Cecilia Huertas Fernández-Espartero

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerization-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MASs). Super-resolution microscopy reveals that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking shows that, during MAS development, integrins become immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also display restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscle cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerization-driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis.

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerisation-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MAS). Super-resolution microscopy revealed that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking showed that, during MAS development, integrins became immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also displayed restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscles cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerisation driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis.

SUMMARY: Decades of research have not yet fully explained the mechanisms of epithelial self-organization and 3D packing. Single-cell analysis of large 3D epithelial libraries is crucial for understanding the assembly and function of whole tissues. Combining 3D epithelial imaging with advanced deep learning segmentation methods is essential for enabling this high-content analysis. We introduce CartoCell, a deep learning-based pipeline that uses small datasets to generate accurate labels for hundreds of whole 3D epithelial cysts. Our method detects the realistic morphology of epithelial cells and their contacts in the 3D structure of the tissue. CartoCell enables the quantification of geometric and packing features at the cellular level. Our Single-cell Cartography approach then maps the distribution of these features on 2D plots and 3D surface maps, revealing cell morphology patterns in epithelial cysts. Additionally, we show that CartoCell can be adapted to other types of epithelial tissues. MOTIVATION: A major bottleneck in developing neural networks for cell segmentation is the need for labor-intensive manual curation in order to develop a training dataset. The present work addresses this limitation by developing an automated image analysis pipeline that utilizes small datasets to generate accurate labels of cells in complex, 3D epithelial contexts. The overall goal is to provide an automatic and feasible method to achieve high-quality epithelial reconstructions and to enable high-content analysis of morphological features, which can improve our understanding of how these tissues self-organize.

Although developmental signalling pathways control tumourigenic growth, the cellular mechanisms that abnormally proliferating cells rely on are still largely unknown. Drosophila melanogaster is a genetically tractable model that is used to study how specific genetic changes confer advantageous tumourigenic traits. Despite recent efforts, the role of deubiquitylating enzymes in cancer is particularly understudied. We performed a Drosophila in vivo RNAi screen to identify deubiquitylating enzymes that modulate Ras^V12-induced hyperplastic growth. We identified the spliceosome core component Prp8 as a crucial regulator of Ras-, EGFR-, Notch- or RET-driven hyperplasia. Loss of prp8 function alone decreased cell proliferation, increased cell death, and affected cell differentiation and polarity. In hyperplasia, Prp8 supported tissue overgrowth independently of caspase-dependent cell death. The depletion of prp8 efficiently blocked Ras-, EGFR- and Notch-driven tumours but, in contrast, enhanced tumours that were driven by oncogenic RET, suggesting a context-specific role in hyperplasia. These data show, for the first time, that Prp8 regulates hyperplasia, and extend recent observations on the potential role of the spliceosome in cancer. Our findings suggest that targeting Prp8 could be beneficial in specific tumour types.

Guided cell migration is a key mechanism for cell positioning in morphogenesis. The current model suggests that the spatially controlled activation of receptor tyrosine kinases (RTKs) by guidance cues would limit Rac activity at the leading edge, which is critical for establishing and maintaining polarized cell protrusions at the front. However, little is known about the mechanisms by which RTKs control the local activation of Rac. Here, using a multidisciplinary approach, we identify the GTP exchange factor (GEF) vav as a key regulator of Rac activity downstream of RTKs in a developmentally regulated cell migration event, that of the Drosophila border cells (BCs). We show that elimination of vav impairs BC migration. Live imaging analysis reveals that vav is required for the stabilization and maintenance of protrusions at the front of the BC cluster. In addition, activation of the PDGF/VEGF-related receptor (PVR) by its ligand the PDGF/PVF1 factor brings about Vav activation by direct interaction with the intracellular domain of PVR. Finally, FRET analyses demonstrate that Vav is required in BCs for the asymmetric distribution of Rac activity at the front. Our results unravel an important role for the Vav proteins as signal transducers that couple signalling downstream of RTKs with local Rac activation during morphogenetic movements.

