bluuebunny/crossref_metadata_2025 huggingface dataset:
Apical recruitment of CFTR in T-84 cells is dependent on cAMP and microtubules but not Ca2+ or microfilaments
Authors: Tousson Albert , Fuller Catherine M. , Benos Dale J.
Published: April 2021

Previous studies from this laboratory have demonstrated that chloride transport induced by forskolin, but not ionomycin, in T84 cells is highly dependent on an intact microtubular network. Using an antibody raised against a region of the R domain of CFTR, we now show by indirect immunofluorescence that forskolin causes relocation of CFTR to the apical domain of T84 cells. T84 cells grown on transparent filters were incubated with agonists and/or cytoskeletal inhibitors prior to fixation, permeabilization, and staining with the antibody. A 30 second stimulation with forskolin (10 μM) caused a twofold increase in relative fluorescence intensity at the apical surface. In contrast, a 30 second exposure to ionomycin (2 μM), had no effect on the distribution of CFTR-related fluorescence. Incubation of the cells with nocodazole (33 μM), a microtubule disrupting agent, prevented the forskolin-induced rise in CFTR fluorescence at the apical surface. However, incubation of the cells with cytochalasin D, an actin inhibitor, was without effect on forskolin-related re-distribution of CFTR-associated fluorescence. In double label experiments using antibodies against both β-tubulin and actin, CFTR-related fluorescence was found to co-localize with the microtubule network, but not with actin filaments. These observations are consistent with the microtubule-dependent acute recruitment of CFTR to the apical plasma membrane of T84 cells in response to elevations in intracellular cAMP.
A nitrogen starvation-induced dormant G0 state in fission yeast: the establishment from uncommitted G1 state and its delay for return to proliferation
Authors: Su Sophia S. Y. , Tanaka Yusuke , Samejima Itaru , Tanaka Kenji , Yanagida Mitsuhiro
Published: April 2021

Fission yeast cells either remain in the mitotic cell cycle or exit to meiotic sporulation from an uncommitted G1 state dependent on the presence or absence of nitrogen source in the medium (Nurse and Bissett, 1981). We examined how heterothallic haploid cells, which cannot sporulate, behave under nitrogen-starvation for longer than 25 days at 26°C. These cells were shown to enter a stable state (designated the dormant G0) with nearly full viability. Maintaining the dormant cells required glucose, suggesting that the cells remained metabolically active although cell division had ceased. They differed dramatically from mitotic and uncommitted G1 cells in heat resistance, and also in cytoplasmic and nuclear morphologies. After nitrogen replenishment, the initial responses of dormant G0 cells were investigated. The kinetics for reentry into the proliferative state were delayed considerably, and the changes in cell shape were enhanced particularly for those recovering from extended nitrogen starvation. A part of the delay could be accounted for by the duration of nuclear decondensation and cell elongation for the first cell division.
Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn
Authors: Cary Leslie A. , Chang James F. , Guan Jun-Lin
Published: April 2021

Cellular interactions with the extracellular matrix proteins play important roles in a variety of biological processes. Recent studies suggest that integrin-mediated cell-matrix interaction can transduce biochemical signals across the plasma membrane to regulate cellular functions such as proliferation, differentiation and migration. These studies have implicated a critical role of focal adhesion kinase (FAK) in integrin-mediated signal transduction pathways. We report here that overexpression of FAK in CHO cells increased their migration on fibronectin. A mutation of the major autophosphorylation site Y397 in FAK abolished its ability to stimulate cell migration, while phosphorylation of Y397 in a kinase-defective FAK by endogenous FAK led to increased migration. We also find that the wild-type and the kinase-defective FAK were associated with Src and Fyn in CHO cells whereas the F397 mutant was not. These results directly demonstrate a functional role for FAK in integrin signaling leading to cell migration. They also provide evidence for the functional significance of FAK/Src complex formation in vivo.
Sequence and expression of Drosophila myristoyl-CoA:protein N-myristoyl transferase: evidence for proteolytic processing and membrane localisation
Authors: Ntwasa Monde , Egerton Mark , Gay Nicholas J.
Published: April 2021

The enzyme N-myristoyl transferase transfers the 14 carbon fatty acid myristate to an N-terminal glycine residue in a small subset of cytoplasmic proteins. Many myristoyl proteins are components of cellular signalling pathways, some of which play important roles during embryonic development, for example protein kinase A. Thus, the function of N-myristoyl transferase is probably essential for embryogenesis and it is of some interest to study the enzyme in an organism with well understood developmental biology. In this paper we report the purification of a processed form of the Drosophila enzyme from peripheral membrane fractions of embryos by affinity chromatography to a protein containing leucine rich repeats. We have also isolated the Drosophila N-myristoyl transferase gene and determined its nucleotide sequence. The predicted amino acid sequence of the Drosophila enzyme is closely related to that of mammalian and fungal N-myristoyl transferases and residues essential for enzyme function are conserved. Our findings indicate that a fraction of Drosphila NMT is bound to the membrane and they are consistent with recent results for the human enzyme. We suggest that N-myristoyl transferase may be recruited to the membrane as part of a translational complex, perhaps by binding to p34 ribosome binding protein, a leucine rich repeat receptor of the microsomal membranes. We have also studied the expression pattern of the gene in the embryo by northern blot analysis and in situ hybridization. The transcripts appear to be uniformly distributed in the pre-cellular embryo but at later stages the RNA is barely detectable with these methods. However, from about stage 14, high levels of transcript are detected in a small number of randomly distributed cells of the central and peripheral nervous system.
Monastral bipolar spindles: implications for dynamic centrosome organization
Authors: Wilson P. G. , Fuller M. T. , Borisy G. G.
Published: April 2021

