During embryonal development, axons of newly differentiated neurons need to properly interconnect and create a functional neuronal network. To achieve this, the cell requires a growth cone. The growth cone is a highly dynamic structure at the end of growing axons that serves both as the navigator and the propeller. Crosstalk between actin and microtubules is vital for proper axonal pathfinding. But the exact mechanism of this cooperation remains unknown. This diploma thesis investigates the possible role of a candidate scaffolding protein called anillin in this process. Anillin has been studied in two human cell lines. SH-SY5Y neuroblastoma cell line was used for overexpression and siRNA knock-down experiments. Anillin overexpression led to perturbed neurite morphology and growth cone dynamics in SH-SY5Y cells, whereas cells with lower anillin expression had fewer neurites. Next, neurons differentiated from human iPSC (induced pluripotent stem cells) expressing endogenous fluorescently tagged anillin were studied. Local dynamic high concentration spots of anillin have been observed at the base of cell protrusions of differentiating neurons. These anillin flares appeared during cell migration, early neurite initiation, and in newly created growth cones. These results suggest that anillin plays a...

La polarisation des cellules est essentielle à la division cellulaire asymétrique, la migration cellulaire, la fonction neuronale et le développement embryonnaire. Elle peut se faire par le mécanisme de localisation des ARNm dans des compartiments cellulaires spécifiques. Bien que le mécanisme de localisation des ARNm soit assez bien défini dans tous les types cellulaires, son implication dans le développement du cancer n'a pas été caractérisée. Une étude de séquençage direct de l'ARN, réalisée sur des cellules métastatiques (HCCLM3) du carcinome hépatocellulaire (CHC) a déterminé qu'au niveau de leurs protrusions, certains ARNm y sont enrichis, alors qu'aux protrusions des cellules CHC non-métastatiques SMMC7721, ces ARNm n’y sont pas enrichis. L'ARNm qui encode pour le facteur de transcription STAT3 figure parmi ceux identifiés comme étant enrichis. La technique du smiFISH a été utilisée pour valider l'enrichissement de l'ARNm STAT3 au niveau des protrusions des cellules CHC associées à un phénotype métastatique. Les résultats obtenus démontrent que l'essai de déplétion de sérum permet d'induire la migration des cellules SMMC7721, HCCLM3 et MHCC97H, qui forment des protrusions de type lamellipode. En parallèle, l'expression de l'α-actinine-GFP a permis de visualiser les régions protrusives et de les délimiter. Les expériences de smiFISH et les mesures d'expression relative par RT-qPCR ont révélé que les cellules métastatiques HCCLM3 et MHCC97H expriment peu d'ARNm STAT3, comparativement aux cellules non-métastatiques SMMC7721. De plus, les quantifications absolues de l'enrichissement démontrent que dans toutes les lignées CHC, les molécules d'ARNm STAT3 uniques localisent majoritairement au niveau des corps cellulaires, et très peu dans les protrusions. Le ratio du nombre de molécules d'ARNm STAT3 uniques localisées dans les protrusions sur le nombre de molécules uniques localisées dans les corps cellulaires est nettement plus petit pour les cellules HCCLM3 et MHCC97H que pour les cellules SMMC7721. La densité de l'ARNm STAT3 / unité de surface dans les protrusions des cellules HCCLM3 et MHCC97H est inférieure à celle des cellules SMMC7721. Ensemble, ces résultats confirment qu'il n'y a pas d'enrichissement de l'ARNm STAT3 aux protrusions des cellules CHC associées à un phénotype métastatique. / Subcellular mRNA localization regulates cell polarization. Asymmetric distribution of transcripts is important in cell division, cell migration, neuronal function and embryonic development. In all cell types, the mechanisms of mRNA localization are well defined, but their involvement in cancer development is still unknown. A recent study has identified protrusion-localized STAT3 mRNA in metastatic hepatocarcinoma cells (HCCLM3). In comparison, the STAT3 mRNA was not found to be enriched in the non-metastatic cells (SMMC7721) protrusions. The enrichment of the STAT3 mRNA at the protrusions of HCC cells associated with a metastatic phenotype was studied by smiFISH. Our results showed that the serum starvation assay induced lamellipodia-based migration in SMMC7721, HCCLM3 and MHCC97H cells. Also, expression of GFP-α-actinin allowed to mark the protrusive regions of migrating HCC cells. The smiFISH results and RT-qPCR quantification revealed that the metastatic cells (HCCLM3 and MHCC97H) express lower levels of STAT3 mRNA compared to the non-metastatic cells (SMMC7721). Absolute quantification of localization and enrichment revealed that STAT3 mRNA mainly localizes in the cell bodies of all HCC cell lines. The ratio of the number of single molecules of STAT3 mRNA localized in the protrusions on the number of single molecules localized in the cell bodies is smaller for the metastatic cells than the non-metastatic cells. In the metastatic cells protrusions, the density of STAT3 mRNA / unit of surface was found the be lower than the non-metastatic cells. Altogether, these results confirm that there is no enrichment of STAT3 mRNA at the protrusions of metastatic HCC cells.

