Model-Based Generation of Synthetic 3D Time-Lapse Sequences of Motile Cells with Growing Filopodia

International audienceThe existence of benchmark datasets is essential to objectively evaluate various image analysis methods. Nevertheless, manual annotations of fluorescence microscopy image data are very laborious and not often practicable, especially in the case of 3D+t experiments. In this work, we propose a simulation system capable of generating 3D time-lapse sequences of single motile cells with filopodial protrusions, accompanied by inherently generated ground truth. The system consists of three globally synchronized modules, each responsible for a separate task: the evolution of filopodia on a molecular level, linear elastic deformation of the entire cell with filopodia, and generation of realistic, time-coherent cell texture. The capability of our system is demonstrated by generating a synthetic 3D time-lapse sequence of a single lung cancer cell with two growing filopodia, visually resembling its real counterpart acquired using a confocal fluorescence microscope

Generative modeling of living cells with SO(3)-equivariant implicit neural representations

Data-driven cell tracking and segmentation methods in biomedical imaging require diverse and information-rich training data. In cases where the number of training samples is limited, synthetic computer-generated data sets can be used to improve these methods. This requires the synthesis of cell shapes as well as corresponding microscopy images using generative models. To synthesize realistic living cell shapes, the shape representation used by the generative model should be able to accurately represent fine details and changes in topology, which are common in cells. These requirements are not met by 3D voxel masks, which are restricted in resolution, and polygon meshes, which do not easily model processes like cell growth and mitosis. In this work, we propose to represent living cell shapes as level sets of signed distance functions (SDFs) which are estimated by neural networks. We optimize a fully-connected neural network to provide an implicit representation of the SDF value at any point in a 3D+time domain, conditioned on a learned latent code that is disentangled from the rotation of the cell shape. We demonstrate the effectiveness of this approach on cells that exhibit rapid deformations (Platynereis dumerilii), cells that grow and divide (C. elegans), and cells that have growing and branching filopodial protrusions (A549 human lung carcinoma cells). A quantitative evaluation using shape features, Hausdorff distance, and Dice similarity coefficients of real and synthetic cell shapes shows that our model can generate topologically plausible complex cell shapes in 3D+time with high similarity to real living cell shapes. Finally, we show how microscopy images of living cells that correspond to our generated cell shapes can be synthesized using an image-to-image model.Comment: Medical Image Analysis 2023 (Submitted

Generative adversarial networks for augmenting training data of microscopic cell images

Generative adversarial networks (GANs) have recently been successfully used to create realistic synthetic microscopy cell images in 2D and predict intermediate cell stages. In the current paper we highlight that GANs can not only be used for creating synthetic cell images optimized for different fluorescent molecular labels, but that by using GANs for augmentation of training data involving scaling or other transformations the inherent length scale of biological structures is retained. In addition, GANs make it possible to create synthetic cells with specific shape features, which can be used, for example, to validate different methods for feature extraction. Here, we apply GANs to create 2D distributions of fluorescent markers for F-actin in the cell cortex of Dictyostelium cells (ABD), a membrane receptor (cAR1), and a cortex-membrane linker protein (TalA). The recent more widespread use of 3D lightsheet microscopy, where obtaining sufficient training data is considerably more difficult than in 2D, creates significant demand for novel approaches to data augmentation. We show that it is possible to directly generate synthetic 3D cell images using GANs, but limitations are excessive training times, dependence on high-quality segmentations of 3D images, and that the number of z-slices cannot be freely adjusted without retraining the network. We demonstrate that in the case of molecular labels that are highly correlated with cell shape, like F-actin in our example, 2D GANs can be used efficiently to create pseudo-3D synthetic cell data from individually generated 2D slices. Because high quality segmented 2D cell data are more readily available, this is an attractive alternative to using less efficient 3D networks

Investigating the roles of Fascin and Rac1 in cell migration, invasion and metastasis

Cell migration and invasion is a central process in embryonic development, wound healing and immune responses. Errors during this process have serious consequences, including mental retardation, vascular disease, tumor formation and metastasis. An understanding of the mechanism by which cells migrate and invade may lead to the development of novel therapeutic strategies for controlling, for example, cancer metastasis. Fascin is an actin-bundling protein involved in filopodia assembly and cancer invasion and metastasis of multiple epithelial cancer types. In this thesis, I have investigated the role of fascin in invadopodia formation, which are invasive finger-like protrusions that cancer cells use to invade into and degrade extracellular matrix. I demonstrated that fascin and its actin bundling activity are required for the assembly of the actin cytoskeleton at invadopodia as well as for the degradation of matrix. Fascin is a very stable component of invadopodia and its presence enhance the stability of actin structures at invadopodia. Furthermore, fascin is required for invasive migration into collagen I-Matrigel gels particularly in cell types that use an elongated mesenchymal type of motility in 3D. These data provide a potential molecular mechanism for how fascin increases the invasiveness of cancer cells. During embryogenesis, melanoblasts proliferate and migrate ventrally through the developing dermis and epidermis as single cells. In the second part of this thesis, I examined the importance of small Rho GTPase Rac1 on melanoblast migration during development. I demonstrate that targeted deletion of Rac1 in the melanoblasts of developing mice causes defects in migration, cell cycle progression and cytokinesis. Rac1 null cells migrate markedly less efficiently, but surprisingly global steering, crossing the dermal/epidermal junction andhoming to hair follicles are normal. Melanoblasts navigate in the epidermis using two classes of protrusion: short stubs and long pseudopods. Short stubs are driven by actin assembly, but unexpectedly are independent of Rac1, Arp2/3 complex, myosin or microtubules. Rac1 positively regulates the frequency of initiation of long pseudopods, which promote migration speed and directional flexibility. Scar/WAVE and Arp2/3 complex drive actin assembly for long pseudopod extension, which is also microtubule dependent. Myosin contractility balances the extension of long pseudopods by effecting retraction and allowing force generation for movement through the complex 3D epidermal environment. In addition, I demonstrated that expression of activated N-Ras did not affect migration and proliferation of melanoblast during embryogensis. However, Rac1 is required for constitutively active N- Ras induced dermal melanocytes survival in mice

