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The accurate timing and execution of organelle biogenesis is crucial for cell physiology. Centriole biogenesis is regulated by Polo-like kinase 4 (Plk4) and initiates in S-phase when a daughter centriole grows from the side of a pre-existing mother. Here, we show that a Plk4 oscillation at the base of the growing centriole initiates and times centriole biogenesis to ensure that centrioles grow at the right time and to the right size. The Plk4 oscillation is normally entrained to the cell-cycle oscillator but can run autonomously of it-potentially explaining why centrioles can duplicate independently of cell-cycle progression. Mathematical modeling indicates that the Plk4 oscillation can be generated by a time-delayed negative feedback loop in which Plk4 inactivates the interaction with its centriolar receptor through multiple rounds of phosphorylation. We hypothesize that similar organelle-specific oscillations could regulate the timing and execution of organelle biogenesis more generally.
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Relojes Biológicos/fisiología , Centriolos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Centrosoma/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Biogénesis de Organelos , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiologíaRESUMEN
Lymphocyte homeostasis and immune surveillance require that T and B cells continuously recirculate between secondary lymphoid organs. Here, we used intravital microscopy to define lymphocyte trafficking routes within the spleen, an environment of open blood circulation and shear forces unlike other lymphoid organs. Upon release from arterioles into the red pulp sinuses, T cells latched onto perivascular stromal cells in a manner that was independent of the chemokine receptor CCR7 but sensitive to Gi protein-coupled receptor inhibitors. This latching sheltered T cells from blood flow and enabled unidirectional migration to the bridging channels and then to T zones, entry into which required CCR7. Inflammatory responses modified the chemotactic cues along the perivascular homing paths, leading to rapid block of entry. Our findings reveal a role for vascular structures in lymphocyte recirculation through the spleen, indicating the existence of separate entry and exit routes and that of a checkpoint located at the gate to the T zone.
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Movimiento Celular/inmunología , Receptores CCR7/inmunología , Bazo/inmunología , Linfocitos T/inmunología , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Humanos , Vigilancia Inmunológica/inmunología , Microscopía Intravital , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Linfocitos/citología , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores CCR7/genética , Receptores CCR7/metabolismo , Transducción de Señal/inmunología , Bazo/citología , Bazo/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue2,3. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs4-11. Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios12. Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.
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Anoicis , Carcinogénesis , Extensiones de la Superficie Celular , Supervivencia Celular , Melanoma , Transducción de Señal , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Septinas/metabolismo , Extensiones de la Superficie Celular/química , Extensiones de la Superficie Celular/metabolismo , Carcinogénesis/genética , Adhesión Celular , Quinasas MAP Reguladas por Señal Extracelular , Fibroblastos , Mutación , Forma de la Célula , Imagenología Tridimensional , Quinasas de Proteína Quinasa Activadas por MitógenosRESUMEN
The repair of DNA double-strand breaks (DSBs) is essential for safeguarding genome integrity. When a DSB forms, the PI3K-related ATM kinase rapidly triggers the establishment of megabase-sized, chromatin domains decorated with phosphorylated histone H2AX (γH2AX), which act as seeds for the formation of DNA-damage response foci1. It is unclear how these foci are rapidly assembled to establish a 'repair-prone' environment within the nucleus. Topologically associating domains are a key feature of 3D genome organization that compartmentalize transcription and replication, but little is known about their contribution to DNA repair processes2,3. Here we show that topologically associating domains are functional units of the DNA damage response, and are instrumental for the correct establishment of γH2AX-53BP1 chromatin domains in a manner that involves one-sided cohesin-mediated loop extrusion on both sides of the DSB. We propose a model in which H2AX-containing nucleosomes are rapidly phosphorylated as they actively pass by DSB-anchored cohesin. Our work highlights the importance of chromosome conformation in the maintenance of genome integrity and demonstrates the establishment of a chromatin modification by loop extrusion.
