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1.
Dev Cell ; 58(20): 2181-2193.e4, 2023 10 23.
Article in English | MEDLINE | ID: mdl-37586367

ABSTRACT

Understanding morphogenesis strongly relies on the characterization of tissue topology and mechanical properties deduced from imaging data. The development of new imaging techniques offers the possibility to go beyond the analysis of mostly flat surfaces and image and analyze complex tissue organization in depth. An important bottleneck in this field is the need to analyze imaging datasets and extract quantifications not only of cell and tissue morphology but also of the cytoskeletal network's organization in an automatized way. Here, we describe a method, called DISSECT, for DisPerSE (Discrete Persistent Structure Extractor)-based Segmentation and Exploration of Cells and Tissues, that offers the opportunity to extract automatically, in strongly deformed epithelia, a precise characterization of the spatial organization of a given cytoskeletal network combined with morphological quantifications in highly remodeled three-dimensional (3D) epithelial tissues. We believe that this method, applied here to Drosophila tissues, will be of general interest in the expanding field of morphogenesis and tissue biomechanics.


Subject(s)
Drosophila , Imaging, Three-Dimensional , Animals , Epithelium/metabolism , Morphogenesis , Imaging, Three-Dimensional/methods
2.
Dev Cell ; 56(5): 687-701.e7, 2021 03 08.
Article in English | MEDLINE | ID: mdl-33535069

ABSTRACT

Epithelial sheets undergo highly reproducible remodeling to shape organs. This stereotyped morphogenesis depends on a well-defined sequence of events leading to the regionalized expression of developmental patterning genes that finally triggers downstream mechanical forces to drive tissue remodeling at a pre-defined position. However, how tissue mechanics controls morphogenetic robustness when challenged by intrinsic perturbations in close proximity has never been addressed. Using Drosophila developing leg, we show that a bias in force propagation ensures stereotyped morphogenesis despite the presence of mechanical noise in the environment. We found that knockdown of the Arp2/3 complex member Arpc5 specifically affects fold directionality while altering neither the developmental nor the force generation patterns. By combining in silico modeling, biophysical tools, and ad hoc genetic tools, our data reveal that junctional myosin II planar polarity favors long-range force channeling and ensures folding robustness, avoiding force scattering and thus isolating the fold domain from surrounding mechanical perturbations.


Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Cell Polarity , Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Embryo, Nonmammalian/cytology , Morphogenesis , Myosin Type II/metabolism , Actin-Related Protein 2-3 Complex/genetics , Animals , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Embryo, Nonmammalian/metabolism , Female , Gene Expression Regulation, Developmental , Male , Myosin Type II/genetics
3.
ACS Appl Mater Interfaces ; 13(9): 11224-11236, 2021 Mar 10.
Article in English | MEDLINE | ID: mdl-33621463

ABSTRACT

The directed self-assembly (DSA) of block copolymers (BCPs) is a powerful method for the manufacture of high-resolution features. Critical issues remain to be addressed for successful implementation of DSA, such as dewetting and controlled orientation of BCP domains through physicochemical manipulations at the BCP interfaces, and the spatial positioning and registration of the BCP features. Here, we introduce novel top-coat (TC) materials designed to undergo cross-linking reactions triggered by thermal or photoactivation processes. The cross-linked TC layer with adjusted composition induces a mechanical confinement of the BCP layer, suppressing its dewetting while promoting perpendicular orientation of BCP domains. The selection of areas of interest with perpendicular features is performed directly on the patternable TC layer via a lithography step and leverages attractive integration pathways for the generation of locally controlled BCP patterns and nanostructured BCP multilayers.

4.
mBio ; 10(4)2019 08 20.
Article in English | MEDLINE | ID: mdl-31431549

ABSTRACT

Single-cell microfluidics is a powerful method to study bacteria and determine their susceptibility to antibiotic treatment. Glass treatment by adhesive molecules is a potential solution to immobilize bacterial cells and perform microscopy, but traditional cationic polymers such as polylysine deeply affect bacterial physiology. In this work, we chemically characterized a class of chitosan polymers for their biocompatibility when adsorbed to glass. Chitosan chains of known length and composition allowed growth of Escherichia coli cells without any deleterious effects on cell physiology. Combined with a machine learning approach, this method could measure the antibiotic susceptibility of a diversity of clinical strains in less than 1 h and with higher accuracy than current methods. Finally, chitosan polymers also supported growth of Klebsiella pneumoniae, another bacterial pathogen of clinical significance.IMPORTANCE Current microfluidic techniques are powerful to study bacteria and determine their response to antibiotic treatment, but they are currently limited by their complex manipulation. Chitosan films are fully biocompatible and could thus be a viable replacement for existing commercial devices that currently use polylysine. Thus, the low cost of chitosan slides and their simple implementation make them highly versatile for research as well as clinical use.


