bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of autofluorescent proteins reveals a novel protein permeable pathway between cells in the lens core
Authors: Shestopalov Valery I. , Bassnett Steven
Published: April 2021

The lens of the eye is composed of concentric layers of tightly packed fiber cells. The oldest fibers, those in the lens core, lose their nuclei and other organelles during terminal differentiation. This is thought to ensure the clarity of the lens. The anucleated core fibers are sustained by gap junction-mediated communication with metabolically active cells near the lens surface. In this study, we expressed autofluorescent proteins and microinjected fluorescent markers to probe cell-to-cell communication in different regions of the developing lens. Our data indicate that a novel cell-cell diffusion pathway becomes patent in the lens core during development. This pathway is remarkable in that it is permeable to proteins and other large molecules and is thus distinct from gap junctions. Diffusion of large molecules probably occurs through regions of membrane fusion observed between neighboring cells in the lens core. Further direct evidence for a continuous plasma membrane system was provided by the observation that exogenous membrane proteins expressed in one core fiber cell were able to diffuse laterally into the membranes of adjacent fibers. Thus, the lens core appears to represent a true syncytium within which both membrane proteins and cytoplasmic proteins freely diffuse. Significantly, the outermost edge of the core syncytium encompasses a shell of nucleated, transcriptionally-competent, fiber cells. This arrangement could facilitate the delivery of newly synthesized protein components to the aged and metabolically quiescent cells in the center of the lens.
HZwint-1, a novel human kinetochore component that interacts with HZW10
Authors: Starr Daniel A. , Saffery Richard , Li Zexiao , Simpson Amanda E. , Choo K. H. Andy , Yen Tim J. , Goldberg Michael L.
Published: April 2021

HZwint-1 (Human ZW10 interacting protein-1) was identified in a yeast two hybrid screen for proteins that interact with HZW10. HZwint-1 cDNA encodes a 43 kDa protein predicted to contain an extended coiled-coil domain. Immunofluorescence studies with sera raised against HZwint-1 protein revealed strong kinetochore staining in nocodazole-arrested chromosome spreads. This signal co-localizes at the kinetochore with HZW10, at a position slightly outside of the central part of the centromere as revealed by staining with a CREST serum. The kinetochore localization of HZwint-1 has been confirmed by following GFP fluorescence in HeLa cells transiently transfected with a plasmid encoding a GFP/HZwint-1 fusion protein. In cycling HeLa cells, HZwint-1 localizes to the kinetochore of prophase HeLa cells prior to HZW10 localization, and remains at the kinetochore until late in anaphase. This localization pattern, combined with the two-hybrid results, suggests that HZwint-1 may play a role in targeting HZW10 to the kinetochore at prometaphase. HZwint-1 was also found to localize to neocentromeres and to the active centromere of dicentric chromosomes. HZwint-1 thus appears to associate with all active centromeres, implying that it plays an important role in correct centromere function.
Talin controls the exit of the integrin α5β1 from an early compartment of the secretory pathway
Authors: Martel Véronique , Vignoud Lucile , Dupé Sandra , Frachet Philippe , Block Marc R. , Albigès-Rizo Corinne
Published: April 2021

Talin is a major cytosolic protein that links the intracellular domains of β1 and β3 integrins to the cytoskeleton. It is required for focal adhesion assembly. However, its downregulation not only slows down cell spreading and organization of focal adhesions but also impairs the maturation of some β1 integrins, including the fibronectin receptor α5β1. To investigate this, we characterized the β1 integrin synthesized in cells expressing talin anti-sense RNA (AT22 cells). We identified a large intracellular pool of β1 integrins that is abnormally accumulated in an earlier compartment of the secretory pathway. In this report, we show that in talin-deficient AT22 cells, the aberrant glycosylation of integrin receptors is accompanied by a delay in the export of the integrin α5β1. In normal cells, talin was found associated with β1 integrins in an enriched membrane fraction containing Golgi and endoplasmic reticulum. Finally, microinjection of anti-talin antibodies resulted in accumulation of the integrins within the cells. These data strongly suggest that talin plays a specific role in the export of newly synthesized integrins. We propose that talin binding to the integrin may disclose a diphenylalanine export signal, which is present in the membrane-proximal GFFKR motif conserved in all integrin α chains.
The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells
Authors: Valenti Giovanna , Procino Giuseppe , Carmosino Monica , Frigeri Antonio , Mannucci Roberta , Nicoletti Ildo , Svelto Maria
Published: April 2021

Phosphorylation by kinases and dephosphorylation by phosphatase markedly affect the biological activity of proteins involved in intracellular signaling. In this study we investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on water permeability properties and on aquaporin2 (AQP2) translocation in AQP2-transfected renal CD8 cells. In CD8 cells both forskolin alone and okadaic acid alone increased the osmotic water permeability coefficient Pf by about 4- to 5-fold. In intact cells, in vivo phosphorylation studies revealed that forskolin stimulation resulted in a threefold increase in AQP2 phosphorylation. In contrast, okadaic acid treatment promoted only a 60% increase in AQP2 phosphorylation which was abolished when this treatment was performed in the presence of 1 μM H89, a specific protein kinase A (PKA) inhibitor. Nevertheless, in this latter condition, confocal microscopy analysis revealed that AQP2 translocated and fused to the apical membrane. Okadaic acid-induced AQP2 translocation was dose dependent having its maximal effect at a concentration of 1 μM. In conclusion, our results clearly indicate that okadaic acid exerts a full forskolin-like effect independent from AQP2 phosphorylation. Thus AQP2 phosphorylation is not essential for water channel translocation in renal cells, indicating that different pathways might exist leading to AQP2 apical insertion and increase in Pf.
Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin
Authors: Thompson H. Cao, H. M. , Krueger E. W. , McNiven M. A.
Published: April 2021