Implicit to all models for mitotic spindle assembly is the view that centrosomes are essentially permanent structures. Yet, immunofluorescence revealed that spindles in larval brains of urchin mutants in Drosophila were frequently monastral but bipolar; the astral pole contained a centrosome while the opposing anastral pole showed neither gamma tubulin nor a radial array of astral microtubules. Thus, mutations in the urchin gene seem to uncouple centrosome organization and spindle bipolarity in mitotic cells. Hypomorphic mutants showed a high frequency of monastral bipolar spindles but low frequencies of polyploidy, suggesting that monastral bipolar spindles might be functional. To test this hypothesis, we performed pedigree analysis of centrosome distribution and spindle structure in the four mitotic divisions of gonial cells. Prophase gonial cells showed two centrosomes, suggesting cells entered mitosis with the normal number of centrosomes and that centrosomes separated during prophase. Despite a high frequency of monastral bipolar spindles, the end products of the four mitotic divisions were equivalent in size and chromatin content. These results indicate that monastral bipolar spindles are functional and that the daughter cell derived from the anastral pole can assemble a functional bipolar spindle in the subsequent cell cycle. Cell proliferation despite high frequencies of monastral bipolar spindles can be explained if centrosome structure in mitotic cells is dynamic, allowing transient and benign disorganization of pericentriolar components. Since urchin proved to be allelic to KLP61F which encodes a kinesin related motor protein (Heck et al. (1993)J. Cell Biol. 123, 665-671), our results suggest that motors influence the dynamic organization of centrosomes.
A mutation in a highly conserved region in brush-border sucrase-isomaltase and lysosomal α-glucosidase results in Golgi retention
Authors: Moolenaar Catharina E. C. , Ouwendijk Joke , Wittpoth Michael , Wisselaar Heleen A. , Hauri Hans-Peter , Ginsel Leo A. , Naim Hassan Y. , Fransen Jack A. M.
Published: April 2021

A point mutation in the cDNA of human intestinal sucrase-isomaltase has been recently identified in phenotype II of congenital sucrase-isomaltase deficiency. The mutation results in a substitution of glutamine by proline at position 1098 (Q1098P) in the sucrase subunit. Expression of this mutant sucrase-isomaltase cDNA in COS-1 cells results in an accumulation of sucrase-isomaltase in the ER, intermediate compartment and the cis-Golgi cisternae similar to the accumulation in phenotype II intestinal cells. An interesting feature of the Q1098P substitution is its location in a region of the sucrase subunit that shares striking similarities with the isomaltase subunit and other functionally related enzymes, such as human lysosomal acid α-glucosi-dase and Schwanniomyces occidentals glucoamylase. We speculated that the QrP substitution in these highly conserved regions may result in a comparable accumula tion. Here we examined this hypothesis using lysosomal α-glucosidase as a reporter gene. Mutagenesis of the glutamine residue at position 244 in the homologous region of α-glucosidase to proline results in a protein that is neither transported to the lysosomes nor secreted extracel-lularly but accumulates in the ER, intermediate compartment and cis-Golgi as a mannose-rich polypeptide similar to mutant sucrase-isomaltase in phenotype II.

We propose that the Q1098P and Q244P mutations (in sucrase-isomaltase and α-glucosidase, respectively) generate structural alterations that are recognized by a control mechanism, operating beyond the ER in the intermediate compartment or cis-Golgi.
VE-cadherin antibody accelerates neutrophil recruitment in vivo
Authors: Gotsch Ursula , Borges Eric , Bosse Roland , Böggemeyer Elena , Simon Markus , Mossmann Horst , Vestweber Dietmar
Published: April 2021

Neutrophils enter sites of inflammation by crossing the endothelial lining of the blood vessel wall. VE-cadherin is an endothelial specific, homophilic adhesion molecule located at the lateral cell surface. We have generated a monoclonal antibody against mouse VE-cadherin which inhibits electrical resistance of endothelial cell monolayers in vitro as well as aggregation of VE-cadherin transfected cells. In vivo, this antibody was found to increase vascular permeability and to accelerate the entry of neutrophils into chemically inflamed mouse peritoneum. Thus, VE-cadherin is essential for the integrity of the endothelial barrier in vivo. Our data suggest that opening of VE-cadherin mediated endothelial cell contacts may be a relevant step during neutrophil extravasation.
Sorting of MHC class II molecules and the associated invariant chain (li) in polarized MDCK cells
Authors: Simonsen Anne , Stang Espen , Bremnes Bjørn , Røe Målfrid , Prydz Kristian , Bakke Oddmund
Published: April 2021

