Anti-GFP Antibody (A121560)

Resistance to radiotherapy and chemotherapy is a common problem in the treatment of cancer in humans and companion animals, including cats. There is thus an urgent need to develop new treatments. Molecularly targeted therapies hold the promise of high specificity and significant cancer-killing effects. Accumulating evidence shows that DNA double-strand break (DSB) repair proteins, which function in Ku-dependent non-homologous DNA-end joining (NHEJ), are potential target molecules for next-generation cancer therapies. Although cancer radioresistance in cats has been previously described, there are no reports on feline Ku-dependent NHEJ. Here, we cloned and sequenced feline XLF cDNA and characterized X-ray repair cross-complementing protein 4-like factor (XLF), which is one of the core NHEJ proteins. We demonstrated that feline XLF localizes to the nuclei of feline cells and that feline XLF immediately accumulates at laser-induced DSB sites in a Ku-dependent manner. Amino acid sequence alignment analysis showed that feline XLF has only 80.9% identity with human XLF protein, while the predicted nuclear localization signal and putative 14-3-3-binding motif are perfectly conserved among human, cat, dog, chimpanzee, and mouse. These findings are consistent with the hypothesis that regulation of subcellular localization is important for the function of XLF. Furthermore, these findings may be useful in clarifying the mechanisms underlying feline Ku-dependent DSB repair and feline cell radioresistance, and possibly facilitate the development of new molecularly targeted therapies that target common proteins in human and feline cancers.

© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Influenza A virus has an eight-partite RNA genome that during viral assembly forms a complex containing one copy of each RNA. Genome assembly is a selective process driven by RNA-RNA interactions and is hypothesized to lead to discrete punctate structures scattered through the cytosol. Here, we show that contrary to the accepted view, formation of these structures precedes RNA-RNA interactions among distinct viral ribonucleoproteins (vRNPs), as they assemble in cells expressing only one vRNP type. We demonstrate that these viral inclusions display characteristics of liquid organelles, segregating from the cytosol without a delimitating membrane, dynamically exchanging material and adapting fast to environmental changes. We provide evidence that viral inclusions develop close to endoplasmic reticulum (ER) exit sites, depend on continuous ER-Golgi vesicular cycling and do not promote escape to interferon response. We propose that viral inclusions segregate vRNPs from the cytosol and facilitate selected RNA-RNA interactions in a liquid environment.

The cochlea is innervated by type I and type II afferent neurons. Type I afferents are myelinated, larger diameter neurons that send a single dendrite to contact a single inner hair cell, whereas unmyelinated type II afferents are fewer in number and receive input from many outer hair cells. This strikingly differentiated innervation pattern strongly suggests specialized functions. Those functions could be investigated with specific genetic markers that enable labeling and manipulating each afferent class without significantly affecting the other. Here three mouse models were characterized and tested for specific labeling of either type I or type II cochlear afferents. Nos1^CreER mice showed selective labeling of type I afferent fibers, Slc6a4-GFP mice labeled type II fibers with a slight preference for the apical cochlea, and Drd2-Cre mice selectively labeled type II afferent neurons nearer the cochlear base. In conjunction with the Th^2A-CreER and CGRPa-EGFP lines described previously for labeling type II fibers, the mouse lines reported here comprise a promising toolkit for genetic manipulations of type I and type II cochlear afferent fibers.

The inwardly rectifying K⁺ channel K_ir4.1 is broadly expressed by CNS glia and deficits in K_ir4.1 lead to seizures and myelin vacuolization. However, the role of oligodendrocyte K_ir4.1 channels in controlling myelination and K⁺ clearance in white matter has not been defined. Here, we show that selective deletion of K_ir4.1 from oligodendrocyte progenitors (OPCs) or mature oligodendrocytes did not impair their development or disrupt the structure of myelin. However, mice lacking oligodendrocyte K_ir4.1 channels exhibited profound functional impairments, including slower clearance of extracellular K⁺ and delayed recovery of axons from repetitive stimulation in white matter, as well as spontaneous seizures, a lower seizure threshold, and activity-dependent motor deficits. These results indicate that K_ir4.1 channels in oligodendrocytes play an important role in extracellular K⁺ homeostasis in white matter, and that selective loss of this channel from oligodendrocytes is sufficient to impair K⁺ clearance and promote seizures.

