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Deep neural networks can improve the quality of fluorescence microscopy images. Previous methods, based on Convolutional Neural Networks (CNNs), require time-consuming training of individual models for each experiment, impairing their applicability and generalization. In this study, we propose a novel imaging-transformer based model, Convolutional Neural Network Transformer (CNNT), that outperforms CNN based networks for image denoising. We train a general CNNT based backbone model from pairwise high-low Signal-to-Noise Ratio (SNR) image volumes, gathered from a single type of fluorescence microscope, an instant Structured Illumination Microscope. Fast adaptation to new microscopes is achieved by fine-tuning the backbone on only 5-10 image volume pairs per new experiment. Results show that the CNNT backbone and fine-tuning scheme significantly reduces training time and improves image quality, outperforming models trained using only CNNs such as 3D-RCAN and Noise2Fast. We show three examples of efficacy of this approach in wide-field, two-photon, and confocal fluorescence microscopy.
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Deep neural networks have been applied to improve the image quality of fluorescence microscopy imaging. Previous methods are based on convolutional neural networks (CNNs) which generally require more time-consuming training of separate models for each new imaging experiment, impairing the applicability and generalization. Once the model is trained (typically with tens to hundreds of image pairs) it can then be used to enhance new images that are like the training data. In this study, we proposed a novel imaging-transformer based model, Convolutional Neural Network Transformer (CNNT), to outperform the CNN networks for image denoising. In our scheme we have trained a single CNNT based "backbone model" from pairwise high-low SNR images for one type of fluorescence microscope (instance structured illumination, iSim). Fast adaption to new applications was achieved by fine-tuning the backbone on only 5-10 sample pairs per new experiment. Results show the CNNT backbone and fine-tuning scheme significantly reduces the training time and improves the image quality, outperformed training separate models using CNN approaches such as - RCAN and Noise2Fast. Here we show three examples of the efficacy of this approach on denoising wide-field, two-photon and confocal fluorescence data. In the confocal experiment, which is a 5×5 tiled acquisition, the fine-tuned CNNT model reduces the scan time form one hour to eight minutes, with improved quality.
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The all-terrain motility of lymphocytes in tissues and tissue-like gels is best described as amoeboid motility. For amoeboid motility, lymphocytes do not require specific biochemical or structural modifications to the surrounding extracellular matrix. Instead, they rely on changing shape and steric interactions with the microenvironment. However, the exact mechanism of amoeboid motility remains elusive. Here, we report that septins participate in amoeboid motility of T cells, enabling the formation of F-actin and α-actinin-rich cortical rings at the sites of cell cortex-indenting collisions with the extracellular matrix. Cortical rings compartmentalize cells into chains of spherical segments that are spatially conformed to the available lumens, forming transient "hourglass"-shaped steric locks onto the surrounding collagen fibers. The steric lock facilitates pressure-driven peristaltic propulsion of cytosolic content by individually contracting cell segments. Our results suggest that septins provide microenvironment-guided partitioning of actomyosin contractility and steric pivots required for amoeboid motility of T cells in tissue-like microenvironments.
Assuntos
Actomiosina , Amoeba , Actomiosina/metabolismo , Septinas/metabolismo , Movimento Celular , Amoeba/metabolismo , Linfócitos T/metabolismoRESUMO
Optical aberrations hinder fluorescence microscopy of thick samples, reducing image signal, contrast, and resolution. Here we introduce a deep learning-based strategy for aberration compensation, improving image quality without slowing image acquisition, applying additional dose, or introducing more optics into the imaging path. Our method (i) introduces synthetic aberrations to images acquired on the shallow side of image stacks, making them resemble those acquired deeper into the volume and (ii) trains neural networks to reverse the effect of these aberrations. We use simulations to show that applying the trained 'de-aberration' networks outperforms alternative methods, and subsequently apply the networks to diverse datasets captured with confocal, light-sheet, multi-photon, and super-resolution microscopy. In all cases, the improved quality of the restored data facilitates qualitative image inspection and improves downstream image quantitation, including orientational analysis of blood vessels in mouse tissue and improved membrane and nuclear segmentation in C. elegans embryos.
