Publications

( 0 )
Brighi, C;Salaris, F;Soloperto, A;Cordella, F;Ghirga, S;de Turris, V;Rosito, M;Porceddu, PF;D'Antoni, C;Reggiani, A;Rosa, A;Di Angelantonio, S.
Cell Death & Disease,
12
(5)
, 1-22
(2021)
Fragile X syndrome (FXS) is a neurodevelopmental disorder, characterized by intellectual disability and sensory deficits, caused by epigenetic silencing of the FMR1 gene and subsequent loss of its protein product, fragile X mental retardation protein (FMRP). Delays in synaptic and neuronal development in the cortex have been reported in FXS mouse models; however, the main goal of translating lab research into pharmacological treatments in clinical trials has been so far largely unsuccessful, leaving FXS a still incurable disease. Here, we generated 2D and 3D in vitro human FXS model systems based on isogenic FMR1 knock-out mutant and wild-type human induced pluripotent stem cell (hiPSC) lines. Phenotypical and functional characterization of cortical neurons derived from FMRP-deficient hiPSCs display altered gene expression and impaired differentiation when compared with the healthy counterpart. FXS cortical cultures show an increased number of GFAP positive cells, likely astrocytes, increased spontaneous network activity, and depolarizing GABAergic transmission. Cortical brain organoid models show an increased number of glial cells, and bigger organoid size. Our findings demonstrate that FMRP is required to correctly support neuronal and glial cell proliferation, and to set the correct excitation/inhibition ratio in human brain development.
Bonnal, RJP;Rossetti, G;Lugli, E;De Simone, M;Gruarin, P;Brummelman, J;Drufuca, L;Passaro, M;Bason, R;Gervasoni, F;Della Chiara, G;D'Oria, C;Martinovic, M;Curti, S;Ranzani, V;Cordiglieri, C;Alvisi, G;Mazza, EMC;Oliveto, S;Silvestri, Y;Carelli, E;Mazzara, S;Bosotti, R;Sarnicola, ML;Godano, C;Bevilacqua, V;Lorenzo, M;Siena, S;Bonoldi, E;Sartore-Bianchi, A;Amatu, A;Veronesi, G;Novellis, P;Alloisio, M;Giani, A;Zucchini, N;Opocher, E;Ceretti, AP;Mariani, N;Biffo, S;Prati, D;Bardelli, A;Geginat, J;Lanzavecchia, A;Abrignani, S;Pagani, M.
Nature immunology,
22
(6)
, 735-745
(2021)
Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.
Sun, YC;Chen, X;Fischer, S;Lu, S;Zhan, H;Gillis, J;Zador, AM.
Nature neuroscience,
24
(6)
, 873-885
(2021)
Functional circuits consist of neurons with diverse axonal projections and gene expression. Understanding the molecular signature of projections requires high-throughput interrogation of both gene expression and projections to multiple targets in the same cells at cellular resolution, which is difficult to achieve using current technology. Here, we introduce BARseq2, a technique that simultaneously maps projections and detects multiplexed gene expression by in situ sequencing. We determined the expression of cadherins and cell-type markers in 29,933 cells and the projections of 3,164 cells in both the mouse motor cortex and auditory cortex. Associating gene expression and projections in 1,349 neurons revealed shared cadherin signatures of homologous projections across the two cortical areas. These cadherins were enriched across multiple branches of the transcriptomic taxonomy. By correlating multigene expression and projections to many targets in single neurons with high throughput, BARseq2 provides a potential path to uncovering the molecular logic underlying neuronal circuits.
Biferali, B;Bianconi, V;Perez, DF;Kronawitter, SP;Marullo, F;Maggio, R;Santini, T;Polverino, F;Biagioni, S;Summa, V;Toniatti, C;Pasini, D;Stricker, S;Di Fabio, R;Chiacchiera, F;Peruzzi, G;Mozzetta, C.
