bluuebunny/crossref_metadata_2025 huggingface dataset:
The kinesin-homologous protein encoded by the Chlamydomonas FLA10 gene is associated with basal bodies and centrioles
Authors: Vashishtha Malini , Walther Zenta , Hall John L.
Published: April 2021

We previously reported that the FLA10 locus on the uni linkage group of Chlamydomonas encodes a kinesin homologous protein, KHP1. The fla10 phenotype, which is a temperature-sensitive defect for flagellar assembly and maintenance, is rescued by transformation with the wild-type KHP1 gene. In the present study we identify the molecular defect associated with the fla10 mutation and examine the subcellular localization of KHP1 throughout the cell cycle. The mutation in the fla10-1 allele consists of a C to A transversion, which alters amino acid 329 in the motor domain of KHP1. This residue and the sequence of the carboxyterminal third of the motor domain in which it is located are highly conserved throughout eukaryotic evolution in a subfamily of kinesin-related proteins from mouse (KIF3), sea urchin (KRP85/95), Xenopus (XKLP3), and Drosophila (KLP68D). These data suggest a conserved function for this family of proteins. Immunofluorescence studies reveal that: (1) in interphase cells KHP1 is associated with basal bodies and with the proximal portion of the flagella; (2) in cells undergoing flagellar regeneration KHP1 occurs in punctate structures that extend to the tip of the developing axoneme; and (3) in dividing cells KHP1 remains associated with centrioles throughout mitosis and localizes to the mitotic spindle. KHP1 is the first kinesin homologous protein to be found in association with basal bodies and centrioles throughout the cell cycle. These observations provide evidence for a direct role of basal bodies in the process of flagellar development, which we propose is based on KHP1 acting as a transporter of flagellar components from the basal bodies out to the distal site of assembly. The localization of KHP1 in mitosis suggests that this protein may play an analogous role in the centriole-based assembly of the mitotic spindle.
Katanin, the microtubule-severing ATPase, is concentrated at centrosomes
Authors: McNally Francis J. , Okawa Katsuya , Iwamatsu Akihiro , Vale Ronald D.
Published: April 2021

The assembly and function of the mitotic spindle involve specific changes in the dynamic properties of microtubules. One such change results in the poleward flux of tubulin in which spindle microtubules polymerize at their kinetochore-attached plus ends while they shorten at their centrosome-attached minus ends. Since free microtubule minus ends do not depolymerize in vivo, the poleward flux of tubulin suggests that spindle microtubules are actively disassembled at or near their centrosomal attachment points. The microtubule-severing ATPase, katanin, has the ability actively to sever and disassemble microtubules and is thus a candidate for the role of a protein mediating the poleward flux of tubulin. Here we determine the subcellular localization of katanin by immunofluorescence as a pre-liminary step in determining whether katanin mediates the poleward flux of tubulin. We find that katanin is highly concentrated at centrosomes throughout the cell cycle. Katanin’s localization is different from that of γ-tubulin in that microtubules are required to maintain the centrosomal localization of katanin. Direct comparison of the localization of katanin and γ-tubulin reveals that katanin is localized in a region surrounding the γ-tubulin-containing pericentriolar region in detergent-extracted mitotic spindles. The centrosomal localization of katanin is consistent with the hypothesis that katanin mediates the disassembly of microtubule minus ends during poleward flux.
Requirement of basement membrane for the suppression of programmed cell death in mammary epithelium
Authors: Pullan Shirley , Wilson James , Metcalfe Anthony , Edwards Gwynneth M. , Goberdhan Nadia , Tilly Jonathan , Hickman John A. , Dive Caroline , Streuli Charles H.
Published: April 2021

Apoptosis is an active mechanism of cell death required for normal tissue homeostasis. Cells require survival signals to avoid the engagement of apoptosis. In the mammary gland, secretory epithelial cells are removed by apoptosis during involution. This cell loss coincides with matrix metalloproteinase activation and basement membrane degradation. In this paper we describe studies that confer a new role for basement membrane in the regulation of cell phenotype. We demonstrate that first passage epithelial cells isolated from pregnant mouse mammary gland die by apoptosis in culture, but that cell death is suppressed by basement membrane. The correct type of extracellular matrix was required, since only a basement membrane, not plastic or a collagen I matrix, lowered the rate of apoptosis. Attachment to a matrix per se was not sufficient for survival, since apoptotic cells were observed when still attached to a collagen I substratum. Experiments with individually isolated cells confirmed the requirement of basement membrane for survival, and demonstrated that survival is enhanced by cell-cell contact. A function-blocking anti-β1 integrin antibody doubled the rate of apoptosis in single cells cultured with basement membrane, indicating that integrin-mediated signals contributed to survival. We examined the cell death-associated genes bcl-2 and bax in mammary epithelia, and found that although the expression of Bcl-2 did not correlate with cell survival, increased levels of Bax were associated with apoptosis. We propose that basement membrane provides a survival stimulus for epithelial cells in vivo, and that loss of interaction with this type of matrix acts as a control point for cell deletions that occur at specific times during development, such as in mammary gland involution.
The effect of wortmannin on the localisation of lysosomal type I integral membrane glycoproteins suggests a role for phosphoinositide 3-kinase activity in regulating membrane traffic late in the endocytic pathway
Authors: Reaves Barbara J. , Bright Nicholas A. , Mullock Barbara M. , Luzio J. Paul
Published: April 2021