Epithelial cells have been found to express MHC class II molecules in vivo and are able to perform class II-restricted antigen presentation. The precise intracellular localization of these molecules in epithelial cells has been a matter of debate. We have analyzed the polarized targeting of human MHC class II molecules and the associated invariant chain (Ii) in stably transfected MDCK cells. The class II molecules are located at the basolateral surface and in intracellular vesicles, both when expressed alone or together with Ii. Ii is located in basolateral endosomes and can internalize through the basolateral plasma membrane domain. We show that the cytoplasmic tail of Ii contains information for basolateral targeting as it is sufficient to redirect the apical protein neuraminidase (NA) to the baso lateral surface. We find that the two leucine-based motifs (LI and ML) in the cytoplasmic tail of Ii are individually sufficient for endosomal sorting and basolateral targeting of Ii in MDCK cells. In addition, basolateral sorting information is located within the 10 membrane-proximal residues of the Ii cytoplasmic tail. As several different signals mediate basolateral sorting of the class II/Ii complex, a polarized distribution of these molecules may be an essential feature of antigen presentation in epithelial cells.
Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins
Authors: Gimona Mario , Lando Zeev , Dolginov Yakov , Vandekerckhove Joel , Kobayashi Ryuji , Sobieszek Apolinary , Helfman David M.
Published: April 2021

Zero-length chemical crosslinking with 1-ethyl-3-[3-(dimethyl amino)propyl]carbodiimide (EDC) indicated an association of the Ca2+-binding protein S100A2 with tropomyosin (TM) in vitro. The mobility of the crosslinked product on SDS-PAGE gels indicated the formation of a 1:1 complex between S100A2 and TM and the interaction was Ca2+ dependent. Monoclonal antibodies were raised against S100A2 and used to determine its cellular localization in the porcine epithelial cell line LLC PK1. It was found that the localization of S100A2 depended on the differentiation state of the cells, being absent from actin stress fibers in sparsely seeded cultures, but present in the actin-containing microvilli characteristic of differentiated cells. Immunoprecipitations of [35S] methionine-labeled extracts using S100A2 as well as TM-specific antibodies failed to co-precipitate TM and S100A2, indicating a transient association between these two molecules in solution. Affinity chromatography of cell extracts on immobilized recombinant TMs, however, confirmed the Ca2+-dependent interaction between S100A2 and both muscle TMs as well as with high and low molecular mass nonmuscle TMs, suggesting that the binding site resides in one of the conserved regions of TM. Our data demonstrate the possible interaction of S100A2 with TM that is not bound to the microfilaments and indicate a differentiation-related function for S100A2 in LLC PK1 cells. The possible functional implications of this interaction are discussed.
The developmentally regulated Drosophila embryonic nuclear lamina protein ‘Young Arrest’ (fs(1)Ya) is capable of associating with chromatin
Authors: Lopez Jacqueline M. , Wolfner Mariana F.
Published: April 2021

The Drosophila fs(1)Ya protein (YA) is an essential component of the early embryonic nuclear lamina. Mutant zygotes lacking functional YA arrest in the first division cycles following fertilization, hence having a ‘Young Arrest’ of their development. The nuclear lamina is thought to act as the structural backbone for the nucleus and to provide anchoring sites for interphase chromosomes. Here, we demonstrate in vitro that YA is not required for the de novo formation of nuclear structures. Since YA’s sequence predicts potential DNA binding motifs, this protein may instead function to connect the lamina and chromosomes, and thus aid in organizing the nucleus. We ectopically expressed YA in polytene cells and demonstrated its association with polytene chromosomes, preferentially at interbands. Furthermore, our in vitro studies indicate that embryonic YA protein is capable of associating with decondensed chromatin. These observations suggest that YA may be required for the interaction between chromatin and the nuclear envelope during early embryogenesis.
Acid modulation of HT29 cell growth and differentiation An in vitro model for Barrett’s esophagus
Authors: Fitzgerald Rebecca C. , Omary M. Bishr , Triadafilopoulos George
Published: April 2021

Cell proliferation and differentiation are influenced by environmental factors, including the extracellular pH. We recently showed, using an ex vivo organ culture system of human mucosal Barrett’s esophageal biopsies, that acid has a highly variable effect on cell proliferation and differentiation depending on the pattern of acid exposure. Study of the mechanisms underlying these dynamic effects of acid on this premalignant intestinal-like epithelium is hampered by lack of an immortalized cell line. We therefore investigated the effect of acid exposure on the human colonic carcinoma cell line HT29, chosen because of its intestinal cell derivation and its ability to differentiate in vitro. HT29 cells exposed to pH 5 medium either continuously (up to 3 weeks), or as a short (1 hour) pulse, were compared with cells cultured at pH 7.4. Villin expression was induced only by long term acid exposure, and correlated with the development of differentiated polarized cells that contain a brush border and microvillus inclusions. Chronic acid exposure arrested cell proliferation, whereas a 1 hour acid-pulse enhanced cell proliferation, as determined by [3H]thymidine incorporation assays and proliferating cell nuclear antigen expression. Serum starvation attenuated the hyperproliferative effect of an acid-pulse. In addition, the doubling time of at least the first cell cycle after an acidpulse was shortened. The Na/H exchanger is likely to play a role since the hyperproliferative acid-induced response was blocked by amiloride; and the activity of the exchanger was increased at acidic pH as determined by 22Na uptake. These results support a role for extracellular pH on cell proliferation and differentiation of HT29 cells. Furthermore, these findings parallel the dynamic effects of acid on Barrett’s esophagus, and suggest that HT29 cells could serve as an in vitro model for studying the mechanism of acid modulation in Barrett’s esophagus.
Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif
Authors: Zlatkine P. , Mehul B. , Magee A. I.
Published: April 2021