Science advances,
7
(23)
(2021)
H3K9 methylation maintains cell identity orchestrating stable silencing and anchoring of alternate fate genes within the heterochromatic compartment underneath the nuclear lamina (NL). However, how cell type-specific genomic regions are specifically targeted to the NL is still elusive. Using fibro-adipogenic progenitors (FAPs) as a model, we identified Prdm16 as a nuclear envelope protein that anchors H3K9-methylated chromatin in a cell-specific manner. We show that Prdm16 mediates FAP developmental capacities by orchestrating lamina-associated domain organization and heterochromatin sequestration at the nuclear periphery. We found that Prdm16 localizes at the NL where it cooperates with the H3K9 methyltransferases G9a/GLP to mediate tethering and silencing of myogenic genes, thus repressing an alternative myogenic fate in FAPs. Genetic and pharmacological disruption of this repressive pathway confers to FAP myogenic competence, preventing fibro-adipogenic degeneration of dystrophic muscles. In summary, we reveal a druggable mechanism of heterochromatin perinuclear sequestration exploitable to reprogram FAPs in vivo.
Birolini, G;Verlengia, G;Talpo, F;Maniezzi, C;Zentilin, L;Giacca, M;Conforti, P;Cordiglieri, C;Caccia, C;Leoni, V;Taroni, F;Biella, G;Simonato, M;Cattaneo, E;Valenza, M.
Brain,
(2021)
Brain cholesterol is produced mainly by astrocytes and is important for neuronal function. Its biosynthesis is severely reduced in mouse models of Huntington’s disease. One possible mechanism is a diminished nuclear translocation of the transcription factor sterol regulatory element binding protein 2 (SREBP2) and, consequently, reduced activation of SREBP-controlled genes in the cholesterol biosynthesis pathway. Here we evaluated the efficacy of a gene therapy based on the unilateral intra-striatal injection of a recombinant adeno-associated virus 2/5 (AAV2/5) targeting astrocytes specifically and carrying the transcriptionally active N-terminal fragment of human SREBP2. Robust hSREBP2 expression in striatal glial cells in R6/2 Huntington’s disease mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed Drd2 transcript levels decline, cleared mutant Huntingtin aggregates and attenuated behavioral deficits. We conclude that glial SREBP2 participates in Huntington’s disease brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of the disease.
Gupta, VK;Nam, S;Yim, D;Camuglia, J;Martin, JL;Sanders, EN;O'Brien, LE;Martin, AC;Kim, T;Chaudhuri, O.
The Journal of cell biology,
220
(8)
(2021)
Epithelial cells undergo striking morphological changes during division to ensure proper segregation of genetic and cytoplasmic materials. These morphological changes occur despite dividing cells being mechanically restricted by neighboring cells, indicating the need for extracellular force generation. Beyond driving cell division itself, forces associated with division have been implicated in tissue-scale processes, including development, tissue growth, migration, and epidermal stratification. While forces generated by mitotic rounding are well understood, forces generated after rounding remain unknown. Here, we identify two distinct stages of division force generation that follow rounding: (1) Protrusive forces along the division axis that drive division elongation, and (2) outward forces that facilitate postdivision spreading. Cytokinetic ring contraction of the dividing cell, but not activity of neighboring cells, generates extracellular forces that propel division elongation and contribute to chromosome segregation. Forces from division elongation are observed in epithelia across many model organisms. Thus, division elongation forces represent a universal mechanism that powers cell division in confining epithelia.
Yoon, B;Kim, M;Jackman, J;Cho, N.
Applied Materials Today,
24
, 101099
(2021)
There is broad interest in developing nanostructured assemblies composed of fatty acids and monoglycerides to inhibit membrane-enveloped pathogens and modulate immune cell behavior. Herein, we investigated the interactions of micellar nanostructures composed of a biologically active monoglyceride, glycerol monolaurate (GML), or its ether-bonded equivalent, 1-O-dodecyl-rac-glycerol (DDG), with cell-membrane-mimicking giant unilamellar vesicles (GUVs). Our findings revealed that GML nanostructures induced fission or fusion depending on the GML concentration and corresponding degree of supramolecular organization, while DDG nanostructures only caused aggregation-like disruption of the GUV outer surface. In specific conditions, the GML nanostructures also triggered pearling instability, which led to dynamic membrane remodeling behavior and the pattern of GML interactions was consistent across simplified and complex membrane compositions. Notably, the spectrum of membrane morphological changes induced by GML nanostructures, including fission, fusion, and pearling behaviors, is appreciably wider than the fission behavior exhibited by fatty acid nanostructures in past studies. Collectively, these findings demonstrate how controlling the supramolecular organization of monoglycerides within nanostructured assemblies can be useful to modulate the type and degree of membrane interactions relevant to biophysical and nanomedicine applications.
