Septins as membrane influencers: direct play or in association with other cytoskeleton partners.

The cytoskeleton comprises three polymerizing structures that have been studied for a long time, actin microfilaments, microtubules and intermediate filaments, plus more recently investigated dynamic assemblies like septins or the endocytic-sorting complex required for transport (ESCRT) complex. These filament-forming proteins control several cell functions through crosstalks with each other and with membranes. In this review, we report recent works that address how septins bind to membranes, and influence their shaping, organization, properties and functions, either by binding to them directly or indirectly through other cytoskeleton elements.

α-Tubulin acetylation on lysine 40 controls cardiac glucose uptake.

Our group previously demonstrated that an excess of nutrients, as observed in diabetes, provokes an increase in cardiac protein acetylation responsible for a reduced insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The acetylated proteins involved in this event have yet not been identified. α-Tubulin is a promising candidate as a major cytoskeleton component involved, among other things, in the translocation of GLUT4-containing vesicles from their intracellular pools toward the plasma membrane. Moreover, α-tubulin is known to be acetylated, Lys40 (K40) being its best characterized acetylated residue. The present work sought to evaluate the impact of α-tubulin K40 acetylation on cardiac glucose entry, with a particular interest in GLUT4 translocation. First, we observed that a mouse model of high-fat diet-induced obesity presented an increase in cardiac α-tubulin K40 acetylation level. We next showed that treatment of insulin-sensitive primary cultured adult rat cardiomyocytes with tubacin, a specific tubulin acetylation inducer, reduced insulin-stimulated glucose uptake and GLUT4 translocation. Conversely, decreasing α-tubulin K40 acetylation by expressing a nonacetylable dominant form of α-tubulin (mCherry α-tubulin K40A mutant) remarkably intensified insulin-induced glucose transport. Finally, mCherry α-tubulin K40A expression similarly improved glucose transport in insulin-resistant cardiomyocytes or after AMP-activated protein kinase activation. Taken together, our study demonstrates that modulation of α-tubulin K40 acetylation level affects glucose transport in cardiomyocytes, offering new putative therapeutic insights regarding modulation of glucose metabolism in insulin-resistant and diabetic hearts.NEW & NOTEWORTHY Acetylation level of α-tubulin on K40 is increased in the heart of a diet-induced mouse model of type 2 diabetes. Pharmacological stimulation of α-tubulin K40 acetylation lowers insulin-mediated GLUT4 vesicles translocation to the plasma membrane, reducing glucose transport. Expressing a nonacetylable dominant form of α-tubulin boosts glucose uptake in both insulin-sensitive and insulin-resistant cardiomyocytes.

Essential role of hyperacetylated microtubules in innate immunity escape orchestrated by the EBV-encoded BHRF1 protein.

Innate immunity constitutes the first line of defense against viruses, in which mitochondria play an important role in the induction of the interferon (IFN) response. BHRF1, a multifunctional viral protein expressed during Epstein-Barr virus reactivation, modulates mitochondrial dynamics and disrupts the IFN signaling pathway. Mitochondria are mobile organelles that move through the cytoplasm thanks to the cytoskeleton and in particular the microtubule (MT) network. MTs undergo various post-translational modifications, among them tubulin acetylation. In this study, we demonstrated that BHRF1 induces MT hyperacetylation to escape innate immunity. Indeed, the expression of BHRF1 induces the clustering of shortened mitochondria next to the nucleus. This "mito-aggresome" is organized around the centrosome and its formation is MT-dependent. We also observed that the α-tubulin acetyltransferase ATAT1 interacts with BHRF1. Using ATAT1 knockdown or a non-acetylatable α-tubulin mutant, we demonstrated that this hyperacetylation is necessary for the mito-aggresome formation. Similar results were observed during EBV reactivation. We investigated the mechanism leading to the clustering of mitochondria, and we identified dyneins as motors that are required for mitochondrial clustering. Finally, we demonstrated that BHRF1 needs MT hyperacetylation to block the induction of the IFN response. Moreover, the loss of MT hyperacetylation blocks the localization of autophagosomes close to the mito-aggresome, impeding BHRF1 to initiate mitophagy, which is essential to inhibiting the signaling pathway. Therefore, our results reveal the role of the MT network, and its acetylation level, in the induction of a pro-viral mitophagy.

Time-modulated excitation for enhanced single-molecule localization microscopy.

