bluuebunny/crossref_metadata_2025 huggingface dataset:
An Analysis of the Postgastrula Differentiation of the Hypomere
Authors: Finnegan Cyril V.
Published: April 2021

A small mass of T. torosa postgastrula (stages 14–21) hypomere with its associated ectoderm, when cultured in vitro, demonstrates cell displacement posteriad along the original antero-posterior axis. Histological examination of these explants indicates that the splanchnic mesoderm disperses and differentiates, as a peritoneum for the most part, with a loose group of haematoblasts residing in the cavity formed. When a small group of endoderm cells was added to the hypomere-ectoderm ball, the results were similar to the above except that splanchnic mesoderm in the vicinity of the endoderm remained as a cell mass rather than dispersing as mesothelium. Late in the second week of culture mitotic figures became more prevalent in the splanchnic mesoderm in the immediate vicinity of the endoderm than elsewhere in the splanchnic cell mass. When epimere (somite) mesoderm was included in the hypomere-ectoderm ball, a nephric duct developed anteriorly and mesoderm cells remained as a cell mass in the vicinity of this duct. The results are discussed as indicating that the endoderm retains a meso-derm mass in its vicinity and thereby assists the differentiation of this mesoderm.
A dissection of the mechanisms generating and stabilizing polarity in mouse 8- and 16-cell blastomeres: the role of cytoskeletal elements
Authors: Johnson Martin H. , Maro Bernard
Published: April 2021

Pairs of 8-cell or 16-cell blastomeres were cultured for up to 9h after their formation from isolated 1/4 or 1/8 blastomeres respectively. Blastomeres were examined for the incidence and orientation of their surface polarity, as assessed by binding of FITC-Con A and by distribution of microvilli, and of their cytoplasmic polarity, as assessed by distribution of cytoplasmic actin, clathrin and a 100kD antigen associated with the lysosomal/acid vesicle fraction of membranous organelles. The effect on polarity of incubating the pairs of cells in taxol, nocodazole, cytochalasin D or in a combination of nocodazole plus cytochalasin D for different parts of the incubation period was examined.

Neither the development nor the stability of the surface polarity in 8-cell blastomeres was blocked by any treatment and only the use of CCD in combination with nocodazole affected the incidence of surface polarity appreciably. However, with some treatments, the form and position of the surface poles were modified. In the presence of microtubule inhibitors surface poles extended over a larger area of the cell surface, while exposure to CCD led to poles that were not opposite to the contact point between cells. In contrast to surface polarity, the development of cytoplasmic polarity was suppressed by both microtubule- and microfilament-inhibiting drugs, which also reversed it rapidly.

In polar 16-cell blastomeres surface polarity was influenced in a similar manner to that of 8cell blastomeres, only the combined use of cytochalasin D and nocodazole having any major effect. Polarization of clathrin in polar 16-cell blastomeres was inhibited almost completely by all drug treatments applied including cytochalasin D. The focal concentration of lysosomal antigen that occurs during the 16-cell stage was reduced only in the continuous presence of nocodazole plus cytochalasin D, but once established was not reversed appreciably by any drug. However, the localization of the lysosomal antigen to the basal region of polarized cells did not seem to occur in the presence of any drug. The dissociation of surface and cytoplasmic polarity revealed in these experiments leads us to conclude that either (1) surface polarity is a prerequisite for the organization of cytoplasmic polarity, and mediates the latter via the cytoskeleton, or (2) surface and cytoplasmic polarity develop by parallel but separate mechanisms.
Cartilage morphogenesis in vitro
Authors: Archer Charles W. , Rooney Paul , Cottrill Christopher P.
Published: April 2021

The morphogenetic capacity of prechondrogenic mesenchyme from two developmentally distinct sources was investigated in high density micromass cultures. We confirmed an earlier report (Weiss & Moscona, 1958) that scleral mesenchyme formed cartilage sheets whilst limb bud mesenchyme formed distinct cartilage nodules. It was thus suggested by these authors that this morphogenesis was tissue type specific. However, by varying cell density at inoculation (which controls cell configuration) and by varying the relative amount of prechondrogenic mesenchyme present in cultures we found that dramatic changes in morphogenesis could be brought about. Viewed in these terms we suggest that cartilage morphogenesis in vitro is dependent on cell configuration and the presence of non-chondrogenic cell types and hence is not necessarily a function of an intrinsic morphogenetic potential of the constituent cells.
X-chromosome deletions in embryo-derived (EK) cell lines associated with lack of X-chromosome inactivation
Authors: Rastan Sohaila , Robertson Elizabeth J.
Published: April 2021

The predictions of a model for the initiation of X-chromosome inactivation based on a single inactivation centre were tested in a cytogenetic study using six different embryo-derived (EK) stem cell lines, each with a different-sized deletion of the distal part of one of the X-chromosomes. Metaphase chromosomes were prepared by the Kanda method from each cell line in the undifferentiated state and after induction of differentiation, and cytogenetic evidence sought for a dark-staining inactive X-chromosome. The results confirm the predictions of the model in that when the inactivation centre is deleted from one of the X-chromosomes neither X present in a diploid cell can be inactivated, and in addition considerably further localize the position of the inactivation centre on the X-chromosome.
Maintenance of function in single epithelial cells spatially isolated from similar cells
Authors: Maddrell S. H. P. , Overton J. A.
Published: April 2021