Ashoka, AH;Klymchenko, AS.
ACS applied materials & interfaces,
13
(24)
, 28889-28898
(2021)
Preparation of bright fluorescent materials based on polymers is hampered by a fundamental problem of aggregation-caused quenching (ACQ) of encapsulated dyes. Here, ultrabright fluorescent polymeric nanofibers and coatings are prepared based on a concept of ionic dye insulation with bulky hydrophobic counterions that overcomes the ACQ problem. It is found that bulky hydrophobic counterion perfluorinated tetraphenylborate can boost >100-fold the fluorescence quantum yields of cationic dye octadecyl rhodamine B at high loading (30 wt %) in biocompatible poly(methyl methacrylate) (PMMA). The concept is applicable to both rhodamine and cyanine dyes, which results in bright fluorescent polymeric materials of four different colors spanning from blue to near-infrared. It allows for preparation of electrospun polymeric nanofibers with >50-fold higher dye loading by mass (30 wt %, >20-fold higher molarity for rhodamine dyes) while preserving good fluorescence quantum yields (31%), which implies drastic improvement in their fluorescence brightness. The counterion-based polymeric materials are also validated as coatings of model medical devices, such as stainless steel fiducials and 3D-printed stents of complex geometry. Spin-coated fluorescent polymeric films loaded with a dye paired with bulky counterions exhibit excellent biocompatibility and low toxicity. Moreover, counterion-modified materials show much better stability against dye leakage in the presence of living cells and a serum-containing medium, compared to materials based on the dye with a small inorganic anion. Overall, by pushing the barriers of ACQ, our counterion approach emerges as a powerful tool to develop ultrabright fluorescent polymeric materials ranging from nano- and macroscale.
Chirivì, M;Maiullari, F;Milan, M;Presutti, D;Cordiglieri, C;Crosti, M;Sarnicola, ML;Soluri, A;Volpi, M;Święszkowski, W;Prati, D;Rizzi, M;Costantini, M;Seliktar, D;Parisi, C;Bearzi, C;Rizzi, R.
International journal of molecular sciences,
22
(11)
(2021)
The immune system is a fine modulator of the tumor biology supporting or inhibiting its progression, growth, invasion and conveys the pharmacological treatment effect. Tumors, on their side, have developed escaping mechanisms from the immune system action ranging from the direct secretion of biochemical signals to an indirect reaction, in which the cellular actors of the tumor microenvironment (TME) collaborate to mechanically condition the extracellular matrix (ECM) making it inhospitable to immune cells. TME is composed of several cell lines besides cancer cells, including tumor-associated macrophages, cancer-associated fibroblasts, CD4+ and CD8+ lymphocytes, and innate immunity cells. These populations interface with each other to prepare a conservative response, capable of evading the defense mechanisms implemented by the host’s immune system. The presence or absence, in particular, of cytotoxic CD8+ cells in the vicinity of the main tumor mass, is able to predict, respectively, the success or failure of drug therapy. Among various mechanisms of immunescaping, in this study, we characterized the modulation of the phenotypic profile of CD4+ and CD8+ cells in resting and activated states, in response to the mechanical pressure exerted by a three-dimensional in vitro system, able to recapitulate the rheological and stiffness properties of the tumor ECM.
Carr, L;Golzio, M;Orlacchio, R;Alberola, G;Kolosnjaj-Tabi, J;Leveque, P;Arnaud-Cormos, D;Rols, MP.