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Roturas del ADN de Doble Cadena , Reparación del ADN , ADN/química , ADN/metabolismo , Conformación de Ácido Nucleico , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteínas Cromosómicas no Histona/metabolismo , ADN/genética , Genoma/genética , Histonas/metabolismo , Humanos , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/metabolismo , Fosforilación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , CohesinasRESUMEN
Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome "maturation" requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd-2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. We show here that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process-peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. We propose that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.
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Centriolos , Proteínas de Drosophila , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Centrosoma/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mitosis , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
RhoU is an atypical member of the Rho family of small G-proteins, which has N- and C-terminal extensions compared to the classic Rho GTPases RhoA, Rac1 and Cdc42, and associates with membranes through C-terminal palmitoylation rather than prenylation. RhoU mRNA expression is upregulated in prostate cancer and is considered a marker for disease progression. Here, we show that RhoU overexpression in prostate cancer cells increases cell migration and invasion. To identify RhoU targets that contribute to its function, we found that RhoU homodimerizes in cells. We map the region involved in this interaction to the C-terminal extension and show that C-terminal palmitoylation is required for self-association. Expression of the isolated C-terminal extension reduces RhoU-induced activation of p21-activated kinases (PAKs), which are known downstream targets for RhoU, and induces cell morphological changes consistent with inhibiting RhoU function. Our results show for the first time that the activity of a Rho family member is stimulated by self-association, and this is important for its activity.
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Neoplasias de la Próstata , Proteínas de Unión al GTP rho , Humanos , Masculino , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photobleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle three-dimensional (3D) imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, an LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150 nm, combined with lower phototoxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1 Hz.
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Imagenología Tridimensional , Iluminación , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , FotoblanqueoRESUMEN
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Molecular biology provides an inspiring proof-of-principle that chemical systems can store and process information to direct molecular activities such as the fabrication of complex structures from molecular components. To develop information-based chemistry as a technology for programming matter to function in ways not seen in biological systems, it is necessary to understand how molecular interactions can encode and execute algorithms. The self-assembly of relatively simple units into complex products1 is particularly well suited for such investigations. Theory that combines mathematical tiling and statistical-mechanical models of molecular crystallization has shown that algorithmic behaviour can be embedded within molecular self-assembly processes2,3, and this has been experimentally demonstrated using DNA nanotechnology4 with up to 22 tile types5-11. However, many information technologies exhibit a complexity threshold-such as the minimum transistor count needed for a general-purpose computer-beyond which the power of a reprogrammable system increases qualitatively, and it has been unclear whether the biophysics of DNA self-assembly allows that threshold to be exceeded. Here we report the design and experimental validation of a DNA tile set that contains 355 single-stranded tiles and can, through simple tile selection, be reprogrammed to implement a wide variety of 6-bit algorithms. We use this set to construct 21 circuits that execute algorithms including copying, sorting, recognizing palindromes and multiples of 3, random walking, obtaining an unbiased choice from a biased random source, electing a leader, simulating cellular automata, generating deterministic and randomized patterns, and counting to 63, with an overall per-tile error rate of less than 1 in 3,000. These findings suggest that molecular self-assembly could be a reliable algorithmic component within programmable chemical systems. The development of molecular machines that are reprogrammable-at a high level of abstraction and thus without requiring knowledge of the underlying physics-will establish a creative space in which molecular programmers can flourish.
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Algoritmos , ADN/química , ADN/síntesis química , Nanotecnología , Reproducibilidad de los ResultadosRESUMEN
Centrioles are composed of a central cartwheel tethered to nine-fold symmetric microtubule (MT) blades. The centriole cartwheel and MTs are thought to grow from opposite ends of these organelles, so it is unclear how they coordinate their assembly. We previously showed that in Drosophila embryos an oscillation of Polo-like kinase 4 (Plk4) helps to initiate and time the growth of the cartwheel at the proximal end. Here, in the same model, we show that CP110 and Cep97 form a complex close to the distal-end of the centriole MTs whose levels rise and fall as the new centriole MTs grow, in a manner that appears to be entrained by the core cyclin-dependent kinase (Cdk)-Cyclin oscillator that drives the nuclear divisions in these embryos. These CP110 and Cep97 dynamics, however, do not appear to time the period of centriole MT growth directly. Instead, we find that changing the levels of CP110 and Cep97 appears to alter the Plk4 oscillation and the growth of the cartwheel at the proximal end. These findings reveal an unexpected potential crosstalk between factors normally concentrated at opposite ends of the growing centrioles, which might help to coordinate centriole growth. This article has an associated First Person interview with the first authors of the paper.
