar needs may underlie convergences among disparate planktotrophic larval forms.
ABSTRACT
Planktonic organisms feed while suspended in water using various hydrodynamic pumping strategies. Appendicularians are a unique group of plankton that use their tail to pump water over mucous mesh filters to concentrate food particles. As ubiquitous and often abundant members of planktonic ecosystems, they play a major role in oceanic food webs. Yet, we lack a complete understanding of the fluid flow that underpins their filtration. Using high-speed, high-resolution video and micro particle image velocimetry, we describe the kinematics and hydrodynamics of the tail in Oikopleura dioica in filtering and free-swimming postures. We show that sinusoidal waves of the tail generate peristaltic pumping within the tail chamber with fluid moving parallel to the tail when filtering. We find that the tail contacts attachment points along the tail chamber during each beat cycle, serving to seal the tail chamber and drive pumping. When we tested how the pump performs across environmentally relevant temperatures, we found that the amplitude of the tail was invariant but tail beat frequency increased threefold across three temperature treatments (5°C, 15°C and 25°C). Investigation into this unique pumping mechanism gives insight into the ecological success of appendicularians and provides inspiration for novel pump designs.
Subject(s)
Ecosystem , Hydrodynamics , Animals , Biomechanical Phenomena , Plankton , Swimming , Water , TailABSTRACT
The nemertean pilidium larva is a long-lived planktotrophic form which is challenging to homologize to other invertebrate larval forms. Here we report a reduced, lecithotrophic pilidium which superficially resembles a trochophore. We document the pilidium-like catastrophic metamorphosis of this larva, including devouring of the larval body. Sequences of COI and 16S rRNA show that this larva belongs to an undescribed lineiform species. This novel larval form highlights the long-standing question, is the trochophore a conserved larval ground-plan or a functional design arrived at by convergence?
Subject(s)
Biological Evolution , Invertebrates/anatomy & histology , Invertebrates/physiology , Metamorphosis, Biological/physiology , Plankton , Animals , Base Sequence , DNA Barcoding, Taxonomic , Electron Transport Complex IV/genetics , Invertebrates/classification , Larva/anatomy & histology , Larva/physiology , Molecular Sequence Data , Oregon , Pacific Ocean , Sequence Analysis, DNA , Species SpecificityABSTRACT
Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskeletal regulators. This phenomenon, termed cortical excitability, results from coupled positive and negative feedback loops of cytoskeletal regulators. The nature of these feedback loops, however, remains poorly understood. We assessed the role of the Rho GAP RGA-3/4 in the cortical excitability that accompanies cytokinesis in both frog and starfish. RGA-3/4 localizes to the cytokinetic apparatus, "chases" Rho waves in an F-actin-dependent manner, and when coexpressed with the Rho GEF Ect2, is sufficient to convert the normally quiescent, immature Xenopus oocyte cortex into a dramatically excited state. Experiments and modeling show that changing the ratio of RGA-3/4 to Ect2 produces cortical behaviors ranging from pulses to complex waves of Rho activity. We conclude that RGA-3/4, Ect2, Rho, and F-actin form the core of a versatile circuit that drives a diverse range of cortical behaviors, and we demonstrate that the immature oocyte is a powerful model for characterizing these dynamics.
Subject(s)
Actins , Cytoskeleton , GTPase-Activating Proteins , Proto-Oncogene Proteins , rho GTP-Binding Proteins , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Cytokinesis , Cytoskeleton/metabolism , GTPase-Activating Proteins/metabolism , Oocytes , Proto-Oncogene Proteins/metabolism , Xenopus , rho GTP-Binding Proteins/metabolismABSTRACT
As the interface between the cell and its environment, the cell cortex must be able to respond to a variety of external stimuli. This is made possible in part by cortical excitability, a behavior driven by coupled positive and negative feedback loops that generate propagating waves of actin assembly in the cell cortex. Cortical excitability is best known for promoting cell protrusion and allowing the interpretation of and response to chemoattractant gradients in migrating cells. It has recently become apparent, however, that cortical excitability is involved in the response of the cortex to internal signals from the cell-cycle regulatory machinery and the spindle during cell division. Two overlapping functions have been ascribed to cortical excitability in cell division: control of cell division plane placement, and amplification of the activity of the small GTPase Rho at the equatorial cortex during cytokinesis. Here, we propose that cortical excitability explains several important yet poorly understood features of signaling during cell division. We also consider the potential advantages that arise from the use of cortical excitability as a signaling mechanism to regulate cortical dynamics in cell division.
Subject(s)
Actins , Cytokinesis , Actins/metabolism , Cell Division , Cytoplasm/metabolism , Signal TransductionABSTRACT
In the October 7th issue of Cell, it is shown that a ring-like structure containing the centrosomal protein centriolin acts as a local recruitment site for the membrane fusion machinery that controls abscission.
