Publications

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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.
Bai, Q;Yang, C;Yang, M;Pei, Z;Zhou, X;Liu, J;Ji, H;Li, G;Wu, M;Qin, Y;Wang, Q;Wu, L.
Analytical chemistry,
94
(6)
, 2901-2911
(2022)
Elucidating the intrinsic relationship between mitochondrial pH (pHm) fluctuation and lipid droplets (LDs) formation is vital in cell physiology. The development of small-molecular fluorescent probes for discrimination and simultaneous visualization of pHm fluctuation toward LDs has not yet been reported. In this work, utilizing pH-driven polarity-reversible hemicyanine and rhodamine derivatives, a multifunctional fluorescent probe is developed for selectively identifying mitochondria and LDs under specific pH values via dual-emission channels. This rapid-response probe, Hcy-Rh, has two distinct chemical structures under acidic and alkaline circumstances. In acidic conditions, Hcy-Rh exhibits good hydrophilicity that can target mitochondria and display an intense red fluorescence. Conversely, the probe becomes lipophilic under weakly alkaline conditions and targets LDs, showing a strong blue emission. In this manner, Hcy-Rh can selectively label mitochondria and LDs, exhibiting red and blue fluorescence, respectively. Moreover, this ratiometric probe is applied to map pHm changes in living cells under the stimulus with FCCP, NAC, and H2O2. The interplay of LD-mitochondria under oleic acid treatment and starvation-induced autophagy has been studied using this probe at different pH values. In a word, Hcy-Rh is a potential candidate for further exploring mitochondria-LD interaction mechanisms under pHm fluctuation. Moreover, the polarity-dependent strategy is valuable for designing other functional biological probes in imaging multiple organelles.
Goodwin, K;Jaslove, JM;Tao, H;Zhu, M;Hopyan, S;Nelson, CM.
iScience,
25
(3)
, 103838
(2022)
Smooth muscle guides the morphogenesis of several epithelia during organogenesis, including the mammalian airways. However, it remains unclear how airway smooth muscle differentiation is spatiotemporally patterned and whether it originates from transcriptionally distinct mesenchymal progenitors. Using single-cell RNA-sequencing of embryonic mouse lungs, we show that the pulmonary mesenchyme contains a continuum of cell identities, but no transcriptionally distinct progenitors. Transcriptional variability correlates with spatially distinct sub-epithelial and sub-mesothelial mesenchymal compartments that are regulated by Wnt signaling. Live-imaging and tension-sensors reveal compartment-specific migratory behaviors and cortical forces and show that sub-epithelial mesenchyme contributes to airway smooth muscle. Reconstructing differentiation trajectories reveals early activation of cytoskeletal and Wnt signaling genes. Consistently, Wnt activation induces the earliest stages of smooth muscle differentiation and local accumulation of mesenchymal F-actin, which influences epithelial morphology. Our single-cell approach uncovers the principles of pulmonary mesenchymal patterning and identifies a morphogenetically active mesenchymal layer that sculpts the airway epithelium.
Trayford, C;Crosbie, D;Rademakers, T;van Blitterswijk, C;Nuijts, R;Ferrari, S;Habibovic, P;LaPointe, V;Dickman, M;van Rijt, S.
ACS Applied Nano Materials,
5
(3)
, 3237-3251
(2022)
Stem cell (SC)-based therapies hold the potential to revolutionize therapeutics by enhancing the body’s natural repair processes. Currently, there are only three SC therapies with marketing authorization within the European Union. To optimize outcomes, it is important to understand the biodistribution and behavior of transplanted SCs in vivo. A variety of imaging agents have been developed to trace SCs; however, they mostly lack the ability to simultaneously monitor the SC function and biodistribution at high resolutions. Here, we report the synthesis and application of a nanoparticle (NP) construct consisting of a gold NP core coated with rhodamine B isothiocyanate (RITC)-doped mesoporous silica (AuMS). The MS layer further contained a thiol-modified internal surface and an amine-modified external surface for dye conjugation. Highly fluorescent AuMS of three different sizes were successfully synthesized. The NPs were non-toxic and efficiently taken up by limbal epithelial SCs (LESCs). We further showed that we can functionalize AuMS with a reactive oxygen species (ROS)-sensitive fluorescent dye using two methods, loading the probe into the mesopores, with or without additional capping by a lipid bilayer, and by covalent attachment to surface and/or mesoporous-functionalized thiol groups. All four formulations displayed a ROS concentration-dependent increase in fluorescence. Further, in an ex vivo SC transplantation model, a combination of optical coherence tomography and fluorescence microscopy was used to synergistically identify AuMS-labeled LESC distribution at micrometer resolution. Our AuMS constructs allow for multimodal imaging and simultaneous ROS sensing of SCs and represent a promising tool for in vivo SC tracing.