The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.
Identification of a novel C-terminal variant of βII spectrin: two isoforms of βII spectrin have distinct intracellular locations and activities
Authors: Hayes Nandini V. L. , Scott Catherine , Heerkens Egidius , Ohanian Vasken , Maggs Alison M. , Pinder Jennifer C. , Kordeli Ekaterini , Baines Anthony J.
Published: April 2021

It is established that variations in the structure and activities of βI spectrin are mediated by differential mRNA splicing. The two βI spectrin splice forms so far identified have either long or short C-terminal regions. Are analogous mechanisms likely to mediate regulation of βII spectrins? Thus far, only a long form of βII spectrin is reported in the literature. Five human expressed sequence tags indicated the existence of a short splice variant of βII spectrin. The occurrence and DNA sequence of the short C-terminal variant was confirmed by analysis of human and rat cDNA. The novel variant lacks a pleckstrin homology domain, and has 28 C-terminal residues not present in the previously recognized longer form. Transcripts of the short C-terminal variant (7.5 and 7.0 kb) were most abundant in tissues originating from muscle and nervous system. Antibodies raised to a unique sequence of short C-terminal variant recognized 240 kDa polypeptides in cardiac and skeletal muscle and in nervous tissue; in cerebellum and forebrain, additional 270 kDa polypeptides were detected. In rat heart and skeletal muscle, both long and short C-terminal forms of βII spectrin localized in the region of the Z line. The central region of the sarcomere, coincident with the M line, was selectively labeled with antibodies to the short C-terminal form. In cerebellum, the short form was not detectable in parallel fibers, structures in which the long form was readily detected. In cultured cerebellar granule neurons, the long form was dominant in neurites, with the short form being most abundant in cell bodies. In vitro, the short form was found to lack the binding activity for the axonal protein fodaxin, which characterizes the C-terminal region of the long form. Subcellular fractionation of brain revealed that the short form was scarcely detectable in post-synaptic density preparations, in which the long form was readily detected. We conclude that variation in the structure of the C-terminal regions of βII spectrin isoforms correlates with their differential intracellular targeting.
Collagen degradation and platelet-derived growth factor stimulate the migration of vascular smooth muscle cells
Authors: Stringa Emanuela , Knäuper Vera , Murphy Gillian , Gavrilovic Jelena
Published: April 2021

Cell migration is a key event in many biological processes and depends on signals from both extracellular matrix and soluble motogenic factors. During atherosclerotic plaque development, vascular smooth muscle cells migrate from the tunica media to the intima through a basement membrane and interstitial collagenous matrix and proliferate to form a neointima. Matrix metalloproteinases have previously been implicated in neointimal formation and in this study smooth muscle cell adhesion and migration on degraded collagen have been evaluated. Vascular smooth muscle cells adhered to native intact collagen type I and to its first degradation by-product, 3/4 fragment (generated by collagenase-3 cleavage), unwound at 35°C to mimic physiological conditions. PDGF-BB pre- treatment induced a fourfold stimulation of smooth muscle cell motility on the collagen 3/4 fragment whereas no increase in smooth muscle cell motility on collagen type I was observed. Cell migration on collagen type I was mediated by α2 integrin, whereas PDGF-BB-stimulated migration on the 3/4 collagen fragment was dependent on αvβ3 integrin. αvβ3 integrin was organised in clusters concentrated at the leading and trailing edges of the cells and was only expressed when cells were exposed to the 3/4 collagen fragment. Tyrphostin A9, an inhibitor of PDGF receptor-β tyrosine kinase activity, resulted in complete abolition of migration of PDGF-BB treated cells on collagen type I and 3/4 fragment. These results strongly support the hypothesis that the cellular migratory response to soluble motogens can be regulated by proteolytic modification of the extracellular matrix.
Expression from the human occludin promoter is affected by tumor necrosis factor α and interferon γ
Authors: Mankertz J. , Tavalali S. , Schmitz H. , Mankertz A. , Riecken E. O. , Fromm M. , Schulzke J. D.
Published: April 2021

The 65 kDa protein occludin is a membrane-spanning part of the epithelial tight junction, which is the main barrier of the paracellular pathway. The function of occludin as part of tight junctions is still poorly understood and even less is known about the regulatory mechanisms that influence occludin gene expression. This study aimed to identify the sequences essential in cis for genomic regulation of tight junction formation and to investigate their funcional role in cytokine-dependent tight junction regulation.

