Publications

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Nasteska, D;Cuozzo, F;Thakker, A;Bakar, R;Westbrook, R;Akerman, I;Cantley, J;Tennant, D;Hodson, D.
bioRxiv,
(2020)
The alpha ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is a hypoxia-inducible factor target that uses molecular oxygen to hydroxylate proline. While PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about effects of this highly conserved enzyme in insulin-secreting ?-cells. Here, we show that deletion of PHD3 specifically in ?-cells (?PHD3KO) is associated with impaired glucose homeostasis in mice fed high fat diet. In the early stages of dietary fat excess, ?PHD3KO islets energetically rewire, leading to defects in the management of pyruvate fate and a shift away from glycolysis. However, ?PHD3KO islets are able to maintain oxidative phosphorylation and insulin secretion by increasing utilization of fatty acids to supply the tricarboxylic acid cycle. This nutrient-sensing switch cannot be sustained and ?PHD3KO islets begin to show signs of failure in response to prolonged metabolic stress, including impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes and insulin secretion. Thus, PHD3 might be a pivotal component of the ?-cell glucose metabolism machinery by suppressing the use of fatty acids as a primary fuel source, under obesogenic and insulin resistant states.SIGNIFICANCE STATEMENTProlyl-4-hydroxylase 3 (PHD3) is involved in the oxygen-dependent regulation of cell phenotype. A number of recent studies have shown that PHD3 might operate at the interface between oxygen availability and metabolism. To understand how PHD3 influences insulin secretion, which depends on intact glucose metabolism, we generated mice lacking PHD3 specifically in pancreatic ?-cells. These mice, termed ?PHD3KO, are apparently normal until fed high fat diet at which point their ?-cells switch to fatty acids as a fuel source. This switch cannot be tolerated and ?-cells in ?PHD3KO mice eventually fail. Thus, PHD3 maintains glucose-stimulated insulin secretion in ?-cells during states of fatty acid excess, such as diabetes and obesity.
Romano, A;Angelini, A;Rossegger, E;Palmara, G;Castellino, M;Frascella, F;Chiappone, A;Chiadò, A;Sangermano, M;Schlögl, S;Roppolo, I.
Macromol Rapid Commun,
41
(10)
, e2000084
(2020)
The light responsivity of ortho-nitrobenzyl esters (o-NBE) is exploited to inscribe µ-scale 2.5D patterns in thiol-ene networks by direct laser writing. For this purpose, a multifunctional thiol and a photosensitive alkene with an o-NBE chromophore are cured upon visible light exposure without inducing a premature photocleavage of the o-NBE links. Once the network is formed, a laser beam source with a wavelength of 375 nm is used for selectively inducing the photocleavage reaction of the o-NBE groups. Positive tone patterns are directly inscribed onto the sample surface without the requirement of a subsequent development step (removing soluble species in an appropriate organic solvent). Along with the realization of dry-developable micropatterns, the chemical surface composition of the exposed areas can be conveniently adjusted since different domains with a tailored content of carboxylic groups are obtained simply by modulating the laser energy dose. In a following step, those are activated and exploited as anchor points for attaching an Alexa-546 conjugated Protein A. Thus, the laser writable thiol-ene networks do not only provide a convenient method for the fabrication of positive tone patterns but also open future prospectives for a wide range of biosensing applications.
Heckman, CA;Biswas, T;Dimick, DM;Cayer, ML.
Biomolecules,
10
(9)
, 1288
(2020)
Protein kinase Cs (PKCs) are activated by lipids in the plasma membrane and bind to a scaffold assembled on the epidermal growth factor (EGF) receptor (EGFR). Understanding how this complex is routed is important, because this determines whether EGFR is degraded, terminating signaling. Here, cells were preincubated in EGF-tagged gold nanoparticles, then allowed to internalize them in the presence or absence of a phorbol ester PKC activator. PKC colocalized with EGF-tagged nanoparticles within 5 min and migrated with EGFR-bearing vesicles into the cell. Two conformations of PKC-epsilon were distinguished by different primary antibodies. One, thought to be enzymatically active, was on endosomes and displayed a binding site for antibody RR (R&D). The other, recognized by Genetex green (GG), was soluble, on actin-rich structures, and loosely bound to vesicles. During a 15-min chase, EGF-tagged nanoparticles entered large, perinuclear structures. In phorbol ester-treated cells, vesicles bearing EGF-tagged nanoparticles tended to enter this endocytic recycling compartment (ERC) without the GG form. The correlation coefficient between the GG (inactive) and RR conformations on vesicles was also lower. Thus, active PKC has a Charon-like function, ferrying vesicles to the ERC, and inactivation counteracts this function. The advantage conferred on cells by aggregating vesicles in the ERC is unclear.