Kaldmäe, M;Vosselman, T;Zhong, X;Lama, D;Chen, G;Saluri, M;Kronqvist, N;Siau, JW;Ng, AS;Ghadessy, FJ;Sabatier, P;Vojtesek, B;Sarr, M;Sahin, C;Österlund, N;Ilag, LL;Väänänen, VA;Sedimbi, S;Arsenian-Henriksson M;Zubarev, RA;Nilsson, L;Koeck, PJB;Rising, A;Abelein, A;Fritz, N;Johansson, J;Lane, DP;Landreh, M.
Structure (London, England : 1993),
(2022)
Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of “life on the edge of solubility.” Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.
Undvall, E;Garofalo, F;Procopio, G;Qiu, W;Lenshof, A;Laurell, T;Baasch, T.
Physical Review Applied,
17
(3)
(2022)
The clinical utility of microfluidic techniques is often hampered by an unsatisfying sample throughput. Here, the effect of inertial forces on acoustofluidic particle sorting at high sample throughputs is investigated experimentally and theoretically. Polystyrene particles are acoustically prefocused to obtain precise trajectories. At increased flow rates it is observed that the particle stream is displaced towards the channel center, and above specific flow settings the particles spill over into the center outlet. This effect, coined the spillover effect, illustrates the complex interplay of viscous and inertial forces inside the microchannel. The effect is due to increased bending of the separatrices at the inlet and outlets and not due to the wall-lift force. The impact of the spillover effect on the separation of two different-sized particles is subsequently studied. Efficient sorting is done for subcritical splitting ratios and flow rates, but for close to critical settings or beyond, there is a breakdown of the acoustofluidic separation.
Genovesi, S;Moro, R;Vignoli, B;De Felice, D;Canossa, M;Montironi, R;Carbone, FG;Barbareschi, M;Lunardi, A;Alaimo, A.
Biomolecules,
12
(2)
(2022)
Metastatic prostate cancer (mPCa) is one of the leading causes of cancer-related mortality in both the US and Europe. Androgen deprivation is the first-line therapy for mPCa; however, resistance to therapy inevitably occurs and the disease progresses to the castration resistant stage, which is uncurable. A definition of novel targeted therapies is necessary for the establishment of innovative and more effective protocols of personalized oncology. We employed genetically engineered mouse models of PCa and human samples to characterize the expression of the TRPM8 cation channel in both hormone naïve and castration resistant tumors. We show that Trpm8 expression marks both indolent (Pten-null) and aggressive (Pten/Trp53 double-null and TRAMP) mouse prostate adenocarcinomas. Importantly, both mouse and human castration-resistant PCa preserve TRPM8 protein expression. Finally, we tested the effect of TRPM8 agonist D-3263 administration in combination with enzalutamide or docetaxel on the viability of aggressive mouse PCa cell lines. Our data demonstrate that D-3263 substantially enhances the pro-apoptotic activity of enzalutamide and docetaxel in TRAMP-C1 e TRAMP-C2 PCa cell lines. To conclude, this study provides the basis for pre-clinical in vivo testing of TRPM8 targeting as a novel strategy to implement the efficacy of standard-of-care treatments for advanced PCa.
Krishnan, N;Swoger, M;Rathbun, LI;Fioramonti, PJ;Freshour, J;Bates, M;Patteson, AE;Hehnly, H.
Life science alliance,
5
(7)
(2022)
The last stage of cell division involves two daughter cells remaining interconnected by a cytokinetic bridge that is cleaved during abscission. Conserved between the zebrafish embryo and human cells, we found that the oldest centrosome moves in a Rab11-dependent manner towards the cytokinetic bridge sometimes followed by the youngest. Rab11-endosomes are organized in a Rab11-GTP dependent manner at the mother centriole during pre-abscission, with Rab11 endosomes at the oldest centrosome being more mobile compared with the youngest. The GTPase activity of Rab11 is necessary for the centrosome protein, Pericentrin, to be enriched at the centrosome. Reduction in Pericentrin expression or optogenetic disruption of Rab11-endosome function inhibited both centrosome movement towards the cytokinetic bridge and abscission, resulting in daughter cells prone to being binucleated and/or having supernumerary centrosomes. These studies suggest that Rab11-endosomes contribute to centrosome function during pre-abscission by regulating Pericentrin organization resulting in appropriate centrosome movement towards the cytokinetic bridge and subsequent abscission.