Using genome walking cloning of occludin-specific human genomic DNA sequences, a 1853 bp DNA fragment containing the transcription start point of occludin cDNA sequences was amplified and sequenced. Subcloning of this fragment in front of the luciferase reporter gene revealed strong expression of enzymatic activity after transfection of the human intestinal cell line HT-29/B6. With subsequent deletions of parts of the promoter fragment, its size was reduced to 280 bp that are necessary and sufficient to mediate promoter activity. Tumor necrosis factor α and another cytokine involved in inflammation, interferon γ, reduced transepithelial resistance in HT-29/B6 cells, which was preceded by a decrease in occludin mRNA expression as revealed by northern blot analysis. Tumor necrosis factor α and interferon γ diminished occludin promoter activity alone and even synergistically, suggesting a genomic regulation of alterations of the paracellular barrier.

In conclusion, proinflammatory cytokines such as tumor necrosis factor α and interferon γ can downregulate the expression of the transmembrane tight junction strand protein occludin, paralleling the barrier disturbance detected electrophysiologically. This could be an important mechanism in gastrointestinal diseases accompanied by barrier defects, for example inflammatory bowel diseases.
Anecdotal recollections of my life as a scientist, by a statue of a mentor
Authors: A Caveman
Published: April 2021

The role of the mentor in advising graduate students and postdoctoral fellows about careers in academia and industry is importantâ€¦
Bi-directional trafficking between the trans-Golgi network and the endosomal/lysosomal system
Authors: Rohn Wolfgang M. , Rouillé Yves , Waguri Satoshi , Hoflack Bernard
Published: April 2021

Protein transport in the secretory and endocytic pathways of eukaryotic cells is mediated by vesicular transport intermediates. Their formation is a tightly controlled multistep process in which coat components are recruited onto specific membranes, and cargo, as well as targeting molecules, become segregated into nascent vesicles. At the trans-Golgi network, two transport systems deliver cargo molecules to the endosomal system. They can be distinguished with regard to coat components that select cargo molecules. AP-1 assembly proteins mediate transport of MPRs and furin, whereas AP-3 adaptors mediate transport of lysosomal membrane glycoproteins to the endosomal/lysosomal system. The molecular basis for protein-specific sorting lies within sorting signals that are present in the cytoplasmic tails of cargo proteins and allow specific interactions with individual coat components. In order to maintain cellular homeostasis, some proteins are retrieved from endosomal compartments and transported back to the trans-Golgi network. Distinct points for protein retrieval exist within the endosomal system, retrieval occurring from either early or late endosomes. Whereas significant progress has been made in recent years in identifying anterograde and retrograde transport pathways, the molecular mechanisms underlying protein sorting and retrieval are only poorly defined. Recently, however, novel vesicle coats (e.g. AP-4) and proteins that might be involved in sorting (e.g. PACS-1 and TIP47) have been described, and the interactions between assembly proteins and sorting signals are becoming increasingly well defined.
Subcellular localization of Bic-D::GFP is linked to an asymmetric oocyte nucleus
Authors: Paré Chantal , Suter Beat
Published: April 2021

Bicaudal-D (Bic-D) is essential for the establishment of oocyte fate and subsequently for polarity formation within the developing Drosophila oocyte. To find out where in the germ cells Bic-D performs its various functions we made transgenic flies expressing a chimeric Bic-D::GFP fusion protein. Once Bic-D::GFP preferentially accumulates in the oocyte, it shows an initial anterior localization in germarial region 2. In the subsequent egg chamber stages 1-6 Bic-D::GFP preferentially accumulates between the oocyte nucleus and the posterior cortex in a focus that is consistently aligned with a crater-like indentation in the oocyte nucleus. After stage 6 Bic-D::GFP fluorescent signal is predominantly found between the oocyte nucleus and the dorso-anterior cortex. During the different phases several genes have been found to be required for the establishment of the new Bic-D::GFP distribution patterns. Dynein heavy chain (Dhc), spindle (spn) genes and maelstrom (mael) are required for the re-localization of the Bic-D::GFP focus from its anterior to its posterior oocyte position. Genes predicted to encode proteins that interact with RNA (egalitarian and orb) are required for the normal subcellular distribution of Bic-D::GFP in the germarium, and another potential RNA binding protein, spn-E, is required for proper transport of Bic-D::GFP from the nurse cells to the oocyte in later oogenesis stages. The results indicate that Bic-D requires the activity of mRNA binding proteins and a negative-end directed microtubule motor to localize to the appropriate cellular domains. Asymmetric subcellular accumulation of Bic-D and the polarization of the oocyte nucleus may reflect the function of this localization machinery in vectorial mRNA localization and in tethering of the oocyte nucleus. The subcellular polarity defined by the Bic-D focus and the nuclear polarity marks some of the first steps in antero-posterior and subsequently in dorso-ventral polarity formation.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs1338.html
Integrin α2β1-dependent EGF receptor activation at cell-cell contact sites
Authors: Yu Xiaochun , Miyamoto Shingo , Mekada Eisuke
Published: April 2021