Bioelectrochemistry,
141
, 107839
(2021)
Three-dimensional (3D) cellular models represent more realistically the complexity of in vivo tumors compared to 2D cultures. While 3D models were largely used in classical electroporation, the effects of nanosecond pulsed electric field (nsPEF) have been poorly investigated. In this study, we evaluated the biological effects induced by nsPEF on spheroid tumor model derived from the HCT-116 human colorectal carcinoma cell line. By varying the number of pulses (from 1 to 500) and the polarity (unipolar and bipolar), the response of nsPEF exposure (10 ns duration, 50 kV/cm) was assessed either immediately after the application of the pulses or over a period lasting up to 6 days. Membrane permeabilization and cellular death occurred following the application of at least 100 pulses. The extent of the response increased with the number of pulses, with a significant decrease of viability, 24 h post-exposure, when 250 and 500 pulses were applied. The effects were highly reduced when an equivalent number of bipolar pulses were delivered. This reduction was eliminated when a 100 ns interphase interval was introduced into the bipolar pulses. Altogether, our results show that nsPEF effects, previously observed at the single cell level, also occur in more realistic 3D tumor spheroids models.
Grisanti, G;Caprini, D;Sinibaldi, G;Scognamiglio, C;Silvani, G;Peruzzi, G;Casciola, CM.
Micromachines,
12
(6)
(2021)
An endothelial-lined blood vessel model is obtained in a PDMS (Polydimethylsiloxane) microfluidic system, where vascular endothelial cells are grown under physiological shear stress, allowing -like maturation. This experimental model is employed for enhanced drug delivery studies, aimed at characterising the increase in endothelial permeability upon microbubble-enhanced ultrasound-induced (USMB) cavitation. We developed a multi-step protocol to couple the optical and the acoustic set-ups, thanks to a 3D-printed insonation chamber, provided with direct optical access and a support for the US transducer. Cavitation-induced interendothelial gap opening is then analysed using a customised code that quantifies gap area and the relative statistics. We show that exposure to US in presence of microbubbles significantly increases endothelial permeability and that tissue integrity completely recovers within 45 min upon insonation. This protocol, along with the versatility of the microfluidic platform, allows to quantitatively characterise cavitation-induced events for its potential employment in clinics.
Mäntylä, E;Ihalainen, T.
Research Square,
(2021)
Cellular forces, mechanics and other physical factors are important co-regulators of normal cell and tissue physiology. These cues are often misregulated in diseases such as cancer, where altered tissue mechanics contribute to the disease progression. Furthermore, intercellular tensile and compressive force related signaling is highlighted in collective cell behavior during development. However, the mechanistic understanding on the role of physical forces in regulation of cellular physiology, including gene expression and signaling, is still lacking. This is partly because studies on the molecular mechanisms of force transmission require easily controllable experimental designs. These approaches should enable both easy mechanical manipulation of cells and, importantly, readouts ranging from microscopy imaging to biochemical assays. To achieve a robust solution for mechanical manipulation of cells, we developed devices built of LEGO bricks allowing cell stretching and compression studies. By using these devices, we show that b-catenin responds differentially to epithelial monolayer stretching and compression, either localizing more to the cell nuclei or cell-cell junctions, respectively. In addition, we show that epithelial compression drives cytoplasmic retention and phosphorylation of transcription coregulator YAP1. We provide a complete part listing and video assembly instructions, allowing other researchers to build and use the devices in cellular mechanics -related studies.
Campaña, M;Davis, T;Cleverdon, E;Bates, M;Krishnan, N;Curtis, E;Childs, M;Morales-Rodriguez, Y;Sieburg, M;Hehnly, H;Luyt, L;Hougland, J.
bioRxiv,
(2021)
Ghrelin O-acyltransferase (GOAT) plays a central role in the maturation and activation of the peptide hormone ghrelin, which performs a wide range of endocrinological signaling roles. Using a tight-binding fluorescent ghrelin-derived peptide designed for high selectivity for GOAT over the ghrelin receptor GHS-R1a, we demonstrate that GOAT interacts with extracellular ghrelin and facilitates ligand cell internalization in both transfected cells and prostate cancer cells endogenously expressing GOAT. Coupled with enzyme mutagenesis, ligand uptake studies provide the first direct evidence supporting interaction of the putative histidine general base within GOAT with the ghrelin peptide acylation site. Our work provides a new understanding of GOAT’s catalytic mechanism, establishes a key step required for autocrine/paracrine ghrelin signaling involving local reacylation by GOAT, and raises the possibility that other peptide hormones may exhibit similar complexity in their intercellular and organismal-level signaling pathways.