Ju, Z;Shen, L;Zhou, M;Luo, J;Yu, Z;Qu, C;Lei, R;Lei, M;Huang, R.
ACS chemical neuroscience,
(2022)
Gut dysbiosis is observed in Alzheimer’s disease (AD) and is frequently associated with AD-induced metabolic dysfunction. However, the extent and specific underlying molecular mechanisms triggered by alterations of gut microbiota composition and function mediating AD-induced metabolic dysfunction in AD remain incompletely uncovered. Here, we indicate that Helicobacter pylori (H. pylori) is abundant in AD patients with relative metabolic dysfunction. Fecal microbiota transplantation from the AD patients promoted metabolic dysfunction in mice and increased gut permeability. H. pylori increased gut permeability through activation of the TLR4/Myd88 inflammation pathway in a p53-dependent manner, leading to metabolic dysfunction. Moreover, p53 deficiency reduced bile acid concentration, leading to an increased abundance of H. pylori colonization. Overall, these data identify H. pylori as a key promoter of AD-induced metabolic dysfunction.
Denha, SA;Atang, AE;Hays, TS;Avery, AW.
Scientific reports,
12
(1)
, 1726
(2022)
Recent structural studies of β-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem calponin homology domains (CH1 and CH2). Here we investigated in Drosophila the significance of the β-spectrin N-terminus, and explored its functional interaction with a CH2-localized L253P mutation that underlies the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). We report that pan-neuronal expression of an N-terminally truncated β-spectrin fails to rescue lethality resulting from a β-spectrin loss-of-function allele, indicating that the N-terminus is essential to β-spectrin function in vivo. Significantly, N-terminal truncation rescues neurotoxicity and defects in dendritic arborization caused by L253P. In vitro studies show that N-terminal truncation eliminates L253P-induced high-affinity actin binding, providing a mechanistic basis for rescue. These data suggest that N-terminal sequences may be useful therapeutic targets for small molecule modulation of the aberrant actin binding associated with SCA5 β-spectrin and spectrin-related disease proteins.
Ho, JCS;Su, WC;Chun Wang, X;Parikh, AN;Liedberg, B.
Langmuir : the ACS journal of surfaces and colloids,
38
(3)
, 1045-1056
(2022)
When a dry mass of certain amphiphiles encounters water, a spectacular interfacial instability ensues: It gives rise to the formation of ensembles of fingerlike tubular protrusions called myelin figures─tens of micrometers wide and tens to hundreds of micrometers long─representing a novel class of nonequilibrium higher-order self-organization. Here, we report that when phase-separating mixtures of unsaturated lipid, cholesterol, and sphingomyelin are hydrated, the resulting myelins break symmetry and couple their compositional degrees of freedom with the extended myelinic morphology: They produce complementary, interlamellar radial gradients of concentrations of cholesterol (and sphingomyelin) and unsaturated lipid, which stands in stark contrast to interlamellar, lateral phase separation in equilibrated morphologies. Furthermore, the corresponding gradients of molecule-specific chemistries (i.e., cholesterol extraction by methyl-β-cyclodextrin and GM1 binding by cholera toxin) produce unusual morphologies comprising compositionally graded vesicles and buckled tubes. We propose that kinetic differences in the information processing of hydration characteristics of individual molecules while expending energy dictate this novel behavior of lipid mixtures undergoing hydration.
Özdağ Acarlı, AN;Klein, T;Egenolf, N;Sommer, C;Üçeyler, N.
