bluuebunny/crossref_metadata_2025 huggingface dataset:
Three microtubule-organizing centres collaborate in a mouse cochlear epithelial cell during supracellularly coordinated control of microtubule positioning
Authors: Henderson Craig G. , Tucker John B. , Mogensen Mette M. , Mackie John B. , Chaplin Mark A. , Slepecky Norma B. , Leckie Lorna M.
Published: April 2021

Large cell surface-associated microtubule bundles that include about 3,000 microtubules assemble in certain epithelial cells called inner pillar cells in the mouse organ of Corti. Microtubule-organizing centres (MTOCs) at both ends and near the middle of each cell act in concert during control of microtubule positioning. In addition, the three cell surface-associated microtubule-organizing centres are involved in coordinating the connection of bundle microtubules to cytoskeletal components in neighbouring cells and to a basement membrane.

The precisely defined locations of the three MTOCs specify the cell surface regions where microtubule ends will finally be anchored. The MTOCs are modified as anchorage proceeds. Substantial fibrous meshworks assemble at the surface sites occupied by the MTOCs and link microtubule ends to cell junctions. This procedure also connects the microtubule bundle to cytoskeletal arrays in neighbouring cells at two of the MTOC sites, and to the basilar membrane (a substantial basement membrane) in the case of the third site. A fourth meshwork that is not positioned at a major MTOC site is involved in connecting one side of the microtubule bundle to the cytoskeletons of two other cell neighbours. The term surfoskelosome is suggested for such concentrations of specialized cytoskeletal materials and junctions at cell surface anchorages for cytoskeletal arrays.

The large microtubule bundle in each cell is composed of two closely aligned microtubule arrays. Bundle assembly begins with nucleation of microtubules by a centrosomal MTOC that is attached to the apical cell surface. These microtubules elongate downwards and the plus ends of many of them are apparently captured by a basal MTOC that is attached to the plasma membrane at the bottom of the cell. In the lower portion of the cell, the microtubule bundle also includes a basal array of microtubules but these elongate in the opposite direction. This investigation provides evidence that they extend upwards from the basal MTOC to be captured by a medial MTOC which is attached to the plasma membrane and situated near the mid-level of the cell. However, there are sub-stantial indications that the basal array’s microtubules are also nucleated by the apically situated centrosomal MTOC, but escape from it, and are translocated downwards for capture of their plus ends by the basal MTOC. If this is the case, then these microtubules continue to elongate after translocation and extend back up to the medial MTOC, which captures their minus ends.
The gene b protein localises to the surface of Leishmania major parasites in the absence of metacyclic stage lipophosphoglycan
Authors: Rangarajan Desikan , Gokool Suzanne , McCrossan Maria V. , Smith Deborah F.
Published: April 2021

The stage specific Gene B protein (GBP) of Leishmania major localises to the surface of infective metacyclic parasites, where it associates with the developmentally regulated surface glycoconjugate, lipophosphoglycan (LPG). This association has been proposed to aid maintenance of GBP on the parasite surface. In this paper, we show that the abundance of GBP on the extracellular meta-cyclic cell surface is in the order of 100,000 copies per cell. This level of expression is comparable to that seen in the intracellular amastigote stage, in which GBP is also localised to the surface, despite the lack of metacyclic stage specific LPG. Furthermore GBP expressed from an episome in avirulent parasites, which are unable to synthesise metacyclic LPG or endogenous GBP, also localises to the parasite surface. These data demonstrate that GBP can maintain a surface localisation in the absence of meta-cyclic LPG, suggesting that it is able to associate with other glycoconjugates on the surface of infective parasites.
Freeze-fracture replica electron microscopy combined with sds digestion for cytochemical labeling of integral membrane proteins: Application to the immunogold labeling of intercellular junctional complexes
Authors: Fujimoto Kazushi
Published: April 2021

We propose a new electron microscopic method, the sodium dodecylsulphate (SDS)-digested freeze-fracture replica labeling technique, to study the two-dimensional distribution of integral membrane proteins in cellular membranes. Unfixed tissue slices were frozen with liquid helium, freeze-fractured, and replicated in a platinum/carbon evaporator. They were digested with 2.5% SDS to solubilize unfractured membranes and cytoplasm. While the detergent dissolved unfractured membranes and cytoplasm, it did not extract fractured membrane halves. After SDS-digestion, the platinum/carbon replicas, along with attached cytoplasmic and exoplasmic membrane halves, were processed for cyto-chemical labeling, followed by electron microscopic observation. As an initial screening, we applied this technique to the immunogold labeling of intercellular junction proteins: connexins (gap junction proteins), occludin (tight junction protein), desmoglein (desmosome protein), and E-cadherin (adherens junction protein). The immunogold labeling was seen superimposed on the image of a fracture face visualized by platinum/carbon shadowing. The immunoreaction was specific, and only the structures where the proteins were expected were labeled. For instance, anti-occludin immunogold complexes were observed immediately adjacent to the tight junction strands on the protoplasmic and exoplasmic fracture faces. No significant levels of gold label were associated with non-tight-junctional regions of plasma membranes. The procedures of the SDS-digested freeze-fracture replica labeling and its potential significance are discussed.
Xenopus lamin b3 has a direct role in the assembly of a replication competent nucleus: Evidence from cell-free egg extracts
Authors: Goldberg Martin , Jenkins Hazel , Allen Terry , Whitfield William G. F. , Hutchison Christopher J.
Published: April 2021

