RESUMO
Cytoplasmic flows are widely emerging as key functional players in development. In early Drosophila embryos, flows drive the spreading of nuclei across the embryo. Here, we combine hydrodynamic modeling with quantitative imaging to develop a two-fluid model that features an active actomyosin gel and a passive viscous cytosol. Gel contractility is controlled by the cell cycle oscillator, the two fluids being coupled by friction. In addition to recapitulating experimental flow patterns, our model explains observations that remained elusive and makes a series of predictions. First, the model captures the vorticity of cytosolic flows, which highlights deviations from Stokes' flow that were observed experimentally but remained unexplained. Second, the model reveals strong differences in the gel and cytosol motion. In particular, a micron-sized boundary layer is predicted close to the cortex, where the gel slides tangentially while the cytosolic flow cannot slip. Third, the model unveils a mechanism that stabilizes the spreading of nuclei with respect to perturbations of their initial positions. This self-correcting mechanism is argued to be functionally important for proper nuclear spreading. Fourth, we use our model to analyze the effects of flows on the transport of the morphogen Bicoid and the establishment of its gradients. Finally, the model predicts that the flow strength should be reduced if the shape of the domain is more round, which is experimentally confirmed in Drosophila mutants. Thus, our two-fluid model explains flows and nuclear positioning in early Drosophila, while making predictions that suggest novel future experiments.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Citosol/metabolismo , Hidrodinâmica , Citoplasma/metabolismo , Proteínas de Drosophila/metabolismoRESUMO
Cytoplasmic flows are widely emerging as key functional players in development. In early Drosophila embryos, flows drive the spreading of nuclei across the embryo. Here, we combine hydrodynamic modeling with quantitative imaging to develop a two-fluid model that features an active actomyosin gel and a passive viscous cytosol. Gel contractility is controlled by the cell cycle oscillator, the two fluids being coupled by friction. In addition to recapitulating experimental flow patterns, our model explains observations that remained elusive, and makes a series of new predictions. First, the model captures the vorticity of cytosolic flows, which highlights deviations from Stokes' flow that were observed experimentally but remained unexplained. Second, the model reveals strong differences in the gel and cytosol motion. In particular, a micron-sized boundary layer is predicted close to the cortex, where the gel slides tangentially whilst the cytosolic flow cannot slip. Third, the model unveils a mechanism that stabilizes the spreading of nuclei with respect to perturbations of their initial positions. This self-correcting mechanism is argued to be functionally important for proper nuclear spreading. Fourth, we use our model to analyze the effects of flows on the transport of the morphogen Bicoid, and the establishment of its gradients. Finally, the model predicts that the flow strength should be reduced if the shape of the domain is more round, which is experimentally confirmed in Drosophila mutants. Thus, our two-fluid model explains flows and nuclear positioning in early Drosophila, while making predictions that suggest novel future experiments.
RESUMO
Metastasisation occurs through the acquisition of invasive and survival capabilities that allow tumour cells to colonise distant sites. While the role of multicellular aggregates in cancer dissemination is acknowledged, the mechanisms that drive the formation of multiclonal cell aggregates are not fully elucidated. Here, we show that cancer cells of different tissue of origins can perform collective directional migration and can actively form heteroclonal aggregates in 3D, through a proliferation-independent mechanism. Coalescence of distant cell clusters is mediated by subcellular actin-rich protrusions and multicellular outgrowths that extend towards neighbouring aggregates. Coherently, perturbation of cytoskeletal dynamics impairs collective migration while myosin II activation is necessary for multicellular movements. We put forward the hypothesis that cluster attraction is mediated by secreted soluble factors. Such a hypothesis is consistent with the abrogation of aggregation by inhibition of PI3K/AKT/mTOR and MEK/ERK, the chemoattracting activity of conditioned culture media and with a wide screening of secreted proteins. Our results present a novel collective migration model and shed light on the mechanisms of formation of heteroclonal aggregates in cancer.