Structured illumination in single-molecule localization microscopy provides new information on the position of molecules and thus improves the localization precision compared to standard localization methods. Here, we used a time-shifted sinusoidal excitation pattern to modulate the fluorescence signal of the molecules whose position information is carried by the phase and recovered by synchronous demodulation. We designed two flexible fast demodulation systems located upstream of the camera, allowing us to overcome the limiting camera acquisition frequency and thus to maximize the collection of photons in the demodulation process. The temporally modulated fluorescence signal was then sampled synchronously on the same image, repeatedly during acquisition. This microscopy, called ModLoc, allows us to experimentally improve the localization precision by a factor of 2.4 in one direction, compared to classical Gaussian fitting methods. A temporal study and an experimental demonstration both show that the short lifetimes of the molecules in blinking regimes impose a modulation frequency in the kilohertz range, which is beyond the reach of current cameras. A demodulation system operating at these frequencies would thus be necessary to take full advantage of this new localization approach. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Inherited Proteoglycan Biosynthesis Defects-Current Laboratory Tools and Bikunin as a Promising Blood Biomarker.

Proteoglycans consist of proteins linked to sulfated glycosaminoglycan chains. They constitute a family of macromolecules mainly involved in the architecture of organs and tissues as major components of extracellular matrices. Some proteoglycans also act as signaling molecules involved in inflammatory response as well as cell proliferation, adhesion, and differentiation. Inborn errors of proteoglycan metabolism are a group of orphan diseases with severe and irreversible skeletal abnormalities associated with multiorgan impairments. Identifying the gene variants that cause these pathologies proves to be difficult because of unspecific clinical symptoms, hardly accessible functional laboratory tests, and a lack of convenient blood biomarkers. In this review, we summarize the molecular pathways of proteoglycan biosynthesis, the associated inherited syndromes, and the related biochemical screening techniques, and we focus especially on a circulating proteoglycan called bikunin and on its potential as a new biomarker of these diseases.

Cytoskeleton and Associated Proteins: Pleiotropic JNK Substrates and Regulators.

This review extensively reports data from the literature concerning the complex relationships between the stress-induced c-Jun N-terminal kinases (JNKs) and the four main cytoskeleton elements, which are actin filaments, microtubules, intermediate filaments, and septins. To a lesser extent, we also focused on the two membrane-associated cytoskeletons spectrin and ESCRT-III. We gather the mechanisms controlling cytoskeleton-associated JNK activation and the known cytoskeleton-related substrates directly phosphorylated by JNK. We also point out specific locations of the JNK upstream regulators at cytoskeletal components. We finally compile available techniques and tools that could allow a better characterization of the interplay between the different types of cytoskeleton filaments upon JNK-mediated stress and during development. This overview may bring new important information for applied medical research.

Cdc42 and its BORG2 and BORG3 effectors control the subcellular localization of septins between actin stress fibers and microtubules.

Cell resistance to taxanes involves several complementary mechanisms, among which septin relocalization from actin stress fibers to microtubules plays an early role. By investigating the molecular mechanism underlying this relocalization, we found that acute paclitaxel treatment triggers the release from stress fibers and subsequent proteasome-mediated degradation of binder of Rho GTPases 2 (BORG2)/Cdc42 effector protein 3 (Cdc42EP3) and to a lesser extent of BORG3/Cdc42EP5, two Cdc42 effectors that link septins to actin in interphase cells. BORG2 or BORG3 silencing not only caused septin detachment from stress fibers but also mimicked the effects of paclitaxel by triggering both septin relocalization to microtubules and significant drug resistance. Conversely, BORG2 or BORG3 overexpression retained septins on actin fibers even after paclitaxel treatment, without affecting paclitaxel sensitivity. We found that drug-induced inhibition of Cdc42 resulted in a drop in BORG2 level and in the relocalization of septins to microtubules. Accordingly, although septins relocalized when overexpressing an inactive mutant of Cdc42, the expression of a constitutively active mutant acted locally at actin stress fibers to prevent septin release, even after paclitaxel treatment. These findings reveal the role of Cdc42 upstream of BORG2 and BORG3 in controlling the interplay between septins, actin fibers, and microtubules in basal condition and in response to taxanes.

Decrease of neuronal FKBP4/FKBP52 modulates perinuclear lysosomal positioning and MAPT/Tau behavior during MAPT/Tau-induced proteotoxic stress.