Subject(s)
Anti-Bacterial Agents/pharmacology , Chitosan/chemistry , Microfluidics/methods , Anti-Bacterial Agents/chemistry , Bacteria/drug effects , Bacterial Adhesion/drug effects , Biocompatible Materials/chemistry , Cell Proliferation/drug effects , Chitosan/classification , Escherichia coli/drug effects , Escherichia coli/growth & development , Glass , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/growth & development , Materials Testing , Microbial Sensitivity Tests
5.
Nat Commun ; 10(1): 2951, 2019 07 04.
Article in English | MEDLINE | ID: mdl-31273212

ABSTRACT

Epithelial-mesenchymal transition (EMT) is an essential process both in physiological and pathological contexts. Intriguingly, EMT is often associated with tissue invagination during development; however, the impact of EMT on tissue remodeling remain unexplored. Here, we show that at the initiation of the EMT process, cells produce an apico-basal force, orthogonal to the surface of the epithelium, that constitutes an important driving force for tissue invagination in Drosophila. When EMT is ectopically induced, cells starting their delamination generate an orthogonal force and induce ectopic folding. Similarly, during mesoderm invagination, cells undergoing EMT generate an apico-basal force through the formation of apico-basal structures of myosin II. Using both laser microdissection and in silico physical modelling, we show that mesoderm invagination does not proceed if apico-basal forces are impaired, indicating that they constitute driving forces in the folding process. Altogether, these data reveal the mechanical impact of EMT on morphogenesis.


Subject(s)
Drosophila melanogaster/embryology , Epithelial-Mesenchymal Transition , Epithelium/embryology , Morphogenesis , Animals , Cell Polarity , Computer Simulation , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Epithelium/metabolism , Mesoderm/cytology , Mesoderm/embryology , Mesoderm/metabolism , Models, Molecular , Myosin Type II/metabolism
6.
J Cell Sci ; 130(1): 97-103, 2017 01 01.
Article in English | MEDLINE | ID: mdl-27034137

ABSTRACT

Border cell migration during Drosophila oogenesis is a potent model to study collective cell migration, a process involved in development and metastasis. Border cell clusters adopt two main types of behaviour during migration: linear and rotational. However, the molecular mechanism controlling the switch from one to the other is unknown. Here, we demonstrate that non-muscle Myosin II (NMII, also known as Spaghetti squash) activity controls the linear-to-rotational switch. Furthermore, we show that the regulation of NMII takes place downstream of guidance receptor signalling and is critical to ensure efficient collective migration. This study thus provides new insight into the molecular mechanism coordinating the different cell behaviours in a migrating cluster.


Subject(s)
Cell Movement , Drosophila Proteins/metabolism , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Myosin Type II/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , Animals , ErbB Receptors/metabolism , Rotation , Time-Lapse Imaging
7.
J Cell Sci ; 128(20): 3720-30, 2015 Oct 15.
Article in English | MEDLINE | ID: mdl-26359299

ABSTRACT

In higher eukaryotes, efficient chromosome congression relies, among other players, on the activity of chromokinesins. Here, we provide a quantitative analysis of kinetochore oscillations and positioning in Schizosaccharomyces pombe, a model organism lacking chromokinesins. In wild-type cells, chromosomes align during prophase and, while oscillating, maintain this alignment throughout metaphase. Chromosome oscillations are dispensable both for kinetochore congression and stable kinetochore alignment during metaphase. In higher eukaryotes, kinesin-8 family members control chromosome congression by regulating their oscillations. By contrast, here, we demonstrate that fission yeast kinesin-8 controls chromosome congression by an alternative mechanism. We propose that kinesin-8 aligns chromosomes by controlling pulling forces in a length-dependent manner. A coarse-grained model of chromosome segregation implemented with a length-dependent process that controls the force at kinetochores is necessary and sufficient to mimic kinetochore alignment, and prevents the appearance of lagging chromosomes. Taken together, these data illustrate how the local action of a motor protein at kinetochores provides spatial cues within the spindle to align chromosomes and to prevent aneuploidy.