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Lymphocytes exit circulation and enter in-tissue guided migration toward sites of tissue pathologies, damage, infection, or inflammation. By continuously sensing and adapting to the guiding chemo-mechano-structural properties of the tissues, lymphocytes dynamically alternate and combine their amoeboid (non-adhesive) and mesenchymal (adhesive) migration modes. However, which mechanisms guide and balance different migration modes are largely unclear. Here we report that suppression of septins GTPase activity induces an abrupt amoeboid-to-mesenchymal transition of T cell migration mode, characterized by a distinct, highly deformable integrin-dependent immune cell contact guidance. Surprisingly, the T cell actomyosin cortex contractility becomes diminished, dispensable and antagonistic to mesenchymal-like migration mode. Instead, mesenchymal-like T cells rely on microtubule stabilization and their non-canonical dynein motor activity for high fidelity contact guidance. Our results establish septin's GTPase activity as an important on/off switch for integrin-dependent migration of T lymphocytes, enabling their dynein-driven fluid-like mesenchymal propulsion along the complex adhesion cues.
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The principal cause of death in cancer patients is metastasis, which remains an unresolved problem. Conventionally, metastatic dissemination is linked to actomyosin-driven cell locomotion. However, the locomotion of cancer cells often does not strictly line up with the measured actomyosin forces. Here, a complementary mechanism of metastatic locomotion powered by dynein-generated forces is identified. These forces arise within a non-stretchable microtubule network and drive persistent contact guidance of migrating cancer cells along the biomimetic collagen fibers. It is also shown that the dynein-powered locomotion becomes indispensable during invasive 3D migration within a tissue-like luminal network formed by spatially confining granular hydrogel scaffolds (GHS) made up of microscale hydrogel particles (microgels). These results indicate that the complementary motricity mediated by dynein is always necessary and, in certain instances, sufficient for disseminating metastatic breast cancer cells. These findings advance the fundamental understanding of cell locomotion mechanisms and expand the spectrum of clinical targets against metastasis.
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Neoplasias da Mama , Dineínas , Humanos , Feminino , Dineínas/metabolismo , Actomiosina/metabolismo , Movimento Celular , HidrogéisRESUMO
Small-molecule inhibitors of PD-L1 are postulated to control immune evasion in tumors similar to antibodies that target the PD-L1/PD-1 immune checkpoint axis. However, the identity of targetable PD-L1 inducers is required to develop small-molecule PD-L1 inhibitors. In this study, using chromatin immunoprecipitation (ChIP) assay and siRNA, we demonstrate that vitamin D/VDR regulates PD-L1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells. We have examined whether a VDR antagonist, MeTC7, can inhibit PD-L1. To ensure that MeTC7 inhibits VDR/PD-L1 without off-target effects, we examined competitive inhibition of VDR by MeTC7, utilizing ligand-dependent dimerization of VDR-RXR, RXR-RXR, and VDR-coactivators in a mammalian 2-hybrid (M2H) assay. MeTC7 inhibits VDR selectively, suppresses PD-L1 expression sparing PD-L2, and inhibits the cell viability, clonogenicity, and xenograft growth of AML cells. MeTC7 blocks AML/mesenchymal stem cells (MSCs) adhesion and increases the efferocytotic efficiency of THP-1 AML cells. Additionally, utilizing a syngeneic colorectal cancer model in which VDR/PD-L1 co-upregulation occurs in vivo under radiation therapy (RT), MeTC7 inhibits PD-L1 and enhances intra-tumoral CD8+T cells expressing lymphoid activation antigen-CD69. Taken together, MeTC7 is a promising small-molecule inhibitor of PD-L1 with clinical potential.
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Metastasis is a principal cause of death in cancer patients, which remains an unresolved fundamental and clinical problem. Conventionally, metastatic dissemination is linked to the actomyosin-driven cell locomotion. However, locomotion of cancer cells often does not strictly line up with the measured actomyosin forces. Here, we identify a complementary mechanism of metastatic locomotion powered by the dynein-generated forces. These forces that arise within a non-stretchable microtubule network drive persistent contact guidance of migrating cancer cells along the biomimetic collagen fibers. We also show that dynein-powered locomotion becomes indispensable during invasive 3D migration within a tissue-like luminal network between spatially confining hydrogel microspheres. Our results indicate that the complementary contractile system of dynein motors and microtubules is always necessary and in certain instances completely sufficient for dissemination of metastatic breast cancer cells. These findings advance fundamental understanding of cell locomotion mechanisms and expand the spectrum of clinical targets against metastasis.
