Publications
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Chen, Y;Chen, X;Baserdem, B;Zhan, H;Li, Y;Davis, MB;Kebschull, JM;Zador, AM;Koulakov, AA;Albeanu, DF.
Cell,
185,
(22),
4117-4134.e28,
(2022)
In most sensory modalities, neuronal connectivity reflects behaviorally relevant stimulus features, such as spatial location, orientation, and sound frequency. By contrast, the prevailing view in the olfactory cortex, based on the reconstruction of dozens of neurons, is that connectivity is random. Here, we used high-throughput sequencing-based neuroanatomical techniques to analyze the projections of 5,309 mouse olfactory bulb and 30,433 piriform cortex output neurons at single-cell resolution. Surprisingly, statistical analysis of this much larger dataset revealed that the olfactory cortex connectivity is spatially structured. Single olfactory bulb neurons targeting a particular location along the anterior-posterior axis of piriform cortex also project to matched, functionally distinct, extra-piriform targets. Moreover, single neurons from the targeted piriform locus also project to the same matched extra-piriform targets, forming triadic circuit motifs. Thus, as in other sensory modalities, olfactory information is routed at early stages of processing to functionally diverse targets in a coordinated manner.
Saelens, JW;Sweeney, MI;Viswanathan, G;Xet-Mull, AM;Jurcic Smith, KL;Sisk, DM;Hu, DD;Cronin, RM;Hughes, EJ;Brewer, WJ;Coers, J;Champion, MM;Champion, PA;Lowe, CB;Smith, CM;Lee, S;Stout, JE;Tobin, DM.
Cell,
185,
(24),
4507-4525.e18,
(2022)
The human pathogen Mycobacterium tuberculosis typically causes lung disease but can also disseminate to other tissues. We identified a M. tuberculosis (Mtb) outbreak presenting with unusually high rates of extrapulmonary dissemination and bone disease. We found that the causal strain carried an ancestral full-length version of the type VII-secreted effector EsxM rather than the truncated version present in other modern Mtb lineages. The ancestral EsxM variant exacerbated dissemination through enhancement of macrophage motility, increased egress of macrophages from established granulomas, and alterations in macrophage actin dynamics. Reconstitution of the ancestral version of EsxM in an attenuated modern strain of Mtb altered the migratory mode of infected macrophages, enhancing their motility. In a zebrafish model, full-length EsxM promoted bone disease. The presence of a derived nonsense variant in EsxM throughout the major Mtb lineages 2, 3, and 4 is consistent with a role for EsxM in regulating the extent of dissemination.
Antonucci, A;Reggente, M;Roullier, C;Gillen, AJ;Schuergers, N;Zubkovs, V;Lambert, BP;Mouhib, M;Carata, E;Dini, L;Boghossian, AA.
Nature nanotechnology,
(2022)
The distinctive properties of single-walled carbon nanotubes (SWCNTs) have inspired the development of many novel applications in the field of cell nanobiotechnology. However, studies thus far have not explored the effect of SWCNT functionalization on transport across the cell walls of prokaryotes. We explore the uptake of SWCNTs in Gram-negative cyanobacteria and demonstrate a passive length-dependent and selective internalization of SWCNTs decorated with positively charged biomolecules. We show that lysozyme-coated SWCNTs spontaneously penetrate the cell walls of a unicellular strain and a multicellular strain. A custom-built spinning-disc confocal microscope was used to image the distinct near-infrared SWCNT fluorescence within the autofluorescent cells, revealing a highly inhomogeneous distribution of SWCNTs. Real-time near-infrared monitoring of cell growth and division reveal that the SWCNTs are inherited by daughter cells. Moreover, these nanobionic living cells retained photosynthetic activity and showed an improved photo-exoelectrogenicity when incorporated into bioelectrochemical devices.
Dolat, L;Carpenter, VK;Chen, YS;Suzuki, M;Smith, EP;Kuddar, O;Valdivia, RH.