© 2018, Larson et al.

The endolysosomal system sustains the reabsorptive activity of specialized epithelial cells. Lysosomal storage diseases such as nephropathic cystinosis cause a major dysfunction of epithelial cells lining the kidney tubule, resulting in massive losses of vital solutes in the urine. The mechanisms linking lysosomal defects and epithelial dysfunction remain unknown, preventing the development of disease-modifying therapies. Here we demonstrate, by combining genetic and pharmacologic approaches, that lysosomal dysfunction in cystinosis results in defective autophagy-mediated clearance of damaged mitochondria. This promotes the generation of oxidative stress that stimulates Ga12/Src-mediated phosphorylation of tight junction ZO-1 and triggers a signaling cascade involving ZO-1-associated Y-box factor ZONAB, which leads to cell proliferation and transport defects. Correction of the primary lysosomal defect, neutralization of mitochondrial oxidative stress, and blockage of tight junction-associated ZONAB signaling rescue the epithelial function. We suggest a link between defective lysosome-autophagy degradation pathways and epithelial dysfunction, providing new therapeutic perspectives for lysosomal storage disorders.

Type II spiral ganglion neurons (SGNs) are small caliber, unmyelinated afferents that extend dendritic arbors hundreds of microns along the cochlear spiral, contacting many outer hair cells (OHCs). Despite these many contacts, type II afferents are insensitive to sound and only weakly depolarized by glutamate release from OHCs. Recent studies suggest that type II afferents may be cochlear nociceptors, and can be excited by ATP released during tissue damage, by analogy to somatic pain-sensing C-fibers. The present work compares the expression patterns among cochlear type II afferents of two genes found in C-fibers: calcitonin-related polypeptide alpha (Calca/Cgrpa), specific to pain-sensing C-fibers, and tyrosine hydroxylase (Th), specific to low-threshold mechanoreceptive C-fibers, which was shown previously to be a selective biomarker of type II versus type I cochlear afferents (Vyas et al., ). Whole-mount cochlear preparations from 3-week- to 2-month-old CGRPa-EGFP (GENSAT) mice showed expression of Cgrpa in a subset of SGNs with type II-like peripheral dendrites extending beneath OHCs. Double labeling with other molecular markers confirmed that the labeled SGNs were neither type I SGNs nor olivocochlear efferents. Cgrpa starts to express in type II SGNs before hearing onset, but the expression level declines in the adult. The expression patterns of Cgrpa and Th formed opposing gradients, with Th being preferentially expressed in apical and Cgrpa in basal type II afferent neurons, indicating heterogeneity among type II afferent neurons. The expression of Th and Cgrpa was not mutually exclusive and co-expression could be observed, most abundantly in the middle cochlear turn.

© 2017 Wiley Periodicals, Inc.

Astrocytes extend highly branched processes that form functionally isolated microdomains, facilitating local homeostasis by redistributing ions, removing neurotransmitters, and releasing factors to influence blood flow and neuronal activity. Microdomains exhibit spontaneous increases in calcium (Ca²⁺), but the mechanisms and functional significance of this localized signaling are unknown. By developing conditional, membrane-anchored GCaMP3 mice, we found that microdomain activity that occurs in the absence of inositol triphosphate (IP3)-dependent release from endoplasmic reticulum arises through Ca²⁺ efflux from mitochondria during brief openings of the mitochondrial permeability transition pore. These microdomain Ca²⁺ transients were facilitated by the production of reactive oxygen species during oxidative phosphorylation and were enhanced by expression of a mutant form of superoxide dismutase 1 (SOD1 G93A) that causes astrocyte dysfunction and neurodegeneration in amyotrophic lateral sclerosis (ALS). By localizing mitochondria to microdomains, astrocytes ensure local metabolic support for energetically demanding processes and enable coupling between metabolic demand and Ca²⁺ signaling events.

Copyright © 2017 Elsevier Inc. All rights reserved.