Addition of wortmannin to normal rat kidney cells caused a redistribution of the lysosomal type I integral membrane proteins lgp 110 and lgp120 to a swollen vacuolar compartment. This compartment did not contain the cation independent mannose 6-phosphate receptor and was depleted in acid hydrolases. It was distinct from another swollen vacuolar compartment containing the cation independent mannose 6-phosphate receptor. The swollen lgp110-positive compartment was accessible to a monoclonal antibody against lgp120 added extracellularly, showing that it had the characteristics of an endosomal compartment. Wortmannin had no gross morphological effect on the trans-Golgi network or lysosomes nor any effect on the delivery to the trans-Golgi network of endocytosed antibodies against the type I membrane protein TGN38. We propose that the observed effects of wortmannin were due to inhibition of membrane traffic between cation independent mannose 6-phosphate receptor-positive late endosomes and the trans-Golgi network and to inhibition of membrane traffic between a novel lgp120-positive, cation independent mannose 6-phosphate receptornegative late endosomal compartment and lysosomes. The effects of wortmannin suggest a function for a phosphatidylinositol 3-kinase(s) in regulating membrane traffic in the late endocytic pathway.
Different distributions of homologous chromosomes in adult human Sertoli cells and in lymphocytes signify nuclear differentiation
Authors: Chandley Ann C. , Speed R. M. , Leitch A. R.
Published: April 2021

Using whole chromosome painting probes for human chro mosomes 3, 7, 8, 13, 17 and 21 and X and the probe pHY2.1 for the Y chromosome coupled with fluorescent in situ hybridization (FISH) analysis, the distribution of chromo somes is reported in nuclei of Sertoli cells of the adult testis and in stimulated blood lymphocytes. The distribution of chromosomes in the two cell types is significantly different. A strong tendency for each pair of homologues to pair is inferred from the observation of only a single detectable signal in the majority of Sertoli cell nuclei. The sex chromosomes, by contrast, give two clearly separated signals. Interphase nuclei in dividing blood lymphocytes, analysed as controls, also show mainly two separated signals for all non-acrocentric autosomal pairs, but acrocentric pairs no. 13 and 21 show some tendency to associate, probably reflecting satellite association.
Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons
Authors: van Steensel Bas , van Binnendijk Erica P. , Hornsby C. Diane , van der Voort Hans T. M. , Krozowski Zygmunt S. , de Kloet E. Ronald , van Driel Roel
Published: April 2021

The glucocorticoid receptor and the mineralocorticoid receptor are hormone-dependent transcription factors. They regulate the excitability of rat hippocampus CA1 neurons in a coordinated fashion. We studied the spatial distribution of these transcription factors in nuclei of CA1 neurons by dual labeling immunocytochemistry and confocal microscopy, combined with novel image resto ration and image analysis techniques. We found that both receptors are concentrated in about one thousand clusters within the nucleus. Some clusters contain either mineralocorticoid receptors or glucocorticoid receptors, but a sig nificant number of clusters contains both receptors. These results indicate that the two receptor types are targeted to specific compartments in the nucleus. The coordinated action of the glucocorticoid and mineralocorticoid receptor on gene expression may be established in a specific set of nuclear domains that contain both receptors.
Microtubule-based peroxisome movement
Authors: Rapp Stephan , Saffrich Rainer , Anton Markus , Jäkle Ursula , Ansorge Wilhelm , Gorgas Karin , Just Wilhelm W.
Published: April 2021

The association of peroxisomes with cytoskeletal structures was investigated both by electron microscopy and by kinetic analysis of peroxisome movement. The morphological studies indicated distinct interactions of peroxisomes with microtubules and frequently revealed multiple contact sites. The kinetic approach utilised microinjection and import of fluorescein-labeled luciferase in order to mark and track peroxisomes in vivo. Peroxisomal motility was analysed by time-lapse imaging and fluorescence microscopy. According to their movement peroxisomes were classified into two groups. Group 1 peroxisomes comprising the majority of organelles at 37°C moved slowly with an average velocity of 0.024±0.012 µm/second whereas the movement of group 2 peroxisomes, 10-15% of the total population, was saltatory exhibiting an average velocity of 0.26±0.17 µm/second with maximal values of more than 2 µm/second. Saltations were completely abolished by the microtubule-depolymerising drug nocodazole and were slightly reduced by about 25% by cytochalasin D which disrupts the actin microfilament system. Double fluorescence labeling of both peroxisomes and microtubules revealed peroxisome saltations linked to distinct microtubule tracks. Cellular depletion of endogenous levels of NTPs as well as the use of 5’-adenylylimidodiphosphate, a nonhydrolysable ATP analog, applied to a permeabilised cell preparation both completely blocked peroxisomal movement. These data suggest an ATPase dependent, microtubule-based mechanism of peroxisome movement. Both the intact and the permeabilised cell system presented in this paper for the first time allow kinetic measurements on peroxisomal motility and thus will be extremely helpful in the biochemical characterisation of the motor proteins involved.
rRNA distribution during microspore development in anthers of Beta vulgarisL.: quantitative in situ hybridization analysis
Authors: Majewska-Sawka Anna , Rodriguez-Garcia Maria Isabel
Published: April 2021

We related changes in the ultrastructural organization of the nucleoli with the results of quantitative in situ hybridizations to characterize rRNA metabolism during the development of microspore mother cells in the sugar beet anther (Beta vulgaris L.). In the course of meiotic prophase and early postmeiotic interphase the morphological characteristics of the nucleoli are typical of low or no transcriptional activity and a low rate of rRNA processing.