Cell host & microbe,
30,
(12),
1685-1700.e10,
(2022)
Invasive microbial pathogens often disrupt epithelial barriers, yet the mechanisms used to dismantle tight junctions are poorly understood. Here, we show that the obligate pathogen Chlamydia trachomatis uses the effector protein TepP to transiently disassemble tight junctions early during infection. TepP alters the tyrosine phosphorylation status of host proteins involved in cytoskeletal regulation, including the filamentous actin-binding protein EPS8. We determined that TepP and EPS8 are necessary and sufficient to remodel tight junctions and that the ensuing disruption of epithelial barrier function promotes secondary invasion events. The genetic deletion of EPS8 renders epithelial cells and endometrial organoids resistant to TepP-mediated tight junction remodeling. Finally, TepP and EPS8 promote infection in murine models of infections, with TepP mutants displaying defects in ascension to the upper genital tract. These findings reveal a non-canonical function of EPS8 in the disassembly of epithelial junctions and an important role for Chlamydia pathogenesis.
Lequyer, J;Philip, R;Sharma, A;Hsu, WH;Pelletier, L.
Nature machine intelligence,
4,
(11),
953-963,
(2022)
Image noise is a common problem in light microscopy. This is particularly true in real-time live-cell imaging applications in which long-term cell viability necessitates low-light conditions. Modern denoisers are typically trained on a representative dataset, sometimes consisting of just unpaired noisy shots. However, when data are acquired in real time to track dynamic cellular processes, it is not always practical nor economical to generate these training sets. Recently, denoisers have emerged that allow us to denoise single images without a training set or knowledge about the underlying noise. But such methods are currently too slow to be integrated into imaging pipelines that require rapid, real-time hardware feedback. Here we present Noise2Fast, which can overcome these limitations. Noise2Fast uses a novel downsampling technique we refer to as ‘chequerboard downsampling’. This allows us to train on a discrete 4-image training set, while convergence can be monitored using the original noisy image. We show that Noise2Fast is faster than all similar methods with only a small drop in accuracy compared to the gold standard. We integrate Noise2Fast into real-time multi-modal imaging applications and demonstrate its broad applicability to diverse imaging and analysis pipelines.
Armbruster, M;Naskar, S;Garcia, JP;Sommer, M;Kim, E;Adam, Y;Haydon, PG;Boyden, ES;Cohen, AE;Dulla, CG.
Nature neuroscience,
25,
(5),
607-616,
(2022)
Astrocytes are glial cells that interact with neuronal synapses via their distal processes, where they remove glutamate and potassium (K+) from the extracellular space following neuronal activity. Astrocyte clearance of both glutamate and K+ is voltage dependent, but astrocyte membrane potential (Vm) is thought to be largely invariant. As a result, these voltage dependencies have not been considered relevant to astrocyte function. Using genetically encoded voltage indicators to enable the measurement of Vm at peripheral astrocyte processes (PAPs) in mice, we report large, rapid, focal and pathway-specific depolarizations in PAPs during neuronal activity. These activity-dependent astrocyte depolarizations are driven by action potential-mediated presynaptic K+ efflux and electrogenic glutamate transporters. We find that PAP depolarization inhibits astrocyte glutamate clearance during neuronal activity, enhancing neuronal activation by glutamate. This represents a novel class of subcellular astrocyte membrane dynamics and a new form of astrocyte-neuron interaction.
Torromino, G;Loffredo, V;Cavezza, D;Sonsini, G;Esposito, F;Crevenna, AH;Gioffrè, M;De Risi, M;Treves, A;Griguoli, M;De Leonibus, E.
Nature communications,
13,
(1),
4194,
(2022)
Incidental memory can be challenged by increasing either the retention delay or the memory load. The dorsal hippocampus (dHP) appears to help with both consolidation from short-term (STM) to long-term memory (LTM), and higher memory loads, but the mechanism is not fully understood. Here we find that female mice, despite having the same STM capacity of 6 objects and higher resistance to distraction in our different object recognition task (DOT), when tested over 1 h or 24 h delays appear to transfer to LTM only 4 objects, whereas male mice have an STM capacity of 6 objects in this task. In male mice the dHP shows greater activation (as measured by c-Fos expression), whereas female mice show greater activation of the ventral midline thalamus (VMT). Optogenetic inhibition of the VMT-dHP pathway during off-line memory consolidation enables 6-object LTM retention in females, while chemogenetic VMT-activation impairs it in males. Thus, removing or enhancing sub-cortical inhibitory control over the hippocampus leads to differences in incidental memory.
