Biology subjects

Manucci, A. C.

Publications and source records attributed to Manucci, A. C..

5 recordsLinked to original sources

bioRxiv2023

The basement membrane regulates the cellular localization and the cytoplasmic interactome of Yes-Associated Protein (YAP) in mammary epithelial cells

The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between ECM cues and cellular responses. We asked whether the basement membrane (BM), a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, non-tumoral cells cultured with a reconstituted BM (rBM) displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB) cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar and carbohydrate metabolism. Collectively, our multifaceted approach unraveled that the BM induces YAP translocation or retention in the cytoplasm of non-tumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.

cell biology↗

bioRxiv2023

A practical and robust method to evaluate metabolic fluxes in pancreatic islets

Aims/hypothesisEfficient mitochondrial oxidative phosphorylation is essential for pancreatic beta cell responses to nutrient levels. Consequently, the evaluation of mitochondrial oxygen consumption and ATP production is important to investigate essential aspects of pancreatic islet pathophysiology. Currently, most studies use cell lines instead of primary islets due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables that are expensive, complicated to use, and poorly reproducible. The aim of this study is to establish a robust and practical method to assess primary islet metabolic fluxes using Extracellular Flux Technology and standard commercial consumables. MethodsPancreatic islets were isolated from 8 to 12-week-old mice and rats, and submitted to a dispersion protocol using trypsin. Dispersed islets were adhered overnight to pre-coated standard Seahorse microplates, and oxygen consumption rates were evaluated using a Seahorse Extracellular Flux Analyzer. We also validated the functionality of dispersed islets by analyzing glucose-stimulated insulin secretion (GSIS) and calcium (Ca2+) influx in response to different modulators by fluorescence microscopy. ResultsWe provide a detailed protocol with all steps necessary to optimize islet isolation and dispersion, in order to achieve a high yield of functional islets and perform metabolic flux analysis. With this method, which requires only a few islets per replicate, both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators (oligomycin, CCCP, antimycin and rotenone) and glucose addition. Both oligomycin and CCCP concentrations were titrated. Our method was also validated by other functional assays, which show these cells present conserved Ca2+ influx and insulin secretion in response to glucose. Conclusions/interpretationWe established a practical and robust method to assess ex vivo islet metabolic fluxes and oxidative phosphorylation. Our findings cover an important gap in primary islet physiology studies, providing a valuable tool we hope is useful to uncover basic beta cell metabolic mechanisms, as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols. Research in context What is already known about this subject?O_LIPancreatic beta cells efficiently couple oxidative phosphorylation and ATP production with insulin secretion; mitochondrial ATP production is crucial for proper insulin secretion. C_LIO_LIMost studies of beta cell respiration use cell lines instead of primary islets, which are a much more robust model to evaluate beta cell function. C_LIO_LIThe few works with primary islet respiration use specific equipment and consumables that are expensive, complicated, and poorly reproducible. C_LI What is the key question?O_LIIs it possible to develop a practical method to evaluate metabolic fluxes and ATP production in isolated islets, using the standard Seahorse Extracellular Flux Technology? C_LI What are the new findings?O_LIWe optimized rodent islet isolation and functional analysis protocols using standard extracellular flux analysis equipment and consumables. C_LIO_LIOur method allows for increased islet yield and robust islet respiration measurements. C_LI How might this impact on clinical practice in the foreseeable future?O_LIQuantitative measurements of metabolic fluxes and oxidative phosphorylation are the cornerstone of new discoveries in beta cells, and can contribute toward the establishment of new cellular protocols, such as for cell transplantation, as well as the development of new pharmacological agents targeted to these cells. C_LI

biochemistry↗

bioRxiv2023

Pten is a disconnecting node in the molecular landscape of the proliferation/quiescence decision during mammary gland acinogenesis

Cell context is key for cell phenotype. Using physiologically relevant models of laminin-rich ECM (lrECM) induction of mammary epithelial cell quiescence and differentiation, we have provided a landscape of the status of key molecular players involved in the proliferation/quiescence decision. Repression of some positive regulators of the cell cycle, such as cyclins and CDKs, occurred already at the mRNA level, whereas negative regulators of the cell cycle, such as Pten and p27, were upregulated only at the protein level. Interestingly, cell cycle arrest occurred despite the active status of Fak, Src and PI3k, because their downstream proliferative signalling pathways were repressed, suggesting the existence of a disconnecting node between upstream and downstream proliferative signalling in quiescent cells. Pten fulfils this role. Inhibition of Pten increased proliferation and restored Akt/mTORC1/2 and Mapk signalling in cells exposed to lrECM. In mice, Pten levels were positively correlated to the basement membrane thickness in the developing mammary epithelia, and Pten localized to the apicolateral membrane of luminal cells both in in ducts and near the nascent lumen in terminal end bud, characteristics consistent with a role for Pten in inducing and sustaining quiescence and tissue architecture. Accordingly, in 3D acininogenesis models, Pten was required for the onset and maintenance of quiescence, cell polarity and lumen assembly. The notion that lrECM-triggered differentiation involves a signalling circuitry with many layers of regulation provides an explanation for the resilience of quiescence within a growth-suppressive microenvironment, and that perturbations in master regulators, such as Pten, could disrupt the quiescent phenotype.

cell biology↗

bioRxiv2021

The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signalling

SARS-CoV-2 non-structural protein 3 (Nsp3) contains a macrodomain that is essential for virus replication and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the removal of ADP-ribose modifications catalysed by host poly(ADP-ribose) polymerases (PARPs). Here, we show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this can be used to screen for cell-active macrodomain inhibitors without the requirement for BSL-3 facilities. This IFN-induced ADP-ribosylation is dependent on the PARP9/DTX3L heterodimer, but surprisingly the expression of Nsp3 macrodomain or PARP9/DTX3L deletion do not impair STAT1 phosphorylation or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyse this end product of IFN signalling, and not to suppress the IFN response itself.

molecular biology↗

bioRxiv2021

Extracellular matrix stiffness regulates degradation of the Hippo kinase MST2 via SCFβTrCP

Tumor microenvironments display disrupted mechanical properties, including altered extracellular matrix (ECM) rigidity. ECM stiffening perturbs cell tensional homeostasis resulting in activation of mechanosensing transcriptional co-activators, such as the Hippo pathway effectors YAP and TAZ. The Hippo pathway plays central roles in development and tumorigenesis, but how the proteostasis of the Hippo kinase MST2 is regulated remains unknown. Here, we show that ECM stiffness induces MST2 degradation via proteasome degradation. MST2 degradation is enhanced in human breast epithelial cells (HMEC) that are cultured in stiffer microenvironments due to integrin and integrin-linked kinase activation. MST2 knockdown resulted in increased nucleus-to-cytoplasm ratio of YAP in physiological and breast tumor rigidities and altered mechanoregulated cellular processes in HMEC. We found that MST2 is ubiquitinated by the SCF{beta}TrCP ubiquitin ligase. Site-directed mutagenesis combined with computational molecular dynamics studies revealed that {beta}TrCP binds MST2 via a noncanonical degradation motif. Our study uncovers the underlying biochemical mechanisms controlling MST2 degradation and demonstrates how changes in the microenvironment rigidity regulate the proteostasis of a central Hippo pathway component.

cell biology↗