Defects of autophagy-lysosomal protein degradation are thought to contribute to the pathogenesis of several neurodegenerative diseases, and the accumulation of aggregation prone proteins such as MAPT/Tau in Alzheimer disease (AD). We previously showed the localization of the immunophilin FKBP4/FKBP52 in the lysosomal system of healthy human neurons suggesting its possible role in lysosome function. We also showed that decreased FKBP4 levels in AD brain neurons correlate with abnormal MAPT accumulation and aggregation. In this study, we demonstrate that FKBP4 decrease in a human neuronal cell line (SH-SY5Y) and in dorsal root ganglion (DRG) neurons from human MAPTP301S transgenic mice affected the function of the autophagy-lysosomal system under MAPT induced proteotoxic stress conditions. We show that acute MAPT accumulation in SH-SY5Y cells induced perinuclear clustering of lysosomes, triggered FKBP4 localization around the clusters and its colocalization with MAPT and MAP1LC3/LC3-positive autophagic vesicles; a similar FKBP4 localization was detected in some AD brain neurons. We demonstrate that FKBP4 decrease altered lysosomal clustering along with MAPT and MAP1LC3 secretion increase. Although ectopic FKBP4 expression could not induce autophagy under our experimental conditions, it prevented MAPT secretion after MAPT accumulation in SH-SY5Y cells implying a regulatory role of FKBP4 on MAPT secretion. Finally, we observe that FKBP4 deficiency decreased MAP1LC3-II expression and provoked MAPT accumulation during long-term stress in mouse DRG neurons. We hypothesize that the abnormal FKBP4 decrease observed in AD brain neurons might hinder autophagy efficiency and contribute to the progression of the tauopathy by modulating MAPT secretion and accumulation during MAPT pathogenesis.Abbreviations: AD: Alzheimer disease; AKT/protein kinase B: AKT serine/threonine kinase; ALP: Autophagy-lysosomal pathway; ATG: autophagy-related; BafA1: bafilomycin A1; CQ: chloroquine; CTSD: cathepsin D; DIV: days in vitro; DRG: dorsal root ganglion neurons; Dox: doxycycline; DNAJC5: DnaJ heat shock protein family (Hsp40) member C5; EL: empty lentiviral vectors; ENO2/NSE: enolase 2, gamma neuronal; FKBP4/FKBP52: FKBP prolyl isomerase 4; FTLD-Tau: frontotemporal lobar degeneration with Tau pathology; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPT/Tau: microtubule associated protein tau; MTT: tetrazolium salt; NFTs: neurofibrillary tangles; RPE-1: retinal pigment epithelial cells; shRNA: small-hairpin ribonucleic acid; SQSTM1/p62: sequestosome 1; SD: standard deviation; SEM: standard error of the mean; SH-SY5Y: human neuroblastoma cells; Sh1 or Sh2: Lentiviral shRNA vectors inducing FKBP4 decrease; SH-52GFP: MAPT/Tau-inducible SH-SY5Y cell line constitutively expressing FKBP4-GFP; TUBB3/ßIII tubulin: tubulin beta 3 class III; UPS: ubiquitin-proteasome system.

BHRF1, a BCL2 viral homolog, disturbs mitochondrial dynamics and stimulates mitophagy to dampen type I IFN induction.

Mitochondria respond to many cellular functions and act as central hubs in innate immunity against viruses. This response is notably due to their role in the activation of interferon (IFN) signaling pathways through the activity of MAVS (mitochondrial antiviral signaling protein) present at the mitochondrial surface. Here, we report that the BHRF1 protein, a BCL2 homolog encoded by Epstein-Barr virus (EBV), inhibits IFNB/IFN-ß induction by targeting the mitochondria. Indeed, we have demonstrated that BHRF1 expression modifies mitochondrial dynamics and stimulates DNM1L/Drp1-mediated mitochondrial fission. Concomitantly, we have shown that BHRF1 is pro-autophagic because it stimulates the autophagic flux by interacting with BECN1/Beclin 1. In response to the BHRF1-induced mitochondrial fission and macroautophagy/autophagy stimulation, BHRF1 drives mitochondrial network reorganization to form juxtanuclear mitochondrial aggregates known as mito-aggresomes. Mitophagy is a cellular process, which can specifically sequester and degrade mitochondria. Our confocal studies uncovered that numerous mitochondria are present in autophagosomes and acidic compartments using BHRF1-expressing cells. Moreover, mito-aggresome formation allows the induction of mitophagy and the accumulation of PINK1 at the mitochondria. As BHRF1 modulates the mitochondrial fate, we explored the effect of BHRF1 on innate immunity and showed that BHRF1 expression could prevent IFNB induction. Indeed, BHRF1 inhibits the IFNB promoter activation and blocks the nuclear translocation of IRF3 (interferon regulatory factor 3). Thus, we concluded that BHRF1 can counteract innate immunity activation by inducing fission of the mitochondria to facilitate their sequestration in mitophagosomes for degradation.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; BCL2: BCL2 apoptosis regulator; CARD: caspase recruitment domain; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CI: compaction index; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride; DDX58/RIG-I: DExD/H-box helicase 58; DNM1L/Drp1: dynamin 1 like; EBSS: Earle's balanced salt solution; EBV: Epstein-Barr virus; ER: endoplasmic reticulum; EV: empty vector; GFP: green fluorescent protein; HEK: human embryonic kidney; IFN: interferon; IgG: immunoglobulin G; IRF3: interferon regulatory factor 3; LDHA: lactate dehydrogenase A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOM: mitochondrial outer membrane; PINK1: PTEN induced kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Serum bikunin isoforms in congenital disorders of glycosylation and linkeropathies.