Xenopus egg extracts which assemble replication competent nuclei in vitro were depleted of lamin B3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were still capable of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that most nuclei assembled in lamin B3-depleted extracts have continuous nuclear envelopes and well formed nuclear pores. However, several consistent differences were observed. Most nuclei were small and only attained diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the assembly of nuclear pores was slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B3-depleted extracts had well formed double unit membranes containing a high density of nuclear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of ‘depleted’ extracts was investigated. While lamin B3 was recovered efficiently from cytosolic and membrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2 did not co-precipitate with lamin B3 during immunodepletion experiments, several protein species did specifically associate with lamin B3 on para-magnetic immunobeads. The major protein species associated with lamin B3 migrated with molecular masses of 102 kDa and 57 kDa, respectively, on one-dimensional poly-acrylamide gels. On two-dimensional O’Farrell gels the mobility of the 102 kDa protein was identical to the mobility of a major nuclear matrix protein, indicating a specific association between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts did not assemble a lamina, judged by indirect immunofluorescence, and failed to initiate semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin B3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.
The Aspergillus nidulans bime (Blocked-In-Mitosis) gene encodes multiple cell cycle functions involved in mitotic checkpoint control and mitosis
Authors: James Steven W. , Mirabito Peter M. , Scacheri Peter C. , Morris N. Ronald
Published: April 2021

The bimE (blocked-in-mitosis) gene appears to function as a negative mitotic regulator because the recessive bimE7 mutation can override certain interphase-arresting treatments and mutations, causing abnormal induction of mitosis. We have further investigated the role of bimE in cell cycle checkpoint control by: (1) coordinately measuring mitotic induction and DNA content of bimE7 mutant cells; and (2) analyzing epistasis relationships between bimE7 and 16 different nim mutations. A combination of cytological and flow cytometric techniques was used to show that bimE7 cells at restrictive temperature (44°C) undergo a normal, although somewhat slower cell cycle prior to mitotic arrest. Most bimE7 cells were fully reversible from restrictive temperature arrest, indicating that they are able to enter mitosis normally, and therefore require bimE function in order to finish mitosis. Furthermore, epistasis studies between bimE7 and mutations in cdc2 pathway components revealed that the induction of mitosis caused by inactivation of bimE requires functional p34cdc2 kinase, and that mitotic induction by bimE7 depends upon several other nim genes whose functions are not yet known. The involvement of bimE in S phase function and mitotic checkpoint control was suggested by three lines of evidence. First, at restrictive temperature the bimE7 mutation slowed the cell cycle by delaying the onset or execution of S phase. Second, at permissive temperature (30°C) the bimE7 mutation conferred enhanced sensitivity to the DNA synthesis inhibitor hydroxyurea. Finally, the checkpoint linking M phase to the completion of S phase was abolished when bimE7 was combined with two nim mutations that cause arrest in G1 or S phase. A model for bimE function based on these findings is presented.
Identification of a molecular chaperone in the eukaryotic flagellum and its localization to the site of microtubule assembly
Authors: Bloch Melissa A. , Johnson Karl A.
Published: April 2021

Monoclonal antibodies that recognize HSP70 family members from evolutionarily divergent organisms were used to identify both constitutively-expressed and stress-inducible HSP70 proteins in the green alga Chlamy-domonas. These monoclonal antibodies also cross-reacted with a 70 kDa flagellar protein that comigrated with the constitutively-expressed HSP70 isoform(s) present in the cell body; this is the first identification of a molecular chaperone within the eukaryotic flagellum. Fractionation experiments demonstrated that much of the flagellar HSP70 was bound to the ‘9+2’ microtubule axoneme. Incubation of isolated axonemes in ATP, but not AMP or AMP-PNP, caused significant release of the previously bound HSP70 as is characteristic of complexed HSP70s. Immuno-fluorescent localization in whole flagella showed that flagellar HSP70 was concentrated at the distal ends of flagella, sites of axonemal assembly in vivo. Extraction of axonemes under ionic conditions known to cause the release of capping structures that link the distal ends of the axonemal microtubules to the flagellar membrane also caused the release of axonemal-bound HSP70. Taken together, these results suggest a model in which an HSP70 chaperone may assist in targeting tubulin and other unassembled axonemal components to the flagellar tip where the chaperone may also participate in the assembly of the ‘9+2’ flagellar axoneme.
A survey of gtp-binding proteins and other potential key regulators of exocytotic secretion in eosinophils : Apparent absence of rab3 and vesicle fusion protein homologues
Authors: Lacy Paige , Thompson Nicki , Tian Min , Solari Roberto , Hide Izumi , Newman Terence M. , Gomperts Bastien D.
Published: April 2021