Several members of the Src family of protein tyrosine kinases have a N-terminal dual acylation motif which specifies their myristoylation and S-acylation. These lipid modifications are necessary for correct intracellular localisation to the plasma membrane and to detergent-resistant glycolipid-enriched membrane domains (GEMs). Using chimaeras of the Lck dual acylation motif with two normally cytosolic proteins (chloramphenicol acetyl transferase and galectin-3), we show here that this motif is sufficient to encode correct lipid modification and to target these chimaeras to the plasma membrane, as demonstrated by subcellular fractionation and confocal immunofluorescence microscopy of transiently transfected COS cells. In addition, the chimaeras are resistant to extraction with cold non-ionic detergent, indicating targeting to GEM subdomains in the plasma membrane. The dual acylation motif has potential for targeting proteins to specific plasma membrane subdomains involved in signalling.
Glucose deprivation-induced cytotoxicity in drug resistant human breast carcinoma MCF-7/ADR cells: role of c-myc and bcl-2 in apoptotic cell death
Authors: Lee Yong J. , Galoforo Sandra S. , Berns Christine M. , Tong William P. , Kim Hyeong Reh C. , Corry Peter M.
Published: April 2021

We investigated the effect of glucose deprivation treatment on clonogenicity in multidrug-resistant human breast carcinoma MCF-7/ADR cells. Survival of MCF-7/ADR cells decreased exponentially up to 8 hours of incubation in the glucose-free medium. The surviving fraction of these cells for 8 hours of glucose-deprivation treatment was 1.5×10−3. Photomicrographs and gel electrophoresis data suggest that glucose deprivation-induced cell death is associated with apoptosis. Data from western and northern blots showed an induction of c-myc gene expression during treatment with glucose-free medium in MCF-7/ADR cells. MCF-7/ADR cells transfected with c-myc antisense oligodeoxynucleotides became resistant to glucose deprivation-induced apoptosis. Overexpression of bcl-2 gene protected MCF-7/ADR cells from this apoptotic cell death. Taken together, these results indicate that c-myc expression is a necessary component of glucose-free medium induced apoptosis and bcl-2 prevents apoptotic death induced by c-myc.
The ‘jaws’ model of tau-microtubule interaction examined in CHO cells
Authors: Preuss U. , Biernat J. , Mandelkow E.M. , Mandelkow E.
Published: April 2021

Tau is a neuronal microtubule-associated protein which promotes microtubule assembly. The C-terminal half of the protein contains three or four tandem repeats that are often considered to be the microtubule binding domain. This view is in conflict with in vitro data showing that the repeat domain binds only weakly to microtubules while the domains flanking the repeats bind strongly, even in the absence of the repeats. This has lead us to propose a ‘jaws’ model of tau whereby the regions flanking the repeats are considered as targetting domains, responsible for positioning tau on the microtubule surface, and the repeats which act as catalytic domains for microtubule assembly. To examine whether this model is appropriate in vivo we generated recombinant tau isoforms and microinjected them into CHO cells. Immunofluorescence microscopy of microtubules and tau shows that binding to microtubules, stabilization of microtubules and formation of bundles is not achieved by tau constructs comprising individual domains, but requires the combination of the flanking regions and the repeat domain. The results show that the jaws model describes the interactions between tau and microtubules in living cells. Since the targetting and catalytic domains are affected differently by phosphorylation the model provides a basis for studying the regulation of the interaction between microtubules and tau or other microtubule-associated proteins.
Oncostatin M induces basic fibroblast growth factor expression in endothelial cells and promotes endothelial cell proliferation, migration and spindle morphology
Authors: Wijelath Errol S. , Carlsen Brian , Cole Toby , Chen Jing , Kothari Sudesh , Hammond William P.
Published: April 2021

Oncostatin M (OSM), a pleiotropic cytokine originally isolated from supernatants of the U937 histiocytic lymphoma cell line, has been shown to have regulatory effects on a wide variety of cultured and tumor cells. We investigated the effects of OSM on basic fibroblast growth factor (bFGF) gene expression in bovine arterial endothelial (BAE) cells. Levels of bFGF mRNA transcripts were low in uninduced BAE cells, were maximal at 8 hours of exposure to OSM, and returned to control levels by 24 hours. Induction of bFGF mRNA transcripts by OSM was dose-dependent. Nuclear transcriptional run-on analysis demonstrated that exposure of BAE cells to OSM stimulated bFGF gene transcription. OSM treatment of BAE cells enhanced the synthesis of bFGF protein as determined by ELISA assays. Immunocytochemistry studies demonstrated the presence of low levels of bFGF protein within the cytoplasm in uninduced cells. After stimulation for 8 hours with OSM there was significant staining for bFGF in the cytoplasm. However, 24 hours after exposure to OSM, bFGF antigen was located only within the nuclei. Western blot analysis demonstrated that OSM stimulated predominantly the synthesis of a 22 kDa form of bFGF. In addition, OSM stimulated endothelial cell proliferation and migration as well as acquisition of a spindle shape. Phosphorothioate antisense oligonucleotide directed against bFGF inhibited OSM induced BAE cell proliferation and spindle shape formation but had only a minimal effect on migration. The levels of the 22 kDa form of bFGF were reduced by antisense treatment indicating that OSM induced proliferation and morphology change is likely to be regulated by intracellular bFGF. Our studies suggest that OSM released at sites of vascular injury could stimulate angiogenesis by inducing bFGF synthesis, endothelial cell proliferation and migration.
A Drosophila Tpr protein homolog is localized both in the extrachromosomal channel network and to nuclear pore complexes
Authors: Zimowska Grażyna , Aris John P. , Paddy Michael R.
Published: April 2021