Antonica, F;Santomaso, L;Pernici, D;Petrucci, L;Aiello, G;Cutarelli, A;Conti, L;Romanel, A;Miele, E;Tebaldi, T;Tiberi, L.
Nature communications,
13,
(1),
4767,
(2022)
Pediatric and adult high-grade gliomas are the most common primary malignant brain tumors, with poor prognosis due to recurrence and tumor infiltration after therapy. Quiescent cells have been implicated in tumor recurrence and treatment resistance, but their direct visualization and targeting remain challenging, precluding their mechanistic study. Here, we identify a population of malignant cells expressing Prominin-1 in a non-proliferating state in pediatric high-grade glioma patients. Using a genetic tool to visualize and ablate quiescent cells in mouse brain cancer and human cancer organoids, we reveal their localization at both the core and the edge of the tumors, and we demonstrate that quiescent cells are involved in infiltration of brain cancer cells. Finally, we find that Harmine, a DYRK1A/B inhibitor, partially decreases the number of quiescent and infiltrating cancer cells. Our data point to a subpopulation of quiescent cells as partially responsible of tumor invasiveness, one of the major causes of brain cancer morbidity.
Hegazi, S;Cheng, AH;Krupp, JJ;Tasaki, T;Liu, J;Szulc, DA;Ling, HH;Rios Garcia, J;Seecharran, S;Basiri, T;Amiri, M;Anwar, Z;Ahmad, S;Nayal, K;Sonenberg, N;Liu, BH;Cheng, HM;Levine, JD;Cheng, HM.
Nature communications,
13,
(1),
1594,
(2022)
Ubiquitin ligases control the degradation of core clock proteins to govern the speed and resetting properties of the circadian pacemaker. However, few studies have addressed their potential to regulate other cellular events within clock neurons beyond clock protein turnover. Here, we report that the ubiquitin ligase, UBR4/POE, strengthens the central pacemaker by facilitating neuropeptide trafficking in clock neurons and promoting network synchrony. Ubr4-deficient mice are resistant to jetlag, whereas poe knockdown flies are prone to arrhythmicity, behaviors reflective of the reduced axonal trafficking of circadian neuropeptides. At the cellular level, Ubr4 ablation impairs the export of secreted proteins from the Golgi apparatus by reducing the expression of Coronin 7, which is required for budding of Golgi-derived transport vesicles. In summary, UBR4/POE fulfills a conserved and unexpected role in the vesicular trafficking of neuropeptides, a function that has important implications for circadian clock synchrony and circuit-level signal processing.
Maqdasy, S;Lecoutre, S;Renzi, G;Frendo-Cumbo, S;Rizo-Roca, D;Moritz, T;Juvany, M;Hodek, O;Gao, H;Couchet, M;Witting, M;Kerr, A;Bergo, MO;Choudhury, RP;Aouadi, M;Zierath, JR;Krook, A;Mejhert, N;Rydén, M.
Nature metabolism,
4,
(2),
190-202,
(2022)
The mechanisms promoting disturbed white adipocyte function in obesity remain largely unclear. Herein, we integrate white adipose tissue (WAT) metabolomic and transcriptomic data from clinical cohorts and find that the WAT phosphocreatine/creatine ratio is increased and creatine kinase-B expression and activity is decreased in the obese state. In human in vitro and murine in vivo models, we demonstrate that decreased phosphocreatine metabolism in white adipocytes alters adenosine monophosphate-activated protein kinase activity via effects on adenosine triphosphate/adenosine diphosphate levels, independently of WAT beigeing. This disturbance promotes a pro-inflammatory profile characterized, in part, by increased chemokine (C-C motif) ligand 2 (CCL2) production. These data suggest that the phosphocreatine/creatine system links cellular energy shuttling with pro-inflammatory responses in human and murine white adipocytes. Our findings provide unexpected perspectives on the mechanisms driving WAT inflammation in obesity and may present avenues to target adipocyte dysfunction.