New principles in collective cell migration during Drosophila organ development

Cell migration drives most developmental processes, wound closure, as well as immune response. It constitutes a hallmark of cancer. Especially in tumour metastasis and development, cells rely on collective cell migration. Much knowledge about the exact mechanics of collective motility was gathered from a few model systems in which migration processes can be precisely analysed and genetically, mechanically, and pharmacologically influenced. This work aims to extend the existing range of such models by establishing a new ex vivo system for collective cell migration. The testis of Drosophila is surrounded by a layer of smooth-like muscles. The precursors of these cells, multinucleated myotubes, have to get to the testis and to migrate toward its distal end during pupal development. Organ-culture conditions were determined, allowing to recapitulate the process ex vivo. Thereby, the mechanical rules by which myotubes migrate could be assessed. Testis myotubes seem to use a lamellipodium-independent migration mode that is based entirely on the dynamics of filopodia-like protrusions. In previous studies, a chemoattractive effect mediated by the fibroblast growth factor receptor (FGFR) Heartless (Htl) was discussed, besides its likely role in regulating the number of myoblasts on the genital disc and its possible role in connecting testis and seminal vesicles. The results obtained in this work oppose a chemoattractive function but rather suggest a general role of Htl in the initiation of cell migration. Mathematical and statistical analysis of migration trajectories in the background of genetic, mechanical, and pharmacological perturbation suggest a self-regulating process. The observed dynamics reveal similarities to contact inhibition of locomotion (CIL) since cell-cell contacts provide crucial information for individual cells to enable directionality. Cells seem to inhibit substrate adhesion in filopodia in a contact-dependent manner. This process appears to be controlled by the Rho-GTPases Rac2 and Cdc42. As a result of the contact-dependent loss of adhesion, there is a net-movement into the cell-free space. At the same time, N-Cadherin seems to ensure that cells maintain adhesion to one another. Therefore, there is no repulsive migration as in CIL. Finally, supracellular RhoA/Rock-dependent actomyosin cables appear to support cohesion by closing gaps in the cell cluster at concavely curved edges. These mechanisms presumably result in a process in which all available space is evenly covered by myotubes. Myotube motility appears to be dependent on proteolytic degradation of the matrix. For this reason, in the future, the newly established ex vivo system could allow studying the collective dynamics of invasive migration in detail

Development of techniques for time-lapse imaging of the dynamics of glial-axonal interactions in the central nervous system

Background: Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualise the various stages of myelination. Methodology/Principal findings: Two approaches have been taken to follow myelination over time i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. The data demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, evidence from time-lapse fluorescence microscopy and confocal microscopy suggest that the oligodendrocytes’ cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs which then extend longitudinally along the axons. From this model it is predicted that these spiral cuffs must extend over each other first before extending to form internodes of myelin. Conclusion: These experiments show the feasibility of visualising the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualisation of an entire internodal length of myelin and the ex vivo approach validating the in vitro data

Cytoskeletal dynamics of Cytotoxic T cells during migration in the tumour microenvironment

Typically, migrating T cells display an elongated polarized shape with a very dynamic leading edge and a uropod in the rear. This ‘amoeboid’ movement guarantees a fast migration driven by the formation of polarized protrusions at the front. The actomyosin cytoskeleton is responsible for the generation of the forces that are involved in this process. This thesis aims to determine what is the effect of T cell migration when different components of the actomyosin cortex were inhibited using a pharmacological approach. We found that the inhibition of each component of the actomyosin cortex, T cells display different conformation of the actin filaments and produce different type of protrusion. Furthermore, T cell migration is an important feature for the killing and clearance of canner cells. It has been reported that T cells can migrate efficiently in any kind of tissue whilst scanning for cognate antigen. On the other hand, it is known that the tumor microenvironment secretes immunosuppressive cytokines such as TGF-β impairing the antitumor activity of T cells. Therefore, we aim to determine how TGF-β affects the migration behavior of T cells and its consequences in the scanning strategy to search their cognate antigen