Certain integrins including α2β1 and α3β1 localize to intercellular binding sites, and thus may participate in cell-cell interactions. We demonstrated here the physical and functional associations of integrin α2β1 with epidermal growth factor receptor (EGFR) at intercellular adhesion sites. Immunoprecipitation with anti-integrin α2 antibodies or anti-integrin β1 antibody resulted in preferential coprecipitation of EGFR from A431 cell lysates, while anti-EGFR antibody coprecipitated integrin α2β1. Chemical crosslinking confirmed the association of integrin α2β1 and EGFR. Colocalization of integrin α2β1 and EGFR at cell-cell contact sites was observed by double immunofluorescence staining of A431 cells. EGF-induced EGFR stimulation did not affect the association of integrin α2β1 and EGFR. However, immunostaining with the antibody specific to activated-EGFR revealed that EGFR localized at cell-cell contact sites are phosphorylated even in serum-depleted conditions, while EGFR localized to other sites is totally dephosphorylated in the same conditions. The EGFR phosphorylation in cell-cell contact sites observed in a serum-depleted culture was abrogated with a function-blocking antibody of integrin α2, but not with a non-function-blocking α2 antibody or function-blocking α3 antibody. Moreover, the EGFR phosphorylation in serum-depleted conditions was not observed in suspended cells, or largely abrogated in sparse cells, indicating that cell-cell adhesion is required for EGFR phosphorylation. These results indicate that integrin α2β1 not only physically associates with EGFR but also functions in serum-independent EGFR activation at cell-cell contact sites. The present results shed a new light on the role of intercellular integrins in cell-cell interactions.
Arrested maturing multivesicular endosomes observed in a Chinese hamster ovary cell mutant, LEX2, isolated by repeated flow-cytometric cell sorting
Authors: Ohashi Masato , Miwako Ishido , Yamamoto Akitsugu , Nagayama Kuniaki
Published: April 2021

Chinese hamster ovary (CHO) cell mutants defective in the disintegration of endocytosed low-density lipoprotein (LDL) were isolated from mutagenized cells by repeated flow-cytometric cell sorting. After seven rounds of cell sorting, we obtained mutant pools, from which nine mutant clones were established. These mutant strains were all recessive, and were categorized into three complementation groups A, B, and C. The previously established CHO mutant, LEX1 (Lysosome-Endosome X1), fell into the complementation group A. One of the newly isolated mutants, LEX2, fell into the complementation group B, and showed slower degradation of RET-LDL than LEX1 cells. LEX2 showed prominence of well-elaborated multivesicular bodies (MVBs), positive for lysosomal glycoprotein-B/cathepsin D and cation-independent mannose 6-phosphate receptor (CI-MPR), yet negative for transferrin receptor or rab7. Endocytosed intact LDL accumulated in these CI-MPR-positive structures starting at 10-15 minutes of internalization and the accumulation reached completion at 20 minutes. Intermixing of separately internalized fluorescent LDLs between the LEX2 MVBs was slow and saturable at a lower level than observed between late endosomes/lysosomes in wild-type or in LEX1 cells. The receptor recycling pathway to the plasma membrane and the acidification of intracellular compartments were normal in LEX2 cells. These results are consistent with the idea that LEX2 cells are defective in the segregation and sequestration of contents at compartments equivalent to the transport intermediates, previously referred to as endosomal carrier vesicles or maturing MVBs. This MVB stage is likely to be an earlier stage than rab7-positive, lysosome-interacting late endosomes observed in LEX1 cells. Thus, LEX1 and LEX2 mutations could be considered as landmarks for these distinct late endocytic stages, and use of these cells in biochemical and molecular genetic analyses would help to understand the as yet unidentified details of late endocytic pathways including the MVB dynamics.
Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBFα/AML/PEBP2α) dependent activation of tissue-specific gene transcription
Authors: Javed Amjad , Guo Bo , Hiebert Scott , Choi Je-Yong , Green Jack , Zhao Shan-Chuan , Osborne Mark A. , Stifani Stefano , Stein Janet L. , Lian Jane B. , van Wijnen André J. , Stein Gary S.
Published: April 2021

The Runt related transcription factors RUNX (AML/CBFα/PEBP2α) are key regulators of hematopoiesis and osteogenesis. Using co-transfection experiments with four natural promoters, including those of the osteocalcin (OC), multi drug resistance (MDR), Rous Sarcoma Virus long terminal repeat (LTR), and bone sialoprotein (BSP) genes, we show that each of these promoters responds differently to the forced expression of RUNX proteins. However, the three RUNX subtypes (i.e. AML1, AML2, and AML3) regulate each promoter in a similar manner. Although the OC promoter is activated in a C terminus dependent manner, the MDR, LTR and BSP promoters are repressed by three distinct mechanisms, either independent of or involving the AML C terminus, or requiring only the conserved C-terminal pentapeptide VWRPY. Using yeast two hybrid assays we find that the C terminus of AML1 interacts with a Groucho/TLE/R-esp repressor protein. Co-expression assays reveal that TLE proteins repress AML dependent activation of OC gene transcription. Western and northern blot analyses suggest that TLE expression is regulated reciprocally with the levels of OC gene expression during osteoblast differentiation. Digital immunofluorescence microscopy results show that TLE1 and TLE2 are both associated with the nuclear matrix, and that a significant subset of each colocalizes with AML transcription factors. This co-localization of TLE and AML proteins is lost upon removing the C terminus of AML family members. Our findings indicate that suppression of AML-dependent gene activation by TLE proteins involves functional interactions with the C terminus of AML at the nuclear matrix in situ. Our data are consistent with the concept that the C termini of AML proteins support activation or repression of cell-type specific genes depending on the regulatory organization of the target promoter and subnuclear localization.
Hrs interacts with SNAP-25 and regulates Ca2+-dependent exocytosis
Authors: Kwong Jeffrey , Roudabush Francine L. , Moore P. Hutton , Montague Michael , Oldham William , Li Yankun , Chin Lih-Shen , Li Lian
Published: April 2021