Lattmann, E;Deng, T;Walser, M;Widmer, P;Rexha-Lambert, C;Prasad, V;Eichhoff, O;Daube, M;Dummer, R;Levesque, M;Hajnal, A.
bioRxiv,
(2021)
Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes over-expressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC functions cell-autonomously in the G1-arrested AC to promote invasion, independently of its role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC acts in the non-proliferating AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.
Chen, Y;Chen, X;Baserdem, B;Zhan, H;Li, Y;Davis, M;Kebschull, J;Zador, A;Koulakov, A;Albeanu, D.
bioRxiv,
(2021)
The structure of neuronal connectivity often provides insights into the relevant stimulus features, such as spatial location, orientation, sound frequency, etc1–6. The olfactory system, however, appears to lack structured connectivity as suggested by reports of broad and distributed connections both from the olfactory bulb to the piriform cortex7–22 and within the cortex23–25. These studies have inspired computational models of circuit function that rely on random connectivity26–33. It remains, nonetheless, unclear whether the olfactory connectivity contains spatial structure. Here, we use high throughput anatomical methods (MAPseq and BARseq)34–38 to analyze the projections of 5,309 bulb and 30,433 piriform cortex output neurons in the mouse at single-cell resolution. We identify previously unrecognized spatial organization in connectivity along the anterior-posterior axis (A-P) of the piriform cortex. We find that both the bulb projections to the cortex and the cortical outputs are not random, but rather form gradients along the A-P axis. Strikingly, these gradients are matched: bulb neurons targeting a given location within the piriform cortex co-innervate extra-piriform regions that receive strong inputs from neurons within that piriform locus. We also identify signatures of local connectivity in the piriform cortex. Our findings suggest an organizing principle of matched direct and indirect olfactory pathways that innervate extra-piriform targets in a coordinated manner, thus supporting models of information processing that rely on structured connectivity within the olfactory system.
Holmgren, M;Sheets, L.
bioRxiv,
(2021)
Noise exposure is particularly stressful to hair-cell mitochondria, which must produce enough energy to meet high metabolic demands as well as regulate local intracellular Ca2+ concentrations. Mitochondrial Inner Membrane Protein 17 (Mpv17) functions as a non-selective channel and plays a role in maintaining mitochondrial homeostasis. In zebrafish, hair cells in mpv17a9/a9 mutants displayed elevated levels of reactive oxygen species (ROS), elevated mitochondrial calcium, hyperpolarized transmembrane potential, and greater vulnerability to neomycin, indicating impaired mitochondrial function. Using a strong water current to overstimulate hair cells in the zebrafish lateral line, we observed mpv17a9/a9 mutant hair cells were more vulnerable to morphological disruption and hair-cell loss than wild type siblings simultaneously exposed to the same stimulus. To determine the role of mitochondrial homeostasis on hair-cell synapse integrity, we surveyed synapse number in mpv17a9/a9 mutants and wild type siblings as well as the sizes of presynaptic dense bodies (ribbons) and postsynaptic densities immediately following stimulus exposure. We observed mechanically injured mpv17a9/a9 neuromasts, while they lost a greater number of hair cells, lost a similar number of synapses per hair cell relative to wild type. Additionally, we quantified the size of hair cell pre- and postsynaptic structures and observed significantly enlarged wild type postsynaptic densities, yet relatively little change in the size of mpv17a9/a9 postsynaptic densities following stimulation. These results suggest impaired hair-cell mitochondrial activity influences synaptic morphology and hair-cell survival but does not exacerbate synapse loss following mechanical injury.
Monmeyran, A;Benyoussef, W;Thomen, P;Dahmane, N;Baliarda, A;Jules, M;Aymerich, S;Henry, N.