Chandrakar, P;Varghese, M;Aghvami, S;Baskaran, A;Dogic, Z;Duclos, G.
arXiv,
(2020)
Spontaneous growth of long-wavelength deformations is a defining feature of active fluids with orientational order. We investigate the effect of biaxial rectangular confinement on the instability of initially shear-aligned 3D isotropic active fluids composed of extensile microtubule bundles and kinesin molecular motors. Under confinement, such fluids exhibit finite-wavelength self-amplifying bend deformations which grow in the plane orthogonal to the direction of the strongest confinement. Both the instability wavelength and the growth rate increase with weakening confinement. These findings are consistent with a minimal hydrodynamic model, which predicts that the fastest growing deformation is set by a balance of active driving and elastic relaxation. Experiments in the highly confined regime confirm that the instability wavelength is set by the balance of active and elastic stresses, which are independently controlled by the concentration of motors and non-motile crosslinkers.
Sun, Y;Chen, X;Fischer, S;Lu, S;Gillis, J;Zador, A.
bioRxiv,
(2020)
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 multi-gene expression and projections to many targets in single neurons with high throughput, BARseq2 provides a path to uncovering the molecular logic underlying neuronal circuits.
Kroeger, H;Grandjean, J;Chiang, W;Bindels, D;Mastey, R;Okalova, J;Nguyen, A;Powers, E;Kelly, J;Grimsey, N;Michaelides, M;Carroll, J;Wiseman, R;Lin, J.
bioRxiv,
(2020)
Dysregulation of the endoplasmic reticulum (ER) Unfolded Protein Response (UPR) is implicated in the pathology of many human diseases associated with ER stress. Inactivating genetic variants in the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital heritable vision loss in patients by an unknown pathomechanism. To investigate this, we generated retinal organoids from patient iPSCs carrying ATF6 disease-causing variants and ATF6 null hESCs generated by CRISPR. Interestingly, we found that cone photoreceptor cells in ATF6 mutant retinal organoids lacked inner and outer segments concomitant with absence of cone phototransduction gene expression; while rod photoreceptors developed normally. Adaptive optics retinal imaging of patients with disease-causing variants in ATF6 also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the phenotypes observed in our retinal organoids. These results reveal that ATF6 is essential for the formation of human cone photoreceptors, and associated absence of cone phototransduction components explains the severe visual impairment in patients with ATF6 -associated retinopathy. Moreover, we show that a selective small molecule ATF6 activator compound restores the transcriptional activity of ATF6 disease-causing variants and stimulates the growth of cone photoreceptors in patient retinal organoids, demonstrating that pharmacologic targeting of ATF6 signaling is a therapeutic strategy that needs to be further explored for blinding retinal diseases.
Logan, G;McCartney, B.
bioRxiv,
(2020)
Cells reposition their nuclei for a diversity of specialized functions through a wide variety of cytoskeletal mechanisms. To complete oogenesis, Drosophila nurse cells employ novel actin cable arrays to reposition their nuclei. During oogenesis, 15 nurse cells connected by ring canals contract to “dump” their cytoplasmic contents into the oocyte. Just prior to dumping, actin cables initiate from the nurse cell cortex and elongate toward their nuclei, pushing them away from the ring canals to prevent obstruction. How the actin cable arrays generate directional nuclear movement is not known. We found regional differences in the actin cable growth rate that are dependent on the differential localization of the actin assembly factors Enabled (Ena) and Diaphanous (Dia). Mislocalization of Ena resulted in actin cable arrays with a uniform growth rate. In the absence of growth rate asymmetry, nuclear relocation was significantly altered and cytoplasmic dumping was incomplete. This novel mechanism for nuclear repositioning relies on the regulated cortical localization of Dia and Ena producing asymmetric actin cable arrays that push the nuclei away from the ring canals, enabling successful oogenesis.Summary statementThis work demonstrates that an asymmetric actin cable array regulated by the differential localization of Diaphanous and Enabled is necessary to reposition nurse cell nuclei and complete oogenesis in Drosophila.
Salvioni, L;Zuppone, S;Andreata, F;Monieri, M;Mazzucchelli, S;Di Carlo, C;Morelli, L;Cordiglieri, C;Donnici, L;De Francesco, R;Corsi, F;Prosperi, D;Vago, R;Colombo, M.