We have found in an insect tissue, the Malpighian tubules of Rhodnius, instances of single epithelial cells which, as the result of a possible error in development, lie within the epithelium some distance from the main population of similar cells. This spatial separation makes it possible to measure the transport abilities of these cells. Their transport abilities were found to be the same as the cells in the main population. This finding shows that the maintenance of function in individual cells of epithelial tissues may not depend on direct contact with other similar cells.
Patterns of peanut agglutinin binding within the developing grasshopper central nervous system
Authors: Burt Richard , Anderson Hilary
Published: April 2021

The location of peanut agglutinin (PNA) binding was investigated in the segmental ganglia of the developing grasshopper embryo. Neuronal processes were stained but cell bodies were not. The first appearance of PNA binding in development was associated with the first neurons to initiate axon outgrowth, the progeny of the MP2 cells. In the early stages of development the location of PNA binding was congruent with that of antibodies against horseradish peroxidase (HRP), which bind to neurons. In more advanced ganglia only a subpopulation of those neuronal processes that bound auti-HRP also bound PNA. The results suggest that PNA-binding sites are present only on those neuronal processes which are still developing and raise the possibility that these molecules may play a rôle in neurite outgrowth and navigation.
The role of glycosaminoglycans in anuran pigment cell migration
Authors: Tucker R. P.
Published: April 2021

The migratory pathways of neural crest-derived pigment cells were examined in two anurans, Xenopus laevis and Discoglossus pictus, and correlated with the distribution of glycosaminoglycans (GAG) in the extracellular matrix (ECM) of these pathways. In Xenopus, melanophores in the trunk reach the dermis by initially migrating ventrally, between the neural tube and somites, and then by migrating through the somites to reach the subectodermal space. In Discoglossus, melanophores, iridophores, and xanthophores migrate laterally over the dorsal margin of the somites to reach the dermis. GAG was identified in the light microscope using alcian blue staining and in the electron microscope using ruthenium red staining. The ECM at the dorsal entrance to the lateral pathway in Xenopus and in young Discoglossus (at a stage prior to invasion by pigment cells) is filled with 25–50 nm chondroitin sulphate proteoglycan aggregates. When this ECM in Xenopus is digested in vivo with chondroitinase ABC, melanophores enter the lateral pathway. In older Discoglossus embryos, the migration of pigment cells into the lateral pathway is correlated with increases in the space between the ectoderm and somites and in the number of hyaluronate microfibrils. These observations suggest that chondroitin sulphate proteoglycan in the subectodermal ECM restricts the migration of pigment cells into the lateral pathway by limiting the amount of space for migration and possibly by acting as a less adhesive migratory substratum than the ventral pathway, and that in Discoglossus hyaluronate opens spaces permitting the migration of pigment cells directly over the dorsal margin of the somites.
Regulation of development in the fully grown mouse oocyte: chromosome-mediated temporal and spatial differentiation of the cytoplasm and plasma membrane
Authors: Blerkom Jonathan Van , Bell Hobart
Published: April 2021

The relationship between nuclear maturation and the differentiation of the cytoplasm and plasma membrane during resumption of arrested meiosis was investigated by culture of GV- and Mil-stage mouse oocytes in the presence and absence of nocodazole. Culture in the presence of nocodazole was associated with dispersal of MI and Mil chromosomes throughout the sub-plasmalemmal cytoplasm. A progression of cortical (thickening of actin filaments) and plasma membrane changes (denudation of microvilli, reduction in cell surface glycoproteins, formation of chromosome-containing evaginations) that normally occurs in proximity to chromosomes associated with intact MI or Mil spindles took place only in those regions of the cortical cytoplasm containing the dispersed subplasmalemmal chromosomes. The dispersion and migration of the chromosomes occurred in an apparently random fashion. Fluorescent probe analysis of normal and treated oocytes indicated a stage-specific association between the spatial distribution of chromosomes and mitochondria. Transfer of individual bivalent chromosomes to untreated oocytes at different stages of maturation and to cytoplasts derived from oocytes anucleated prior to GVB demonstrated (1) the necessity of chromosomes for cytoplasmic and plasma membrane differentiation, and (2) that the capacity of the cytoplasm and plasma membrane to differentiate in response to the presence of a chromosome is acquired prior to GVB.
The formation of axonal projections of the mesencephalic trigeminal neurones in chick embryos
Authors: Hiscock Jennifer , Straznicky Charles
Published: April 2021

Horseradish peroxidase-wheat germ agglutinin conjugate (HRP) was injected into masticatory and eye muscles of 6-to 15-day-old chick embryos and posthatched chicks to establish the timetable of axonal outgrowth and distribution of central and peripheral terminations of mesencephalic trigeminal neurones (MTN).