Assuntos
Neoplasias , Fosfatidilinositol 3-Quinases , Humanos , Movimento Celular , Actinas/metabolismoRESUMO
Compelling evidence shows that cancer persister cells represent a major limit to the long-term efficacy of targeted therapies. However, the phenotype and population dynamics of cancer persister cells remain unclear. We developed a quantitative framework to study persisters by combining experimental characterization and mathematical modeling. We found that, in colorectal cancer, a fraction of persisters slowly replicates. Clinically approved targeted therapies induce a switch to drug-tolerant persisters and a temporary 7- to 50-fold increase of their mutation rate, thus increasing the number of persister-derived resistant cells. These findings reveal that treatment may influence persistence and mutability in cancer cells and pinpoint inhibition of error-prone DNA polymerases as a strategy to restrict tumor recurrence.
Assuntos
Neoplasias Colorretais , Taxa de Mutação , Antibacterianos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Humanos , Dinâmica PopulacionalRESUMO
In most metazoans, early embryonic development is characterized by rapid division cycles that pause before gastrulation at the midblastula transition (MBT).1 These cleavage divisions are accompanied by cytoskeletal rearrangements that ensure proper nuclear positioning. However, the molecular mechanisms controlling nuclear positioning are not fully elucidated. In Drosophila, early embryogenesis unfolds in a multinucleated syncytium. Nuclei rapidly move across the anterior-posterior (AP) axis at cell cycles 4-6 in a process driven by actomyosin contractility and cytoplasmic flows.2,3 In shackleton (shkl) mutants, this axial spreading is impaired.4 Here, we show that shkl mutants carry mutations in the cullin-5 (cul-5) gene. Live imaging experiments show that Cul-5 is downstream of the cell cycle but is required for cortical actomyosin contractility. The nuclear spreading phenotype of cul-5 mutants can be rescued by reducing Src activity, suggesting that a major target of cul-5 is Src kinase. cul-5 mutants display gradients of nuclear density across the AP axis that we exploit to study cell-cycle control as a function of the N/C ratio. We found that the N/C ratio is sensed collectively in neighborhoods of about 100 µm, and such collective sensing is required for a precise MBT, in which all the nuclei in the embryo pause their division cycle. Moreover, we found that the response to the N/C ratio is slightly graded along the AP axis. These two features can be linked to Cdk1 dynamics. Collectively, we reveal a new pathway controlling nuclear positioning and provide a dissection of how nuclear cycles respond to the N/C ratio.
Assuntos
Proteínas Culina , Drosophila , Actomiosina/metabolismo , Animais , Ciclo Celular/fisiologia , Proteínas Culina/metabolismo , Drosophila/genética , Embrião não Mamífero , Desenvolvimento Embrionário/genéticaRESUMO
Cancer adoptive cell therapy (ACT) with HLA-independent tumor killer lymphocytes is a promising approach, with intrinsic features potentially addressing crucial tumor-escape mechanisms of checkpoint inhibitors. Cytokine-induced Killer (CIK) and Natural Killer (NK) lymphocytes share similar tumor-killing mechanisms, with preclinical evidence of intense activity against multiple solid tumors and currently testing in clinical studies. To improve the effective clinical translation of such ACT approaches, several fundamental questions still need to be addressed within appropriate preclinical contexts, capable of overcoming limitations imposed by most traditional two-dimensional assays. Here, we developed a novel experimental approach to explore, dissect, and visualize the interactions of CIK and NK lymphocytes with melanoma tumors in vitro in 3D. Primary melanoma cells were assembled into small tumors that were dispersed in a 3D matrix and challenged with patient-derived CIK or the NK-92 cell line. By means of imaging-based methods, we reported, visualized, and quantitatively measured the recruitment of CIK and NK on the 3D targets, their infiltration, and cytotoxic activity. Our results support the effective tumor recruitment and tumor infiltration by CIK and NK. Such features appeared dependent on the specific geometric aspects of the environment but can be explained in terms of directional migration toward the tumor, without invoking major feedback components. Overall, our 3D platform allows us to monitor the processes of tumor recruitment, infiltration, and killing by means of live measurements, revealing important kinetic aspects of ACT with CIK and NK against melanoma.