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Centriolos , Proteínas Asociadas a Microtúbulos , Fosfoproteínas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Drosophila/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
ZBP1 is an interferon-induced cytosolic nucleic acid sensor that facilitates antiviral responses via RIPK3. Although ZBP1-mediated programmed cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1-induced inflammatory signaling pathway mediated by K63- and M1-linked ubiquitin chains, which depends on RIPK1 and RIPK3 as scaffolds independently of cell death. In human HT29 cells, ZBP1 associated with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. ZBP1-induced K63- and M1-linked ubiquitination of RIPK1 and ZBP1 to promote TAK1- and IKK-mediated inflammatory signaling and cytokine production. Inhibition of caspase activity suppressed ZBP1-induced cell death but enhanced cytokine production in a RIPK1- and RIPK3 kinase activity-dependent manner. Lastly, we provide evidence that ZBP1 signaling contributes to SARS-CoV-2-induced cytokine production. Taken together, we describe a ZBP1-RIPK3-RIPK1-mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspases, which may induce inflammation when ZBP1 is activated below the threshold needed to trigger a cell death response.
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Muerte Celular , Proteínas de Unión al ARN , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Humanos , Citocinas , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal , Ubiquitina , Proteínas de Unión al ARN/genética , Células HT29 , InflamaciónRESUMEN
Purpose: When admitted to the hospital, individuals with celiac disease rely on food handlers for provision of safe, uncontaminated gluten-free meals. We aimed to assess the knowledge of gluten-free diet (GFD) amongst individuals involved in meal preparation for patients.Methods: A questionnaire with 10 demographic and 35 test items to assess knowledge of GFD, including workplace scenarios encountered in meal preparation, was administered to food handlers including cooks, utility workers, dietary technicians, and supervisors in 2 tertiary care, university-affiliated hospitals. A score of ≥28 of 35 (≥80%) was considered a "pass".Results: A total of 72 individuals completed the study, mean age 40.3 ± 1.6 years, 75% female. Only 42 (56.8%) scored ≥80% and achieved a pass. The average score was 75.9% ± 13.4%, range 25.7%-100%. The supervisors had significantly higher scores (87.9% ± 11.4%) than utility workers (73.0% ± 11.4%; P = 0.01) and cooks (71.7% ± 14.5%; P = 0.01). Cooks had the lowest scores with 80% scoring <80%. Females scored higher than males (77.8% vs. 68.8%; P = 0.02).Conclusions: There are significant differences in GFD knowledge amongst various groups involved in food preparation in hospitals. The gaps identified in knowledge can potentially compromise the safety of patients with celiac disease. Targeted interventions to educate hospital food handlers about GFD are warranted. Registered Dietitians can play an important role in providing this education.
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Enfermedad Celíaca , Dieta Sin Gluten , Masculino , Humanos , Femenino , Adulto , Encuestas y Cuestionarios , Hospitales , ComidasRESUMEN
We have used two different live-cell fluorescent protein markers to monitor the formation and localization of double-strand breaks (DSBs) in budding yeast. Using GFP derivatives of the Rad51 recombination protein or the Ddc2 checkpoint protein, we find that cells with three site-specific DSBs, on different chromosomes, usually display 2 or 3 foci that may coalesce and dissociate. This motion is independent of Rad52 and microtubules. Rad51-GFP, by itself, is unable to repair DSBs by homologous recombination in mitotic cells, but is able to form foci and allow repair when heterozygous with a wild type Rad51 protein. The kinetics of formation and disappearance of a Rad51-GFP focus parallels the completion of site-specific DSB repair. However, Rad51-GFP is proficient during meiosis when homozygous, similar to rad51 "site II" mutants that can bind single-stranded DNA but not complete strand exchange. Rad52-RFP and Rad51-GFP co-localize to the same DSB, but a significant minority of foci have Rad51-GFP without visible Rad52-RFP. We conclude that co-localization of foci in cells with 3 DSBs does not represent formation of a homologous recombination "repair center," as the same distribution of Ddc2-GFP foci was found in the absence of the Rad52 protein.