Subject(s)
Biological Clocks/physiology , Chromosome Segregation/physiology , Chromosomes, Fungal/metabolism , Kinesins/metabolism , Models, Biological , Schizosaccharomyces/metabolism , Chromosomes, Fungal/genetics , Kinesins/genetics , Schizosaccharomyces/genetics
9.
Nature ; 518(7538): 245-8, 2015 Feb 12.
Article in English | MEDLINE | ID: mdl-25607361

ABSTRACT

Epithelium folding is a basic morphogenetic event that is essential in transforming simple two-dimensional epithelial sheets into three-dimensional structures in both vertebrates and invertebrates. Folding has been shown to rely on apical constriction. The resulting cell-shape changes depend either on adherens junction basal shift or on a redistribution of myosin II, which could be driven by mechanical signals. Yet the initial cellular mechanisms that trigger and coordinate cell remodelling remain largely unknown. Here we unravel the active role of apoptotic cells in initiating morphogenesis, thus revealing a novel mechanism of epithelium folding. We show that, in a live developing tissue, apoptotic cells exert a transient pulling force upon the apical surface of the epithelium through a highly dynamic apico-basal myosin II cable. The apoptotic cells then induce a non-autonomous increase in tissue tension together with cortical myosin II apical stabilization in the surrounding tissue, eventually resulting in epithelium folding. Together our results, supported by a theoretical biophysical three-dimensional model, identify an apoptotic myosin-II-dependent signal as the initial signal leading to cell reorganization and tissue folding. This work further reveals that, far from being passively eliminated as generally assumed (for example, during digit individualization), apoptotic cells actively influence their surroundings and trigger tissue remodelling through regulation of tissue tension.


Subject(s)
Apoptosis , Cell Polarity , Drosophila melanogaster/cytology , Drosophila melanogaster/embryology , Epithelial Cells/cytology , Epithelium/embryology , Morphogenesis , Adherens Junctions/chemistry , Adherens Junctions/metabolism , Animals , Cell Shape , Epithelial Cells/metabolism , Models, Biological , Myosin Type II/metabolism
10.
PLoS One ; 8(7): e68309, 2013.
Article in English | MEDLINE | ID: mdl-23874582

ABSTRACT

The study of vesicles in suspension is important to understand the complicated dynamics exhibited by cells in in vivo and in vitro. We developed a computer simulation based on the boundary-integral method to model the three dimensional gravity-driven sedimentation of charged vesicles towards a flat surface. The membrane mechanical behavior was modeled using the Helfrich Hamiltonian and near incompressibility of the membrane was enforced via a model which accounts for the thermal fluctuations of the membrane. The simulations were verified and compared to experimental data obtained using suspended vesicles labelled with a fluorescent probe, which allows visualization using fluorescence microscopy and confers the membrane with a negative surface charge. The electrostatic interaction between the vesicle and the surface was modeled using the linear Derjaguin approximation for a low ionic concentration solution. The sedimentation rate as a function of the distance of the vesicle to the surface was determined both experimentally and from the computer simulations. The gap between the vesicle and the surface, as well as the shape of the vesicle at equilibrium were also studied. It was determined that inclusion of the electrostatic interaction is fundamental to accurately predict the sedimentation rate as the vesicle approaches the surface and the size of the gap at equilibrium, we also observed that the presence of charge in the membrane increases its rigidity.


Subject(s)
Glass/chemistry , Lipids/chemistry , Computer Simulation , Microscopy, Fluorescence , Static Electricity , Surface Properties
11.
J Cell Biol ; 196(6): 757-74, 2012 Mar 19.
Article in English | MEDLINE | ID: mdl-22412019

ABSTRACT

In fission yeast, erroneous attachments of spindle microtubules to kinetochores are frequent in early mitosis. Most are corrected before anaphase onset by a mechanism involving the protein kinase Aurora B, which destabilizes kinetochore microtubules (ktMTs) in the absence of tension between sister chromatids. In this paper, we describe a minimal mathematical model of fission yeast chromosome segregation based on the stochastic attachment and detachment of ktMTs. The model accurately reproduces the timing of correct chromosome biorientation and segregation seen in fission yeast. Prevention of attachment defects requires both appropriate kinetochore orientation and an Aurora B-like activity. The model also reproduces abnormal chromosome segregation behavior (caused by, for example, inhibition of Aurora B). It predicts that, in metaphase, merotelic attachment is prevented by a kinetochore orientation effect and corrected by an Aurora B-like activity, whereas in anaphase, it is corrected through unbalanced forces applied to the kinetochore. These unbalanced forces are sufficient to prevent aneuploidy.