Here we report structural, molecular, and biochemical characterizations of Bx34, a Drosophila melanogaster nuclear coiled-coil protein which is localized to extrachromosomal and extranucleolar spaces in the nuclear interior and which is homologous to the mammalian nuclear pore complex protein Tpr. In the nuclear interior, Bx34 is excluded from chromosomes and the nucleolus and generally localizes to regions between these structures and the nuclear periphery. This distribution matches the ‘extrachromosomal channel network’ described previously. In the nuclear periphery, Bx34 localizes on or near nuclear pore complexes. Biochemically, Bx34 isolates exclusively with the nuclear matrix fraction. The Bx34 cDNA sequence predicts a large protein (262 kDa) with two distinct structural domains. The Bx34 N-terminal 70% (180 kDa) is predicted to form an extended region of coiled-coil, while the C-terminal 30% (82 kDa) is predicted to be unstructured and acidic. Bx34 shows moderate sequence identity over its entire length to the mammalian nuclear pore complex protein ‘Tpr’ (28% amino acid identity and 50% similarity). Furthermore, several of the sequence motifs and biochemical similarities between Bx34 and Tpr are sufficiently striking that it is likely that Bx34 and Tpr are functionally related. The Bx34 gene exists in a single copy in region 48C of chromosome 2R. The localization of coiledcoil Bx34 to both the nuclear interior and nuclear pore complexes and its sequence similarity to a known nuclear pore complex protein leads to speculations about a role for Bx34 in nucleo-cytoplasmic transport which we can test using molecular genetic approaches.
The transcriptional basis of chromosome pairing
Authors: Cook P. R.
Published: April 2021

Pairing between homologous chromosomes is essential for successful meiosis; generally only paired homologs recombine and segregate correctly into haploid germ cells. Homologs also pair in some somatic cells (e.g. in diploid and polytene cells of Drosophila). How homologs find their partners is a mystery. First, I review some explanations of how they might do so; most involve base-pairing (i.e. DNA-DNA) interactions. Then I discuss the remarkable fact that chromosomes only pair when they are transcriptionally active. Finally, I present a general model for pairing based upon the DNA-protein interactions involved in transcription. Each chromosome in the haploid set has a unique array of transcription units strung along its length. Therefore, each chromatin fibre will be folded into a unique array of loops associated with clusters of polymerases and transcription factors; only homologs share similar arrays. As these loops and clusters, or transcription factories, move continually, they make and break contact with others. Correct pairing would be nucleated when a promoter in a loop tethered to one factory binds to a homologous polymerizing site in another factory, before transcription stabilizes the association. This increases the chances that adjacent promoters will bind to their homologs, so that chromosomes eventually become zipped together with their partners. Pairing is then the inevitable consequence of transcription of partially-condensed chromosomes.
Lateral mobility of plasma membrane lipids in bull spermatozoa: heterogeneity between surface domains and rigidification following cell death
Authors: Ladha Shab , James Peter S. , Clark David C. , Howes Elizabeth A. , Jones Roy
Published: April 2021

Compartmentalization of surface membrane antigens into discrete regions or domains is a characteristic feature of differentiated cells. In mammalian spermatozoa at least 5 surface domains are known, implying the presence of barriers or boundaries within the plasma membrane. Using the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusibility of fluorescent lipid analogues 1,1’-dihexadecyl-3,3,3’3’-tetramethylindo-carbocyanine (DiIC16) and 5-(N-octa-decanoyl) aminofluorescein (ODAF), we have investigated lipid topology and dynamics in the plasma membrane of ejaculated bull spermatozoa. Contrary to reports in the literature, we have found that DiIC16 stains only dead or damaged spermatozoa whereas ODAF intercalates into the plasma membrane of both live and dead cells, each type showing a distinctive staining pattern. FRAP analysis with ODAF revealed that diffusion coefficients on live spermatozoa are significantly faster on the acrosome and postacrosome (29.3×10−9 cm2/second) than on the midpiece and principal piece (11.8× 10−9 cm2/second). Recovery (R) is >90% in all domains. ODAF diffusion also shows regionalized temperature-sensitivity with a 4-fold increase over the sperm head and a 1.8-fold increase on the tail between 20°C and 37°C. Remarkably, dead or permeabilized spermatozoa rapidly develop a large immobile phase (R<25%) over the whole plasma membrane. This rigidification is temperature insensitive and irreversible suggesting major changes in the physical state of membrane lipids. It is concluded that lipid diffusion in the plasma membrane of live bull spermatozoa is rapid and varies significantly between surface domains. Following permeabilization or cell death, however, a large immobile phase develops indicating substantial changes in membrane lipid disposition.
Keratin intermediate filament dynamics in cell heterokaryons reveals diverse behaviour of different keratins
Authors: Paramio Jesús M. , Casanova M. Llanos , Alonso Angel , Jorcano José L.
Published: April 2021