Synaptosome-associated protein of 25 kDa (SNAP-25) is a neuronal membrane protein essential for synaptic vesicle exocytosis. To investigate the mechanisms by which SNAP-25 mediates neurosecretion, we performed a search for proteins that interact with SNAP-25 using a yeast two-hybrid screen. Here, we report the isolation and characterization of a SNAP-25-interacting protein that is the rat homologue of mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Hrs specifically interacts with SNAP-25, but not SNAP-23/syndet. The association of Hrs and SNAP-25 is mediated via coiled-coil interactions. Using an Hrs-specific antibody, we have shown that Hrs is highly enriched in brain, where it codistributes with SNAP-25 in most brain regions. Subcellular fractionation studies demonstrate that in brain, Hrs exists in both cytosolic and membrane-associated pools. Studies using indirect immunofluorescence and confocal microscopy reveal that, in addition to early endosomes, Hrs is also localized to large dense-core secretory granules and synaptic-like microvesicles in nerve growth factor-differentiated PC12 cells. Moreover, overexpression of Hrs in PC12 cells inhibits Ca2+-dependent exocytosis. These results suggest that Hrs is involved in regulation of neurosecretion through interaction with SNAP-25.
Functional analysis of CLIP-115 and its binding to microtubules
Authors: Hoogenraad Casper C. , Akhmanova Anna , Grosveld Frank , Zeeuw Chris I. De , Galjart Niels
Published: April 2021

Cytoplasmic linker proteins (CLIPs) bind to microtubules and are proposed to link this cytoskeletal network to other intracellular structures. We are interested in CLIP-115, since this protein is enriched in neuronal dendrites and may operate in the control of brain-specific organelle translocations. Each CLIP monomer is characterized by two microtubule-binding (MTB) motifs, surrounded by basic, serine-rich regions. This head domain is connected to the C-terminal tail through a long coiled-coil structure. The MTB domains are conserved as a single domain in other proteins involved in microtubule based transport and dynamics, such as p150Glued. Here we provide evidence that efficient binding of CLIP-115 to microtubules is sensitive to phosphorylation and is not mediated by the conserved MTB domains alone, but requires the presence of the basic, serine rich regions in addition to the MTB motifs. In transfected COS-1 cells, CLIP-115 initially accumulates at the distal ends of microtubules and coincides with CLIP-170, indicating that both proteins mark growing microtubule ends. However, when expressed at higher levels, CLIP-115 and -170 affect the microtubule network differently. This might be partly due to the divergent C-termini of the two proteins. We demonstrate that, similar to CLIP-170, CLIP-115 forms homodimers, which, at least in vitro, are linked by disulfide bridges. Cysteine391 of CLIP-115, however, is specific in that it controls the microtubule bundling capacity of certain mutant CLIP-115 molecules. Therefore, both similar and specific mechanisms appear to regulate the conformation of CLIPs as well as their binding to microtubules.
Evidence for a myogenic stem cell that is exhausted in dystrophic muscle
Authors: Heslop L. , Morgan J. E. , Partridge T. A.
Published: April 2021

Injection of the myotoxin notexin, was found to induce regeneration in muscles that had been subjected to 18 Gy of radiation. This finding was unexpected as irradiation doses of this magnitude are known to block regeneration in dystrophic (mdx) mouse muscle. To investigate this phenomenon further we subjected mdx and normal (C57Bl/10) muscle to irradiation and notexin treatment and analysed them in two ways. First by counting the number of newly regenerated myofibres expressing developmental myosin in cryosections of damaged muscles. Second, by isolating single myofibres from treated muscles and counting the number of muscle precursor cells issuing from these over 2 day and 5 day periods. After irradiation neither normal nor dystrophic muscles regenerate to any significant extent. Moreover, single myofibres cultured from such muscles produce very few muscle precursor cells and these undergo little or no proliferation. However, when irradiated normal and mdx muscles were subsequently treated with notexin, regeneration was observed. In addition, some of the single myofibres produced rapidly proliferative muscle precursor cells when cultured. This occurred more frequently, and the myogenic cells proliferated more extensively, with fibres cultured from normal compared with dystrophic muscles. Even after 25 Gy, notexin induced some regeneration but no proliferative myogenic cells remained associated with the muscle fibres. Thus, skeletal muscles contain a number of functionally distinct populations of myogenic cells. Most are radiation sensitive. However, some survive 18 Gy as proliferative myogenic cells that can be evoked by extreme conditions of muscle damage; this population is markedly diminished in muscles of the mdx mouse. A small third population survives 25 Gy and forms muscle but not proliferative myogenic cells.
Meltrin γ (ADAM-9) mediates cellular adhesion through α6β1 integrin, leading to a marked induction of fibroblast cell motility
Authors: Nath Deepa , Slocombe Patrick M. , Webster Ailsa , Stephens Paul E. , Docherty Andrew J. P. , Murphy Gillian
Published: April 2021