Adv Therap,
3
(8)
, 2000007
(2020)
Systemic chemotherapy has not significantly reduced clinical demand for triple negative breast cancer (TNBC) treatments. To address the need for more effective therapy, the use of nonviral nanoparticles is explored to deliver suicide gene therapy as valuable alternative to protect nucleic acids in the bloodstream and improve their tumor uptake. Biocompatible cationic lipid nanoparticles are developed as a novel delivery system of a suicide plasmid gene encoding saporin. Active targeting is accomplished by taking advantage of nanoparticle functionalization with U11 peptide, designed to be directed toward urokinase plasminogen activator receptor, limiting off?target toxicity. The antitumor effect of U11?lipid?protamine?DNA (U11?LPD) nanoparticles are tested in TBNC cells, showing a strong prevalence of targeted versus nontargeted nanoparticles in terms of uptake kinetics and proliferation inhibition. Transfection of green fluorescent protein (GFP) plasmid in MDA?MB?231 cells is demonstrated. U11?LPD nanoparticles administered by retro bulbar injection exhibit excellent tumor tropism in TNBC orthotopic xenograft mice and effectively transfect TNBC cells with saporin plasmid resulting in tumor mass reduction. No systemic toxicity or organ damage is discovered after repeated treatments with nanoparticles. The findings suggest that systemic administration of targeted LPD nanoparticles to deliver saporin safely allows for active inhibition of cancer progression even in the absence of specific promoter gene sequences.
Seabright, AP;Fine, NHF;Barlow, JP;Lord, SO;Musa, I;Gray, A;Bryant, JA;Banzhaf, M;Lavery, GG;Hardie, DG;Hodson, DJ;Philp, A;Lai, YC.
FASEB J,
34
(5)
, 6284-6301
(2020)
Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.
Swoger, M;Gupta, S;Charrier, E;Bates, M;Hehnly, H;Patteson, A.
bioRxiv,
(2020)
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 amongst 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 the vimentin cytoskeletal network is a physical modulator of how cells sense and respond to mechanical properties of their extracellular environment.
Roberts, JZ;Holohan, C;Sessler, T;Fox, J;Crawford, N;Riley, JS;Khawaja, H;Majkut, J;Evergren, E;Humphreys, LM;Ferris, J;Higgins, C;Espona-Fiedler, M;Moynagh, P;McDade, SS;Longley, DB.
Cell Death Differ,
27
(9)
, 2726-2741
(2020)
TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2’s pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1’s DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1’s recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)’s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.
Campanella, R;Guarnaccia, L;Cordiglieri, C;Trombetta, E;Caroli, M;Carrabba, G;La Verde, N;Rampini, P;Gaudino, C;Costa, A;Luzzi, S;Mantovani, G;Locatelli, M;Riboni, L;Navone, SE;Marfia, G.
Cells,
9
(2)
, 294
(2020)
: Circulating platelets (PLTs) are able to affect glioblastoma (GBM) microenvironment by supplying oncopromoter and pro-angiogenic factors. Among these mediators, sphingosine-1-phophate (S1P) has emerged as a potent bioactive lipid enhancing cell proliferation and survival. Here, we investigated the effect of “tumor education”, characterizing PLTs from GBM patients in terms of activation state, protein content, and pro-angiogenic potential. PLTs from healthy donors (HD-PLTs) and GBM patients (GBM-PLTs) were collected, activated, and analyzed by flow cytometry, immunofluorescence, and Western blotting. To assess the pro-angiogenic contribution of GBM-PLTs, a functional cord formation assay was performed on GBM endothelial cells (GECs) with PLT-releasate. GBM-PLTs expressed higher positivity for P-selectin compared to HD-PLTs, both in basal conditions and after stimulation with adenosine triphosphate (ADP) and thrombin receptor activating peptide (TRAP). PLTs showed higher expression of VEGFR-1, VEGFR-2, VWF, S1P, S1PR1, SphK1, and SPNS. Interestingly, increased concentrations of VEGF and its receptors VEGFR1 and VEGFR2, VWF, and S1P were found in GBM-PLT-releasate with respect to HD-PLTs. Finally, GBM-PLT-releasate showed a pro-angiogenic effect on GECs, increasing the vascular network’s complexity. Overall, our results demonstrated the contribution of PLTs to GBM angiogenesis and aggressiveness, advancing the potential of an anti-PLT therapy and the usefulness of PLT cargo as predictive and monitoring biomarkers.
González, G;Baruffaldi, D;Martinengo, C;Angelini, A;Chiappone, A;Roppolo, I;Pirri, CF;Frascella, F.