We set out to identify potential key regulators of exocytotic fusion in the eosinophil, in the knowledge that granule exocytosis can be stimulated in these cells by intracellular application of nonhydrolyzable analogues of guanosine triphosphate, with Ca2+ acting as a modulator of guanine nucleotide-dependent secretion. To screen for GTP-binding proteins, guinea pig eosinophils were purified from peritoneal washings and subjected to western blotting analysis using specific immune sera raised against recombinant proteins or consensus peptide sequences within proteins of interest. We found a number of heterotrimeric G proteins (Gαi3, Gαo, Gαq11, Gαs and Gβ subunits) and members of the small GTP-binding proteins expressed in eosinophils. Two subtypes of G-protein alpha subunits (Gαi1 and Gαz) could not be detected. Separation of sub-cellular organelles from homogenized eosinophils by density gradient centrifugation revealed that all of the detected GTP-binding proteins were mainly expressed in fractions containing peak plasma membrane and Golgi marker enzyme activities, while Gβ subunits were also detected in secretory granule fractions. However, isoforms of Rab3, a putative GTP-binding regulator of exocytotic fusion, were undetectable in eosinophils. Neither, with the exception of syntaxin-3, could we detect any of the proteins belonging to the proposed synaptic vesicle fusion complex (SNAP-25; synaptobrevin (VAMP) and its non-neuronal homologue, cellubrevin; synaptophysin; synaptotagmin). The results from this study, based on western blotting, suggest that eosinophils express a different class of exocytotic fusion complex proteins from those found in neuronal tissues, although a number of potential candidates fulfilling the role of GE were identified in this important inflammatory cell.
Lung surfactant protein A (SP-A) activates a phosphoinositide/calcium signaling pathway in alveolar macrophages
Authors: Ohmer-Schröck Daniela , Schlatterer Christina , Plattner Helmut , Schlepper-Schäfer Jutta
Published: April 2021

Lung surfactant protein A (SP-A), the main protein component of lung surfactant which lines the alveoli, strongly enhances serum-independent phagocytosis of bacteria by rat alveolar macrophages. We tested if the effect of SP-A is due to interaction with the macrophages or to opsonization of the bacteria. In phagocytosis assays with fluorescein isothiocyanate labeled bacteria, SP-A had no opsonic effect on Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, but enhanced phagocytosis by acting only on the macrophages. We characterized this activation mechanism. With single cell measurements of fura-2 loaded cells we demonstrate that SP-A raises the intracellular free calcium ion concentration 6 to 8 seconds after addition. This calcium mobilization is dosedependent in that increased SP-A concentrations lead to a higher percentage of responding cells. Additionally, SP-A leads to a dosedependent and transient generation of inositol 1,4,5-trisphosphate. Release of intracellular stored calcium by SP-A is a prerequisite for its stimulatory effect on phagocytosis, since SP-A-induced enhancement of phagocytosis can be impaired by prior addition of thapsigargin, a Ca2+-ATPase inhibitor that leads to depletion of intracellular calcium stores.

We conclude that SP-A activates a phosphoinositide/calcium signaling pathway in alveolar macrophages leading to enhanced serumindependent phagocytosis of bacteria.
The role of macrophage receptors in adhesion and uptake of Leishmania mexicana amastigotes
Authors: Peters Christopher , Aebischer Toni , Stierhof York-Dieter , Fuchs Manuela , Overath Peter
Published: April 2021

Amastigotes of the protozoan parasite Leishmania proliferate in phagolysosomes of mammalian macrophages. Propagation of the infection is considered to occur by hostcell rupture and uptake of released parasites by uninfected macrophages. In this study, the kinetics of binding of L. mexicana mexicana amastigotes to COS cells and to COS cells transfected with three different macrophage receptors (FcRII-B2, receptor for the Fc-domain of immunoglobulins; CR3, complement type 3 receptor and the mannose receptor) is compared to the rate of adhesion to peritoneal macrophages. Amastigotes isolated from macrophages cultivated in vitro bind with slow, sigmoid kinetics to COS cells expressing either of the three receptors, or to peritoneal macrophages. In contrast, amastigotes isolated from mouse lesions bind with rapid, hyperbolic kinetics to COS cells expressing the Fc receptor or to peritoneal macrophages but with slow, sigmoid kinetics to COS cells expressing the CR3 or the mannose receptor. As shown by immunofluorescence experiments, lesion-derived amastigotes contain host-derived immunoglobulins (Ig) but no complement component 3 at their surface. It is concluded that amastigotes contain no intrinsic ligand at their surface, which enables high-affinity interactions with macrophages. Opsonization by specific Ig may be of relevance in vivo because firstly, in cryosections of mouse lesions extracellular amastigotes containing surface Ig can be detected and, secondly, B cell-deficient mice reconstituted with parasite-specific Ig show a modest increase in the rate of lesion development. In addition, it is shown that amastigotes are internalized by COS cells and grow in large para-sitophorous vacuoles similar to those observed in macrophages.
Synthesis and assembly of human β1 gap junctions in BHK cells by DNA transfection with the human β1 cDNA
Authors: Kumar Nalin M. , Friend Daniel S. , Gilula Norton B.
Published: April 2021