To study the dynamics of keratin intermediate filaments, we fused two different types of epithelial cells (PtK2 and BMGE+H) and studied how the keratins from the parental cells recombine and copolymerize to form the heterokaryon cytoskeleton. The behaviour of the keratins during this process was followed by immunofluorescence using specific antibodies. After fusion, the parental cytoskeletons undergo a depolymerization process most apparent in the region adjacent to the fusion area. The depolymerized subunits spread throughout the heterokaryon and copolymerize into a new hybrid cytoskeleton. The complete process is very rapid, occurring in 3-4 hours, thus demonstrating the highly dynamic nature of the keratin cytoskeleton. Although newly synthesised subunits contribute to the formation of the hybrid cytoskeleton, the process takes place with similar kinetics in the absence of protein synthesis, showing the dynamic nature of the keratins from pre-existing cytoskeletons. During this process, specific keratins behave differently. Keratins K8, K18, K5 and K10 are mobilised from the parental cytoskeletons and reassemble rapidly into the hybrid cytoskeleton (3-6 hours), whereas K14 requires a substantially longer period (9-24 hours). Thus, different keratins, even when they form part of the same heterodimeric/tetrameric complexes, as is the case for K5 and K14, exhibit different dynamics. This suggests that individual polypeptides or homopolymeric complexes rather than exclusively heterodimeric/ tetrameric subunits, as is currently thought, can also take part in keratin intermediate filament assembly and dynamics. Biochemical analysis performed in the absence of protein synthesis revealed greater amounts of K5 than of K14 in the soluble pool of BMGE+H cells. Crosslinking and immunoprecipitation experiments indicated an excess of monomeric K5, as well as of K5/K14 heterodimers and K5 homodimers in the soluble pool. These results are in agreement with the different dynamic behaviour of these keratins observed in immunofluorescence. On the contrary, the phosphorylation levels of K5 and K14 are similar in both the soluble pool and the polymerized fraction, suggesting that phosphorylation does not play an important role in the different dynamics displayed by these two proteins. In summary, our results demonstrate that, following fusion, the keratin intermediate filament network reshapes rather rapidly and that keratins are highly dynamic proteins, although this mobility depends on each particular polypeptide.
Internalization of prolactin receptor and prolactin in transfected cells does not involve nuclear translocation
Authors: Perrot-Applanat Martine , Gualillo Oreste , Buteau Helene , Edery Marc , Kelly Paul A.
Published: April 2021

Prolactin (PRL) interacts with a specific, well characterized plasma membrane receptor (PRLR) that is coupled to signal transduction pathways involving Jak2, Fyn, and MAP kinases, and signal transducers and activators of transcription (STAT). Although a few previous studies have indicated nuclear translocation of PRL in IL-2 stimulated T lymphocytes, PRL-dependent Nb2 lymphoma cell lines and 235-1 lactotrophs, the mechanisms of nuclear targeting remain unknown and conflicting results have been reported concerning the putative nuclear translocation of the PRLR. We therefore decided to investigate nuclear translocation of PRLR and PRL in various cell lines transfected with an expression plasmid encoding PRLR, using confocal laser microscopy. We have constructed various cDNAs of the long and short forms of the rat PRLR containing an oligonucleotide encoding a Flag epitope inserted either just before the N-terminal amino acid or in the C-terminal end of the mature receptor (named N-terminal or C-terminal Flag-tagged PRLR). The corresponding receptors function as the PRLR in transfected cells : they are expressed at the plasma membrane and in compartments of the secretory pathway, they bind PRL with normal affinity (Kd= 4×10−10M) and have the same capacity to stimulate the transcriptional activity of a milk protein (β-casein) gene as wild-type PRLR. In addition, the tagged receptors are much more efficiently immunodetected using anti-Flag antibodies, as compared to anti-PRL antibodies (U5 or U6). Immunofluorescence combined with detailed confocal laser microscopy showed that addition of PRL (0 to 12 hours) to COS-7, CHO and NIH-3T3 transfected fibroblasts induces rapid internalization of the receptor (long form), without any translocation to the nucleus. Using PRL-R tagged both in the N-terminal or C-terminal regions of the mature receptor excludes the possibility of a cleaved fragment which could have been subsequently imported into the nucleus. An absence of nuclear translocation of PRLR was also observed in a 293 cell line stably expressing the receptor, and in physiological targets for PRL, i.e. in Nb2 lymphoma cells expressing the Nb2 form of the receptor or in BGME mammary gland epithelial cells upon overexpression of a Flag-tagged PRLR. Similarly, the short form of the PRLR was not detected in nuclei of transfected COS cells upon PRL treatment. Clearly, our results provide evidence that internalization of the plasma membrane PRLR does not lead to nuclear translocation of the receptor, or part of it, in most fibroblasts and epithelial cells at physiological concentrations of PRL. Also, in co-local-ization experiments, PRL was internalized without nuclear translocation. Activation of STATs transcription factors and MAP kinases, as well as translocation of these proteins to the nucleus following their phosphorylation, probably remains the intracellular mechanism coupling stimulation to nuclear events.
Sphingolipid organization in biomembranes: what physical studies of model membranes reveal
Authors: Brown Rhoderick E.
Published: April 2021