Carvelli, A;Setti, A;Desideri, F;Galfrè, SG;Biscarini, S;Santini, T;Colantoni, A;Peruzzi, G;Marzi, MJ;Capauto, D;Di Angelantonio, S;Ballarino, M;Nicassio, F;Laneve, P;Bozzoni, I.
The EMBO journal,
e108918,
(2022)
The transition from dividing progenitors to postmitotic motor neurons (MNs) is orchestrated by a series of events, which are mainly studied at the transcriptional level by analyzing the activity of specific programming transcription factors. Here, we identify a post-transcriptional role of a MN-specific transcriptional unit (MN2) harboring a lncRNA (lncMN2-203) and two miRNAs (miR-325-3p and miR-384-5p) in this transition. Through the use of in vitro mESC differentiation and single-cell sequencing of CRISPR/Cas9 mutants, we demonstrate that lncMN2-203 affects MN differentiation by sponging miR-466i-5p and upregulating its targets, including several factors involved in neuronal differentiation and function. In parallel, miR-325-3p and miR-384-5p, co-transcribed with lncMN2-203, act by repressing proliferation-related factors. These findings indicate the functional relevance of the MN2 locus and exemplify additional layers of specificity regulation in MN differentiation.
Musardo, S;Therin, S;Pelucchi, S;D'Andrea, L;Stringhi, R;Ribeiro, A;Manca, A;Balducci, C;Pagano, J;Sala, C;Verpelli, C;Grieco, V;Edefonti, V;Forloni, G;Gardoni, F;Meli, G;Di Marino, D;Di Luca, M;Marcello, E.
Molecular therapy : the journal of the American Society of Gene Therapy,
(2022)
The development of new therapeutic avenues that target the early stages of Alzheimer’s disease (AD) is urgently necessary. A disintegrin and metalloproteinase domain 10 (ADAM10) is a sheddase that is involved in dendritic spine shaping and limits the generation of amyloid-β. ADAM10 endocytosis increases in the hippocampus of AD patients, resulting in the decreased postsynaptic localization of the enzyme. To restore this altered pathway, we developed a cell-permeable peptide (PEP3) with a strong safety profile that is able to interfere with ADAM10 endocytosis, upregulating the postsynaptic localization and activity of ADAM10. After extensive validation, experiments in a relevant animal model clarified the optimal timing of the treatment window. PEP3 administration was effective for the rescue of cognitive defects in APP/PS1 mice only if administered at an early disease stage. Increased ADAM10 activity promoted synaptic plasticity, as revealed by changes in the molecular compositions of synapses and the spine morphology. Even though further studies are required to evaluate efficacy and safety issues of long-term administration of PEP3, these results provide preclinical evidence to support the therapeutic potential of PEP3 in AD.
Miluzio, A;Cuomo, A;Cordiglieri, C;Donnici, L;Pesce, E;Bombaci, M;Conti, M;Fasciani, A;Terracciano, L;Manganaro, L;Toccafondi, M;Scagliola, A;Oliveto, S;Ricciardi, S;Grifantini, R;De Francesco, R;Abrignani, S;Manfrini, N;Biffo, S.
EBioMedicine,
87,
104390,
(2022)
The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood.Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach.We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation.We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection.This work was supported by an unrestricted grant from “Fondazione Romeo ed Enrica Invernizzi” to Stefano Biffo and by AIRC under MFAG 2021 – ID. 26178 project – P.I. Manfrini Nicola.
Gabryelczyk, B;Sammalisto, FE;Gandier, JA;Feng, J;Beaune, G;Timonen, JVI;Linder, MB.
Materials today. Bio,
17,
100492,
(2022)
Recombinant expression of proteins destined to form biological materials often results in poor production yields or loss of their function due to premature aggregation. Recently, liquid-liquid phase separation has been proposed as a mechanism to control protein solubility during expression and accumulation in the cytoplasm. Here, we investigate this process in vivo during the recombinant overexpression of the mimetic spider silk mini-spidroin NT2RepCT in Escherichia coli. The protein forms intracellular liquid-like condensates that shift to a solid-like state triggered by a decrease in their microenvironmental pH. These features are also maintained in the purified sample in vitro both in the presence of a molecular crowding agent mimicking the bacterial intracellular environment, and during a biomimetic extrusion process leading to fiber formation. Overall, we demonstrate that characterization of protein condensates inside E. coli could be used as a basis for selecting proteins for both materials applications and their fundamental structure-function studies.