Gap junctional communication is important in many phys-iological processes, including growth control, patterning, and the synchronization of cell-to-cell activities. It has been difficult to study the synthesis and assembly of gap junctions due to their low abundance. To overcome this limitation, baby hamster kidney cells (BHK) have been transfected with a human β1 (Cx32) connexin cDNA construct. Expression was placed under the control of the mouse metallothionein promoter that can be induced by heavy metals. The transfected cells were characterized by DNA, RNA and protein analysis, as well as by scrape loading to detect functional channels. Functional β1 connexin was detected only in cells transfected with β1 connexin cDNA in the correct orientation (β1-BHK). Analysis of the cells by light microscopic immunocyto-chemistry indicated that β1 connexin antigen was localized to the plasma membrane and to several intracellular compartments. Characterization with thin section electron microscopy revealed extensive areas of assembled double membrane gap junctions between cells (on the cell surface), in the endoplasmic reticulum (ER), and the nuclear envelope. This unusual intracellular distribution for assembled gap junction protein was confirmed by freeze fracture analysis, which revealed large particle aggregates, characteristic of gap junction plaques, on the fracture faces of all these membranes. The presence of gap junction particle aggregates in the ER suggests that the oligomer-ization of connexin can occur at its site of synthesis. Further, the process of assembly into double membrane junction structures in intracellular membranes may be driven by connexin protein concentration.
A tumor-associated glycosylation change in the glucose transporter GLUT1 controlled by tumor suppressor function in human cell hybrids
Authors: Kitagawa Takayuki , Tsuruhara Yasuko , Hayashi Masahiro , Endo Tamao , Stanbridge Eric J.
Published: April 2021

Studies of human cell hybrids have provided evidence that the tumorigenicity of a cervical carcinoma (HeLa) is under the control of a putative tumor suppressor on chromosome 11. Using these human cell hybrids, we found a tumor-associated glycosylation change in the glucose transporter GLUT1, which is an N-linked glycoprotein at the plasma membrane. The non-tumorigenic HeLa × fibroblast cell hybrid CGL1 and the normal diploid fibroblast WI38 expressed the 50-55 kDa GLUT1, whereas in a tumorigenic segregant hybrid, CGL4, as well as in parental HeLa cells, GLUT1 glycosylation was altered and its molecular mass was about 70 kDa. However, the altered GLUT1 glycosylation was not observed in SV40-transformed WI38 cells, suggesting a correlation between this glycosylation change and a putative tumor suppressor function. Further investigations using glycosidases, glycosylation inhibitors and lectin-affinity chromatography demonstrated that the tumor-associated glycosylation change in GLUT1 was mainly due to the increase in N-acetyl-lactosamine repeats in the N-linked oligosaccharides. In accordance with the altered glycosylation, affinity for 2-deoxyglucose in the tumorigenic CGL4 cells increased 2-fold, but there was little change in the Vmax. These results suggest there may be a functional role for the modulation by glycosylation of GLUT1 in the tumorigenic behavior of CGL4 and HeLa cells.
Cell spreading and the regulation of ornithine decarboxylase
Authors: Morrison Ron F. , Seidel Edward R.
Published: April 2021

The aim of this study was to investigate the effect of cell spreading on the induction of ornithine decarboxylase and the rate of putrescine uptake in anchorage-dependent and anchorage-independent cells. Plating non-transformed IEC-6 epithelial cells at high versus low cell density restricted cell spreading from 900 μm2 to approximately 140 μm2, blunted the transient induction of ornithine decarboxylase activity from 202 to 32 pmol 14CO2/mg protein per hour and reduced the rate of [14C]putrescine uptake from 46 to 23 pmol/105 cells per hour. The mean spreading area of the cell population was controlled by coating tissue culture dishes with the nonadhesive polymer, polyHEMA. Ornithine decarboxylase activity and putrescine uptake correlated with cell spreading with minimal spreading (263 μm2) corresponding to an 83% decrease in ornithine decarboxylase activity and 51% decrease in the rate of putrescine uptake. Adding the RGD peptide, Gly-Arg-Gly-Glu-Ser-Pro to the medium of sparsely plated cells resulted in rapid reductions in cell spreading concomitant with dose-dependent decreases in ornithine decarboxylase activity and putrescine uptake. Finally, minimizing cell spreading by depriving cells of sub-stratum contact completely abolished serum-induced increases in ornithine decarboxylase and reduced the rate of putrescine uptake by 47%. In contrast to IEC-6 cells, ornithine decarboxylase of neoplastic HTC-116 cells was constitutively expressed with basal and stimulated activity (193 and 982 pmol 14CO2/mg protein per hour, respec-tively) completely independent of cell adhesion. Putrescine uptake, however, was abolished in the absence of cell adhesion. These data suggest that the induction of ornithine decarboxylase activity and the rate of putrescine uptake correlate with spreading of anchorage-dependent IEC-6 cells and that ornithine decarboxylase activity, but not putrescine uptake, appears to be independent of spreading of neoplastic HTC-116 cells.
Microtubule organization and the distribution of γ-tubulin in spermatogenesis of a beetle, Tenebrio molitor (Tenebrionidae, Coleoptera, Insecta)
Authors: Wolf Klaus Werner , Joshi Harish C.
Published: April 2021