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Blockade of epidermal growth factor receptor (EGFR) causes tumor regression in some patients with metastatic colorectal cancer (mCRC). However, residual disease reservoirs typically remain even after maximal response to therapy, leading to relapse. Using patient-derived xenografts (PDXs), we observed that mCRC cells surviving EGFR inhibition exhibited gene expression patterns similar to those of a quiescent subpopulation of normal intestinal secretory precursors with Paneth cell characteristics. Compared with untreated tumors, these pseudodifferentiated tumor remnants had reduced expression of genes encoding EGFR-activating ligands, enhanced activity of human epidermal growth factor receptor 2 (HER2) and HER3, and persistent signaling along the phosphatidylinositol 3-kinase (PI3K) pathway. Clinically, properties of residual disease cells from the PDX models were detected in lingering tumors of responsive patients and in tumors of individuals who had experienced early recurrence. Mechanistically, residual tumor reprogramming after EGFR neutralization was mediated by inactivation of Yes-associated protein (YAP), a master regulator of intestinal epithelium recovery from injury. In preclinical trials, Pan-HER antibodies minimized residual disease, blunted PI3K signaling, and induced long-term tumor control after treatment discontinuation. We found that tolerance to EGFR inhibition is characterized by inactivation of an intrinsic lineage program that drives both regenerative signaling during intestinal repair and EGFR-dependent tumorigenesis. Thus, our results shed light on CRC lineage plasticity as an adaptive escape mechanism from EGFR-targeted therapy and suggest opportunities to preemptively target residual disease.
Assuntos
Neoplasias Colorretais , Fosfatidilinositol 3-Quinases , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Receptores ErbB , Humanos , Recidiva Local de Neoplasia , Neoplasia Residual , Celulas de Paneth , FenótipoRESUMO
The activation of the majority of AGC kinases is regulated by two phosphorylation events on two conserved serine/threonine residues located on the activation loop and on the hydrophobic motif, respectively. In AGC kinase family, phosphomimetic substitutions with aspartate or glutamate, leading to constitutive activation, have frequently occurred at the hydrophobic motif site. On the contrary, phosphomimetic substitutions in the activation loop are absent across the evolution of AGC kinases. This observation is explained by the failure of aspartate and glutamate to mimic phosphorylatable serine/threonine in this regulatory site. By detailed 3D structural simulations of RSK2 and further biochemical evaluation in cells, we show that the phosphomimetic residue on the activation loop fails to form a critical salt bridge with R114, necessary to reorient the αC-helix and to activate the protein. By a phylogenetic analysis, we point at a possible coevolution of a phosphorylatable activation loop and the presence of a conserved positively charged amino acid on the αC-helix. In sum, our analysis leads to the unfeasibility of phosphomimetic substitution in the activation loop of RSK and, at the same time, highlights the peculiar structural role of activation loop phosphorylation.
Assuntos
Substituição de Aminoácidos , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Motivos de Aminoácidos , Ativação Enzimática , Evolução Molecular , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mimetismo Molecular , Fosforilação , Filogenia , Estrutura Secundária de Proteína , Proteínas Quinases S6 Ribossômicas 90-kDa/genéticaRESUMO
Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes.
Assuntos
Células Endoteliais/metabolismo , Neovascularização Fisiológica , Pericitos/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Células Endoteliais/citologia , Camundongos , Camundongos Transgênicos , Pericitos/citologiaRESUMO
Cell-substrate interactions can modulate cellular behaviors in a variety of biological contexts, including development and disease. Light-responsive materials have been recently proposed to engineer active substrates with programmable topographies directing cell adhesion, migration, and differentiation. However, current approaches are affected by either fabrication complexity, limitations in the extent of mechanical stimuli, lack of full spatio-temporal control, or ease of use. Here, a platform exploiting light to plastically deform micropatterned polymeric substrates is presented. Topographic changes with remarkable relief depths in the micron range are induced in parallel, by illuminating the sample at once, without using raster scanners. In few tens of seconds, complex topographies are instructed on demand, with arbitrary spatial distributions over a wide range of spatial and temporal scales. Proof-of-concept data on breast cancer cells and normal kidney epithelial cells are presented. Both cell types adhere and proliferate on substrates without appreciable cell damage upon light-induced substrate deformations. User-provided mechanical stimulation aligns and guides cancer cells along the local deformation direction and constrains epithelial colony growth by biasing cell division orientation. This approach is easy to implement on general-purpose optical microscopy systems and suitable for use in cell biology in a wide variety of applications.