Subject(s)
Chromosome Segregation/physiology , Chromosomes, Fungal/metabolism , Kinetochores/metabolism , Microtubules/metabolism , Schizosaccharomyces/metabolism , Aurora Kinases , Chromatids/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Schizosaccharomyces/ultrastructure , Stochastic Processes
12.
Cell Div ; 6: 22, 2011 Dec 12.
Article in English | MEDLINE | ID: mdl-22152157

ABSTRACT

BACKGROUND: Multicellular tumor spheroids are models of increasing interest for cancer and cell biology studies. They allow considering cellular interactions in exploring cell cycle and cell division mechanisms. However, 3D imaging of cell division in living spheroids is technically challenging and has never been reported. RESULTS: Here, we report a major breakthrough based on the engineering of multicellular tumor spheroids expressing an histone H2B fluorescent nuclear reporter protein, and specifically designed sample holders to monitor live cell division dynamics in 3D large spheroids using an home-made selective-plane illumination microscope. CONCLUSIONS: As illustrated using the antimitotic drug, paclitaxel, this technological advance paves the way for studies of the dynamics of cell divion processes in 3D and more generally for the investigation of tumor cell population biology in integrated system as the spheroid model.

13.
PLoS One ; 5(5): e10634, 2010 May 13.
Article in English | MEDLINE | ID: mdl-20498706

ABSTRACT

The plus-end microtubule binding proteins (+TIPs) play an important role in the regulation of microtubule stability and cell polarity during interphase. In S. pombe, the CLIP-170 like protein Tip1, together with the kinesin Tea2, moves along the microtubules towards their plus ends. Tip1 also requires the EB1 homolog Mal3 to localize to the microtubule tips. Given the requirement for Tip1 for microtubule stability, we have investigated its role during spindle morphogenesis and chromosome movement. Loss of Tip1 affects metaphase plate formation and leads to the activation of the spindle assembly checkpoint. In the absence of Tip1 we also observed the appearance of lagging chromosomes, which do not influence the normal rate of spindle elongation. Our results suggest that S. pombe Tip1/CLIP170 is directly or indirectly required for correct chromosome poleward movement independently of Mal3/EB1.


Subject(s)
Cell Polarity , Chromosomes, Fungal/metabolism , Heat-Shock Proteins/metabolism , Intermediate Filament Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/metabolism , Cell Polarity/drug effects , Gene Deletion , Kinetochores/drug effects , Kinetochores/metabolism , Metaphase/drug effects , Mitosis/drug effects , Phenotype , Protein Transport/drug effects , Schizosaccharomyces/drug effects , Spindle Apparatus/drug effects , Spindle Apparatus/metabolism , Thiabendazole/pharmacology
14.
J Cell Biol ; 187(3): 399-412, 2009 Nov 02.
Article in English | MEDLINE | ID: mdl-19948483

ABSTRACT

Faithful segregation of sister chromatids requires the attachment of each kinetochore (Kt) to microtubules (MTs) that extend from opposite spindle poles. Merotelic Kt orientation is a Kt-MT misattachment in which a single Kt binds MTs from both spindle poles rather than just one. Genetic induction of merotelic Kt attachment during anaphase in fission yeast resulted in intra-Kt stretching followed by either correction or Kt disruption. Laser ablation of spindle MTs revealed that intra-Kt stretching and merotelic correction were dependent on MT forces. The presence of multiple merotelic chromosomes linearly antagonized the spindle elongation rate, and this phenomenon could be solved numerically using a simple force balance model. Based on the predictions of our mechanical model, we provide in vivo evidence that correction of merotelic attachment in anaphase is tension dependent and requires an Ase1/Prc1-dependent mechanism that prevents spindle collapse and thus asymmetric division and/or the appearance of the cut phenotype.


Subject(s)
Anaphase , Kinetochores/metabolism , Microtubule-Associated Proteins/physiology , Schizosaccharomyces pombe Proteins/physiology , Spindle Apparatus/physiology , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Microtubules/physiology , Models, Biological , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces/ultrastructure , Schizosaccharomyces pombe Proteins/metabolism , Spindle Apparatus/metabolism
15.
Mol Biol Cell ; 19(4): 1646-62, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18256284

ABSTRACT

In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. We have studied chromosome recapture in the fission yeast Schizosaccharomyces pombe. We show by live cell analysis that lost kinetochores interact laterally with intranuclear microtubules (INMs) and that both microtubule depolymerization (end-on pulling) and minus-end-directed movement (microtubule sliding) contribute to chromosome retrieval to the spindle pole body (SPB). We find that the minus-end-directed motor Klp2 colocalizes with the kinetochore during its transport to the SPB and contributes to the effectiveness of retrieval by affecting both end-on pulling and lateral sliding. Furthermore, we provide in vivo evidence that Dam1, a component of the DASH complex, also colocalizes with the kinetochore during its transport and is essential for its retrieval by either of these mechanisms. Finally, we find that the position of the unattached kinetochore correlates with the size and orientation of the INMs, suggesting that chromosome recapture may not be a random process.