The present study focuses on the restructuring of the microtubule (MT) cytoskeleton and microtubule-organizing centres (MTOCs) throughout spermatogenesis of a darkling beetle, Tenebrio molitor (Tenebrionidae, Coleoptera, Insecta). To this end, serial ultrathin sections through male germ cells were studied using transmission electron microscopy. Additionaly, spindles and young sper-matids were isolated from testes under MT-stabilizing con-ditions and doubly labeled with antibodies against β- and γ-tubulin. The latter is a tubulin isoform detected in MTOCs of a wide variety of species. The observations suggest that microtubules may be nucleated from sites with and without high γ-tubulin content and that these sites do not necessarily possess canonical centrosomes. In a prominent cytoplasmic MT system of primary spermato-cytes in prophase, microtubule nucleation apparently occurs in the absence of immunologically detectable γ-tubulin. At the poles of the meiotic spindles, MTs are directly inserted into γ-tubulin-containing material and this connection is considered responsible for their nucleation. The interzone spindle MTs of telophase cells contain γ-tubulin and this may confer stability to them. Finally, manchette MTs of spermatids originate in the vicinity of the acrosome precursor but are not inserted into this body. The acrosome precursor is surrounded by a membrane and is clearly detected by the antibody against γ-tubulin.
Coupling of [ca2+]i and ciliary beating in cultured tracheal epithelial cells
Authors: Salathe Matthias , Bookman Richard J.
Published: April 2021

The molecular mechanisms responsible for the regulation of ciliary beating frequency (CBF) are only partially characterized. To determine whether elevation of intracellular Ca2+ ([Ca2+]i) can cause an increase in CBF, we measured CBF and Ca2+ in single cells. Ovine tracheal epithelial cells, obtained by dissociation with protease, were grown in primary culture for 1 to 28 days in a mucus-free system. CBF of a single cilium was measured by digital video phase-contrast microscopy and on-line Fourier-transform analysis. Changes in [Ca2+]i from single cells were determined with fura-2, using ratio imaging video microscopy. Activation of a muscarinic pathway with 10 μM ACh (acetylcholine) increased [Ca2+]i from 53±9 nM (mean ± s.e.m.) to 146±12 nM or to 264±51% above initial baseline. In the same cells, ACh increased CBF from a baseline of 7±0.5 Hz to 9±0.2 Hz or to 31±2.8% above baseline (n=14). The elevations of both [Ca2+]i and CBF were transient and relaxed back to an elevated plateau (10/14 cells) as long as ACh was present. To elevate [Ca2+]i by mechanisms independent of a G-protein-coupled receptor, we measured [Ca2+]i and CBF of the same cells in extracellular solutions with either 0 Ca2+ (+ 1 mM EGTA) or 10 mM Ca2+. Both signals rose and fell with similar kinetics in response to changing [Ca2+]o, suggesting that changes in [Ca2+]i alone can modulate CBF. In a second independent manipulation, cells were treated with 1 μM thapsigargin, an irreversible inhibitor of the endoplasmic reticulum Ca2+-ATPase. Upon thapsigargin addition, 37 of 42 cells showed a transient [Ca2+]i increase and, as measured in different experiments, 8 of 9 cells showed a transient increase in CBF. Interestingly, application of ACh after cells were treated with thapsigargin produced decreases in both [Ca2+]i and CBF in 8/8 cells. Lastly, after 1-3 days in culture, addition of 10 μM ACh often produced [Ca2+]i oscillations rather than transients in [Ca2+]i. Measurements of CBF in these cells showed frequency modulation of CBF with the same peak-to-peak time interval as the Ca2+ oscillation. These results show that: (1) CBF can be measured from a single cilium and monitored on-line to track changes; (2) CBF and [Ca2+]i can be measured in the same single cell; (3) transient changes in [Ca2+]i (induced by 4 different manipulations) are associated with kinetically similar changes in CBF; and (4) [Ca2+]i oscillations are coupled to frequency modulation of ciliary beating. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of CBF in mammalian tracheal epithelial cells.
Mg-160, a membrane sialoglycoprotein of the medial cisternae of the rat golgi apparatus, binds basic fibroblast growth factor and exhibits a high level of sequence identity to a chicken fibroblast growth factor receptor
Authors: Gonatas Jacqueline O. , Mourelatos Zissimos , Stieber Anna , Lane William S. , Brosius Jurgen , Gonatas Nicholas K.
Published: April 2021

We report the primary structure of MG-160, a 160 kDa membrane sialoglycoprotein residing in the medial cisternae of the Golgi apparatus of rat neurons, pheochromocytoma (PC-12), and several other cells. The cDNA encodes a polypeptide of 1,171 amino acids with an Mr of 133,403. An intralumenal cleavable signal peptide is followed by a Pro-Gln-rich segment and 16 contiguous, approx. 60-residue-long, regularly spaced cysteine-rich segments showing sequence identities ranging from 15 to 35%. The lumenal domain is followed by a single membrane spanning domain and a short carboxy-terminal cytoplasmic tail. The protein contains 5 potential NXT glycosylation sites.

The sequence of MG-160 shows no homologies with enzymes and other membrane proteins of the Golgi apparatus. MG-160 displays a so far unique feature for a membrane protein of the Golgi apparatus: namely, an upstream open reading frame (uORF), encoding 58 amino acids, located in front of the major open reading frame (ORF). Most vertebrate mRNAs containing uORF or AUG codons in front of the major ORF encode growth factors and cell surface receptors (Geballe and Morris 1994). In that regard a 90% identity between the primary structure of MG-160 and a receptor for acidic and basic fibroblast growth factors (CFR), isolated from chicken embryos (Burrus et. al., 1992), may be relevant.