The ADAMs (A Disintegrin and Metalloprotease Domains) are a family of membrane-anchored proteins that play a role in fertilisation, myoblast fusion and ectodomain shedding of cell surface proteins. Meltrin γ (ADAM-9) is a widely expressed member of this family and is involved in the shedding of heparin binding epidermal growth factor. Here we report that meltrin γ can function as a cell adhesion molecule via its disintegrin domain. Using solid-phase binding assays and antibody inhibition experiments, we demonstrate that a murine meltrin γ-Fc (Melγ-Fc) fusion protein binds to the integrin α6β1 on the surface of fibroblast cell lines, HT1080 and Wehi 164 in a specific manner. Since α6β1 is important for the motility of several cell types on laminin, cell migration studies using time-lapse video microscopy were performed. Cells adhering to Melγ-Fc displayed a rounded morphology and a marked increase (eight-to tenfold) in their motility compared to that on laminin. Furthermore, the p160 ROCK kinase inhibitor Y-27632 specifically reduced the migration of cells on meltrin γ but had no effect on migration of cells on laminin, whilst the general tyrosine phoshorylation inhibitor, genistein, inhibited cell migration on both substrates. These results together suggest that meltrin γ may play a role in regulating the motility of cells by binding to α6β1 integrin and this may be important during a variety of biological and pathological processes.
Formation of glycine receptor clusters and their accumulation at synapses
Authors: Meier Jochen , Meunier-Durmort Claire , Forest Claude , Triller Antoine , Vannier Christian
Published: April 2021

The glycine receptor is highly enriched in microdomains of the postsynaptic neuronal surface apposed to glycinergic afferent endings. There is substantial evidence suggesting that the selective clustering of glycine receptor at these sites is mediated by the cytoplasmic protein gephyrin. To investigate the formation of postsynaptic glycine receptor domains, we have examined the surface insertion of epitope-tagged receptor α subunits in cultured spinal cord neurons after gene transfer by polyethylenimine-adenofection. Expression studies were also carried out using the non-neuronal cell line COS-7. Immunofluorescence microscopy was performed using wild-type isoforms and an α mutant subunit bearing the gephyrin-binding motif of the β subunit. In COS-7 cells, transfected glycine receptor α subunits had a diffuse surface distribution. Following cotransfection with gephyrin, only the mutant subunit formed cell surface clusters.

In contrast, in neurons all subunits were able to form cell surface clusters after transfection. These clusters were not colocalized with detectable endogenous gephyrin, and the GlyR β subunit could not be detected in transfected cells. Therefore, exogenous receptors were not assembled as heteromeric complexes. A quantitative analysis demonstrated that newly synthesized glycine receptor progressively populated endogenous gephyrin clusters, since association of both proteins increased as a function of time after the onset of receptor synthesis. This phenomenon was accelerated when glycine receptor contained the gephyrin-binding domain.

Together with previous results, these data support a two-step model for glycinergic synaptogenesis whereby the gephyrin-independent formation of cell surface clusters precedes the gephyrin-mediated postsynaptic accumulation of clusters.
Do growth and cell division rates determine cell size in multicellular organisms?
Authors: Coelho Carmen M. , Leevers Sally J.
Published: April 2021

Studies in yeast have provided some clues to how cell size might be determined in unicellular eukaryotes; yet little attention has been paid to this issue in multicellular organisms. Reproducible cell sizes might be achieved in the dividing cells of multicellular organisms by the coordination of growth with cell division. Recently, mutations in genes encoding homologues of components of the mammalian insulin/phosphoinositide 3-kinase signalling pathway have been shown to affect organ growth and cell size during Drosophila melanogaster imaginal disc development. The data suggest that signalling through this pathway alters cell size because it primarily affects the growth of these organs (i.e. their increase in mass) and does not have a proportional impact on cell division. These observations are in keeping with the hypothesis that growth and cell division are regulated independently, and that cell size is just a consequence of the rate at which tissues grow and the cells within them divide. However, signalling through this pathway can affect cell cycle phasing and at least influence cell division. These interactions may provide a means of coordinating growth and cell division, such that cells divide only when they are above a minimum size.
Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor
Authors: Vandenbulcke Franck , Nouel Dominique , Vincent Jean-Pierre , Mazella Jean , Beaudet Alain
Published: April 2021

The neuropeptide neurotensin (NT) is known to be internalized in a receptor-mediated fashion into its target cells. To gain insight into the mechanisms underlying this process, we monitored in parallel the migration of the NT1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalization was prevented by hypertonic sucrose, potassium depletion and cytosol acidification, demonstrating that it proceeded via clathrin-coated pits. Within 0-30 minutes, fluo-NT accumulated together with its receptor in Acridine Orange-positive, acidic organelles. These organelles concentrated transferrin and immunostained positively for rab 5A, therefore they were early endosomes. After 30-45 minutes, the ligand and its receptor no longer colocalized. Fluo-NT was first found in rab 7-positive late endosomes and later in a nonacidic juxtanuclear compartment identified as the Trans-Golgi Network (TGN) by virtue of its staining for syntaxin 6. This juxtanuclear compartment also stained positively for rab 7 and for the TGN/pericentriolar recycling endosome marker rab 11, suggesting that the ligand could have been recruited to the TGN from either late or recycling endosomes. By that time, internalized receptors were detected in Lamp-1-immunoreactive lysosomes. These results demonstrate that neurotensin/ NT1 receptor complexes follow a recycling cycle that is unique among the G protein-coupled receptors studied to date, and provide the first evidence for the targeting of a nonendogenous protein from endosomes to the TGN.
Intracellular distribution and mobilization of unesterified cholesterol in adipocytes: triglyceride droplets are surrounded by cholesterol-rich ER-like surface layer structures
Authors: Prattes Susanne , Hörl Gerd , Hammer Astrid , Blaschitz Astrid , Graier Wolfgang F. , Sattler Wolfgang , Zechner Rudolf , Steyrer Ernst
Published: April 2021