However, we found evidence of an apparent increase in the relative numbers of 18 S rRNA transcripts in some stages of microsporogenesis. This was found in both the nucleoli and cytoplasm of pachytene meiocytes, and in later stages there was a spectacular accumulation of rRNA transcripts in nucleoli of the tetrad cells. Quantitative data are analyzed in the light of morphometric findings in the cells and their compartments to elucidate the degree to which changes in cell size are related to changes in labeling density and distribution. The results are discussed in terms of rRNA synthesis, transport and degradation as processes involved in the regulation of rRNA within microsporocytes and microspores.
Localization of kinesin superfamily proteins to the connecting cilium of fish photoreceptors
Authors: Beech Peter L. , Pagh-Roehl Kathryn , Noda Yasuko , Hirokawa Nobutaka , Burnside Beth , Rosenbaum Joel L.
Published: April 2021

Kinesin superfamily proteins (KIFs) are probable motors in vesicular and non-vesicular transport along microtubular tracks. Since a variety of KIFs have been recently identified in the motile flagella of Chlamydomonas, we sought to ascertain whether KIFs are also associated with the connecting cilia of vertebrate rod photoreceptors. As the only structural link between the rod inner segment and the photosensitive rod outer segment, the connecting cilium is thought to be the channel through which all material passes into and out of the outer segment from the rod cell body. We have performed immunological tests on isolated sunfish rod inner-outer segments (RIS-ROS) using two antibodies that recognize the conserved motor domain of numerous KIFs (anti-LAGSE, a peptide antibody, and anti-Klp1 head, generated against the N terminus of Chlamydomonas Klp1) as well as an antibody specific to a neuronal KIF, KIF3A. On immunoblots of RIS-ROS, LAGSE antibody detected a prominent band at ∼117 kDa, which is likely to be kinesin heavy chain, and Klp1 head antibody detected a single band at ∼170 kDa; KIF3A antibody detected a polypeptide at ∼85 kDa which co-migrated with mammalian KIF3A and displayed ATP-dependent release from rod cytoskeletons. Immunofluorescence localizations with anti-LAGSE and anti-Klp1 head antibodies detected epitopes in the axoneme and ellipsoid, and immunoelectron microscopy with the LAGSE antibody showed that the connecting cilium region was particularly antigenic. Immunofluorescence with anti-KIF3A showed prominent labelling of the connecting cilium and the area surrounding its basal body; the outer segment axoneme and parts of the inner segment coincident with microtubules were also labelled. We propose that these putative kinesin superfamily proteins may be involved in the translocation of material between the rod inner and outer segments.
The vacuolar proton pump of Dictyostelium discoideum: Molecular cloning and analysis of the 100 kDa subunit
Authors: Liu Tongyao , Clarke Margaret
Published: April 2021

The vacuolar proton pump is a highlyconserved multimeric enzyme that catalyzes the translocation of protons across the membranes of eukaryotic cells. Its largest subunit (95-116 kDa) occurs in tissue and organellespecific isoforms and thus may be involved in targeting the enyzme or modulating its function. In amoebae of Dictyostelium discoideum, proton pumps with a 100 kDa subunit are found in membranes of the contractile vacuole complex, an osmoregulatory organelle. We cloned the cDNA that encodes this 100 kDa protein and found that its sequence predicts a protein 45% identical (68% similar) to the corresponding mammalian proton pump subunit. Like the mammalian protein, the predicted Dictyostelium sequence contains six possible transmembrane domains and a single consensus sequence for N-linked glycosylation. Southern blot analysis detected only a single gene, which was designated vatM. Using genomic DNA and degenerate oligonucleotides based on conserved regions of the protein as primers, we generated products by polymerase chain reaction that included highly variable regions of this protein family. The cloned products were identical in nucleotide sequence to vatM, arguing that Dictyostelium cells contain only a single isoform of this proton pump subunit. Consistent with this interpretation, the amino acid sequences of peptides derived from a protein associated with endosomal membranes (Adessu et al. (1995) J. Cell Sci. 108, 3331-3337) match the predicted sequence of the protein encoded by vatM. Thus, a single isoform of the 100 kDa proton pump subunit appears to serve in both the contractile vacuole system and the endosomal/lysosomal system of Dictyostelium, arguing that this subunit is not responsible for regulating the differing abundance and function of proton pumps in these two compartments. Gene targeting experiments suggest that this subunit plays important (possibly essential) roles in Dictyostelium cells.
SCO-spondin: a new member of the thrombospondin family secreted by the subcommissural organ is a candidate in the modulation of neuronal aggregation
Authors: Gobron Stéphane , Monnerie Hubert , Meiniel Robert , Creveaux Isabelle , Lehmann Werner , Lamalle David , Dastugue Bernard , Meiniel Annie
Published: April 2021