Nanomaterials (Basel),
10
(9)
, 1788
(2020)
Light-based 3D printing techniques could be a valuable instrument in the development of customized and affordable biomedical devices, basically for high precision and high flexibility in terms of materials of these technologies. However, more studies related to the biocompatibility of the printed objects are required to expand the use of these techniques in the health sector. In this work, 3D printed polymeric parts are produced in lab conditions using a commercial Digital Light Processing (DLP) 3D printer and then successfully tested to fabricate components suitable for biological studies. For this purpose, different 3D printable formulations based on commercially available resins are compared. The biocompatibility of the 3D printed objects toward A549 cell line is investigated by adjusting the composition of the resins and optimizing post-printing protocols; those include washing in common solvents and UV post-curing treatments for removing unreacted and cytotoxic products. It is noteworthy that not only the selection of suitable materials but also the development of an adequate post-printing protocol is necessary for the development of biocompatible devices.
Moroz, LL;Romanova, DY;Nikitin, MA;Sohn, D;Kohn, AB;Neveu, E;Varoqueaux, F;Fasshauer, D.
Sci Rep,
10
(1)
, 13020
(2020)
Nitric oxide (NO) is a ubiquitous gaseous messenger, but we know little about its early evolution. Here, we analyzed NO synthases (NOS) in four different species of placozoans-one of the early-branching animal lineages. In contrast to other invertebrates studied, Trichoplax and Hoilungia have three distinct NOS genes, including PDZ domain-containing NOS. Using ultra-sensitive capillary electrophoresis assays, we quantified nitrites (products of NO oxidation) and L-citrulline (co-product of NO synthesis from L-arginine), which were affected by NOS inhibitors confirming the presence of functional enzymes in Trichoplax. Using fluorescent single-molecule in situ hybridization, we showed that distinct NOSs are expressed in different subpopulations of cells, with a noticeable distribution close to the edge regions of Trichoplax. These data suggest both the compartmentalized release of NO and a greater diversity of cell types in placozoans than anticipated. NO receptor machinery includes both canonical and novel NIT-domain containing soluble guanylate cyclases as putative NO/nitrite/nitrate sensors. Thus, although Trichoplax and Hoilungia exemplify the morphologically simplest free-living animals, the complexity of NO-cGMP-mediated signaling in Placozoa is greater to those in vertebrates. This situation illuminates multiple lineage-specific diversifications of NOSs and NO/nitrite/nitrate sensors from the common ancestor of Metazoa and the preservation of conservative NOS architecture from prokaryotic ancestors.
Hudson, A;Loughran, G;Szabo, N;Wills, N;Atkins, J;Cooley, L.
bioRxiv,
(2020)
Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3’ of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue- and cell-specific dynamic stop codon redefinition in Drosophila.
Deng, T;Stempor, P;Appert, A;Daube, M;Ahringer, J;Hajnal, A;Lattmann, E.
PLoS Genet,
16
(3)
, e1008470
(2020)
Cell invasion allows cells to migrate across compartment boundaries formed by basement membranes. Aberrant cell invasion is a first step during the formation of metastases by malignant cancer cells. Anchor cell (AC) invasion in C. elegans is an excellent in vivo model to study the regulation of cell invasion during development. Here, we have examined the function of egl-43, the homolog of the human Evi1 proto-oncogene (also called MECOM), in the invading AC. egl-43 plays a dual role in this process, firstly by imposing a G1 cell cycle arrest to prevent AC proliferation, and secondly, by activating pro-invasive gene expression. We have identified the AP-1 transcription factor fos-1 and the Notch homolog lin-12 as critical egl-43 targets. A positive feedback loop between fos-1 and egl-43 induces pro-invasive gene expression in the AC, while repression of lin-12 Notch expression by egl-43 ensures the G1 cell cycle arrest necessary for invasion. Reducing lin-12 levels in egl-43 depleted animals restored the G1 arrest, while hyperactivation of lin-12 signaling in the differentiated AC was sufficient to induce proliferation. Taken together, our data have identified egl-43 Evi1 as an important factor coordinating cell invasion with cell cycle arrest.
Riley, J;Hehnly, H;Castañeda, C.
bioRxiv,
(2020)
Mutations in Ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells is unknown. Here, we employ live-cell imaging with photokinetic analysis to investigate how five patient-derived ALS-linked mutations in UBQLN2 impact stress-induced UBQLN2 condensate assembly and condensate material properties. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, cells transfected with mutant UBQLN2 contain fewer stress-induced UBQLN2 condensates than those with wild-type UBQLN2. Most strikingly, ectopically expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.
Valanciunaite, J;Kempf, E;Seki, H;Danylchuk, DI;Peyriéras, N;Niko, Y;Klymchenko, AS.