RESUMO
The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm. Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclo Celular/fisiologia , Divisão do Núcleo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Actomiosina/metabolismo , Animais , Núcleo Celular/metabolismo , Citocinese/fisiologia , Citoplasma , Citoesqueleto/metabolismo , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/fisiologia , Microtúbulos/metabolismo , Mitose , Miosina Tipo II/metabolismo , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
Transcription factor TFEB is thought to control cellular functions-including in the vascular bed-primarily via regulation of lysosomal biogenesis and autophagic flux. Here, we report that TFEB also orchestrates a non-canonical program that controls the cell cycle/VEGFR2 pathway in the developing vasculature. In endothelial cells, TFEB depletion halts proliferation at the G1-S transition by inhibiting the CDK4/Rb pathway. TFEB-deficient cells attempt to compensate for this limitation by increasing VEGFR2 levels at the plasma membrane via microRNA-mediated mechanisms and controlled membrane trafficking. TFEB stimulates expression of the miR-15a/16-1 cluster, which limits VEGFR2 transcript stability and negatively modulates expression of MYO1C, a regulator of VEGFR2 trafficking to the cell surface. Altered levels of miR-15a/16-1 and MYO1C in TFEB-depleted cells cause increased expression of plasma membrane VEGFR2, but in a manner associated with low signaling strength. An endothelium-specific Tfeb-knockout mouse model displays defects in fetal and newborn mouse vasculature caused by reduced endothelial proliferation and by anomalous function of the VEGFR2 pathway. These previously unrecognized functions of TFEB expand its role beyond regulation of the autophagic pathway in the vascular system.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Proliferação de Células , Embrião de Mamíferos/citologia , Endotélio Vascular/citologia , Regulação da Expressão Gênica no Desenvolvimento , Neovascularização Fisiológica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/fisiologia , Endotélio Vascular/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Somatic activating mutations within the PIK3CA gene have been recently detected in sporadic lymphatic and venous malformations, and in vascular malformations (VM) associated to overgrowth syndromes, such as CLOVES and Klippel-Trenaunay syndrome. Although VM are often limited to specific tissue areas and can be well treated, in extended or recurrent lesions novel therapeutic approaches are needed. We generated a mouse model of VM by local expression of PIK3CA-activating mutation in endothelial cells. PIK3CA-driven lesions are characterized by large areas of hemorrhage, hyperplastic vessels, infiltrates of inflammatory cells, and elevated endothelial cell density. Such vascular lesions are ameliorated by administration of dual PI3K/mTOR inhibitor, BEZ235, and mTOR inhibitor, Everolimus. Unexpectedly, the expression of PIK3CA-activating mutations in human endothelial cells results in both increased proliferation rates and senescence. Moreover, active forms of PIK3CA strongly promote the angiogenic sprouting. Treatment with PI3K/mTOR inhibitors restores normal endothelial cell proliferation rate and reduces the amount of senescent cells, whereas treatment with Akt inhibitor is less effective. Our findings reveal that PIK3CA mutations have a key role in the pathogenesis of VM and PIK3CA-driven experimental lesions can be effectively treated by PI3K/mTOR inhibitors.
Assuntos
Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Serina-Treonina Quinases TOR/antagonistas & inibidores , Malformações Vasculares/genética , Animais , Bovinos , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Embrião de Mamíferos , Células Endoteliais , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Cordão Umbilical , Malformações Vasculares/metabolismo , Malformações Vasculares/patologiaRESUMO
Rational target therapy of cancer would benefit from the identification of new targets that can be easily inhibited by small molecules. An increasing amount of evidence hints at 3-phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1) as an intriguing and underexplored target for cancer therapy. Several reports show that PDK1 expression is dysregulated in multiple cancer types. Furthermore PDK1 is implicated in signaling pathways frequently altered in cancer, such as PI3K/Akt, Ras/MAPK and Myc. PDK1 targeting has been proven to be effective in experimental models harboring alterations of these pathways. In this paper we review PDK1 main biochemical mechanisms, its alterations in cancer and interactions with relevant cancer pathways. A potential role of PDK1 in tumor microenvironment is also discussed.