A number of cues are known to influence neuronal devel­opment including growth factors, cell-adhesion molecules, components of the extracellular matrix and guidance molecules. In this study, we present molecular and func­tional evidence that SCO-spondin, a novel relative of the thrombospondin family, could also be involved in neuronal development by modulating cell aggregative mechanisms. SCO-spondin corresponds to glycoproteins secreted by the subcommissural organ (SCO), an ependymal differen­tiation of the vertebrate brain located at the entrance to the Sylvian aqueduct. A cDNA clone of 2.6 kb, isolated from a bovine SCO cDNA library, was shown to be specifically and highly expressed in the bovine SCO by in situ hybridization and was subsequently sequenced. Analysis of the deduced amino acid sequence reveals the presence of four conserved domains known as thrombospondin (TSP) type I repeats. To account for the homology with thrombospondins and F- spondin, this secreted glycoprotein was called SCO- spondin. Two potent binding sites to glycosaminoglycan (BBXB) and to cytokine (TXWSXWS) are also found in the TSP type I repeats. The deduced amino acid sequence exhibits three other conserved domains called low density lipoprotein (LDL) receptor type A repeats. The possibility of SCO-spondin involvement in neuronal development as a component of the extracellular matrix is discussed regarding these molecular features. The idea of a modula­tion of cell-cell and/or cell-matrix interaction is further supported by the anti-aggregative effect observed on cultured neuronal cells of material solubilized from Reissner’s fiber. That Reissner’s fiber, the condensed secretory product of the SCO present along the whole spinal cord can be a potent morphogenetical structure is an important concept for the analysis of the molecular mech­anisms leading to spinal cord differentiation.
Activation of the Xenopuscyclin degradation machinery by full-length cyclin A
Authors: Jones Craig , Smythe Carl
Published: April 2021

The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase. In many cell types, cyclin A-associated kinase activity peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear. Previous work has suggested that while cyclin B is capable of triggering cyclin destruction in Xenopus cellfree systems, cyclin A-associated kinase is not able to support this function. Here we have expressed a fulllength human cyclin A in Escherichia coli and purified the protein to homogeneity by virtue of an N-terminal histidine tag. We have found that when added to Xenopus cellfree extracts free of cyclin B and incapable of protein synthesis, the temporal pattern of cyclin A-associated cdc2 kinase activity showed distinct differences that were dependent on the concentration of cyclin A added. When cyclin A was added to a concentration that generated levels of cdc2 kinase activity capable of inducing nuclear envelope breakdown, the histone H1 kinase activity profile was biphasic, consisting of an activation phase followed by an inactivation phase. Inactivation was found to be due to cyclin destruction, which was prevented by mos protein.

Cyclin destruction was followed by nuclear reassembly and an additional round of DNA replication, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system. It has been suggested that the evolutionary recruitment of cyclin A into an S phase function may have necessitated the loss of an original mitotic ability to activate the cyclin destruction pathway. The results presented here indicate that cyclin A has not lost the ability to activate its own destruction and that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating that there are not distinct cyclin A and cyclin B destruction pathways. Thus the ordered progression of the cell cycle requires the careful titration of cyclin A concentration in order to avoid activation of the cyclin destruction pathway before sufficient active cyclin B/cdc2 kinase has accumulated.
Meiotic spindle organization in fertilized Drosophila oocyte: Presence of centrosomal components in the meiotic apparatus
Authors: Riparbelli Maria Giovanna , Callaini Giuliano
Published: April 2021

We examined spindle reorganization during the completion of meiosis in fertilized and unfertilized oocytes of Drosophila using indirect immunofluorescence and laser scanning confocal microscopy. The results defined a complex pathway of spindle assembly during resumption of meiosis, and revealed a transient array of microtubules radiating from the equatorial region of the spindle towards discrete foci in the egg cortex. A monastral array of microtubules was observed between twin metaphase II spindles in fertilized and unfertilized eggs. These microtubules originated from diskshaped material stained with Rb188 antibody specific for an antigen associated with the centrosome of Drosophila embryos. The Drosophila egg, therefore, contains a maternal pool of centrosomal components undetectable in mature inactivated oocytes. These components nucleate microtubules in a monastral array after activation, but are unable to organize bipolar spindles.
Metallopeptidase inhibitors induce an up-regulation of endothelin-converting enzyme levels and its redistribution from the plasma membrane to an intracellular compartment
Authors: Barnes K. , Shimada K. , Takahashi M. , Tanzawa K. , Turner A.J.
Published: April 2021

Endothelin-converting enzyme is a phosphoramidon-sensitive membrane metallopeptidase that catalyses the final step in biosynthesis of the potent vasoactive endothelin peptides. Immunomagnetic separation technology and immunohistochemistry have been used to demonstrate the co-localisation of endothelin-converting enzyme with the established ectoenzyme, aminopeptidase N, on the surface of endothelial cells. Unlike aminopeptidase N, however, endothelin-converting enzyme is seen to associate in clusters on the plasma membrane which can be distinguished from caveolae both biochemically and immunologically. Pre-treatment of endothelial cells with the metallopeptidase inhibitors phosphoramidon or thiorphan in the range 0.01-100 microM produced a dose-dependent increase in the levels of endothelin-converting enzyme protein and its accumulation in an intracellular compartment. No corresponding change in the levels of endothelin-converting enzyme-1 mRNA was detected under these conditions, nor in the levels of the closely related metalloenzyme, endopeptidase-24.11. The phosphoramidon and thiorphan-dependent increase is not due to direct inhibition of endothelin-converting enzyme not endopeptidase-24.11 but, rather, to an inhibition of the selective turnover of endothelin-converting enzyme protein.
Integrin dynamics on the tail region of migrating fibroblasts
Authors: Palecek Sean P. , Schmidt Christine E. , Lauffenburger Douglas A. , Horwitz Alan F.
Published: April 2021