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Microtubules (MTs) and MT motor proteins form active 3D networks made of unstretchable cables with rod-like bending mechanics that provide cells with a dynamically changing structural scaffold. In this study, we report an antagonistic mechanical balance within the dynein-kinesin microtubular motor system. Dynein activity drives the microtubular network inward compaction, while isolated activity of kinesins bundles and expands MTs into giant circular bands that deform the cell cortex into discoids. Furthermore, we show that dyneins recruit MTs to sites of cell adhesion, increasing the topographic contact guidance of cells, while kinesins antagonize it via retraction of MTs from sites of cell adhesion. Actin-to-microtubule translocation of septin-9 enhances kinesin-MT interactions, outbalances the activity of kinesins over that of dyneins, and induces the discoid architecture of cells. These orthogonal mechanisms of MT network reorganization highlight the existence of an intricate mechanical balance between motor activities of kinesins and dyneins that controls cell 3D architecture, mechanics, and cell-microenvironment interactions.
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Dineínas , Cinesinas , Dineínas/metabolismo , Actinas/metabolismo , Septinas/metabolismo , Microtúbulos/metabolismoRESUMO
Contractile forces within the planar interface between T cell and antigen-presenting surface mechanically stimulate T cell receptors (TCR) in the mature immune synapses. However, the origin of mechanical stimulation during the initial, i.e., presynaptic, microvilli-based TCR activation in the course of immune surveillance remains unknown and new tools to help address this problem are needed. In this work, we develop nucleic acid nanoassembly (NAN)-based technology for functionalization of hydrogels using isothermal toehold-mediated reassociation of RNA/DNA heteroduplexes. Resulting platform allows for regulation with NAN linkers of 3D force momentum along the TCR mechanical axis, whereas hydrogels contribute to modulation of 2D shear modulus. By utilizing different lengths of NAN linkers conjugated to polyacrylamide gels of different shear moduli, we demonstrate an efficient capture of human T lymphocytes and tunable activation of TCR, as confirmed by T-cell spreading and pY foci.
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Hidrogéis/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/genética , Células Apresentadoras de Antígenos/efeitos dos fármacos , DNA/química , DNA/farmacologia , Humanos , Hidrogéis/química , Ativação Linfocitária/genética , Linfócitos/metabolismo , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/farmacologia , RNA/química , RNA/genética , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , Linfócitos T/efeitos dos fármacosRESUMO
We demonstrate residual channel attention networks (RCAN) for the restoration and enhancement of volumetric time-lapse (four-dimensional) fluorescence microscopy data. First we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy four-dimensional super-resolution data, enabling image capture of over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables resolution enhancement equivalent to, or better than, other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling ~2.5-fold lateral resolution enhancement using stimulated emission depletion microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy data as ground truth, achieving improvements of ~1.9-fold laterally and ~3.6-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluation and further enhancement of network performance.
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Microscopia de Fluorescência/métodos , Algoritmos , Aprendizado Profundo , Processamento de Imagem Assistida por ComputadorRESUMO
Instant structured illumination microscopy (iSIM) allows for rapid multicolor three-dimensional fluorescence imaging at levels of resolution approaching twice the diffraction limit. Here we briefly describe the theory of iSIM and outline a typical hardware setup. We also provide step-by-step guides for generating a cellular-based fluorescent standard, obtaining a multicolor image with iSIM, and the post-processing steps of de-striping and deconvolution using freely distributed software to minimize time and expense. A "Notes" section is also given to inform the reader of the limitations and considerations for the methods shown. Also discussed are alternative methods, quality control checks, and considerations for two-camera alignment.
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Fibroblastos/citologia , Imageamento Tridimensional/instrumentação , Microscopia de Fluorescência/instrumentação , Animais , Linhagem Celular , Camundongos , SoftwareRESUMO
Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.