Assuntos
Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Genes myc , Humanos , Sistema de Sinalização das MAP Quinases , Neoplasias/genética , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Transdução de Sinais , Microambiente Tumoral , Proteínas ras/metabolismoRESUMO
Extrusion of apoptotic cells from epithelial tissues requires orchestrated morphological rearrangements of the apoptotic cell and its neighbors. However, the connections between the apoptotic cascade and events leading to extrusion are not fully understood. Here, we characterize an apoptotic extrusion apical actin ring (EAAR) that is assembled within the apoptotic cell and drives epithelial extrusion. Caspase-mediated cleavage of myotonic dystrophy kinase-related CDC42-binding kinase-α (MRCKα) triggers a signaling pathway that leads to the assembly of EAAR that pulls actin bundles, resulting in the compaction and removal of the cell body. We provide a detailed portrait of the EAAR including F-actin flow, the contribution of myosin contraction, and actin polymerization at bundles' terminals when the product of MRCKα cleavage is expressed. These results add to our understanding of the mechanisms controlling the process of epithelial extrusion by establishing a causal relationship between the triggering events of apoptosis, the activation of MRCKα, and its subsequent effects on the dynamics of actomyosin cytoskeleton rearrangement.
Assuntos
Actomiosina/metabolismo , Apoptose/fisiologia , Caspases/metabolismo , Células Epiteliais/metabolismo , Miotonina Proteína Quinase/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Células CACO-2 , Miosinas Cardíacas/metabolismo , Linhagem Celular , Cães , Células HEK293 , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Centro Organizador dos Microtúbulos/fisiologia , Cadeias Leves de Miosina/metabolismo , Miosinas/metabolismo , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismoRESUMO
Dissecting the cellular signaling that governs the motility of eukaryotic cells is one of the fundamental tasks of modern cell biology, not only because of the large number of physiological processes in which cell migration is crucial, but even more so because of the pathological ones, in particular tumor invasion and metastasis. Cell migration requires the coordination of at least four major processes: polarization of intracellular signaling, regulation of the actin cytoskeleton and membrane extension, focal adhesion and integrin signaling and contractile forces generation and rear retraction. Among the molecular components involved in the regulation of locomotion, the phosphatidylinositol-3-kinase (PI3K) pathway has been shown to exert fundamental role. A pivotal node of such pathway is represented by the serine/threonine kinase 3-phosphoinositide-dependent protein kinase-1 (PDPK1 or PDK1). PDK1, and the majority of its substrates, belong to the AGC family of kinases (related to cAMP-dependent protein kinase 1, cyclic Guanosine monophosphate-dependent protein kinase and protein kinase C), and control a plethora of cellular processes, downstream either to PI3K or to other pathways, such as RAS GTPase-MAPK (mitogen-activated protein kinase). Interestingly, PDK1 has been demonstrated to be crucial for the regulation of each step of cell migration, by activating several proteins such as protein kinase B/Akt (PKB/Akt), myotonic dystrophy-related CDC42-binding kinases alpha (MRCKα), Rho associated coiled-coil containing protein kinase 1 (ROCK1), phospholipase C gamma 1 (PLCγ1) and ß3 integrin. Moreover, PDK1 regulates cancer cell invasion as well, thus representing a possible target to prevent cancer metastasis in human patients. The aim of this review is to summarize the various mechanisms by which PDK1 controls the cell migration process, from cell polarization to actin cytoskeleton and focal adhesion regulation, and finally, to discuss the evidence supporting a role for PDK1 in cancer cell invasion and dissemination.
RESUMO
How cell growth and proliferation are orchestrated in living tissues to achieve a given biological function is a central problem in biology. During development, tissue regeneration and homeostasis, cell proliferation must be coordinated by spatial cues in order for cells to attain the correct size and shape. Biological tissues also feature a notable homogeneity of cell size, which, in specific cases, represents a physiological need. Here, we study the temporal evolution of the cell-size distribution by applying the theory of kinetic fragmentation to tissue development and homeostasis. Our theory predicts self-similar probability density function (PDF) of cell size and explains how division times and redistribution ensure cell size homogeneity across the tissue. Theoretical predictions and numerical simulations of confluent non-homeostatic tissue cultures show that cell size distribution is self-similar. Our experimental data confirm predictions and reveal that, as assumed in the theory, cell division times scale like a power-law of the cell size. We find that in homeostatic conditions there is a stationary distribution with lognormal tails, consistently with our experimental data. Our theoretical predictions and numerical simulations show that the shape of the PDF depends on how the space inherited by apoptotic cells is redistributed and that apoptotic cell rates might also depend on size.