Cell migration is a complex process that can be considered as a repeated cycle of lamellipod extension and attachment, cytoskeletal contraction, and tail detachment. While lamellipodial and cytoskeletal phenomena are currently the focus of considerable research on cell migration, under many conditions locomotion appears to be ratelimited by events at the cell rear, especially release of cell/substratum adhesions. To study the mechanism of tail detachment, we have developed a novel experimental system that permits observation of integrin dynamics on the ventral surface of migrating fibroblasts. Photoactivatable caged fluorescein is coupled to a nonadhesionperturbing antiaviaN-β1 integrin subunit antibody, which labels integrins on chicken fibroblasts migrating on a laminincoated glass coverslip. Ultraviolet light is focused through a pinhole to photoactivate the caged fluorophore in a 10-µmdiameter spot at the rear of a polarized cell. The fate of integrins initially present in this spot is monitored using a cooled CCD camera to follow the movement of fluorescent intensity as a function of time over a 2 to 3 hour period.

We find that a substantial fraction of the integrins is left behind on the substratum as the cell detaches and locomotes, while another fraction collects into vesicles which are transported along the cell body as the cell migrates. As aggregates rip from the cell membrane, the integrincytoskeletal bonds are preferentially fractured resulting in 81±15% of the integrin remaining attached to the substratum. We additionally find that adhesions sometimes disperse into integrins which can form new adhesions at other locations in the cell. Adhesions along the cell edge can release from the substrate and translocate with the cell. They either disperse in the cell membrane, rip from the cell membrane and remain attached to the substratum, or form a new aggregate. These observations indicate that the behavior of integrins at the cell rear is much more dynamic than previously appreciated, suggesting that an important locus for regulation of motility may reside in this region.
A high molecular mass non-muscle tropomyosin isoform stimulates retrograde organelle transport
Authors: Pelham Robert J. , Lin Jim Jung-Ching , Wang Yuli
Published: April 2021

Although nonmuscle tropomyosins (TM) have been implicated in various cellular functions, such as stabilization of actin filaments and possibly regulation of organelle transport, their physiological role is still poorly understood. We have probed the role of a high molecular mass isoform of human fibroblast TM, hTM3, in regulating organelle transport by microinjecting an excess amount of bacteriallyexpressed protein into normal rat kidney (NRK) epithelial cells. The microinjection induced the dramatic retrograde translocation of organelles into the perinuclear area. Microinjection of hTM5, a low molecular mass isoform, had no effect on organelle distribution. Fluorescent staining indicated that hTM3 injection stimulated the retrograde movement of both mitochondria and lysosomes. Moreover, both myosin I and cytoplasmic dynein were found to redistribute with the translocated organelles to the perinuclear area, indicating that these organelles were able to move along both microtubules and actin filaments. The involvement of microtubules was further suggested by the partial inhibiton of hTM3-induced organelle movement by the microtubuledepolymerizing drug nocodazole. Our results, along with previous genetic and antibody microinjection studies, suggest that hTM3 may be involved in the regulation of organelle transport.
Orientation and directed migration of cultured corneal epithelial cells in small electric fields are serum dependent
Authors: Zhao M. , Agius-Fernandez A. , Forrester J.V. , McCaig C.D.
Published: April 2021

Reorientation and migration of cultured bovine corneal epithelial cells (CECs) in an electric field were studied. Electric field application was designed to model the laterally directed, steady direct current electric fields which arise in an injured corneal epithelium. Single cells cultured in media containing 10% foetal bovine serum showed significant galvanotropism, reorienting to lie perpendicular to electric field vector with a threshold field strength of less than 100 mV/mm. Cells cultured in serum-free medium showed no reorientation until 250 mV/mm. Addition of EGF, bFGF or TGF-beta 1 singly or in combination to serum free medium significantly restored the reorientation response at low field strengths. Both the mean translocation rate and directedness of cell migration were serum dependent. Cultured in medium with serum or serum plus added EGF, single cells showed obvious cathodal migration at 100 mV/mm. Increasing electric field strength enhanced the cathodal directedness of single cell migration. Supplementing serum free medium with growth factors restored the cathodal directed migration of single cells and highest directedness was found for the combination of EGF and TGF-beta 1. Corneal epithelial sheets also migrated towards the cathode in electric fields. Serum or individual growth factors stimulated CEC motility (randomly directed). Applied fields did not further augment migration rates but added a vector to stimulated migration. Electric fields which are present in wounded cornea interact with other environmental factors and may impinge on CECs migration during wound healing. Therapies which combine the application of growth factors and electric fields may be useful clinically.
Cell-free assembly of rough and smooth endoplasmic reticulum
Authors: Lavoie Christine , Lanoix Joël , Kan Frederick W. K. , Paiement Jacques
Published: April 2021

Smooth endoplasmic reticulum assembly was studied in a cell-free system using thin-section and freeze-fracture electron microscopy. Incubation of rat hepatocyte rough and smooth microsomes in the presence of ATP, GTP, cytosol (Xenopus egg) and an ATP-regenerating system led to assembly of membrane networks comprising a central core of interconnecting smooth tubules continuous with peripherally located rough membrane cisternae. Glucose-6-phosphatase cytochemistry confirmed the endoplasmic reticulum origin of the reconstituted membranes. When both ATP and GTP were omitted from the incubation medium, or when GTP was replaced by a variety of nucleotide analogues, including GTPγS, membrane aggregates contained only unfused microsomes. The presence of GTP alone stimulated assembly of rough membrane cisternae but had no effect on smooth membranes. Smooth tubule formation occurred independent of cytosol and an ATP-regenerating system, but did require GTP and ATP.