Subject(s)
Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Biological Transport, Active , Dyneins/genetics , Dyneins/metabolism , Genes, Fungal , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Microtubule-Associated Proteins/genetics , Microtubules/metabolism , Models, Biological , Molecular Motor Proteins/genetics , Molecular Motor Proteins/metabolism , Multiprotein Complexes , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Sister Chromatid Exchange/genetics , Sister Chromatid Exchange/physiology , Spindle Apparatus/genetics , Spindle Apparatus/metabolism
16.
Environ Sci Technol ; 41(15): 5466-70, 2007 Aug 01.
Article in English | MEDLINE | ID: mdl-17822118

ABSTRACT

Heavy metals are known to be significant pollutants in sludges produced by wastewater treatment. A newtechnique to make the metallic contaminants migrate is to submit such sludges to a slow freezing in order to purify the frozen region and to concentrate impurities in the non-frozen zone. In this paper, we apply this method to model contaminated sludges consisting of clayey suspensions charged with a heavy metal (zinc). The effect of some physicochemical parameters (zinc and clay concentrations, pH) on the effectiveness of the method is experimentally investigated. The pH is the most relevant parameter and the highest purification rate (approximately 80%) is obtained for pH approximately 9.


Subject(s)
Refuse Disposal/methods , Sewage , Zinc/isolation & purification , Bentonite/chemistry , Freezing , Hydrogen-Ion Concentration , Solubility , Solutions
17.
Biol Cell ; 99(11): 627-37, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17561805

ABSTRACT

BACKGROUND INFORMATION: In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. For faithful segregation of sister chromatids, each sister kinetochore must attach to microtubules that extend to opposite poles (chromosome bi-orientation). At the metaphase-anaphase transition, cohesion between sister chromatids is removed, and each sister chromatid is pulled to opposite poles of the cell by microtubule-dependent forces. RESULTS: We have studied the role of the minus-end-directed motor protein dynein by analysing kinetochore dynamics in fission yeast cells deleted for the dynein heavy chain (Dhc1) or the light chain (Dlc1). In these mutants, we found an increased frequency of cells showing defects in chromosome segregation, which leads to the appearance of lagging chromosomes and an increased rate of chromosome loss. By following simultaneously kinetochore dynamics and localization of the checkpoint protein Mad2, we provide evidence that dynein function is not necessary for spindle-assembly checkpoint inactivation. Instead, we have demonstrated that loss of dynein function alters chromosome segregation and activates the Mad2-dependent spindle-assembly checkpoint. CONCLUSIONS: These results show an unexpected role for dynein in the control of chromosome segregation in fission yeast, most probably operating during the process of bi-orientation during early mitosis.


Subject(s)
Chromosome Segregation/physiology , Chromosomes, Fungal/metabolism , Dyneins/metabolism , Mitosis/physiology , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Anaphase/physiology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatids/genetics , Chromatids/metabolism , Chromosomes, Fungal/genetics , Dyneins/genetics , Gene Deletion , Kinetochores/metabolism , Mad2 Proteins , Metaphase/physiology , Microtubules/genetics , Microtubules/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Spindle Apparatus/genetics , Spindle Apparatus/metabolism
18.
Water Res ; 37(10): 2520-4, 2003 May.
Article in English | MEDLINE | ID: mdl-12727265

ABSTRACT

Radial freezing experiments on wastewater models were conducted in the presence of imposed stirring in order to remove impurities. The studied samples (dilute Na-montmorillonite suspensions charged with nitrates and with zinc or lead) were placed inside a cylindrical annulus, cooled at a controlled temperature around -7 degrees C at its inner wall which rotated around a vertical axis. The freezing front propagated toward the still outer wall which was maintained at a constant temperature around +1 degrees C. Thanks to stirring, considerable purification rates up to 99.97% were attained. It was also demonstrated that combining radial freezing and stirring ended in residual concentrations which agreed with drinking water standards.


Subject(s)
Lead/isolation & purification , Nitrates/isolation & purification , Waste Disposal, Fluid/methods , Water Purification/methods , Zinc/isolation & purification , Freezing , Water Movements
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