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Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular/genética , Roturas del ADN de Doble Cadena , Recombinasa Rad51/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteínas de Saccharomyces cerevisiae/genética , Daño del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas Fluorescentes Verdes/genética , Recombinación Homóloga/genética , Cinética , Meiosis/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Atherosclerosis is characterized by the pathological accumulation of cholesterol-laden macrophages in the arterial wall. Atherosclerosis is also the main underlying cause of CVDs, and its development is largely driven by elevated plasma cholesterol. Strong epidemiological data find an inverse association between plasma ß-carotene with atherosclerosis, and we recently showed that ß-carotene oxygenase 1 (BCO1) activity, responsible for ß-carotene cleavage to vitamin A, is associated with reduced plasma cholesterol in humans and mice. In this study, we explore whether intact ß-carotene or vitamin A affects atherosclerosis progression in the atheroprone LDLR-deficient mice. Compared with control-fed Ldlr-/- mice, ß-carotene-supplemented mice showed reduced atherosclerotic lesion size at the level of the aortic root and reduced plasma cholesterol levels. These changes were absent in Ldlr-/- /Bco1-/- mice despite accumulating ß-carotene in plasma and atherosclerotic lesions. We discarded the implication of myeloid BCO1 in the development of atherosclerosis by performing bone marrow transplant experiments. Lipid production assays found that retinoic acid, the active form of vitamin A, reduced the secretion of newly synthetized triglyceride and cholesteryl ester in cell culture and mice. Overall, our findings provide insights into the role of BCO1 activity and vitamin A in atherosclerosis progression through the regulation of hepatic lipid metabolism.
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Aterosclerosis/metabolismo , Lípidos/química , Hígado/química , Vitamina A/metabolismo , beta Caroteno/metabolismo , Animales , Aterosclerosis/patología , Células Cultivadas , Femenino , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/deficiencia , Receptores de LDL/metabolismo , beta-Caroteno 15,15'-Monooxigenasa/deficiencia , beta-Caroteno 15,15'-Monooxigenasa/metabolismoRESUMEN
The actin filament (F-actin) bundling protein fascin-1 is highly enriched in many metastatic cancers. Fascin's contribution to metastasis have been ascribed to its enhancement of cell migration and invasion. However, mouse genetic studies clearly point to functions also in tumorigenesis, yet without mechanistic underpinnings. Here, we show that fascin expression promotes the formation of a non-canonical signaling complex that enables anchorage-independent proliferation. This complex shares similarities to focal adhesions and we refer to them as pseudo-adhesion signaling scaffolds (PASS). PASS are enriched with tyrosine phosphorylated proteins and require fascin's F-actin-bundling activity for its assembly. PASS serve as hubs for the Rac1/PAK/JNK proliferation signaling axis, driven by PASS-associated Rac-specific GEFs. Experimental disruption of either fascin or RacGEF function abrogates sustained proliferation of aggressive cancers in vitro and in vivo . These results add a new molecular element to the growing arsenal of metabolic and oncogenic signaling programs regulated by the cytoskeleton architecture.
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The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to the opposite side of the mouse embryo. To characterize the molecular bases for the unique properties of the AVE, we combined single-cell RNA sequencing of the VE prior to and during AVE migration with phosphoproteomics, high-resolution live-imaging, and short-term lineage labeling and intervention. This identified the transient nature of the AVE with attenuation of "anteriorizing" gene expression as cells migrate and the emergence of heterogeneities in transcriptional states relative to the AVE's position. Using cell communication analysis, we identified the requirement of semaphorin signaling for normal AVE migration. Lattice light-sheet microscopy showed that Sema6D mutants have abnormalities in basal projections and migration speed. These findings point to a tight coupling between transcriptional state and position of the AVE and identify molecular controllers of AVE migration.