Subject(s)
Cell Cycle Proteins/metabolism , Membrane Fusion/physiology , Animals , Cell Division/physiology , Humans , Secretory Vesicles/metabolismABSTRACT
Cytokinesis in animal cells results from the assembly and constriction of a circumferential array of actin filaments and myosin-2. Microtubules of the mitotic apparatus determine the position at which the cytokinetic actomyosin array forms, but the molecular mechanisms by which they do so remain unknown. The small GTPase RhoA has previously been implicated in cytokinesis. Using four-dimensional microscopy and a probe for active RhoA, we show that active RhoA concentrates in a precisely bounded zone before cytokinesis and is independent of actin assembly. Cytokinetic RhoA activity zones are common to four echinoderm species, the vertebrate Xenopus laevis, and the highly asymmetric cytokinesis accompanying meiosis. Microtubules direct the formation and placement of the RhoA activity zone, and the zone is repositioned after physical spindle displacement. We conclude that microtubules specify the cytokinetic apparatus via a dynamic zone of local RhoA activity.
Subject(s)
Cytokinesis/physiology , Embryo, Nonmammalian/metabolism , Microtubules/metabolism , rhoA GTP-Binding Protein/metabolism , Actin Cytoskeleton/metabolism , Animals , Cleavage Stage, Ovum/cytology , Cleavage Stage, Ovum/physiology , Embryo, Nonmammalian/cytology , Female , Intracellular Signaling Peptides and Proteins/metabolism , Meiosis/physiology , Myosins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sea Urchins , Species Specificity , Spindle Apparatus/metabolism , Starfish , Strongylocentrotus purpuratus , Xenopus laevisABSTRACT
MCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs.
Subject(s)
Cell Compartmentation/physiology , Cell Polarity/physiology , Kinesins/metabolism , Microtubules/metabolism , Animals , CHO Cells , Cricetinae , Green Fluorescent Proteins , HeLa Cells , Humans , Microscopy, Video , Microtubules/ultrastructure , Phosphorylation , Polymers/metabolism , Protein Structure, Tertiary/physiology , Protein Transport/physiology , Recombinant Fusion Proteins/metabolismABSTRACT
We describe methods and techniques for introduction of molecular probes in the form of synthetic mRNA by rapid repetitive microinjection into oocytes or early embryos of echinoderms and various invertebrates. Construct assembly is followed by standard kit-based in vitro mRNA synthesis, with slight modifications to optimize expression and clean-up. Variations of a basic microinjection procedures are detailed for echinoderms: starfish oocytes (Patiria miniata or other species), purple urchin (Strongylocentrotus purpuratus) and sand dollar (Dendraster excentricus) zygotes, with notes included for other invertebrate eggs and embryos as well.
Subject(s)
Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/physiology , Microinjections/methods , Molecular Probes/administration & dosage , Oocytes/cytology , RNA, Messenger/administration & dosage , Animals , Sea Urchins/genetics , Starfish/geneticsABSTRACT
BACKGROUND: Many gene networks used by developing organisms have been conserved over long periods of evolutionary time. Why is that? We showed previously that a model of the segment polarity network in Drosophila is robust to parameter variation and is likely to act as a semiautonomous patterning module. Is this true of other networks as well? RESULTS: We present a model of the core neurogenic network in Drosophila. Our model exhibits at least three related pattern-resolving behaviors that the real neurogenic network accomplishes during embryogenesis in Drosophila. Furthermore, we find that it exhibits these behaviors across a wide range of parameter values, with most of its parameters able to vary more than an order of magnitude while it still successfully forms our test patterns. With a single set of parameters, different initial conditions (prepatterns) can select between different behaviors in the network's repertoire. We introduce two new measures for quantifying network robustness that mimic recombination and allelic divergence and use these to reveal the shape of the domain in the parameter space in which the model functions. We show that lateral inhibition yields robustness to changes in prepatterns and suggest a reconciliation of two divergent sets of experimental results. Finally, we show that, for this model, robustness confers functional flexibility. CONCLUSIONS: The neurogenic network is robust to changes in parameter values, which gives it the flexibility to make new patterns. Our model also offers a possible resolution of a debate on the role of lateral inhibition in cell fate specification.