Bikunin (Bkn) isoforms are serum chondroitin sulfate (CS) proteoglycans synthesized by the liver. They include two light forms, that is, the Bkn core protein and the Bkn linked to the CS chain (urinary trypsin inhibitor [UTI]), and two heavy forms, that is, pro-α-trypsin inhibitor and inter-α-trypsin inhibitor, corresponding to UTI esterified by one or two heavy chains glycoproteins, respectively. We previously showed that the Western-blot analysis of the light forms could allow the fast and easy detection of patients with linkeropathy, deficient in enzymes involved in the synthesis of the initial common tetrasaccharide linker of glycosaminoglycans. Here, we analyzed all serum Bkn isoforms in a context of congenital disorders of glycosylation (CDG) and showed very specific abnormal patterns suggesting potential interests for their screening and diagnosis. In particular, genetic deficiencies in V-ATPase (ATP6V0A2-CDG, CCDC115-CDG, ATP6AP1-CDG), in Golgi manganese homeostasis (TMEM165-CDG) and in the N-acetyl-glucosamine Golgi transport (SLC35A3-CDG) all share specific abnormal Bkn patterns. Furthermore, for each studied linkeropathy, we show that the light abnormal Bkn could be further in-depth characterized by two-dimensional electrophoresis. Moreover, besides being interesting as a specific biomarker of both CDG and linkeropathies, Bkn isoforms' analyses can provide new insights into the pathophysiology of the aforementioned diseases.

Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue.

The stress-induced c-Jun N-terminal kinase (JNK) controls microtubule dynamics by enhancing both microtubule growth and rescues. Here, we show that upon cell stress, JNK directly phosphorylates the microtubule rescue factor CLIP-170 in its microtubule-binding domain to increase its rescue-promoting activity. Phosphomimetic versions of CLIP-170 enhance its ability to promote rescue events in vitro and in cells. Furthermore, while phosphomimetic mutations do not alter CLIP-170's capability to form comets at growing microtubule ends, both phosphomimetic mutations and JNK activation increase the occurrence of CLIP-170 remnants on the microtubule lattice at the rear of comets. As the CLIP-170 remnants, which are potential sites of microtubule rescue, display a shorter lifetime when CLIP-170 is phosphorylated, we propose that instead of acting at the time of rescue occurrence, CLIP-170 would rather contribute in preparing the microtubule lattice for future rescues at these predetermined sites.

Insight into microtubule nucleation from tubulin-capping proteins.

Nucleation is one of the least understood steps of microtubule dynamics. It is a kinetically unfavorable process that is templated in the cell by the γ-tubulin ring complex or by preexisting microtubules; it also occurs in vitro from pure tubulin. Here we study the nucleation inhibition potency of natural or artificial proteins in connection with their binding mode to the longitudinal surface of α- or ß-tubulin. The structure of tubulin-bound CopN, a Chlamydia protein that delays nucleation, suggests that this protein may interfere with two protofilaments at the (+) end of a nucleus. Designed ankyrin repeat proteins that share a binding mode similar to that of CopN also impede nucleation, whereas those that target only one protofilament do not. In addition, an αRep protein predicted to target two protofilaments at the (-) end does not delay nucleation, pointing to different behaviors at both ends of the nucleus. Our results link the interference with protofilaments at the (+) end and the inhibition of nucleation.

Septin 9 has Two Polybasic Domains Critical to Septin Filament Assembly and Golgi Integrity.

Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins interact with PIs and then assemble and shape membranes is poorly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similar to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association is not dependent on these PB domains, but on putative PB-adjacent amphipathic helices. The presence of PB domains guarantees protein enrichment in PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings offer further insight into the role of septins in organelle morphology.

