Biology subjects

La Marca, J. E.

Publications and source records attributed to La Marca, J. E..

4 recordsLinked to original sources

bioRxiv2025

Actin cytoskeletal deregulation, caused by RhoGEF2 overexpression, induces cell competition dependent on Ptp10D, Crumbs, and the Hippo signaling pathway.

In Drosophila larval epithelial tissues, cells containing mutations in the apico-basal polarity proteins, Scrib, Dlg or Lgl, are eliminated by cell competition when surrounded by wild-type cells. In these polarity-impaired cells, signaling mediated by the receptor-type tyrosine phosphatase Ptp10D upon engagement with its ligand Sas in the surrounding wild-type cells triggers cell competition via EGFR pathway inhibition and JNK pathway activation, which induces apoptosis of the mutant cells. Here, we investigate whether directly triggering cytoskeletal deregulation (which usually occurs downstream of cell polarity disruptions) is sufficient to trigger their elimination by cell competition via the Sas-Ptp10D signaling system. We show that actin cytoskeleton deregulated cells (as induced by RhoGEF2 overexpression (RhoGEF2OE)) are eliminated when surrounded by wild-type cells, and that Ptp10D knockdown increases RhoGEF2OE clone growth, revealing the importance of Ptp10D in the elimination of RhoGEF2OE cells. Mechanistically, in clones that are moderately overexpressing RhoGEF2OE, Ptp10D knockdown rescued cell elimination by reducing Hippo signaling. In this setting, JNK and EGFR-Ras signaling were not affected (in contrast to what occurs in apico-basal mutant cells), suggesting that Sas-Ptp10D may regulate the Hippo pathway directly in RhoGEF2OE cells. We also found that mutations in the apical cell polarity protein, Crb, partially rescued the elimination of RhoGEF2OE clones, showing that Crb normally plays a role in RhoGEF2OE clone elimination. In this setting, in which RhoGEF2OE is highly overexpressed, JNK and Hippo signaling were elevated while EGFR-Ras signaling was reduced, and crb loss normalized these pathways. crb mutant cells also showed reduced abundance of and apical membrane localization of Ptp10D, suggesting that Crb may play an important role in Sas-Ptp10D mediated cell competition. Thus, actin cytoskeleton deregulation, caused by RhoGEF2OE, results in clone elimination dependent on Crb, Ptp10D, and Hippo signaling. Altogether, our results reveal that Ptp10D is acting more broadly in cell competition to trigger the elimination of actin cytoskeleton deregulated loser cells, as well as polarity-impaired cells.

developmental biology↗

bioRxiv2024

Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening

Cas12a is a gene-editing tool that simplifies multiplexed gene targeting through its RNase activity, enabling maturation of individual crRNAs from a pre-crRNA-encoding RNA. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene-editing in cells from enAsCas12aKI mice. To test in vivo activity, we transduced haematopoietic stem cells from E-MycT/+;enAsCas12aKI/+animals with Trp53-targeting pre-crRNAs followed by transplantation into irradiated recipient animals. Tumour development was accelerated and TRP53 protein lost. We generated compact, genome-wide Cas12a knockout libraries targeting each gene with four guide RNAs encoded on two (Menuetto) or one (Scherzo) vector. Introducing these libraries into E-MycT/+;enAsCas12aKI/+lymphoma cells followed by treatment with an MCL-1 inhibitor (S63845) or TRP53-inducer (nutlin-3a) identified known and novel drug resistance genes. Finally, we demonstrate simultaneous gene knockouts (Trp53 or combined Bax/Bak) and activation (Cd19) in primary T cells and mouse dermal fibroblasts from crosses of our enAsCas12a and CRISPR activation models (dCas9a-SAM). Our enAsCas12a mouse model and accompanying libraries enhance genome engineering capabilities and complements current CRISPR technologies.

molecular biology↗

bioRxiv2023

The Drosophila Tumour Suppressor Lgl and Vap33 activate the Hippo pathway by a dual mechanism, involving RtGEF/Git/Arf79F and inhibition of the V-ATPase.

The tumour suppressor, Lethal (2) giant larvae (Lgl), is an evolutionarily conserved protein that was discovered in the vinegar fly, Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity and tissue architecture. Our previous studies have revealed a new role for Lgl in linking cell polarity and tissue growth through regulation of the Notch (proliferation and differentiation) and the Hippo (negative tissue growth control) signalling pathways. Moreover, Lgl regulates vesicle acidification, via the Vacuolar ATPase (V-ATPase), and we showed that Lgl inhibits V-ATPase activity through Vap33 (a Vamp (v-SNARE)-associated protein, involved in endo-lysosomal trafficking) to regulate the Notch pathway. However, how Lgl acts to regulate the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Using an in vivo affinity-purification approach we found that Vap33 binds to the actin cytoskeletal regulators RtGEF (Pix, a Rho-type guanine nucleotide exchange factor) and Git (G protein-coupled receptor kinase interacting ArfGAP), which also bind to the Hpo protein kinase, and are involved in the activation of the Hippo pathway. Vap33 genetically interacts with RtGEF and Git in Hippo pathway regulation. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether our data suggests that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism, 1) through interaction with RtGEF/Git/Arf79F, and 2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth.

cell biology↗

bioRxiv2022

A Drosophila in vivo chemical screen reveals that combination drug treatment targeting MEK and DGKα mitigates Ras-driven polarity-impaired tumourigenesis.

The RAS oncogene and upregulation of the RAS signalling pathway is highly prevalent in human cancer, and therefore, therapeutically targeting the RAS pathway is a common treatment in cancer. However, RAS pathway upregulation is not sufficient to drive malignant cancer, since senescence mechanisms prevent cancer progression. Thus, additional mutations, such as mutations that prevent senescence or alter the tissue architecture (cell polarity), are required for RAS-driven tumour progression. Moreover, targeting RAS-driven cancers with RAS pathway inhibitors can often lead to undesirable side-effects and to drug resistance. Thus, identifying compounds that synergise with RAS-pathway inhibitors would enable lower doses of the RAS pathway inhibitors to be used and also decrease the acquisition of drug resistance. Here, in a boutique chemical screen using a Drosophila model of Ras-driven cell polarity-impaired cancer, we have identified compounds that reduce tumour burden by synergising with subtherapeutic doses of the RAS pathway inhibitor, Trametinib, which inhibits mitogen-activated kinase kinase (MEK). Analysis of one of the hits from the screen, Ritanserin, which targets serotonin receptors and diacy glycerol kinase alpha (DGK), revealed that DGK was the critical target in its synergism with Trametinib. We show that human mammary epithelial cells harbouring the H-RAS oncogene and knockdown of the cell polarity gene, SCRIB, are also sensitive to treatment with low doses of Trametinib and DGK inhibition. Mechanistically, DGK inhibition synergises with Trametinib by inhibiting MEK and mTOR activity. Altogether, our results provide evidence that targeting RAS-driven human cancers with RAS pathway and DGK inhibitors will be an effective combination therapy.

cancer biology↗