Recent cell biological studies suggest that sphingolipids and cholesterol may cluster in biomembranes to form raft-like microdomains. Such lipid domains are postulated to function as platforms involved in the lateral sorting of certain proteins during their trafficking within cells as well as during signal transduction events. Here, the physical interactions that occur between cholesterol and sphingolipids in model membrane systems are discussed within the context of microdomain formation. A model is presented in which the role of cholesterol is refined compared to earlier models.
RagA is a functional homologue of S. cerevisiae Gtr1p involved in the Ran/Gsp1-GTPase pathway
Authors: Hirose Eiji , Nakashima Nobutaka , Sekiguchi Takeshi , Nishimoto Takeharu
Published: April 2021

Human RagA and RagB is reported to be 52% identical to a putative GTPase of Saccharomyces cerevisiae, Gtr1p. According to the reported nucleotide sequence, we amplified human RagA and RagBs cDNAs from the human B cell cDNA library with PCR. Both cDNAs rescued a cold sensitivity of S. cerevisiae, gtr1-11. Furthermore, we introduced into the cloned human RagA cDNA, the mutation ‘T21L’ corresponding to the gtr1-11 mutation which has been reported to suppress not only all of rcc1−, temperature-sensitive mutants of Ran/Gsp1p GTPase GDP/GTP-exchanging factor, but also rna1-1, a temperature-sensitive mutant of Ran/Gsp1p GTPase-activating protein. The resulting RagAgtr1-11 cDNA partially, but significantly, suppressed both rcc1− and rna1-1 mutations. These results indicated that RagA and RagBs are functional homologues of S. cervisiae Gtr1p. Interestingly, while wild-type human RagA and RagBs were localized within the cytoplasm, similar to S. cerevisiae Gtr1p, the mutated human RagAgtr1-11 corresponding to a dominant negative form of RagA was distributed in discrete speckles in the nucleus, being localized side by side with SC-35, a non-snRNP of the splicing complex. In contrast, a dominant positive form of RagA, Q66L was localized in the cytoplasm. Thus, RagA was suggested to shuttle between the cytoplasm and the nucleus, depending on the bound nucleotide state.
Platelet-derived growth factor (PDGF)-induced actin rearrangement is deregulated in cells expressing a mutant Y778F PDGF β-receptor
Authors: Ruusala Aino , Sundberg Christian , Arvidsson Ann-Kristin , Rupp-Thuresson Eva , Heldin Carl-Henrik , Claesson-Welsh Lena
Published: April 2021

Platelet-derived growth factor-stimulated actin rearrangement and edge ruffle formation have previously been shown to be dependent on activation of phosphatidylinositol 3′-kinase, the activity of which also is important for directed migration of cells. This lipid kinase binds to phosphorylated tyrosine residues Y740 and Y751 in the kinase insert of the human platelet-derived growth factor β-receptor. We examined the role of two other tyrosine residues in the kinase insert of this receptor, Y775 and Y778, for ligand-induced actin rearrangement. Both were shown to be phosphorylation sites; Y775 was only marginally phosphorylated in cells expressing the wild-type β-receptor, whereas Y778 was phosphorylated at higher stoichiometry. Mutant receptors Y775F, Y778F and Y775/778F were active kinases and mediated proliferative responses when expressed in porcine aortic endothelial cells. Fluorescence staining of actin in platelet-derived growth factor-stimulated PAE cells revealed that Y778 is involved in regulation of the actin cytoskeleton since the cells contained, apart from edge ruffles and circular ruffles, a novel type of giant ruffle on the dorsal side of the cell, which consisted of irregular multilayered actin structures. Mutation at Y778 had no effect on activation of phosphatidylinositol 3′-kinase, nor on the GTPase activating protein of Ras and phospholipase Cγ, and the extent of directed migration towards platelet-derived growth factor of these cells was not changed. We conclude that actin rearrangement is regulated in part by Y778 in the platelet-derived growth factor β-receptor, potentially through binding of a novel signaling molecule to this site.
In vivo association of lamins with nucleic acids in Drosophila melanogaster
Authors: Rzepecki Ryszard , Bogachev Sergei S. , Kokoza Elena , Stuurman Nico , Fisher Paul A.
Published: April 2021