Immunoreactivity for MG-160 has been detected in the Golgi apparatus of neural and other cells of 2-day-old chicken embryos and adult chicken; furthermore, recombinant human basic fibroblast growth factor (bFGF) binds MG-160 purified from rat brain. MG-160 shows no sequence similarity with members of the family of fibroblast growth factor receptors (FGFR) involved in signal transduction. These findings are consistent with the hypothesis that MG-160 is involved in the traffic and processing of endogenous or autocrine FGFs. This is the first example of an intrinsic membrane protein of the Golgi apparatus which binds a growth factor and may be involved in its regulation.
The Schizosaccharomyces Pombe Hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis
Authors: Al-Khodairy Fahad , Enoch Tamar , Hagan Iain M. , Carr Antony M.
Published: April 2021

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called ‘check-points’, mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs).

To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.
Sequence and tissue distribution of the human integrin α8 subunit: a β1-associated α subunit expressed in smooth muscle cells
Authors: Schnapp Lynn M. , Breuss Johannes M. , Ramos Daniel M. , Sheppard Dean , Pytela Robert
Published: April 2021

Integrins are a major family of cell adhesion molecules involved in cell-cell and cell-extracellular matrix interactions. Each integrin is a heterodimeric glycoprotein composed of an α and a β subunit. We now report the cDNA sequence and distribution of a new human integrin α subunit. This sequence is 78% identical to the previously reported chicken α8 integrin sequence. Thus, we have designated this subunit as human α8. By northern blot analysis, an α8 probe detects two mRNA species of approximately 6.5 and 4.0 kb in neuroglioma H4 cells. An anti-α8 polyclonal antibody precipitates a protein complex containing the β1 subunit associated with the putative α8 subunit, which has an apparent molecular mass of 180 kDa (non-reduced) or 155 kDa and 25 kDa (reduced). Immunohistochemistry with anti-α8 polyclonal antibody in adult rat tissues shows prominent staining in vascular and visceral smooth muscle. In addition, the antibody strongly stained kidney mesangial cells and a cell type in the alveolar wall of the lungs, most likely corresponding to alveolar myofibroblasts. These results suggest that in adult mammalian tissues, α8 is predominantly expressed in smooth muscle and smooth muscle-like contractile cells.
Mutation of the predicted p34cdc2 phosphorylation sites in numa impair the assembly of the mitotic spindle and block mitosis
Authors: Compton Duane A. , Luo Chenghua
Published: April 2021

NuMA is a 236 kDa intranuclear protein that is distributed into each daughter cell during mitosis through association with the pericentrosomal region of the mitotic spindle. NuMA’s interaction with the microtubules of the mitotic spindle is mediated through its 45 kDa carboxyl-terminal globular tail, and there is indirect evidence suggesting that NuMA’s interaction with the mitotic spindle is controlled in a mitosis-specific manner. Consistent with this evidence is the fact that all four of the predicted p34cdc2 consensus phosphorylation sites in the NuMA protein are located in the carboxyl-terminal globular domain, and we demonstrate here that NuMA is phosphorylated in a mitosis-specific fashion in vivo. To test if the predicted p34cdc2 phosphorylation sites are necessary for NuMA’s mitosis-specific interaction with the mitotic spindle, we have introduced mutations into the human NuMA cDNA that convert these predicted p34cdc2 phosphorylation sites from threonine or serine residues into alanine residues, and subsequently determined the cell cycle-dependent localization of these altered NuMA proteins following their expression in tissue culture cells. While none of these specific mutations in the NuMA sequence alters the faithful targeting of the protein into the interphase nucleus, mutation of threonine residue 2040 alone or in combination with mutations in other potential p34cdc2 phosphorylation sites abolishes NuMA’s ability to associate normally with the microtubules of the mitotic spindle. Instead of binding to the mitotic spindle these mutant forms of NuMA concentrate at the plasma membrane of the mitotic cell. Cells expressing these mutant forms of NuMA have disorganized mitotic spindles, fail to complete cytokinesis normally, and assemble micronuclei in the subsequent interphase. These data suggest that NuMA’s interaction with the microtubules of the mitotic spindle is controlled by cell cycle-dependent phosphorylation in addition to differential subcellular compartmentalization, and the characteristics of the dominant negative phenotype induced by these mutant forms of NuMA support a role for NuMA in the organization of the mitotic spindle apparatus.
Centriolar cycle of fused cells
Authors: Manandhar G. , Onishchenko Galina E.
Published: April 2021