In addition to their central role in triglyceride storage, fat cells are a primary depot of unesterified cholesterol (FC) in the body. In comparison, peripheral cells contain very little FC. This difference in adipocytes versus peripheral tissues is inconsistent with the current theory of cholesterol homeostasis. Attempting to resolve this discrepancy, we examined intracellular storage sites of FC in murine 3T3-F442A adipocytes. Using the cholesterol-binding antibiotic, filipin, in combination with high resolution fluorescence microscopy, intense fluorescent staining characteristically decorated the periphery of triglyceride droplets (TGD) as well as the plasma membrane (PM) of fat cells. Filipin-staining was not visible inside the lipid droplets. Purification of TGD by subcellular fractionation demonstrated that the rise in total FC content of adipocytes upon differentiation was attributable to an increase in TGD-FC, which contributed up to one third of the total cellular FC. The protein component of purified TGD from cultured adipocytes as well as from murine adipocytes obtained from fresh tissues contained the lumenal endoplasmic reticulum (ER) immunoglobulin binding protein (BiP) and the integral ER membrane protein calnexin. Efflux experiments using the extracellular FC acceptors β-cyclodextrin or apolipoprotein A-I demonstrated that TGD-associated FC was releasable from TGD. Whereas FC efflux from adipocytes was unaffected in the presence of brefeldin A or monensin, the secretion of a control protein, lipoprotein lipase, was effectively reduced.

In summary, our findings identify the TGD surface layer as primary intracellular storage site for FC within adipocytes. We suggest that the structural role of ER-resident proteins in this adipocyte TGD envelope has been previously neglected. Our findings support the suggestion that an ER-like structure, albeit of modified lipid composition, constitutes the lipid droplets’ surface layer. Finally, the efflux process of FC from adipocytes upon extracellular stimulation with β-cyclodextrin provides evidence for an energy-dependent intracellular trafficking route between the TGD-FC pool and the PM-FC sites which is distinct from the secretory pathway of proteins.
Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking
Authors: Paulson Alicia F. , Lampe Paul D. , Meyer Rita A. , TenBroek Erica , Atkinson Michael M. , Walseth Timothy F. , Johnson Ross G.
Published: April 2021

Given the rapid turnover of connexin proteins, gap junction (GJ) assembly represents an important means of regulating the extent of GJ communication between cells. This report describes an increase in the level of GJ assembly within one hour following treatment with cAMP-elevating reagents or low density lipoprotein (LDL). Dye transfer methods and freeze-fracture with electron microscopy were used to assay junctional permeability and structure, respectively, subsequent to the dissociation, recovery and reaggregation of Novikoff hepatoma cells. Reaggregating cells in the presence of agents that increase cAMP levels (8-Br-cAMP, forskolin and IBMX) enhanced both dye transfer rates between cells and the extent of GJ formation 2-to 3-fold. These data and studies with the protein kinase A inhibitor, H-89, indicate that cAMP signaling plays a key role in enhanced assembly. The response to LDL parallels that to cAMP and relies on the activity of both adenylyl cyclase and protein kinase A. Immunoblot analysis revealed no change in the level of connexin43 (Cx43) or its phosphorylation states over a period of 2.5 hours. However, three agents (brefeldin A, monensin and nocodazole), that inhibit intracellular membrane trafficking by different mechanisms, all blocked the enhanced assembly of GJs when triggered by either elevated cAMP or exposure to LDL. Related studies, which employed trafficking inhibitors at different stages in GJ assembly, suggested that Cx43 trafficking during enhanced assembly is regulated, in part, by cell contact. Intracellular sources of Cx43 were characterized by colabeling for several markers of cytoplasmic membrane systems. We conclude that an increase in GJ assembly: (i) occurs rapidly in the presence of elevated cAMP or LDL, (ii) does not require an increase in Cx43 levels or major changes in Cx43 phosphorylation and (iii) is dependent upon the trafficking of Cx43 from intracellular storage sites.
Activation-induced nuclear translocation of RING3
Authors: Guo Ning , Faller Douglas V. , Denis Gerald V.
Published: April 2021