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Movimento Celular/fisiologia , Linfócitos T/imunologia , Linfócitos T/fisiologia , Microambiente Tumoral/imunologia , Microambiente Tumoral/fisiologia , Animais , Fenômenos Biomecânicos , Células Cultivadas , Matriz Extracelular/imunologia , Matriz Extracelular/fisiologia , Técnicas de Inativação de Genes , Engenharia Genética , Humanos , Camundongos , Camundongos Transgênicos , Microtúbulos/fisiologia , Modelos Biológicos , Nanoestruturas , Fatores de Troca de Nucleotídeo Guanina Rho/antagonistas & inibidores , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/fisiologia , Evasão Tumoral/imunologia , Evasão Tumoral/fisiologiaRESUMO
We present a reproducible protocol for fabrication of polyacrylamide (PAA) hydrogel-based nano-patterns and nano-textures with a wide range of elastic rigidities to study fundamental cell behaviors, such as mechanosensitivity and motility. We explore the benefits of this protocol by successfully testing the compatibility of the PAA platforms with super-resolution microscopy, which is largely unavailable with platforms of nano-scale textures made from different polymers. We also utilized soft and rigid nano-textures to study the mechanosensing basis of T cell behavior and phenotype. For complete information on the generation and use of this protocol, please refer to Tabdanov et al. (2018b).
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Resinas Acrílicas , Engenharia Celular/métodos , Movimento Celular , Nanotecnologia/métodos , Linhagem Celular Tumoral , HumanosRESUMO
Contact guidance refers to the ability of cells to sense the geometrical features of the microenvironment and respond by changing their shape and adopting the appropriate orientation. Inhibition and ablation of nonmuscle myosin 2 (NM2) paralogues have demonstrated their importance for contact guidance. However, the specific roles of the NM2 paralogues have not been systematically studied. In this work we use micropatterned substrates to examine the roles of NM2A and NM2B and to elucidate the relationship of the microenvironment, actomyosin, and microtubules in contact guidance. We show that contact guidance is preserved following loss of NM2B and that expression of NM2A alone is sufficient to establish an appropriate orientation of the cells. Loss of NM2B and overexpression of NM2A result in a prominent cell polarization that is found to be linked to the increased alignment of microtubules with the actomyosin scaffold. Suppression of actomyosin with blebbistatin reduces cell polarity on a flat surface, but not on a surface with contact guidance cues. This indicates that the lost microtubule-actomyosin interactions are compensated for by microtubule-microenvironment interactions, which are sufficient to establish cell polarity through contact guidance.
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Comunicação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Actomiosina/metabolismo , Animais , Polaridade Celular , Forma Celular , Fibroblastos/metabolismo , Camundongos , Microtúbulos/metabolismo , Fibras de Estresse/metabolismoRESUMO
Cancer cell migration through and away from tumors is driven in part by migration along aligned extracellular matrix, a process known as contact guidance (CG). To concurrently study the influence of architectural and mechanical regulators of CG sensing, we developed a set of CG platforms. Using flat and nanotextured substrates with variable architectures and stiffness, we show that CG sensing is regulated by substrate stiffness and define a mechanical role for microtubules and actomyosin-microtubule interactions during CG sensing. Furthermore, we show that Arp2/3-dependent lamellipodia dynamics can compete with aligned protrusions to diminish the CG response and define Arp2/3- and Formins-dependent actin architectures that regulate microtubule-dependent protrusions, which promote the CG response. Thus, our work represents a comprehensive examination of the physical mechanisms influencing CG sensing.
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Actomiosina/metabolismo , Neoplasias da Mama/fisiopatologia , Adesão Celular , Comunicação Celular , Movimento Celular , Matriz Extracelular/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Feminino , Humanos , Pseudópodes/fisiologia , Células Tumorais CultivadasRESUMO
RNA with site-specific modification is a useful tool for RNA biology studies. However, generating kilobase (kb) -long RNA with internal modification at a site distant from RNA termini remains challenging. Here we report an enhanced splint ligation technique, proximal disruptor aided ligation (ProDAL), which allows adequate efficiency toward this purpose. The key to our approach is using multiple DNA oligonucleotides, 'proximal disruptors', to target the RNA substrate sequence next to the ligation site. The binding of disruptors helps to free the ligation site from intramolecular RNA basepairing, and consequently promotes more efficient formation of the pre-ligation complex and a higher overall ligation yield. We used naturally occurring 1.0 kb renilla and 1.9 kb firefly luciferase mRNA sequences to test the efficacy of our approach. ProDAL yielded 9-14% efficiency for the ligation between two RNA substrates, both of which were between 414 and 1313 nucleotides (nt) long. ProDAL also allowed similarly high efficiency for generating kb-long RNA with site-specific internal modification by a simple three-part ligation between two long RNA substrates and a modification-carrying RNA oligonucleotide. In comparison, classical splint ligation yielded a significantly lower efficiency of 0-2% in all cases. We expect that ProDAL will benefit studies involving kb-long RNAs, including translation, long non-coding RNAs, RNA splicing and modification, and large ribonucleoprotein complexes.