Hamed, MM;Taniguchi, K;Duncan, MC.
Methods in molecular biology (Clifton, N.J.),
2557,
83-98,
(2023)
Membrane traffic at the Golgi and endosomes plays many critical roles in the polarization and the morphogenesis of epithelial tissues. Studies into the roles of traffic in morphogenesis in mammals are often complicated by early embryonic lethality of mutations in membrane traffic as well as the inherent difficulty in imaging developing embryos posed by their size and location. Increasingly, human pluripotent stem cell (hPSC)-derived embryo- and organ-like systems (e.g., embryoids, organoids) provide a useful platform to illuminate the requirements of traffic in human embryonic tissue morphogenesis because these in vitro models are highly amenable to fluorescence microscopy and provide the ability to examine the role of essential genes not possible with animal studies. Here, we present a method to generate hPSC-cysts, a 3-D hPSC-based model of human epiblast lumen formation. This system provides unique opportunities to examine the role of membrane traffic during epithelial morphogenesis. We also present methods to process hPSC-cysts for immunofluorescence and staining with commonly used fluorescence labels useful for detecting defects in polarization and morphogenesis caused by defects in membrane traffic.
Puri, D;Fang, X;Allison, KR.
iScience,
26,
(1),
105795,
(2023)
Biofilms are surface-attached multicellular microbial communities. Their genetics have been extensively studied, but the cell-scale morphogenetic events of their formation are largely unknown. Here, we recorded the entirety of morphogenesis in Escherichia coli, and discovered a previously unknown multicellular self-assembly process. Unattached, single-cells formed 4-cell rosettes which grew into constant-width chains. After ∼10 cell generations, these multicellular chains attached to surfaces and stopped growing. Chains remained clonal throughout morphogenesis. We showed that this process generates biofilms, which we found are composed of attached clonal chains, aligned in parallel. We investigated genetics of chain morphogenesis: Ag43 facilitates rosette formation and clonality; type-1 fimbriae and curli promote stability and configuration; and extracellular polysaccharide production facilitates attachment. Our study establishes that E. coli, a unicellular organism, can follow a multistage, clonal, genetically-regulated, rosette-initiated multicellular life cycle. These findings have implications for synthetic biology, multicellular development, and the treatment and prevention of bacterial diseases.
Sgritta, M;Vignoli, B;Pimpinella, D;Griguoli, M;Santi, S;Bialowas, A;Wiera, G;Zacchi, P;Malerba, F;Marchetti, C;Canossa, M;Cherubini, E.
iScience,
26,
(1),
105728,
(2023)
In Neurodevelopmental Disorders, alterations of synaptic plasticity may trigger structural changes in neuronal circuits involved in cognitive functions. This hypothesis was tested in mice carrying the human R451C mutation of Nlgn3 gene (NLG3R451C KI), found in some families with autistic children. To this aim, the spike time dependent plasticity (STDP) protocol was applied to immature GABAergic Mossy Fibers (MF)-CA3 connections in hippocampal slices from NLG3R451C KI mice. These animals failed to exhibit STD-LTP, an effect that persisted in adulthood when these synapses became glutamatergic. Similar results were obtained in mice lacking the Nlgn3 gene (NLG3 KO mice), suggesting a loss of function. The loss of STD-LTP was associated with a premature shift of GABA from the depolarizing to the hyperpolarizing direction, a reduced BDNF availability and TrkB phosphorylation at potentiated synapses. These effects may constitute a general mechanism underlying cognitive deficits in those forms of Autism caused by synaptic dysfunctions.
Sun, Q;Wang, Y;Ji, H;Sun, X;Xie, S;Chen, L;Li, S;Zeng, W;Chen, R;Tang, Q;Zuo, J;Hou, L;Hosaka, K;Lu, Y;Liu, Y;Ye, Y;Yang, Y.