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Tissue homeostasis and the emergence of disease are controlled by changes in the proportions of resident and recruited cells, their organization into cellular neighbourhoods, and their interactions with acellular tissue components. Highly multiplexed tissue profiling (spatial omics) 1 makes it possible to study this microenvironment in situ , usually in 4-5 micron thick sections (the standard histopathology format) 2 . Microscopy-based tissue profiling is commonly performed at a resolution sufficient to determine cell types but not to detect subtle morphological features associated with cytoskeletal reorganisation, juxtracrine signalling, or membrane trafficking 3 . Here we describe a high-resolution 3D imaging approach able to characterize a wide variety of organelles and structures at sub-micron scale while simultaneously quantifying millimetre-scale spatial features. This approach combines cyclic immunofluorescence (CyCIF) imaging 4 of over 50 markers with confocal microscopy of archival human tissue thick enough (30-40 microns) to fully encompass two or more layers of intact cells. 3D imaging of entire cell volumes substantially improves the accuracy of cell phenotyping and allows cell proximity to be scored using plasma membrane apposition, not just nuclear position. In pre-invasive melanoma in situ 5 , precise phenotyping shows that adjacent melanocytic cells are plastic in state and participate in tightly localised niches of interferon signalling near sites of initial invasion into the underlying dermis. In this and metastatic melanoma, mature and precursor T cells engage in an unexpectedly diverse array of juxtracrine and membrane-membrane interactions as well as looser "neighbourhood" associations 6 whose morphologies reveal functional states. These data provide new insight into the transitions occurring during early tumour formation and immunoediting and demonstrate the potential for phenotyping of tissues at a level of detail previously restricted to cultured cells and organoids.
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In crowded microenvironments, migrating cells must find or make a path. Amoeboid cells are thought to find a path by deforming their bodies to squeeze through tight spaces. Yet, some amoeboid cells seem to maintain a near-spherical morphology as they move. To examine how they do so, we visualized amoeboid human melanoma cells in dense environments and found that they carve tunnels via bleb-driven degradation of extracellular matrix components without the need for proteolytic degradation. Interactions between adhesions and collagen at the cell front induce a signaling cascade that promotes bleb enlargement via branched actin polymerization. Large blebs abrade collagen, creating feedback between extracellular matrix structure, cell morphology, and polarization that enables both path generation and persistent movement.
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Projection imaging accelerates volumetric interrogation in fluorescence microscopy, but for multi-cellular samples, the resulting images may lack contrast, as many structures and haze are summed up. Here, we demonstrate rapid projective light-sheet imaging with parameter selection (props) of imaging depth, position and viewing angle. This allows us to selectively image different sub-volumes of a sample, rapidly switch between them and exclude background fluorescence. Here we demonstrate the power of props by functional imaging within distinct regions of the zebrafish brain, monitoring calcium firing inside muscle cells of moving Drosophila larvae, super-resolution imaging of selected cell layers, and by optically unwrapping the curved surface of a Drosophila embryo. We anticipate that props will accelerate volumetric interrogation, ranging from subcellular to mesoscopic scales.
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Drosophila , Pez Cebra , Animales , Microscopía Fluorescente/métodos , Encéfalo/ultraestructura , LarvaRESUMEN
Cell segmentation is the fundamental task. Only by segmenting, can we define the quantitative spatial unit for collecting measurements to draw biological conclusions. Deep learning has revolutionized 2D cell segmentation, enabling generalized solutions across cell types and imaging modalities. This has been driven by the ease of scaling up image acquisition, annotation and computation. However 3D cell segmentation, which requires dense annotation of 2D slices still poses significant challenges. Labelling every cell in every 2D slice is prohibitive. Moreover it is ambiguous, necessitating cross-referencing with other orthoviews. Lastly, there is limited ability to unambiguously record and visualize 1000's of annotated cells. Here we develop a theory and toolbox, u-Segment3D for 2D-to-3D segmentation, compatible with any 2D segmentation method. Given optimal 2D segmentations, u-Segment3D generates the optimal 3D segmentation without data training, as demonstrated on 11 real life datasets, >70,000 cells, spanning single cells, cell aggregates and tissue.