Subject(s)
Computer Simulation , Drosophila melanogaster/embryology , Models, Neurological , Nerve Net/embryology , Nerve Net/metabolism , Proteins , Alleles , Animals , Biological Evolution , Cell Differentiation , Cell Lineage , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Homeostasis , Nerve Net/cytology , Neurons/cytology , Neurons/metabolism , Recombination, Genetic , Repressor Proteins/metabolismABSTRACT
BACKGROUND: Nemertean embryos undergo equal spiral cleavage, and prior fate-mapping studies showed that some also exhibit key aspects of spiralian lineage-based fate specification, including specification of the primary trochoblasts, which differentiate early as the core of the prototroch of the spiralian trochophore larva. Yet it remains unclear how the nemertean pilidium larva, a long-lived planktotroph that grows substantially as it builds a juvenile body from isolated rudiments, develops within the constraints of spiral cleavage. RESULTS: We marked single cells in embryos of the pilidiophoran Maculaura alaskensis to show that primary, secondary, and accessory trochoblasts, cells that would make the prototroch in conventional spiralian trochophores (1q2, 1q12, and some descendants of 2q), fully account for the pilidium's primary ciliary band, but without undergoing early cleavage arrest. Instead, the primary ciliary band consists of many small, albeit terminally differentiated, cells. The trochoblasts also give rise to niches of indefinitely proliferative cells ("axils") that sustain continuous growth of the larval body, including new ciliated band. Several of the imaginal rudiments that form the juvenile body arise from the axils: in particular, we show that cephalic imaginal disks originate from 1a2 and 1b12 and that trunk imaginal disks likely originate from 2d. CONCLUSIONS: The pilidium exhibits a familiar relation between identified blastomeres and the primary ciliated band, but the manner in which these cells form this organ differs fundamentally from the way equivalent cells construct the trochophore's prototroch. Also, the establishment, by some progeny of the putative trochoblasts, of indeterminate stem cell populations that give rise to juvenile rudiments, as opposed to an early cleavage arrest, implies a radical alteration in their developmental program. This transition may have been essential to the evolution of a maximally indirect developing larval form-the pilidium-among nemerteans.
ABSTRACT
Gametogenesis in animal oocytes reduces the diploid genome content of germline precursors to a haploid state in gametes by discarding ¾ of the duplicated chromosomes through a sequence of two meiotic cell divisions called meiosis I and II. The assembly of the microtubule-based spindle structure that mediates this reduction in genome content remains poorly understood compared to our knowledge of mitotic spindle assembly and function. In this review, we consider the diversity of oocyte meiotic spindle assembly and structure across animal phylogeny, review recent advances in our understanding of how animal oocytes assemble spindles in the absence of the centriole-based microtubule-organizing centers that dominate mitotic spindle assembly, and discuss different models for how chromosomes are captured and moved to achieve chromosome segregation during oocyte meiotic cell division.
Subject(s)
Meiosis , Oocytes/cytology , Oocytes/physiology , Spindle Apparatus/physiology , Animals , Caenorhabditis elegans , Centrosome/metabolism , Centrosome/ultrastructure , Chromosomes/metabolism , Female , Kinetochores/physiology , Microtubules/metabolism , Microtubules/ultrastructure , Spindle Apparatus/ultrastructureABSTRACT
Emergence of the cytokinetic Rho zone that orchestrates formation and ingression of the cleavage furrow had been explained previously via microtubule-dependent cortical concentration of Ect2, a guanine nucleotide exchange factor for Rho. The results of a recent publication now demonstrate that, en route from resting cortex to fully established furrow, there lies a regime of cortical excitability in which Rho activity and F-actin play the roles of the prototypical activator and inhibitor, respectively. This cortical excitability is manifest as dramatic traveling waves on the cortex of oocytes and embryos of frogs and starfish. These waves are initiated by autocatalytic activation of Rho at the wave front and extinguished by F-actin-dependent inhibition at their back. It is still unclear how propagating excitable Rho-actin waves give rise to the stable co-existence of Rho activity and F-actin density in the static cleavage furrow during cytokinesis. It is possible that some central spindle-associated signaling molecule simply turns off the inhibition of Rho activity by F-actin. However, mathematical modeling suggests a distinct scenario in which local "re-wiring" of the Rho-actin coupling in the furrow is no longer necessary. Instead, the model predicts that the continuously rising level of Ect2 produces in the furrow a qualitatively new stable steady state that replaces excitability and brings about the stable co-existence of high Rho activity and dense F-actin despite the continuing inhibition of Rho by F-actin.
Subject(s)
Cytokinesis , Actins/metabolism , Animals , Humans , rho GTP-Binding Proteins/metabolismABSTRACT
Cell division in all eukaryotes depends on function of the spindle, a microtubule-based structure that segregates chromosomes to generate daughter cells in mitosis or haploid gametes in meiosis. Spindle size adapts to changes in cell size and shape, which vary dramatically across species and within a multicellular organism, but the nature of scaling events and their underlying mechanisms are poorly understood. Cell size variations are most pronounced in early animal development, as egg diameters range from tens of microns up to millimeters across animal phyla, and decrease several orders of magnitude during rapid reductive divisions. During early embryogenesis in the model organisms X. laevis and C. elegans, the spindle scales with cell size [1, 2], a phenomenon regulated by molecules that modulate microtubule dynamics [3-6], as well as by limiting cytoplasmic volume [7, 8]. However, it is not known to what extent spindle scaling is conserved across organisms and among different cell types. Here we show that in a range of metazoan phyla, mitotic spindle length decreased with cell size across an â¼30-fold difference in zygote size. Maximum spindle length varied, but linear spindle scaling occurred similarly in all species once embryonic cell diameter reduced to 140 µm. In contrast, we find that the female meiotic spindle does not scale as closely to egg size, adopting a more uniform size across species that most likely reflects its specialized function. Our analysis reveals that spindle morphometrics change abruptly, within one cell cycle, at the transition from meiosis to mitosis in most animals.