Ultrastructure of centrioles of fused cells containing heterophasic interphase nuclei, premature chromosome condensation, and a telophase-like nucleus were studied. The study indicated that the heterophasic cellular environments contributed by the different cell partners exert a mutually opposite effect on the structure of centrioles. The G1-cell partner suppresses replication of S-centrioles. Asynchronous replications of G1 and S-stage centrioles were observed in some G1-S, G1-G2 and S-G2 fused cells. In interphase-mitotic fused cells, centrioles of interphase stages underwent mitotic activation when their nuclei were induced to premature chromosome condensation. Daughter centrioles of G1-, S-, and G2-stages were also capable of mitotic activation independently if they were separated from their mother centriole. Inactive centrioles were observed in some cells containing G1-premature chromosome condensation. When mitotic nuclei were induced to telophase-like nucleus formation, their centrioles were also inactivated. Concomitant events of induced nuclear and centriolar changes suggest that they might have been controlled by the heterophasic cytoplasmic factors through similar pathways.
Coupling between gamma irradiation, p53 induction and the apoptotic response depends upon cell type in vivo
Authors: Midgley Carol A. , Owens Bronwyn , Briscoe Cecilie V. , Thomas David Brynmor , Lane David P. , Hall Peter A.
Published: April 2021

The accumulation of p53 protein following whole body irradiation of adult mice was studied using a new polyclonal antibody to mouse p53. While dramatic accumulation of the protein was apparent in splenocytes, thymocytes and osteocytes no p53 protein accumulation was detected in the hepatocytes of the irradiated mouse. Thus the upstream initiating signals that control the induction of p53 are controlled in a tissue specific manner. While massive apoptosis accompanies p53 induction in thymocytes and splenocytes it is not seen in the osteocytes. Thus the downstream consequences of p53 induction are also tightly controlled. These results have profound significance for an understanding of the role of the p53 tumour suppression pathway in different tissues.
The mechanism of nuclear transport of natural or artificial transport substrates in digitonin-permeabilized cells
Authors: Cserpán Imre , Udvardy Andor
Published: April 2021

Characterization of nuclear protein transport in digitonin-permeabilized cells revealed that the number of the nuclear localization signal sequences (NLS) within the transport substrate basically influences the mechanism of the transport reaction.

Phycoerythrine-NLS transport substrate carrying a maximum of 4-5 conjugated NLSs/subunit, or Bsp methyl-transferase-NLS fusion protein were efficiently transported into the nuclei of digitonin-permeabilized cultured cells without any exogenously added cytosolic protein. All the characteristic properties of in vivo nuclear transport are faithfully reproduced with these transport substrates: (i) the transport requires a functional NLS in the transported protein, a transport-incompetent mutant NLS being ineffective; (ii) the transport is energy dependent; (iii) the wild type nuclear localization peptide efficiently competes for transport, while the transport-incompetent mutant peptide does not; and (iv) wheat germ agglutinin inhibits this transport reaction. Nuclear transport observed with these substrates was not due to any damage of the nuclear membrane or inefficient extraction of the cytosolic proteins during the permeabilization of the cells. The nuclear transport was proportional to the number of conjugated NLSs.

Nuclear transport of phycoerythrine carrying 7-8 conjugated NLSs/subunit required the addition of exogenous cytosolic proteins. This transport also fulfilled all the characteristic properties of an authentic nuclear transport.

Nuclear transport with different combinations of transport substrates further supported the assumption that distinct transport mechanisms operate for different substrates. From a mixture of PE-NLS7-8 and Bsp methyltransferase-NLS, the highly conjugated substrate was completely retained in the cytoplasm in the absence of exogenous cytosol, while Bsp methyltransferase-NLS was efficiently transported. Exogenous cytosol promoted the nuclear transport of the highly conjugated substrate.
Colocalization of a high molecular mass phosphoprotein of the nuclear matrix (p255) with spliceosomes
Authors: Bisotto Sandra , Lauriault Pascal , Duval Marie , Vincent Michel
Published: April 2021

It was previously demonstrated that monoclonal antibody CC-3 binds to a phosphorylation dependent epitope present on a 255 kDa nuclear protein (p255). We show here that in interphase cells, p255 distributes to typical nuclear speckles that correspond to the localization of spliceosome components as revealed by antibodies to the m3G cap of snRNAs or to the non-snRNP splicing factor SC-35. Immunofluorescence and immunoblot studies indicated that p255 is resistant to extraction with non-ionic detergents, nucleases and high ionic strength buffers and may thus be defined biochemically as a nuclear matrix phosphoprotein. To determine the nature of the association of p255 with the nuclear structure, its distribution was studied at different stages of the cell cycle and after the cells were treated with nucleases or heat shocked. We found that the antigen diffused into the cytoplasm during metaphase but was reorganized into cytoplasmic speckles during anaphase-telophase transition, where it colocalized with SC-35. Nuclear matrix preparations that were digested with DNases and RNases showed that interphasic p255 still localized to nuclear speckles even though snRNA and snRNP antigens were removed. Heat-shocked cells labelled with monoclonal antibody CC-3 exhibited more rounded and less interconnected speckles, identical to those decorated by anti-SC-35 antibody under such conditions. These results indicate that p255 and SC-35 are present in the same nuclear structures, to which they are more tightly bound than the snRNP antigens. They further suggest that both proteins are implicated in spliceosome assembly or attachment.
Cyclic AMP modulates the rate of ‘constitutive’ exocytosis of apical membrane proteins in Madin-Darby canine kidney cells
Authors: Brignoni Mirtha , Pignataro Omar P. , Rodriguez Marcelo L. , Alvarez Adriana , Vega-Salas Dora E. , Rodriguez-Boulan Enrique , Salas Pedro J. I.
Published: April 2021