Lamin B receptor (LBR), an inner nuclear membrane (INM) protein, contributes to the functional integrity of the nucleus by tethering heterochromatin to the nuclear envelope. We have previously reported that the depletion of embryonic large molecule derived from yolk sac (ELYS; also known as AHCTF1), a component of the nuclear pore complex, from cells perturbs the localization of LBR to the INM, but little is known about the underlying molecular mechanism. In this study, we found that the depletion of ELYS promoted LBR phosphorylation at the residues known to be phosphorylated by cyclin-dependent kinase (CDK) and serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2, respectively). These phosphorylation events were most likely to be counter-balanced by protein phosphatase 1 (PP1), and the depletion of PP1 from cells consistently caused the mislocalization of LBR. These observations point to a new mechanism regulating the localization of LBR, which is governed by an ELYS-mediated phosphorylation network. This phosphorylation-dependent coordination between INM proteins and the nuclear pore complex might be important for the integrity of the nucleus.

© 2016. Published by The Company of Biologists Ltd.

Acoustic information propagates from the ear to the brain via spiral ganglion neurons that innervate hair cells in the cochlea. These afferents include unmyelinated type II fibers that constitute 5 % of the total, the majority being myelinated type I neurons. Lack of specific genetic markers of type II afferents in the cochlea has been a roadblock in studying their functional role. Unexpectedly, type II afferents were visualized by reporter proteins induced by tyrosine hydroxylase (TH)-driven Cre recombinase. The present study was designed to determine whether TH-driven Cre recombinase (TH-2A-CreER) provides a selective and reliable tool for identification and genetic manipulation of type II rather than type I cochlear afferents. The "TH-2A-CreER neurons" radiated from the spiral lamina, crossed the tunnel of Corti, turned towards the base of the cochlea, and traveled beneath the rows of outer hair cells. Neither the processes nor the somata of TH-2A-CreER neurons were labeled by antibodies that specifically labeled type I afferents and medial efferents. TH-2A-CreER-positive processes partially co-labeled with antibodies to peripherin, a known marker of type II afferents. Individual TH-2A-CreER neurons gave off short branches contacting 7-25 outer hair cells (OHCs). Only a fraction of TH-2A-CreER boutons were associated with CtBP2-immunopositive ribbons. These results show that TH-2A-CreER provides a selective marker for type II versus type I afferents and can be used to describe the morphology and arborization pattern of type II cochlear afferents in the mouse cochlea.

The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Proliferation of cells under hypoxia is facilitated by metabolic adaptation, mediated by the transcriptional activator Hypoxia Inducible Factor-1 (HIF-1). HIF-1a, the inducible subunit of HIF-1 is regulated by oxygen as well as by oxygen-independent mechanisms involving phosphorylation. We have previously shown that CK1d phosphorylates HIF-1a in its N-terminus and reduces its affinity for its heterodimerization partner ARNT. To investigate the importance of this mechanism for cell proliferation under hypoxia, we visually monitored HIF-1a interactions within the cell nucleus using the in situ proximity ligation assay (PLA) and fluorescence recovery after photobleaching (FRAP). Both methods show that CK1d-dependent modification of HIF-1a impairs the formation of a chromatin binding HIF-1 complex. This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation. Inhibition of CK1d increases lipid droplet formation and proliferation of both cancer and normal cells specifically under hypoxia and in an HIF-1a- and lipin-1-dependent manner. These data reveal a novel role for CK1d in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.

Copyright © 2015 Elsevier Inc. All rights reserved.

In overloaded and regenerating muscle, the generation of new myonuclei depends on muscle satellite cells (MuSCs). Because MuSC behaviors in these two environments have not been considered separately, MuSC behaviors in overloaded muscle remain unexamined. Here, we show that most MuSCs in overloaded muscle, unlike MuSCs in regenerating muscle, proliferate in the absence of MyoD expression. Mechanistically, MuSCs in overloaded muscle sustain the expression of Heyl, a Notch effector gene, to suppress MyoD expression, which allows effective MuSC proliferation on myofibers and beneath the basal lamina. Although Heyl-knockout mice show no impairment in an injury model, in a hypertrophy model, their muscles harbor fewer new MuSC-derived myonuclei due to increased MyoD expression and diminished proliferation, which ultimately causes blunted hypertrophy. Our results show that sustained HeyL expression is critical for MuSC proliferation specifically in overloaded muscle, and thus indicate that the MuSC-proliferation mechanism differs in overloaded and regenerating muscle.

© 2019, Fukuda et al.