Muscle & nerve,
65
(4)
, 471-479
(2022)
Schwann cell clusters have been described at the murine dermis-epidermis border. We quantified dermal Schwann cells in the skin of patients with small-fiber neuropathy (SFN) compared with healthy controls to correlate with the clinical phenotype.Skin punch biopsies from the lower legs of 28 patients with SFN (11 men, 17 women; median age, 54 [range, 19-73] years) and 9 healthy controls (five men, four women, median age, 34 [range, 25-69] years) were immunoreacted for S100 calcium-binding protein B as a Schwann cell marker, protein-gene product 9.5 as a pan-neuronal marker, and CD207 as a Langerhans cell marker. Intraepidermal nerve fiber density (IENFD) and subepidermal Schwann cell counts were determined.Skin samples of patients with SFN showed lower IENFD (P < .05), fewer Schwann cells per millimeter (P < .01), and fewer Schwann cell clusters per millimeter (P < .05) than controls. When comparing SFN patients with reduced (n = 13; median age, 53 [range, 19-73] years) and normal distal (n = 15, median age, 54 [range, 43-68] years) IENFD, the number of solitary Schwann cells per millimeter (p < .01) and subepidermal nerve fibers associated with Schwann cell branches (P < .05) were lower in patients with reduced IENFD. All three parameters correlated positively with distal IENFD (P < .05 to P < .01), whereas no correlation was found between Schwann cell counts and clinical pain characteristics.Our data raise questions about the mechanisms underlying the interdependence of dermal Schwann cells and skin innervation in SFN. The temporal course and functional impact of Schwann cell presence and kinetics need further investigation.
Baudon, A;Clauss-Creusot, E;Darbon, P;Patwell, R;Grinevich, V;Charlet, A.
STAR protocols,
3
(1)
, 101159
(2022)
Astrocytes are glial cells that exhibit calcium signaling-mediated activity. Here, we present a protocol to monitor and manipulate astrocyte calcium activity from mouse amygdala slices. In the first part of this protocol, we describe the procedure of astrocyte calcium imaging. In the second part, we detail how to disrupt astrocyte calcium activity by patch-clamp-mediated loading of BAPTA. These two approaches are presented separately but they can also be used simultaneously to monitor the effects of disruption on an astrocyte network. For complete details on the use and execution of this protocol, please refer to Wahis et al. (2021).
Swoger, M;Gupta, S;Charrier, EE;Bates, M;Hehnly, H;Patteson, AE.
ACS applied bio materials,
5
(2)
, 552-561
(2022)
The ability of cells to take and change shape is a fundamental feature underlying development, wound repair, and tissue maintenance. Central to this process is physical and signaling interactions between the three cytoskeletal polymeric networks: F-actin, microtubules, and intermediate filaments (IFs). Vimentin is an IF protein that is essential to the mechanical resilience of cells and regulates cross-talk among the cytoskeleton, but its role in how cells sense and respond to the surrounding extracellular matrix is largely unclear. To investigate vimentin’s role in substrate sensing, we designed polyacrylamide hydrogels that mimic the elastic and viscoelastic nature of in vivo tissues. Using wild-type and vimentin-null mouse embryonic fibroblasts, we show that vimentin enhances cell spreading on viscoelastic substrates, even though it has little effect in the limit of purely elastic substrates. Our results provide compelling evidence that vimentin modulates how cells sense and respond to their environment and thus plays a key role in cell mechanosensing.
Torrente, D;Su, E;Schielke, G;Warnock, M;Stevenson, T;Mann, K;Vivien, D;Lawrence, D.
SSRN Electronic Journal,
(2022)
The protease tissue plasminogen activator (tPA) is linked to diverse functions in the central nervous system. Here we characterize the expression and localization of tPA in the substantia nigra (SN) and explore the role of tPA in dopaminergic neuron degeneration in a human α-synuclein (hα-SYN) mouse model of Parkinson’s disease. We found that striatal GABAergic neurons send tPA + axons that innervate the SN proximal to dopaminergic neuronal cell bodies and axons. tPA deficiency protected dopaminergic neurons from degeneration and reversed behavioral deficits induced by hα-SYN-induced neurotoxicity. tPA’s action was independent of its proteolytic activity, and could be blocked by treatment with Glunomab, a neutralizing antibody that selectively inhibits tPA interaction with N-methyl-D-aspartate receptor-1. Both tPA deficiency and Glunomab treatment prevented neuronal degeneration, and reduced microglia activation and T-cell infiltration. Together, these data demonstrate a previously unrecognized pathway promoting dopaminergic neuronal degeneration and suggest a potential therapeutic intervention with Glunomab.