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During the epithelial-to-mesenchymal transition, the intracellular cytoskeleton undergoes severe reorganization which allows epithelial cells to transition into a motile mesenchymal phenotype. Among the different cytoskeletal elements, the intermediate filaments keratin (in epithelial cells) and vimentin (in mesenchymal cells) have been demonstrated to be useful and reliable histological markers. In this study, we assess the potential invasiveness of six human breast carcinoma cell lines and two mouse fibroblasts cells lines through single cell migration assays in confinement. We find that the keratin and vimentin networks behave mechanically the same when cells crawl through narrow channels and that vimentin protein expression does not strongly correlate to single cells invasiveness. Instead, we find that what determines successful migration through confining spaces is the ability of cells to mechanically switch from a substrate-dependent stress fibers based contractility to a substrate-independent cortical contractility, which is not linked to their tumor phenotype.

In tissue development, wound healing and aberrant cancer progression cell–cell interactions drive mixing and segregation of cellular composites. However, the exact nature of these interactions is unsettled. Here we study the dynamics of packed, heterogeneous cellular systems using wound closure experiments. In contrast to previous cell sorting experiments, we find non-universal sorting behavior. For example, monolayer tissue composites with two distinct cell types that show low and high neighbor exchange rates (i.e., MCF-10A & MDA-MB-231) produce segregated domains of each cell type, contrary to conventional expectation that the construct should stay jammed in its initial configuration. On the other hand, tissue compounds where both cell types exhibit high neighbor exchange rates (i.e., MDA-MB-231 & MDA-MB-436) produce highly mixed arrangements despite their differences in intercellular adhesion strength. The anomalies allude to a complex multi-parameter space underlying these sorting dynamics, which remains elusive in simpler systems and theories merely focusing on bulk properties. Using cell tracking data, velocity profiles, neighborhood volatility, and computational modeling, we classify asymmetric interfacial dynamics. We indicate certain understudied facets, such as the effects of cell death & division, mechanical hindrance, active nematic behavior, and laminar & turbulent flow as their potential drivers. Our findings suggest that further analysis and an update of theoretical models, to capture the diverse range of active boundary dynamics which potentially influence self-organization, is warranted.

The vertebrate embryonic limb is a key model in elucidating the genetic basis underlying the three dimensional morphogenesis of structures. Despite the wealth of insights that have been generated from this model, many long-standing questions remain. For example, it has been known for over 70 years that the apical ectodermal ridge (AER) of the embryonic limb is essential for distal outgrowth and patterning of the adjacent limb mesenchyme. The mechanisms whereby the AER does accomplish outgrowth and patterning are still poorly understood. We propose that secreted FGFs from the AER activate Wnt5a expression in gradient fashion, which in turn provides an instructional cue to direct outgrowth in the direction of increasing Wnt5a expression (i.e. toward the distal tip of the limb). In vivo and in vitro models were used to test this hypothesis. We placed Wnt5a expressing L-cell implants into stage 23 chick limb buds and demonstrate that labeled mesenchyme cells grow toward the source of Wnt5a. Purified Wnt5a soaked heparin bead implants have only a marginal effect on directed growth of the adjacent mesenchyme, whereas a greater effect was seen with beads soaked in Wnt5a conditioned media. Using an in vitro model where cultured limb mesenchyme cells were subjected to a gradient of conditioned Wnt5a media or purified Wnt5a, we show no specific migratory direction. However, clusters of cells tended to move toward the source of Wnt5a indicating that it might be necessary for the cells to be in complete contact to respond to the Wnt5a signal. Taken together, our results suggest that Wnt5a is sufficient to direct limb mesenchyme. This finding has given support to a new model of limb development proposed by our lab and referred to as the Mesenchyme Recruitment Model.

Understanding cell migration is a key step in unraveling many physiological phenomena and predicting several pathologies, such as cancer metastasis. In particular, confinement has been proven to be a key factor in the cellular migration strategy choice. As our insight in the field improves, new tools are needed in order to empower biologists’ analysis capabilities. In this framework, microfluidic devices have been used to engineer the mechanical and spatial stimuli and to investigate cellular migration response in a more controlled way. In this work, we will review the existing technologies employed in the realization of microfluidic cellular migration assays, namely the soft lithography of PDMS and hydrogels and femtosecond laser micromachining. We will give an overview of the state of the art of these devices, focusing on the different geometrical configurations that have been exploited to study specific aspects of cellular migration. Our scope is to highlight the advantages and possibilities given by each approach and to envisage the future developments in in vitro migration studies under spatial confinement in microfluidic device

The study of cellular migration dynamics and strategies plays a relevant role in the understanding of both physiological and pathological processes. An important example could be the link between cancer cell motility and tumor evolution into metastatic stage. These strategies can be strongly influenced by the extracellular environment and the consequent mechanical constrains. In this framework, the possibility to study the behavior of single cells when subject to specific topological constraints could be an important tool in the hands of biologists. Two-photon polymerization is a sub-micrometric additive manufacturing technique that allows the fabrication of 3D structures in biocompatible resins, enabling the realization of ad hoc biochips for cell motility analyses, providing different types of mechanical stimuli. In our work, we present a new strategy for the realization of multilayer microfluidic lab-on-a-chip constructs for the study of cell motility which guarantees complete optical accessibility and the possibility to freely shape the migration area, to tailor it to the requirements of the specific cell type or experiment. The device includes a series of micro-constrictions that induce different types of mechanical stress on the cells during their migration. We show the realization of different possible geometries, in order to prove the versatility of the technique. As a proof of concept, we present the use of one of these devices for the study of the motility of murine neuronal cancer cells under high physical confinement, highlighting their peculiar migration mechanisms.

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