Early mitochondrial fragmentation is a potential in vitro biomarker of environmental stress.

Mitochondria are essential dynamic organelles that ordinarily balance between fragmentation and fusion. Under stress conditions, a shift toward fragmentation or hyper-fusion is observed as a pro-survival reaction. Fragmentation of mitochondria occurs within minutes or hours after the beginning of the stress and occurs in response to a large number of stress stimuli, including those triggered by environmental contaminants. In this study, we tested whether the change in the mitochondrial phenotype, from tubular to fragmented, could be used as a potential environmental stress biomarker in cells and compared this response with the standard MTT-based viability assay. Firstly, we show that mitochondrial fragmentation induced by selected stressors not only increases with concentrations, but also correlates positively with the cytotoxicity. Secondly, we found that the mitochondrial fragmentation that occurs in the first hour of stress correlated with the viability measured after a 24-h stress, allowing the establishment of a linear relation between mitochondrial fragmentation at 1 h and the predictable associated cytotoxicity of environmental contaminants alone or in mixture. In conclusion, we have succeeded in developing a model of predictable 24 h-cytotoxicity given mitochondrial fragmentation at 1 h with a set of chemicals. This model has been successful applied to three environmental toxicants and to a set of two chemical mixtures. We thus propose that mitochondrial fragmentation is a response that could be used as an early in vitro biomarker of environmental stress for toxicants alone or in mixture.

Septin filament coalignment with microtubules depends on SEPT9_i1 and tubulin polyglutamylation, and is an early feature of acquired cell resistance to paclitaxel.

Cancer cell resistance to taxanes is a complex, multifactorial process, which results from the combination of several molecular and cellular changes. In breast cancer cells adapted to long-term paclitaxel treatment, we previously identified a new adaptive mechanism that contributes to resistance and involves high levels of tubulin tyrosination and long-chain polyglutamylation coupled with high levels of septin expression, especially that of SEPT9_i1. This in turn led to higher CLIP-170 and MCAK recruitment to microtubules to enhance microtubule dynamics and therefore counteract the stabilizing effects of taxanes. Here, we explored to which extent this new mechanism alone could trigger taxane resistance. We show that coupling septins (including SEPT9_i1) overexpression together with long-chain tubulin polyglutamylation induce significant paclitaxel resistance in several naive (taxane-sensitive) cell lines and accordingly stimulate the binding of CLIP-170 and MCAK to microtubules. Strikingly, such resistance was paralleled by a systematic relocalization of septin filaments from actin fibers to microtubules. We further show that this relocalization resulted from the overexpression of septins in a context of enhanced tubulin polyglutamylation and reveal that it could also be promoted by an acute treatment with paclitaxel of sensitve cell displaying a high basal level of SEPT9_i1. These findings point out the functional importance and the complex cellular dynamics of septins in the onset of cell resistance to death caused by microtubule-targeting antimitotic drugs of the taxane family.

Microtubule reorientation in the blue spotlight: Cutting and CLASPing at dynamic hot spots.

Microtubule reorientation into a longitudinal network during the phototropic response in Arabidopsis thaliana depends on their severing by katanin at crossovers. Lindeboom et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201805047) show that at newly generated plus ends, the anti-catastrophe activity of CLASP is essential for further growth.

Functional differences of short and long isoforms of spastin harboring missense mutation.

Mutations of the SPG4 (SPAST) gene encoding for spastin protein are the main causes of hereditary spastic paraplegia. Spastin binds to microtubules and severs them through the enzymatic activity of its AAA domain. Several missense mutations located in this domain lead to stable, nonsevering spastins that decorate a subset of microtubules, suggesting a possible negative gain-of-function mechanism for these mutants. Of the two main isoforms of spastin, only mutations of the long isoform, M1, are supposed to be involved in the onset of the pathology, leaving the role of the ubiquitously expressed shorter one, M87, not fully investigated and understood. Here, we show that two isoforms of spastin harboring the same missense mutation bind and bundle different subsets of microtubules in HeLa cells, and likely stabilize them by increasing the level of acetylated tubulin. However, only mutated M1 has the ability to interact with wild-type M1, and decorates a subset of perinuclear microtubules associated with the endoplasmic reticulum that display higher resistance to microtubule depolymerization and increased intracellular ionic strength, compared with those decorated by mutated M87. We further show that only mutated M1 decorates microtubules of proximal axons and dendrites, and strongly impairs axonal transport in cortical neurons through a mechanism likely independent of the microtubule-severing activity of this protein.