Assuntos
Divisão Celular , Tamanho Celular , Células Epiteliais/metabolismo , Homeostase , Modelos Biológicos , Animais , Cães , Células Epiteliais/citologia , Epitélio/metabolismo , Células Madin Darby de Rim CaninoRESUMO
The blastema is a mass of progenitor cells that enables regeneration of amputated salamander limbs or fish fins. Methodology to label and track blastemal cell progeny has been deficient, restricting our understanding of appendage regeneration. Here, we created a system for clonal analysis and quantitative imaging of hundreds of blastemal cells and their respective progeny in living adult zebrafish undergoing fin regeneration. Amputation stimulates resident cells within a limited recruitment zone to reset proximodistal (PD) positional information and assemble the blastema. Within the newly formed blastema, the spatial coordinates of connective tissue progenitors are predictive of their ultimate contributions to regenerated skeletal structures, indicating early development of an approximate PD pre-pattern. Calcineurin regulates size recovery by controlling the average number of progeny divisions without disrupting this pre-pattern. Our longitudinal clonal analyses of regenerating zebrafish fins provide evidence that connective tissue progenitors are rapidly organized into a scalable blueprint of lost structures.
Assuntos
Nadadeiras de Animais/fisiologia , Regeneração , Peixe-Zebra/fisiologia , Animais , Calcineurina/metabolismo , Feminino , Masculino , Células-Tronco , Proteínas de Peixe-Zebra/metabolismoRESUMO
Current fate mapping and imaging platforms are limited in their ability to capture dynamic behaviors of epithelial cells. To deconstruct regenerating adult epithelial tissue at single-cell resolution, we created a multicolor system, skinbow, that barcodes the superficial epithelial cell (SEC) population of zebrafish skin with dozens of distinguishable tags. With image analysis to directly segment and simultaneously track hundreds of SECs in vivo over entire surface lifetimes, we readily quantified the orchestration of cell emergence, growth, repositioning, and loss under homeostatic conditions and after exfoliation or appendage amputation. We employed skinbow-based imaging in conjunction with a live reporter of epithelial stem cell cycle activity and as an instrument to evaluate the effects of reactive oxygen species on SEC behavior during epithelial regeneration. Our findings introduce a platform for large-scale, quantitative in vivo imaging of regenerating skin and reveal unanticipated collective dynamism in epithelial cell size, mobility, and interactions.
Assuntos
Código de Barras de DNA Taxonômico/métodos , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Nadadeiras de Animais/lesões , Nadadeiras de Animais/fisiologia , Animais , Animais Geneticamente Modificados , Células Epiteliais/fisiologia , Epitélio/lesões , Epitélio/fisiologia , Homeostase , Modelos Animais , Espécies Reativas de Oxigênio/metabolismo , Regeneração/genética , Regeneração/fisiologia , Pele/lesões , Pigmentação da Pele/genética , Pigmentação da Pele/fisiologiaRESUMO
BACKGROUND: Tankyrases are poly(adenosine diphosphate)-ribose polymerases that contribute to biological processes as diverse as modulation of Wnt signaling, telomere maintenance, vesicle trafficking, and microtubule-dependent spindle pole assembly during mitosis. At interphase, polarized reshaping of the microtubule network fosters oriented cell migration. This is attained by association of adenomatous polyposis coli with the plus end of microtubules at the cortex of cell membrane protrusions and microtubule-based centrosome reorientation towards the migrating front. RESULTS: Here we report a new function for tankyrases, namely, regulation of directional cell locomotion. Using a panel of lung cancer cell lines as a model system, we found that abrogation of tankyrase activity by two different, structurally unrelated small-molecule inhibitors (one introduced and characterized here for the first time) or by RNA interference-based genetic silencing weakened cell migration, invasion, and directional movement induced by the motogenic cytokine hepatocyte growth factor. Mechanistically, the anti-invasive outcome of tankyrase inhibition could be ascribed to sequential deterioration of the distinct events that govern cell directional sensing. In particular, tankyrase blockade negatively impacted (1) microtubule dynamic instability; (2) adenomatous polyposis coli plasma membrane targeting; and (3) centrosome reorientation. CONCLUSIONS: Collectively, these findings uncover an unanticipated role for tankyrases in influencing at multiple levels the interphase dynamics of the microtubule network and the subcellular distribution of related polarity signals. These results encourage the further exploration of tankyrase inhibitors as therapeutic tools to oppose dissemination and metastasis of cancer cells.