A 32P-labeling strategy was developed to study the interaction(s) in tissue culture cells between proteins and nucleic acids. Interphase and mitotic nuclear lamins were studied in Drosophila Kc cells. After bromodeoxyuridine incorporation and in vivo photo-crosslinking with 366 nm light, it was found that interphase lamins were associated with nucleic acid. Interactions with DNA as well as RNA were detected. In contrast, interaction of nucleic acids with mitotic lamin was not observed. Photo-crosslinking in the presence of antibiotics distamycin and/or chromomycin suggested that interphase lamins interacted with both A-T-rich DNA and G-C-rich DNA; interactions with G-C-rich DNA predominated. These results have implications for understanding the interphase organization of the higher eukaryotic cell nucleus as well as the transition of cells from interphase to mitosis. A model of nuclear organization, consistent with our results, is proposed.
Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus
Authors: Röttger Sabine , White Jamie , Wandall Hans H. , Olivo Jean-Christophe , Stark Annika , Bennett Eric P. , Whitehouse Caroline , Berger Eric G. , Clausen Henrik , Nilsson Tommy
Published: April 2021

O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19). However, upon relocation of chimeric GalNAc-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficiencies indicating that all components required for initiation of O-glycosylation are present in the ER except for polypeptide GalNAc-transferases.
T cell syncytia induced by HIV release T cell chemoattractants: demonstration with a newly developed single cell chemotaxis chamber
Authors: Shutt Damon C. , Jenkins Leslie M. , Carolan Edward J. , Stapleton Jack , Daniels Karla J. , Kennedy Ronald C. , Soll David R.
Published: April 2021

A chemotaxis chamber has been developed to analyze both the velocity and the directionality of individual T cells in gradients of high molecular mass molecules over long periods of time. Employing this chamber, it is demonstrated that syncytia induced by HIV in SUP-T1 cell cultures release two T cell chemoattractants with approximate molecular masses of 30 and 120 kDa. Neither uninfected single cells nor polyethylene glycol-induced syncytia release detectable chemoattractant, suggesting that these chemoattractants are linked to HIV infection. Soluble gp120 functions as a T cell chemoattractant and the addition of anti-gp120 antibody to syncytium-conditioned medium blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. The addition of anti-CD4 antibody to syncytium-conditioned medium also blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. These results demonstrate that HIV-induced T cell syncytia release a low and a high molecular mass T cell chemoattractant, and suggest that the high molecular mass factor is gp120 and that it functions through the CD4 receptor.
Ret finger protein is a normal component of PML nuclear bodies and interacts directly with PML
Authors: Cao Tongyu , Duprez Estelle , Borden Katherine L. B. , Freemont Paul S. , Etkin Laurence D.
Published: April 2021

The ret finger protein (rfp) is a member of the B-box zinc finger gene family many of which may function in growth regulation and in the appropriate context become oncogenic. Members of this family are nuclear proteins that possess a characteristic tripartite motif consisting of the RING and B-box zinc binding domains and a coiled-coil domain. The promyelocytic leukemia gene (PML), another B-box family member, produces a protein product that is detected within punctate nuclear structures called PML nuclear bodies (NBs) or PML oncogenic domains (PODs). These NBs are complex structures that consist of a number of different proteins many of which have yet to be identified. In the disease acute promyelocytic leukemia (APL) a fusion protein, PML-RARA, is produced through the t(15:17) translocation. In APL the morphology of the NBs is altered. We report that rfp co-localizes with PML in a subset of the PML NBs and that it interacts directly with PML. This interaction is mediated through the rfp B-box and the distal two coils. In contrast, homomultimerization of rfp preferentially involves the B-box and the proximal coil. The association of rfp with the PML NBs is altered by mutations that affect rfp/PML interaction and in NB4 cells that are derived from APL patients. When treated with retinoic acid, rfp reassociates with the NBs in a pattern similar to non APL cells. Additionally, we found that rfp colocalizes with PML-RARA protein produced in APL patients. These results suggest that rfp, along with the other known/unknown components of PML NBs, have an important role in regulating cellular growth and differentiation.
Clustering of pericentromeres initiates in step 9 of spermiogenesis of the rat (Rattus norvegicus) and contributes to a well defined genome architecture in the sperm nucleus
Authors: Meyer-Ficca Mirella , Müller-Navia Jutta , Scherthan Harry
Published: April 2021

Fluorescence in situ hybridization with centromeric, telomeric and whole chromosome paint probes was used to study nuclear topology in epididymal sperm as well as spermatids from testis tissue sections of the rat. Pericentromeric regions of 9 chromosomes of the rat (n=21) were labeled with a satellite I specific DNA probe. Pericentromeres showed few tandem associations in spermatids of steps 1-8 of spermiogenesis. At step 9, pericentromeric regions associated to form an elongated cluster in the spermatid nucleus. This arrangement was also seen in the sperm nucleus. FISH with telomere probes revealed numerous, variably arranged signals in round and elongated spermatids as well as sperm nuclei. Telomere signals showed a tendency for pairwise association which was more pronounced in elongated spermatid and epididymal sperm nuclei. FISH to DTT treated sperm suggested that telomeres reside at the periphery and that pericentromeres are located in the nuclear interior. Chromosome painting with rat chromosome 2 and 12 specific microdissection library probes showed that these chromosomes predominantly occupy compact and variably shaped territories during spermatid maturation. In elongated epididymal sperm nuclei chromosome 2 and 12 territories took up specific positions. We suppose that the associations of pericentromeres during step 9 render a well defined nuclear topology which facilitates the ordered compaction of the genome at subsequent stages.