Anal Chem,
92
(9)
, 6512-6520
(2020)
Solvatochromic dyes enable sensing and imaging of biomolecular organization in living systems by monitoring local polarity (lipophilicity), but most such dyes suffer from limited brightness, photostability, lack of a convenient spectral range, and limited sensitivity to polarity. Moreover, the presence of an electron acceptor group, typically a carbonyl, in its push-pull structure raises concerns about its potential chemical reactivity within the biological environment. In order to achieve robust bioimaging, we synthesized a push-pull pyrene probe bearing a ketone acceptor group (PK) and compared it with a recently developed aldehyde analogue (PA). We found that in live cells the aldehyde analogue PA transforms slowly (in ?100 min) into blue-emissive species, assigned to in situ formation of an imine analogue, whereas the PK probe is stable in the presence of primary amines and inside cells. Like the parent PA, the new probe shows strong solvatochromism and an emission color response to lipid order in membranes (ordered vs disordered liquid phases), while its blue-shifted absorption is more optimal for excitation with 400 nm light sources. In live cells, the PK probe enables high-contrast polarity mapping of organelles using two-color ratiometric detection, suggesting that polarity increases in the following order: lipid droplets < plasma membranes < endoplasmic reticulum. In the zebrafish embryo, polarity imaging with the PK probe reveals a new dimension in visualizing the organization of tissues-lipophilicity distribution, where biomembranes, lipid droplets, cells, yolk, extracellular space, and newly formed organs are revealed by specific emission wavelengths of the probe. The newly developed probe and the proposed approach of polarity mapping open new opportunities for bioimaging at the cellular and animal level.
Muñoz-Castañeda, R;Zingg, B;Matho, K;Wang, Q;Chen, X;Foster, N;Narasimhan, A;Li, A;Hirokawa, K;Huo, B;Bannerjee, S;Korobkova, L;Park, C;Park, Y;Bienkowski, M;Chon, U;Wheeler, D;Li, X;Wang, Y;Kelly, K;An, X;Attili, S;Bowman, I;Bludova, A;Cetin, A;Ding, L;Drewes, R;D’Orazi, F;Elowsky, C;Fischer, S;Galbavy, W;Gao, L;Gillis, J;Groblewski, P;Gou, L;Hahn, J;Hatfield, J;Hintiryan, H;Huang, J;Kondo, H;Kuang, X;Lesnar, P;Li, X;Li, Y;Lin, M;Liu, L;Lo, D;Mizrachi, J;Mok, S;Naeemi, M;Nicovich, P;Palaniswamy, R;Palmer, J;Qi, X;Shen, E;Sun, Y;Tao, H;Wakemen, W;Wang, Y;Xie, P;Yao, S;Yuan, J;Zhu, M;Ng, L;Zhang, L;Lim, B;Hawrylycz, M;Gong, H;Gee, J;Kim, Y;Peng, H;Chuang, K;Yang, X;Luo, Q;Mitra, P;Zador, A;Zeng, H;Ascoli, G;Huang, Z;Osten, P;Harris, J;Dong, H.
bioRxiv,
(2020)
An essential step toward understanding brain function is to establish a cellular-resolution structural framework upon which multi-scale and multi-modal information spanning molecules, cells, circuits and systems can be integrated and interpreted. Here, through a collaborative effort from the Brain Initiative Cell Census Network (BICCN), we derive a comprehensive cell type-based description of one brain structure – the primary motor cortex upper limb area (MOp-ul) of the mouse. Applying state-of-the-art labeling, imaging, computational, and neuroinformatics tools, we delineated the MOp-ul within the Mouse Brain 3D Common Coordinate Framework (CCF). We defined over two dozen MOp-ul projection neuron (PN) types by their anterograde targets; the spatial distribution of their somata defines 11 cortical sublayers, a significant refinement of the classic notion of cortical laminar organization. We further combine multiple complementary tracing methods (classic tract tracing, cell type-based anterograde, retrograde, and transsynaptic viral tracing, high-throughput BARseq, and complete single cell reconstruction) to systematically chart cell type-based MOp input-output streams. As PNs link distant brain regions at synapses as well as host cellular gene expression, our construction of a PN type resolution MOp-ul wiring diagram will facilitate an integrated analysis of motor control circuitry across the molecular, cellular, and systems levels. This work further provides a roadmap towards a cellular resolution description of mammalian brain architecture.
Fattinger, SA;Böck, D;Di Martino, ML;Deuring, S;Samperio Ventayol, P;Ek, V;Furter, M;Kreibich, S;Bosia, F;Müller-Hauser, AA;Nguyen, BD;Rohde, M;Pilhofer, M;Hardt, WD;Sellin, ME.
PLoS Pathog,
16
(5)
, e1008503
(2020)
Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host’s gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through “discreet-invasion”. This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context.