Shen, L;Lin, C;Lu, W;He, J;Wang, Q;Huang, Y;Zheng, X;Wang, Z.
Research Square,
(2022)
Colorectal cancer (CRC) is a common malignant cancer worldwide. Although the molecular mechanism of CRC carcinogenesis has been studied extensively, the details remain unclear. Small nucleolar RNAs (snoRNAs) have recently been reported to have essential functions in carcinogenesis, although their roles in CRC pathogenesis are largely unknown. In this study, we found that the H/ACA snoRNA SNORA24 was up-regulated in various cancers, including CRC. SNORA24 expression was significantly associated with age and history of colon polyps in CRC patient cohorts, with high expression associated with a decreased 5-year overall survival. Our results indicated that the oncogenic function of SNORA24 is mediated by promoting G1/S phase transformation, cell proliferation, colony formation and growth of xenograft tumors. Furthermore, SNORA24 knockdown induced massive apoptosis. RNA-sequencing and Gene Ontology (GO) enrichment analyses were performed to explore its downstream targets. Finally, we confirmed that SNORA24 regulates p53 protein stability in a proteasomal degradation pathway. Our study clarifies the oncogenic role of SNORA24 in CRC and advance the current model of the role of the p53 pathway in this process.
Zaccaria, C;Mancinelli, M;Vignoli, B;Auslender, I;Bettotti, P;Malkoc, A;Canossa, M;Pavesi, L.
Optogenetics and Optical Manipulation 2022,
(2022)
To study the brain and the related neuronal network activity, many attempts were made to design and develop platforms able to induce and record neuronal signals. However, many brain processes – like memory formation and storage – and diseases – like amnesia or epilepsy – need more basic studies. For these, a bottom-up approach is needed, starting from 2D in-vitro neuronal cultures. In this work, we will present two experimental systems able to optogenetically interact with 2D neuronal networks with patternized light. One system consists in a Digital Light Projector (DLP) integrated in a microscope setup, which can illuminate neurons from the top; the other, is a compact and transportable photonic chip, properly designed to illuminate neurons plated on its surface.
Erbacher, C;Britz, S;Dinkel, P;Klein, T;Sauer, M;Stigloher, C;Üçeyler, N.
bioRxiv,
(2022)
Traditionally, peripheral sensory neurons hold the monopole of transducing external stimuli. Current research moves epidermal keratinocytes into focus as sensors and transmitters of nociceptive and non-nociceptive sensations, tightly interacting with intraepidermal nerve fibers at the neuro-cutaneous unit. In animal models, epidermal cells establish close contacts and ensheath sensory neurites. However, ultrastructural morphological and mechanistic data examining the human keratinocyte-nociceptor interface are sparse. We investigated this exact interface in human skin applying super-resolution array tomography, expansion microscopy, and structured illumination microscopy. We show keratinocyte ensheathment of nociceptors and connexin 43 plaques at keratinocyte-nociceptor contact sites in healthy native skin. We further derived a fully human co-culture system, modeling ensheathment and connexin 43 plaques in vitro. Unraveling human intraepidermal nerve fiber ensheathment and interaction sites marks a milestone in research at the neuro-cutaneous unit. These findings are mind-changers on the way to decipher the mechanisms of cutaneous nociception.
Rosito, M;Sanchini, C;Gosti, G;Moreno, M;Panfilis, S;Giubettini, M;Debellis, D;Catalano, F;Peruzzi, G;Marotta, R;Indrieri, A;Leonibus, E;Ragozzino, D;Ruocco, G;Angelantonio, S;Bartolini, F.
bioRxiv,
(2022)
Microglia reactivity triggered by proinflammatory signaling entails a large-scale remodeling of cellular geometry, but the role of the microtubule cytoskeleton during these changes remains unexplored. Here we show that reactive proinflammatory microglia provide a heretofore unique example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state microglia nucleate microtubules at Golgi outposts, proinflammatory signaling induces recruitment of nucleating material nearby the centrosome and inhibition of de novo formed pericentrosomal microtubule-organizing centers enhances NLRP3 inflammasome activation and secretion of the interleukin IL-1β. Our results demonstrate that remodeling of the microtubule cytoskeleton is a hallmark of proinflammatory microglia reactivity and suggest that pericentrosomal microtubule nucleating material maturation may offer a valuable target to modulate cytokine-mediated inflammatory responses in chronic disease and tissue injury.