Omission of ATP, or replacement of this nucleotide with a variety of analogues, including ATPγS, prevented tubule formation but did not affect the assembly of the rough membrane cisternae. Morphometric studies revealed sequential formation of rough membrane cisternae (0-60 minutes) followed by appearance of interconnecting smooth tubules (>60 minutes). The amount of rough membrane cisternae per membrane network diminished with time after 60 minutes; that of smooth tubules increased. Thus GTP is required for reconstitution of rough membrane cisternae, both GTP and ATP are required for smooth tubule formation, and assembly of smooth tubules occurs as an outgrowth (i.e. via tubulation) from rough membranes.
The chicken lysozyme gene 5′ MAR and the Drosophila histone SAR are electroelutable from encapsulated and digested nuclei
Authors: Hempel Katrin , Strätling Wolf H.
Published: April 2021

Cultured chicken cells were encapsulated in agarose microbeads, lysed in a near-physiological buffer and resulting encapsulated nuclei were digested with a restriction enzyme and electroeluted. After removal of ∼97% of the chromatin, the nuclear lamina, residual nucleoli and an internal nuclear network remained. The majority of nascent RNA was also recovered in digested and electroe-luted nuclei. Surprisingly, however, the chicken lysozyme gene 5′ MAR was quantitatively electroeluted from digested nuclei of expressing and non-expressing cells, as well as the promoter region and the coding sequence. When encapsulated nuclei were digested partially, the proportion of elutable 5′ MAR chromatin was comparable to that of elutable bulk chromatin. Furthermore, after digestion of encapsulated nuclei from Drosophila Kc cells, the histone SAR was electroeluted to the same extent as bulk chromatin. We conclude that the lysozyme gene 5′ MAR and the histone SAR are not permanently attached to a nuclear matrix or scaffold.
A cdc2-like kinase distinct from cdk5 is associated with neurofilaments
Authors: Starr Robyn , Hall Frederick L. , Monteiro Mervyn J.
Published: April 2021

An immunoprecipitation assay was used to identify protein kinases which are physically associated with neurofilaments (NF) in mouse brain extracts. Using this approach, we show that a cdc2-related kinase is associated with NF. The cdc2-related kinase was found to be distinct from cdk5 and the authentic cdc2 by a number of criteria. Firstly, it has a molecular mass on SDS-PAGE gels of 34 kDa, similar to that of cdc2, but differing from cdk5 (31 kDa). Secondly, it is not recognized by an antibody specific for cdk5. Thirdly, it is recognized by an antibody raised against the C-terminal region of authentic cdc2, but not by an antibody specific for the PSTAIRE motif. Using immunoblotting, we further show that the cdc2-related kinase copurifies with NF isolated from rat tissues. In vitro kinase assays further demonstrated that immunoprecipitated cdc2-related kinase phosphorylates recombinant NF-H protein. Phos-phorylation of NF-H by the cdc2-like activity was not affected by 3 μM olomoucine but was inhibited by 10 μM of this kinase inhibitor. Phosphoamino acid analysis of in vitro phosphorylated NF-H indicates that the immunoprecipitated cdc2-related kinase phosphorylates serine residues.
Transport pathway, maturation, and targetting of the vesicular stomatitis virus glycoprotein in skeletal muscle fibers
Authors: Rahkila Paavo , Alakangas Anne , Väänänen Kalervo , Metsikkö Kalervo
Published: April 2021

We have infected isolated skeletal muscle fibers with the vesicular stomatitis virus or the mutant tsO45, whose glycoprotein is blocked in the endoplasmic reticulum at 39°C. Immunofluorescence analysis for the viral glycoprotein indicated that the fibers were infected over their entire length at a virus dose of 109/ml. When we infected the myofibers with the tsO45 mutant at 39°C, the viral glyco-protein appeared to be localised to the terminal cisternae of the sarcoplasmic reticulum. Upon shifting the cultures to the permissive temperature, 32°C, in the presence of dinitrophenol, which blocks vesicular transport, the viral glycoprotein proceeded to completely fill the sarcoplasmic reticulum. Thus, both the endoplasmic reticulum located at the terminal cisternae of the sarcoplasmic reticulum, and the entire endoplasmic and sarcoplasmic reticulum appeared to be continuous. Shifting the culture temperature from 39°C to 20°C, resulted in prominent perinuclear staining throughout the fibers, accompanied by the appearance of distinct bright dots between the nuclei. Electron microscopic immunoperoxidase labeling indicated that these bright structures represented the Golgi apparatus. When either the tsO45-infected or wild-type virus-infected fibers were incubated at 32°C, the viral glycoprotein showed a staining pattern that consisted of double rows of punctate fluorescence. Immunogold labeling showed that the viral glycoprotein was present in both the transverse tubules as well as the endoplasmic/sarcoplasmic reticulum endomembranes. In addition, extensive viral budding was observed in the transverse tubules. Metabolic labeling experiments revealed that only half of the glycoprotein was processed in the Golgi, and this processed form had become incorporated into the budding viral particles. Thus, the processed viral glycoprotein was targeted to the transverse tubules. The other half of the glycoprotein remained endo-glycosidase H-sensitive, suggesting its retention in the endo-plasmic/sarcoplasmic reticulum endomembranes.
Modulation of osteoblast behaviour by tenascin
Authors: Mackie Eleanor J. , Ramsey Susan
Published: April 2021