bioRxiv,
(2021)
Multispecies microbial adherent communities are widespread in nature and organisms but the principles of their assembly and development remain unclear. Yet, the demand to understand and predict the responses of such living communities to environmental changes is increasing, calling for new approaches. Here, we test the possibility to establish a simplified but relevant model of multispecies biofilm in a laboratory setup enabling in situ real-time monitoring of the community development and control of the environmental parameters in order to decipher the mechanisms underlying the formation of the community. Using video-microscopy and species combinatorial approach, we assess the global and individual species spatiotemporal development in millifluidic channels under constant flow of nutrients. Based on quantitative measurements of expansion kinetics, local dynamics and spatial distribution, we demonstrate that the four chosen species (Bacillus thuringiensis, Pseudomonas fluorescens, Kocuria varians and Rhodocyclus sp.) form a dynamical community that deterministically reaches its equilibrium after about 30 hours of growth. We evidence the emergence of complexity in this simplified community as reported by spatial heterogeneity rise and non-monotonic developmental kinetics. We find interspecies interactions consisting in competition for resources — in particular oxygen — and both direct and indirect physical interactions but no positive feedback. Thereby, we introduce a model of multispecies adherent community where effective couplings result from individual species quest for fitness optimization in a moving and heterogenous environment. This control and the understanding of this simplified experimental model shall open new avenues to apprehend adherent bacterial communities behavior in a context of rapid global change.
Janská, L;Anandi, L;Kirchberger, N;Marinkovic, Z;Schachtner, L;Guzelsoy, G;Carmona-Fontaine, C.
bioRxiv,
(2021)
There is an urgent need for accurate, scalable, and cost-efficient models of the complexity and heterogeneity of the tumor microenvironment. Here, we detail how to fabricate and use the Metabolic Microenvironment Chamber (MEMIC) – a 3D-printed ex vivo model of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is the accessibility to the blood stream that provides key resources such as oxygen and nutrients. While some tumor cells have direct access to these resources, many others must survive under progressively more ischemic environments as they reside further from the vasculature. The MEMIC is designed to simulate the differential access to nutrients and allows co-culturing different cell types, such as tumor and immune cells. This system is optimized for live imaging and other microscopy-based approaches and it is a powerful tool to study tumor features such as the effect of nutrient scarcity on tumor-stroma interactions. Due to its adaptable design and full experimental control, the MEMIC can provide novel insights into the tumor microenvironment that would be difficult to obtain via other methods. As a proof of principle, we show that cells can sense gradual changes in metabolite concentration, and tune intracellular cell signaling to form multicellular spatial patterns of cell proliferation. We also show that ischemic macrophages reduce epithelial features in neighboring tumor cells highlighting the power of this system to study cell-cell interactions and non-cell autonomous effects of the metabolic microenvironment. We propose that the MEMIC can be easily adapted to study early development, ischemic stroke, and other systems where multiple cell types interact within heterogeneous environments.
Satoh, A;Fujioka, Y;Kashiwagi, S;Yoshida, A;Fujioka, M;Sasajima, H;Nanbo, A;Amano, M;Ohba, Y.
Cell Press,
(2021)
Intracellular organelles of mammalian cells communicate with each other during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identified voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 was found to tether endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and to promote clathrin-independent endocytosis as well as endosome maturation at membrane contact sites. With a newly developed optogenetics system to induce mitochondrion-endosome association, we found that, in addition to its structural role in such association, the pore function of VDAC2 is also required for the promotion of endosome maturation. Our findings thus uncover a previously unappreciated role of mitochondrion-endosome association in the regulation of endocytosis and endosome maturation.HighlightsThe mitochondrial protein VDAC2 binds PI3K and tethers endosomes to mitochondriaVDAC2 promotes clathrin-independent endocytosisVDAC2-PI3K interaction induces acidification of endosomes associated with mitochondriaThe pore function of VDAC2 also contributes to endosome maturation at contact sites
Baruffaldi, D;Pirri, C;Frascella, F.
Bioprinting,
22
, e00135
(2021)
Traditional in vitro culture models are unable to fully reflect the organ microenvironment, due to differences in terms of cell morphology, protein expression, cell-cell and cell-matrix interactions, and drug response. In contrast, the flexibility of bioprinting modes allows for the deposition of cell-containing biomaterials in any free-form-inspired 3D structures on chip. The main purpose of this study was to design and optimize commercially available Carbopol-based 3D printing formulations, because of their many advantages, such as low-cost, the ability to produce clear and stable gels, and the water thickening. For this purpose, three different Carbopol gels (EDT 2020 NF, Ultrez 10 NF and NF-980) were tested in terms of printability and biocompatibility, with lung cancer epithelial (A549) and normal lung fibroblast (MRC-5) cells. This study demonstrates that Carbopol is a promising candidate for the 3D printing of cell-laden constructs, both in terms of rheology and printing performance.