Stationary‐to‐migratory transitions of epithelial cells have a key role in development and tumour progression. Border cell migration is a powerful system in which to investigate this transition in living organisms. Here, we identify the Ste20‐like kinase misshapen ( msn ) as a novel regulator of border‐cell migration in Drosophila . Expression of msn in border cells is independent of the transcription factor slow border cells and of inputs from all pathways that are known to control border‐cell migration. The msn gene functions to modulate the levels and/or distribution of Drosophila E‐cadherin to promote the invasive migratory behaviour of border cells.

In the subfamily Arvicolinae (Cricetidae, Rodentia) the satellite DNA Msat-160 has been so far described in only some species from the genus Microtus and in one species from another genus, Chionomys nivalis. Here we cloned and characterized this satellite in two new arvicoline species, Microtus (Terricola) savii and Arvicola amphibius (terrestris). We have also demonstrated, by PCR and FISH, its existence in the genomes of several other species from both genera. These results suggest that Msat-160 already occurred in the common ancestor of the four genera/subgenera of Arvicolinae (Microtus, Chionomys, Arvicola, and Terricola). In Arvicola and Terricola, Msat-160 showed the basic monomer length of 160 bp, although a higher-order repeat (HORs) of 640 bp could have been probably replacing the original monomeric unit in A. a. terrestris. Msat-160 was localized by FISH mostly on the pericentromeric regions of the chromosomes, but the signal intensity and the number of carrier chromosomes varied extremely even between closely related species, resulting in a species-specific pattern of chromosomal distribution of this satellite. Such a variable pattern most likely is a consequence of a rapid amplification and contraction of particular repeats in the pericentromeric regions of chromosomes. In addition, we proposed that the rapid variation of pericentromeric repeats is strictly related to the prolific species radiation and diversification of karyotypes that characterize Arvicolinae lineage. Finally, we performed phylogenetic analysis in this group of related species based on Msat-160 that results to be in agreement with previously reported phylogenies, derived from other molecular markers.

Several karyotypic forms have been previously described in populations of the vole species Microtus thomasi from Greece. In particular, the karyomorphs Microtus thomasi ‘thomasi’ and ‘atticus’ differ in X chromosome morphology, being acrocentric and subtelocentric, respectively. Furthermore, remarkable heterochromatin content variability has been described in sex chromosomes of both karyomorphs. Genomic DNA digestion with AluI allowed us to clone an 884 bp long repeated DNA sequence (Mth-Alu900) from the karyomorph M. thomasi ‘atticus’. This repeated DNA is AT rich and seems to be organized mainly as a dimer of the 884-bp unit, which presents three simple repeats (CAAAT, CAGAT and CAGAC) that constitute 80% of the total unit length. This repeated DNA is exclusive to M. thomasi, since it is absent from the genome of other studied Arvicolinae species. The chromosomal location of Mth-Alu900 was analyzed on M. thomasi ‘thomasi’ and M. thomasi ‘atticus’ karyomorphs, with different sex chromosome constitution. It was mainly located on the pericentromeric heterochromatin of most autosomes and X chromosomes on both karyomorphs. Results are also discussed in relation to karyotypic and sex chromosome variations in M. thomasi. To our knowledge, Mth-Alu900 constitutes a new – the third discovered so far – pericentromeric repeated DNA sequence described in Microtus species.

The chromosomal distribution of mobile genetic elements is scarcely known in Arvicolinae species, but could be of relevance to understand the origin and complex evolution of the sex chromosome heterochromatin. In this work we cloned two retrotransposon sequences, L1 and SINE-B1, from the genome of Chionomys nivalis and investigated their chromosomal distribution on several arvicoline species. Our results demonstrate first that both retroelements are the most abundant repeated DNA sequences in the genome of these species. L1 elements, in most species, are highly accumulated in the sex chromosomes compared to the autosomes. This favoured L1 insertion could have played an important role in the origin of the enlarged heterochromatic blocks existing in the sex chromosomes of some Microtus species. Also, we propose that L1 accumulation on the X heterochromatin could have been the consequence of different, independent and rapid amplification processes acting in each species. SINE elements, however, were completely lacking from the constitutive heterochromatin, either in autosomes or in the heterochromatic blocks of sex chromosomes. These data could indicate that some SINE elements are incompatible with the formation of heterochromatic complexes and hence are necessarily missing from the constitutive heterochromatin.