The extracellular matrix protein tenascin is secreted by osteoblasts but absent from mineralized bone matrix. The current study was undertaken to test the hypothesis that tenascin regulates osteoblast behaviour. Three osteoblast-like cell lines UMR-106, ROS-17/2.8 (rat) and SAOS-2 (human) were used to investigate the role of tenascin in osteoblast morphology, differentiation and proliferation. Two of three cell lines adhered specifically to tenascin, remaining round and failing to spread. Tenascin as a sub-stratum stimulated alkaline phosphatase activity (a marker of osteoblast differentiation) in two of three cell lines. Moreover, anti-tenascin in the medium caused a reduction in alkaline phosphatase levels in all three cell lines. Anti-tenascin also inhibited collagen synthesis, an important osteoblast function. Since it seemed possible that tenascin may exert its effects on cell function through its ability to cause cell rounding, the ability of cell shape change alone to influence alkaline phosphatase levels was investigated. Cells were incubated in the presence of cytochalasin D and alkaline phosphatase levels assayed. Alkaline phosphatase activity was not elevated by cytochalasin D treatment, indicating that cell rounding alone is insufficient to mimic the effect of tenascin. Anti-tenascin caused a slight increase in proliferation of SAOS-2 cells, indicating that tenascin is itself inhibitory. In ROS 17/2.8 and UMR-106 cells, in contrast, proliferation was inhibited by anti-tenascin. The results presented here indicate that tenascin is able to stimulate osteoblastic differentiation and that endogenous tenascin helps to maintain the functional state of cultured osteoblast-like cells.
Establishment of hepatic cell polarity in the rat hepatoma-human fibroblast hybrid WIF-B9: A biphasic phenomenon going from a simple epithelial polarized phenotype to an hepatic polarized one
Authors: Decaens C. , Rodriguez P. , Bouchaud C. , Cassio D.
Published: April 2021

By immunofluorescence and freeze fracture methods, we have studied the establishment of hepatic cell polarity in WIF-B9 cells, a subclone of the WIF-B rat hepatoma-derived hybrid cell line. As previously shown (Ihrke et al. (1993)J. Cell Biol. 123, 1761-1775; Shanks et al. (1994)J. Cell Sci. 107, 813-825), these cells are a suitable model for in vitro studies of various hepatic functions, particularly polarity: in confluent cultures, the majority of cells form bile canaliculus-like structures; membrane domains are settled, according to plasma membrane protein localization similar to rat hepatocytes in situ. We here report that the establishment of WIF-B9 cell polarity is a slow progressive biphasic phenomenon. During the first days of culture, the majority of cells do not make bile canaliculus-like structures. However, they display a polarity similar to that of simple epithelial cells: apical membrane proteins and villin are found at the cell apex; basolateral ones, excluded from this area, are expressed in the remaining membrane area; the tight junction-associated protein ZO-1 and actin are concentrated at the boundary of these two poles, whereas E-cadherin is present at the lateral pole just under the apex. With time in culture, the number of cells expressing this simple epithelial polarized phenotype decreases progressively and, after 10-15 days, depending on the plating density, nearly all the cells express the typical hepatic polarized phenotype. The expression of these two pheno-types is mutually exclusive. Freeze-fracture replicas of both types of polarized cells show either macula occludens, fascia occludens (simple epithelial polarity) or zonula occludens (hepatic polarity), associated with gap junctions. In this last case, two or three continuous strands are generally present all around the bile canaliculus-like structures.
The unique proline-rich domain of parotid proline-rich proteins functions in secretory sorting
Authors: Stahl Lisa E. , Wright Rhonda L. , Castle J. David , Castle Anna M.
Published: April 2021

When expressed in pituitary AtT-20 cells, parotid proline-rich proteins enter the regulated pathway. Because the short N-terminal domain of a basic proline-rich protein is necessary for efficient export from the ER, it has not been possible to evaluate the role of this polypeptide segment as a sorting signal for regulated secretion. We now show that addition of the six-amino acid propeptide of proparathyroid hormone to the proline-rich protein, and especially to a deletion mutant lacking the N-terminal domain, dramatically accelerates intracellular transport of these polypeptides. Under these conditions the chimeric deletion mutant is stored as effectively as the full-length protein in dense core granules. The propeptide does not function as a sorting signal in AtT-20 cells as it does not reroute a constitutively secreted reporter protein to the regulated pathway. During transit, the propeptide is cleaved from the chimeric polypeptides such that the original structures of the full-length and the deletion mutant proline-rich proteins are reestablished. We have also found that the percentage stimulated secretion of the proline-rich proteins increases incrementally (almost twofold) as their level of expression is elevated. The increase reflects an enrichment of these polypeptides in the granule pool and its incremental nature suggests that sorting of proline-rich proteins involves an aggregation-based process. Because we can now rule out contributions to sorting by both N- and C-terminal segments of the proline-rich protein, we deduce that the unique proline-rich domain is responsible for storage. Thus at least some of the determinants of sorting for regulated secretion are protein-specific rather than universal.
Localised and global cytosolic Ca2+ changes in neutrophils during engagement of Cd11b/CD18 integrin visualised using confocal laser scanning reconstruction
Authors: Pettit E. J. , Hallett M. B.
Published: April 2021