RING3 is a novel protein kinase linked to human leukaemia. Its Drosophila homologue female sterile homeotic is a developmental regulator that interacts genetically with trithorax, a human homologue of which is also associated with leukaemia. The RING3 structure contains two mutually related bromodomains that probably assist in the remodelling of chromatin and thereby affect transcription. Consistent with this hypothesis, a RING3-like protein has been identified in the mouse Mediator complex, where it is associated with transcription factors. We show that, whilst RING3 is constitutively localised to the nucleus of exponentially growing HeLa cells, it is delocalised throughout serum-starved fibroblasts. We use immunostaining and confocal microscopy to demonstrate that RING3 translocates to the fibroblast nucleus upon serum stimulation. After translocation, RING3 participates in nuclear protein complexes that include E2F proteins; it transactivates the promoters of several important mammalian cell cycle genes that are dependent on E2F, including dihydrofolate reductase, cyclin D1, cyclin A and cyclin E. We use site-directed mutagenesis of a putative nuclear localisation motif to show that the activation-induced nuclear localisation and consequent transcriptional activity of RING3 depends on a monopartite, classical nuclear localisation sequence. These observations refine and extend the mechanism by which RING3 contributes to E2F-regulated cell cycle progression. Deregulation of this mechanism may be leukaemogenic.
The protease-activated receptor-2 upregulates keratinocyte phagocytosis
Authors: Sharlow E. R. , Paine C. S. , Babiarz L. , Eisinger M. , Shapiro S. , Seiberg M.
Published: April 2021

The protease-activated receptor-2 (PAR-2) belongs to the family of seven transmembrane domain receptors, which are activated by the specific enzymatic cleavage of their extracellular amino termini. Synthetic peptides corresponding to the tethered ligand domain (SLIGRL in mouse, SLIGKV in human) can activate PAR-2 without the need for receptor cleavage. PAR-2 activation is involved in cell growth, differentiation and inflammatory processes, and was shown to affect melanin and melanosome ingestion by human keratinocytes. Data presented here suggest that PAR-2 activation may regulate human keratinocyte phagocytosis. PAR-2 activation by trypsin, SLIGRL or SLIGKV increased the ability of keratinocytes to ingest fluorescently labeled microspheres or E. coli K-12 bioparticles. This PAR-2 mediated increase in keratinocyte phagocytic capability correlated with an increase in actin polymerization and α-actinin reorganization, cell surface morphological changes and increased soluble protease activity. Moreover, addition of serine protease inhibitors downmodulated both the constitutive and the PAR-2 mediated increases in phagocytosis, suggesting that serine proteases mediate this functional activity in keratinocytes. PAR-2 involvement in keratinocyte phagocytosis is a novel function for this receptor.
Translocation of protein tyrosine phosphatase Pez/PTPD2/PTP36 to the nucleus is associated with induction of cell proliferation
Authors: Wadham Carol , Gamble Jennifer R. , Vadas Mathew A. , Khew-Goodall Yeesim
Published: April 2021

Pez is a non-transmembrane tyrosine phosphatase with homology to the FERM (4.1, ezrin, radixin, moesin) family of proteins. The subcellular localisation of Pez in endothelial cells was found to be regulated by cell density and serum concentration. In confluent monolayers Pez was cytoplasmic, but in cells cultured at low density Pez was nuclear, suggesting that it is a nuclear protein in proliferating cells. This notion is supported by the loss of nuclear Pez when cells are serum-starved to induce quiescence, and the rapid return of Pez to the nucleus upon refeeding with serum to induce proliferation. Vascular endothelial cells normally exist as a quiescent confluent monolayer but become proliferative during angiogenesis or upon vascular injury. Using a ‘wound’ assay to mimic these events in vitro, Pez was found to be nuclear in the cells that had migrated and were proliferative at the ‘wound’ edge. TGFβ, which inhibits cell proliferation but not migration, inhibited the translocation of Pez to the nucleus in the cells at the ‘wound’ edge, further strengthening the argument that Pez plays a role in the nucleus during cell proliferation. Together, the data presented indicate that Pez is a nuclear tyrosine phosphatase that may play a role in cell proliferation.
Novel generation of human satellite DNA-based artificial chromosomes in mammalian cells
Authors: Csonka Erika , Cserpán Imre , Fodor Katalin , Holló Gyöngyi , Katona Róbert , Keresö Judit , Praznovszky Tünde , Szakál Barnabás , Telenius Adèle , deJong Gary , Udvardy Andor , Hadlaczky Gyula
Published: April 2021

An in vivo approach has been developed for generation of artificial chromosomes, based on the induction of intrinsic, large-scale amplification mechanisms of mammalian cells. Here, we describe the successful generation of prototype human satellite DNA-based artificial chromosomes via amplification-dependent de novo chromosome formations induced by integration of exogenous DNA sequences into the centromeric/rDNA regions of human acrocentric chromosomes. Subclones with mitotically stable de novo chromosomes were established, which allowed the initial characterization and purification of these artificial chromosomes. Because of the low complexity of their DNA content, they may serve as a useful tool to study the structure and function of higher eukaryotic chromosomes. Human satellite DNA-based artificial chromosomes containing amplified satellite DNA, rDNA, and exogenous DNA sequences were heterochromatic, however, they provided a suitable chromosomal environment for the expression of the integrated exogenous genetic material. We demonstrate that induced de novo chromosome formation is a reproducible and effective methodology in generating artificial chromosomes from predictable sequences of different mammalian species. Satellite DNA-based artificial chromosomes formed by induced large-scale amplifications on the short arm of human acrocentric chromosomes may become safe or low risk vectors in gene therapy.