The hypothalamus plays a key role in the detection of energy substrates to regulate energy homeostasis. Tanycytes, the hypothalamic ependymo-glia, are located at a privileged position to integrate multiple peripheral inputs. We observed that tanycytes produce and secrete Fgf21 and are located close to Fgf21-sensitive neurons. Fasting, likely via the increase in circulating fatty acids, regulates this central Fgf21 production. Tanycytes store palmitate in lipid droplets and oxidize it, leading to the activation of a reactive oxygen species (ROS)/p38-MAPK signaling pathway, which is essential for tanycytic Fgf21 expression upon palmitate exposure. Tanycytic Fgf21 deletion triggers an increase in lipolysis, likely due to impaired inhibition of key neurons during fasting. Mice deleted for tanycytic Fgf21 exhibit increased energy expenditure and a reduction in fat mass gain, reminiscent of a browning phenotype. Our results suggest that tanycytes sense free fatty acids to maintain body lipid homeostasis through Fgf21 signaling within the hypothalamus.

Copyright © 2019 Elsevier Inc. All rights reserved.

Delta like non-canonical Notch ligand 1 (Dlk1) is a paternally expressed gene which is also known as preadipocyte factor 1 (Pref-1). The accumulation of adipocytes and expression of Dlk1 in regenerating muscle suggests a correlation between fat accumulation and Dlk1 expression in the muscle. Additionally, mice overexpressing Dlk1 show increased muscle weight, while Dlk1-null mice exhibit decreased body weight and muscle mass, indicating that Dlk1 is a critical factor in regulating skeletal muscle mass during development. The muscle regeneration process shares some features with muscle development. However, the role of Dlk1 in regeneration processes remains controversial. Here, we show that mesenchymal progenitors also known as adipocyte progenitors exclusively express Dlk1 during muscle regeneration. Eliminating developmental effects, we used conditional depletion models to examine the specific roles of Dlk1 in muscle stem cells or mesenchymal progenitors. Unexpectedly, deletion of Dlk1 in neither the muscle stem cells nor the mesenchymal progenitors affected the regenerative ability of skeletal muscle. In addition, fat accumulation was not increased by the loss of Dlk1. Collectively, Dlk1 plays essential roles in muscle development, but does not greatly impact regeneration processes and adipogenic differentiation in adult skeletal muscle regeneration.

Influenza A is a rapidly evolving virus that is successful in provoking periodic epidemics and occasional pandemics in humans. Viral assembly is complex as the virus incorporates an eight-partite genome of RNA (in the form of viral ribonucleoproteins, vRNPs), and viral genome assembly - with its implications to public health - is not completely understood. It has previously been reported that vRNPs are transported to the cell surface on Rab11-containing vesicles by using microtubules but, so far, no molecular motor has been assigned to the process. Here, we have identified KIF13A, a member of the kinesin-3 family, as the first molecular motor to efficiently transport vRNP-Rab11 vesicles during infection with influenza A. Depletion of KIF13A resulted in reduced viral titers and less accumulation of vRNPs at the cell surface, without interfering with the levels of other viral proteins at sites of viral assembly. In addition, when overexpressed and following two separate approaches to displace vRNP-Rab11 vesicles, KIF13A increased levels of vRNP at the plasma membrane. Together, our results show that KIF13A plays an important role in the transport of influenza A vRNPs, a crucial step for viral assembly.This article has an associated First Person interview with the first author of the paper.

© 2017. Published by The Company of Biologists Ltd.

Influenza A virus assembly is an unclear process, whereby individual virion components form an infectious particle. The segmented nature of the influenza A genome imposes a problem to assembly because it requires packaging of eight distinct RNA particles (vRNPs). It also allows genome mixing from distinct parental strains, events associated with influenza pandemic outbreaks. It is important to public health to understand how segmented genomes assemble, a process that is dependent on the transport of components to assembly sites. Previously, it has been shown that vRNPs are carried by recycling endosome vesicles, resulting in a change of Rab11 distribution. Here, we describe that vRNP binding to recycling endosomes impairs recycling endosome function, by competing for Rab11 binding with family-interacting proteins, and that there is a causal relationship between Rab11 ability to recruit family-interacting proteins and Rab11 redistribution. This competition reduces recycling sorting at an unclear step, resulting in clustering of single- and double-membraned vesicles. These morphological changes in Rab11 membranes are indicative of alterations in protein and lipid homeostasis during infection. Vesicular clustering creates hotspots of the vRNPs that need to interact to form an infectious particle.