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RNA/química , RNA/síntese químicaRESUMO
UV-induced DNA damage causes repression of RNA synthesis. Following the removal of DNA lesions, transcription recovery operates through a process that is not understood yet. Here we show that knocking-out of the histone methyltransferase DOT1L in mouse embryonic fibroblasts (MEF(DOT1L)) leads to a UV hypersensitivity coupled to a deficient recovery of transcription initiation after UV irradiation. However, DOT1L is not implicated in the removal of the UV-induced DNA damage by the nucleotide excision repair pathway. Using FRAP and ChIP experiments we established that DOT1L promotes the formation of the pre-initiation complex on the promoters of UV-repressed genes and the appearance of transcriptionally active chromatin marks. Treatment with Trichostatin A, relaxing chromatin, recovers both transcription initiation and UV-survival. Our data suggest that DOT1L secures an open chromatin structure in order to reactivate RNA Pol II transcription initiation after a genotoxic attack.
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Cromatina/genética , Dano ao DNA/genética , Metiltransferases/genética , Animais , Cromatina/efeitos da radiação , Reparo do DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase , Ácidos Hidroxâmicos/farmacologia , Hipersensibilidade , Camundongos , Camundongos Knockout , RNA Polimerase II/metabolismo , Ativação Transcricional , Raios UltravioletaRESUMO
Poly-(ADP-ribose) glycohydrolase (PARG) is a catabolic enzyme that cleaves ADP-ribose polymers synthesized by poly-(ADP-ribose) polymerases. Here, transcriptome profiling and differentiation assay revealed a requirement of PARG for retinoic acid receptor (RAR)-mediated transcription. Mechanistically, PARG accumulates early at promoters of RAR-responsive genes upon retinoic acid treatment to promote the formation of an appropriate chromatin environment suitable for transcription. Silencing of PARG or knockout of its enzymatic activity maintains the H3K9me2 mark at the promoter of the RAR-dependent genes, leading to the absence of preinitiation complex formation. In the absence of PARG, we found that the H3K9 demethylase KDM4D/JMJD2D became PARsylated. Mutation of two glutamic acids located in the Jumonji N domain of KDM4D inhibited PARsylation. PARG becomes dispensable for ligand-dependent transcription when either a PARP inhibitor or a non-PARsylable KDM4D/JMJD2D mutant is used. Our results define PARG as a coactivator regulating chromatin remodeling during RA-dependent gene expression.
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Glicosídeo Hidrolases/metabolismo , Receptores do Ácido Retinoico/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Montagem e Desmontagem da Cromatina , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Ácido Glutâmico , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/genética , Células HeLa , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Metilação , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Interferência de RNA , Receptores do Ácido Retinoico/efeitos dos fármacos , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptor alfa de Ácido Retinoico , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , Tretinoína/farmacologiaRESUMO
TFIIH is organized into a seven-subunit core associated with a three-subunit Cdk-activating kinase (CAK) module. TFIIH has roles in both transcription initiation and DNA repair. During the last 15 years, several studies have been conducted to identify the composition of the TFIIH complex involved in DNA repair. Recently, a new technique combining chromatin immunoprecipitation and western blotting resolved the hidden nature of the TFIIH complex participating in DNA repair. Following the recruitment of TFIIH to the damaged site, the CAK module is released from the core TFIIH, and the core subsequently associates with DNA repair factors. The release of the CAK is specifically driven by the recruitment of the DNA repair factor XPA and is required to promote the incision/excision of the damaged DNA. Once the DNA lesions have been repaired, the CAK module returns to the core TFIIH on the chromatin, together with the release of the repair factors. These data highlight the dynamic composition of a fundamental cellular factor that adapts its subunit composition to the cell needs.