Madin-Darby canine kidney and other epithelial cell lines (e.g. Caco-2, MCF-10A and MCF-7) develop intracellular vacuoles composed of apical membrane displaying microvilli (VACs) when impaired from forming normal cell-to-cell contacts. In a previous publication, we showed that VACs are rapidly exocytosed upon treatment with 8-Br-3’,5’-cyclic adenosine monophosphate (8-Br-cAMP), a membrane-permeable analog of cAMP, and that this exocytosis correlates with variations in the cellular cAMP concentration in response to the cell-cell contacts. In the present work, we tested the hypothesis that cAMP may be a positive modulator of the ‘constitutive’ exocytic pathway. To mimic conditions in cells with incomplete intercellular contacts, the intracellular levels of cAMP were decreased by means of two independent approaches: (i) pores were induced in the plasma membrane with the polypeptidic antibiotic subtilin, thus allowing small molecules (including cAMP) to permeate and move out of the cytoplasm; and (ii) adenylate cyclase and protein kinase A were blocked with specific inhibitors. In all cases, the intracellular levels of cAMP were measured and, in porated cells, equilibrated to simulate the corresponding physiological intracellular concentrations. The decrease in cAMP within the physiological range resulted in a decreased rate of transport of an apical marker of the constitutive pathway (influenza virus hemagglutinin) from the trans -Golgi network to the apical plasma membrane. Likewise, the delivery of a number of cellular apical proteins to the plasma membrane was retarded at low cAMP concentrations. The inhibitors of adenylate cyclase failed to block basolateral delivery of vesicular stomatitis virus G protein. This differential modulatory effect may represent a differentiation-dependent control of the insertion of apical membrane in epithelial cells.
Expression of cartilage-specific molecules is retained on long-term culture of human articular chondrocytes
Authors: Kolettas Evangelos , Buluwela Lakjaya , Bayliss Michael T. , Muir Helen I.
Published: April 2021

Normal human adult articular chondrocytes were used to determine how the chondrocyte phenotype is modulated by culture conditions following long-term culture. We report here for the first time that human articular chondrocytes have a lifespan in the range of 34-37 population doublings. While chondrocytes cultured as monolayers displayed a fibroblastoid morphology and grew faster, those cultured as suspensions over agarose adopted a round morphology and formed clusters of cells reminiscent of chondrocyte differentiation in intact cartilage, with little or no dNa synthesis. These morphologies were independent of the age of the culture. Despite, these morphological differences, however, chondrocytes expressed markers at mRNA and protein levels characteristic of cartilage: namely, types II and IX collagens and the large aggregating proteoglycans, aggrecan, versican and link protein, but not syndecan, under both culture conditions. However, they also expressed type I collagen α1(I) and α2(I) chains. It has been suggested that expression of collagen α1(I) by chondrocytes cultured as monolayers is a marker of the loss of the chondrocyte phenotype. However, we show here, using reverse transcriptase/polymerase chain reaction, that normal fresh intact human articular cartilage expresses collagen α1(I). The data show that following long-term culture human articular chondrocytes retain their differentiated characteristics and that cell shape does not correlate with the expression of the chondrocyte phenotype. It is proposed that loss of the chondrocyte phenotype is marked by the loss of one or more cartilage-specific molecules rather than by the appearance of non-cartilage-specific molecules.
Multiple mRNAs encode the avian lysosomal membrane protein LAMP-2, resulting in alternative transmembrane and cytoplasmic domains
Authors: Hatem Christine L. , Gough Nancy R. , Fambrough Douglas M.
Published: April 2021

Lysosomal membranes are enriched in extensively glycosylated transmembrane proteins, LAMP-1 and LAMP-2. LAMP-1 proteins have been characterized from several mammalian species and from chickens, but no nonmammalian homologues of LAMP-2 have been described, and no splice variants of either protein have been reported. Here we report the characterization of three cDNA clones encoding chicken LAMP-2. The nucleotide sequences of the cDNAs diverge at their 3’ ends within the open reading frame, resulting in sequences that code for three different transmembrane and cytoplasmic domains. Southern analysis suggests that a single gene encodes the common region of chicken LAMP-2. The position of the divergence and the identity of the common sequence are consistent with alternative splicing of 3’ exons. Analysis of the mRNAs present in adult chicken tissues suggests tissue-specific expression of the three chicken LAMP-2 variants, with LAMP-2b expressed primarily in the brain. The cytoplasmic domain of LAMP-type proteins contains the targeting signal for directing these molecules to the lysosome. Using chimeras consisting of the lumenal domain of chicken LEP100 (a LAMP-1) and the transmembrane and cytoplasmic domains of the LAMP-2 variants, we demonstrate in transfected mouse L cells that all three LAMP-2 carboxyl-terminal regions are capable of targeting the chimeric proteins to lysosomes. Levels of expression, subcellular distribution, and glycosylation of the LAMP proteins have all been shown to change with differentiation in mammalian cells and to be correlated with metastatic potential in certain tumor cell lines. Alternative splicing of the LAMP-2 transcript may play a role in these changes.