© 2016. Published by The Company of Biologists Ltd.

X-linked Charcot-Marie-Tooth disease (CMTX1) results from numerous mutations in the GJB1 gene encoding the gap junction protein connexin32 (Cx32) and is one of the commonest forms of inherited neuropathy. Owing to the expression of Cx32 not only in Schwann cells but also in oligodendrocytes, a subset of CMT1X patients develops central nervous system (CNS) clinical manifestations in addition to peripheral neuropathy. While most GJB1 mutations appear to cause peripheral neuropathy through loss of Cx32 function, the cellular mechanisms underlying the CNS manifestations remain controversial. A novel start codon GJB1 mutation (p.Met1Ile) has been found in a CMT1X patient presenting with recurrent episodes of transient encephalomyelitis without apparent signs of peripheral neuropathy. In order to clarify the functional consequences of this mutation, we examined the cellular expression of two different constructs cloned from genomic DNA including the mutated start codon. None of the cloned constructs resulted in detectable expression of Cx32 by immunocytochemistry or immunoblot, although mRNA was produced at normal levels. Furthermore, co-expression with the other major oligodendrocyte connexin, Cx47, had no negative effect on GJ formation by Cx47. Finally, lysosomal and proteasomal inhibition in cells expressing the start codon mutant constructs failed to recover any detection of Cx32 as a result of impaired protein degradation. Our results indicate that the Cx32 start codon mutation is equivalent to a complete loss of the protein with failure of translation, although transcription is not impaired. Thus, complete loss of Cx32 function is sufficient to produce CNS dysfunction with clinical manifestations.

Astrocytes perform crucial supportive functions, including neurotransmitter clearance, ion buffering, and metabolite delivery. They can also influence blood flow and neuronal activity by releasing gliotransmitters in response to intracellular Ca(2+) transients. However, little is known about how astrocytes are engaged during different behaviors in vivo. Here we demonstrate that norepinephrine primes astrocytes to detect changes in cortical network activity. We show in mice that locomotion triggers simultaneous activation of astrocyte networks in multiple brain regions. This global stimulation of astrocytes was inhibited by alpha-adrenoceptor antagonists and abolished by depletion of norepinephrine from the brain. Although astrocytes in visual cortex of awake mice were rarely engaged when neurons were activated by light stimulation alone, pairing norepinephrine release with light stimulation markedly enhanced astrocyte Ca(2+) signaling. Our findings indicate that norepinephrine shifts the gain of astrocyte networks according to behavioral state, enabling astrocytes to respond to local changes in neuronal activity.

Copyright © 2014 Elsevier Inc. All rights reserved.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of humans that uses a type III secretion (T3S) system to manipulate host cells through the delivery of effector proteins into their cytosol and membranes. The function of T3S systems depends on small bacterial cytosolic chaperone-like proteins, which bind T3S substrates and ensure their appropriate secretion. To find novel T3S chaperone-substrate complexes of C. trachomatis we first searched its genome for genes encoding proteins with features of T3S chaperones. We then systematically tested for interactions between candidate chaperones and chlamydial T3S substrates by bacterial two-hybrid. This revealed interactions between Slc1 (a known T3S chaperone) or CT584 and several T3S substrates. Co-immunoprecipitation after protein expression in Yersinia enterocolitica and protein overlay binding assays indicated that Slc1 interacted with the N-terminal region of the known T3S substrates Tarp (a previously described substrate of Slc1), CT694, and CT695, and that CT584 interacted with a central region of CT082, which we identified as a C. trachomatis T3S substrate using Y. enterocolitica as a heterologous system. Further T3S assays in Yersinia indicated that Slc1 or CT584 increased the amount of secreted Tarp, CT694, and CT695, or CT082, respectively. Expression of CT584 increased the intra-bacterial stability of CT082, while Slc1 did not affect the stability of its substrates. Overall, this indicated that in C. trachomatis Slc1 is a chaperone of multiple T3S substrates and that CT584 is a chaperone of the newly identified T3S substrate CT082.