A confocal laser scanning technique was used to provide optical sections in the vertical plane through living neutrophils during engagement of integrin and shape change. This has permitted the visualisation of cytosolic free Ca2+ rises localised to the sites of integrin immobilisation. These localised Ca2+ changes resulted from the release of Ca2+ from intracellular stores, and were not inhibited by removal of extracellular Ca2+ or by the Ca2+ channel blocking nickel ions. After integrin engagement and initial cell shape changes, a second rapid phase of cell spreading occurred, which was accompanied by global Ca2+ signalling bursts that continued sporadically after maximum spreading. This global signalling was driven mainly by Ca2+ influx from the extracellular medium. We propose that the localised and global Ca2+ signalling triggered by integrin engagement results from a common underlying mechanism and these signals are important for neutrophil shape change and extravasation.
Formation of crystalloid endoplasmic reticulum in COS cells upon overexpression of microsomal aldehyde dehydrogenase by cDNA transfection
Authors: Yamamoto Akitsugu , Masaki Ryuichi , Tashiro Yutaka
Published: April 2021

When rat liver microsomal aldehyde dehydrogenase (msALDH) was overexpressed in COS-1 cells by cDNA transfection, large granular structures containing both msALDH and endogenous protein disulfide isomerase appeared (Masaki et al. (1994)J. Cell Biol. 126, 1407-1420). Confocal laser microscopy revealed that these granular structures are dispersed throughout the cytoplasm. Electron microscopy showed that the structures are composed of regularly arranged crystalloid smooth endoplasmic reticulum (ER). The formation of the crystalloid ER was accompanied by a remarkable proliferation of smooth ER, which appeared occasionally continuous to the rough ER. We suggest that the smooth ER, proliferated from the rough ER, is transformed and assembled into the crystalloid ER by head-to-head association of the msALDH molecules on the apposed smooth ER membranes.

In order to understand the molecular mechanism of the crystalloid ER formation, we asked which portions of the msALDH molecules are needed for the crystalloid ER formation by expressing deletion mutants or chimera protein of msALDH in COS-1 cells. The overexpression of msALDH molecules lacking the stem region preceding the membrane spanning region, although they were exclusively localized in the ER, did not induce the formation of crystalloid ER. More detailed analysis showed that the amino acid sequence FFLL, located in the stem region, is necessary to form the crystalloid ER. The chimera protein containing the last 35 amino acids of msALDH at the carboxyl terminus of chloramphenicol acetyltransferase was localized to the ER, but did not induce the formation of the crystalloid ER. These results suggest that at least two regions, the bulky amino-terminal region and the FFLL sequence in the stem region of msALDH molecules are required for the formation of the crystalloid ER.
Porcine aortic endothelial gap junctions: identification and permeation by caged InsP3
Authors: Carter T. D. , Chen X. Y. , Carlile G. , Kalapothakis E. , Ogden D. , Evans W. H.
Published: April 2021

Gap junction channels permit the direct intercellular transfer of ions and small molecules and allow electrotonic coupling within tissues. Porcine aortic endothelial cells were extensively coupled, as assessed by gap junctional transfer of Lucifer yellow and the fluorescent calcium indicators fluo-3 and furaptra, but were not permeable to rhodamine B isothiocyanate-dextran 10S. The subunit composition of gap junction channels of porcine aortic endothelial cells was characterised using both northern blot analysis and RT-PCR techniques. Messenger RNA encoding connexins 37 and 43, but not 26, 32 or 40, were found in freshly isolated and cultured porcine aortic endothelial cells. Western blots using antipeptide antibodies raised to unique sequences of connexins 37, 40 and 43 showed the presence of connexins 37 and 43, but no connexin 40 was detected. Immunostaining with anticonnexin 43 antibodies showed extensive punctate fluorescent decoration of contacting membranes, whilst antibodies to connexin 37 showed predominantly intracellular staining. Caged InsP3 was found to readily permeate endothelial gap junctions. These results show that primary cultures of porcine aortic endothelial cells express connexin 37 and 43, and provide strong evidence that the second messenger molecule InsP3 can permeate porcine endothelial gap junctions.
Distribution of guanylate cyclase within photoreceptor outer segments
Authors: Hallett Mark A. , Delaat Joan L. , Arikawa Kentaro , Schlamp Cassandra L. , Kong Fansheng , Williams David S.
Published: April 2021

Guanylate cyclases play an essential role in the recovery of vertebrate photoreceptor cells after light activation. Here, we have investigated how one such guanylate cyclase, RetGC-1, is distributed within light- and dark-adapted rod photoreceptor cells. Guanylate cyclase activity partitioned with the photoreceptor outer segment (OS) cytoskeleton in a light-sensitive manner. RetGC-1 was found to bind actin filaments in actin blot overlays, suggesting a mechanism for its association with the OS cytoskeleton. In retinal sections, this enzyme was immunodetected only in the OSs, where it appeared to be distributed throughout the disk membranes.