Cell death & disease,
13,
(8),
724,
(2022)
Nasopharyngeal carcinoma (NPC) clinical trials show that antiangiogenic drugs (AADs) fail to achieve the expected efficacy, and combining AAD with chemoradiotherapy does not show superiority over chemoradiotherapy alone. Accumulating evidence suggests the intrinsic AAD resistance in NPC patients with poorly understood molecular mechanisms. Here, we describe NPC-specific FGF-2 expression-triggered, VEGF-independent angiogenesis as a mechanism of AAD resistance. Angiogenic factors screening between AAD-sensitive cancer type and AAD-resistant NPC showed high FGF-2 expression in NPC in both xenograft models and clinical samples. Mechanistically, the FGF-2-FGFR1-MYC axis drove endothelial cell survival and proliferation as an alternative to VEGF-VEGFR2-MYC signaling. Genetic knockdown of FGF-2 in NPC tumor cells reduced tumor angiogenesis, enhanced AAD sensitivity, and reduced pulmonary metastasis. Moreover, lenvatinib, an FDA recently approved multi-kinase inhibitor targeting both VEGFR2 and FGFR1, effectively inhibits the tumor vasculature, and exhibited robust anti-tumor effects in NPC-bearing nude mice and humanized mice compared with an agent equivalent to bevacizumab. These findings provide mechanistic insights on FGF-2 signaling in the modulation of VEGF pathway activation in the NPC microenvironment and propose an effective NPC-targeted therapy by using a clinically available drug.
Meka, DP;Kobler, O;Hong, S;Friedrich, CM;Wuesthoff, S;Henis, M;Schwanke, B;Krisp, C;Schmuelling, N;Rueter, R;Ruecker, T;Betleja, E;Cheng, T;Mahjoub, MR;Soba, P;Schlüter, H;Fornasiero, EF;Calderon de Anda, F.
Cell reports,
39,
(3),
110686,
(2022)
Microtubule (MT) modifications are critical during axon development, with stable MTs populating the axon. How these modifications are spatially coordinated is unclear. Here, via high-resolution microscopy, we show that early developing neurons have fewer somatic acetylated MTs restricted near the centrosome. At later stages, however, acetylated MTs spread out in soma and concentrate in growing axon. Live imaging in early plated neurons of the MT plus-end protein, EB3, show increased displacement and growth rate near the MTOC, suggesting local differences that might support axon selection. Moreover, F-actin disruption in early developing neurons, which show fewer somatic acetylated MTs, does not induce multiple axons, unlike later stages. Overexpression of centrosomal protein 120 (Cep120), which promotes MT acetylation/stabilization, induces multiple axons, while its knockdown downregulates proteins modulating MT dynamics and stability, hampering axon formation. Collectively, we show how centrosome-dependent MT modifications contribute to axon formation.
Viloria, K;Nasteska, D;Ast, J;Hasib, A;Cuozzo, F;Heising, S;Briant, LJB;Hewison, M;Hodson, DJ.
Diabetes,
(2022)
GC-globulin (GC), or vitamin D-binding protein, is a multifunctional protein involved in transport of circulating vitamin 25(OH)D and fatty acids, as well as actin-scavenging. In the pancreatic islets, the gene encoding GC, GC, is highly-localized to glucagon-secreting α cells. Despite this, the role of GC in α cell function is poorly understood. We previously showed that GC is essential for α cell morphology, electrical activity and glucagon secretion. We now show that loss of GC exacerbates α cell failure during metabolic stress. High fat diet-fed GC-/- mice have basal hyperglucagonemia, which is associated with decreased α cell size, impaired glucagon secretion and Ca2+ fluxes, and changes in glucose-dependent F-actin remodelling. Impairments in glucagon secretion can be rescued using exogenous GC to replenish α cell GC levels, increase glucagon granule area and restore the F-actin cytoskeleton. Lastly, GC levels decrease in α cells of donors with type 2 diabetes, which is associated with changes in α cell mass, morphology and glucagon expression. Together, these data demonstrate an important role for GC in α cell adaptation to metabolic stress.