HRP-labelled MTN first appeared on the 10th day of incubation and by the 14th day most of MTN became labelled, indicating that axonic outgrowth to peripheral targets occurred between the 10th and 14th days of incubation. Peripheral targets included the pterygoideus lateralis and medialis, protractor quadratus, pseudotemporalis superficialis and profundus, and adductor mandibulae of jaw-closing muscles. HRP-filled central axonic processes of MTN were identified first on the 13th day of incubation and they terminated exclusively in the motor nucleus of the trigeminal nerve.

Between the 10th and 13th days of incubation, at the peak of naturally occurring cell death in the MTN pool, a consistently lower percentage of neurones could be labelled with HRP than in older embryos and in posthatched chicks. This finding suggests that many MTN die before establishing contact with peripheral targets.
Regeneration of mammary glands in vivo from isolated mammary ducts
Authors: Ormerod E. Jane , Rudland Philip S.
Published: April 2021

Rat mammary ducts, free of buds, can alone regenerate complete mammary trees when transplanted into the interscapular fat pads of syngeneic host rats. All the main mammary cell types are identified within such outgrowths. Epithelial cells, which show the presence of milk fat globule membrane antigens and microvilli on their luminal surfaces, line the ducts. Basal cells surrounding the ducts show characteristic features of myoepithelial cells: immunoreactive actin and keratin within the cytoplasm, myofilaments, pinocytotic vesicles and hemidesmosomal attachments to the basement membrane. Cells within the end buds and lateral buds, however, show few if any cytoplasmic myofilaments and are relatively undifferentiated in appearance. Intermediate morphologies between these cells and myoepithelial cells are seen nearer the ducts. In this respect they exactly resemble the cap cells found in terminal end buds (TEBs) of normal mammary glands. Occasional epithelial cells within alveolar buds show the presence of immunoreactive casein, which is a product of secretory alveolar cells in the normal rat mammary gland. Dissected terminal end buds can regenerate similar ductal outgrowths. Thus, ductal tissue alone can generate all the major mammary cell types seen in the normal gland, including the cap cells.
Epithelial–mesenchymal tissue interactions guiding otic capsule formation: the role of the otocyst
Authors: McPhee Joseph R. , Van De Water Thomas R.
Published: April 2021

The otocyst is the epithelial anlage of the membranous labyrinth which interacts with surrounding cephalic mesenchyme to form an otic capsule. A series of in vitro studies was performed to gain a better understanding of the epithelial–mesenchymal interactions involved in this process. Parallel series of otocyst/mesenchyme (0/M) and isolated periotic mesenchyme (M) explants provided morphological and biochemical data to define the role of the otocyst in organizing and directing formation of its cartilaginous otic capsule. Explants were made from mouse embryos ranging in age from 10 to 14 days of gestation, and organ cultured under identical conditions until the chronological equivalent of 16 days of gestation. Expression of chrondrogenesis was determined by both histology and biochemistry. The in vitro behaviour of periotic mesenchyme explanted either with or without an otocyst supports several hypotheses that explain aspects of otic capsule development. The results indicate that (a) prior to embryonic day 12 the otocyst alone is not sufficient to stimulate chondrogenesis of the otic capsule within O/M explants; (b) the otocyst acts as an inductor of capsule chondrogenesis within O/M explants between embryonic days 12 to 13; (c) isolated mesenchyme within M explants taken from 13-day-old embryos are capable of initiating in vitro chondrogenesis, but without expressing capsule morphology in the absence of the otocyst; and (d) the isolated mesenchyme of M explants obtained from 14-day-old embryos expresses both chondrogenesis and otic capsule morphology in the absence of the otocyst. These findings suggest that the otocyst acts as an inductor of chondrogenesis of periotic mesenchyme tissue between embryonic days 11 to 13, and controls capsular morphogenesis between embryonic days 13 to 14 in the mouse embryo.
Regulation of extraembryonic calcium mobilization by the developing chick embryo
Authors: Tuan Rocky S. , Ono Tamao
Published: April 2021

During development, the chick embryo mobilizes the calcium it needs from two extra-embryonic sources, first the yolk and then the eggshell. Since previous studies have strongly suggested that vitamins D and K may regulate chick embryonic calcium metabolism, we have examined here how these vitamins might be involved in regulating the calcium mobilization processes. We used as our experimental system chick embryos which were maintained in longterm in vitro culture in the absence of the eggshell. Our results showed that exogenous vitamin D3, in the form of the active 1,25-dihydroxylated metabolite, was hypercalcaemic in both control embryos and the calcium-deficient, shell-less embryos. Since the eggshell was absent in the latter, the vitamin D-induced hypercalcaemia must involve mobilization of calcium from the yolk and, or, the embryonic skeleton. The latter was unlikely since concomitant hyper-phosphataemia was not observed. By radiolabelling the yolk with 45Ca2+ and subsequently monitoring its distribution, we showed that vitamin D3 stimulated yolk calcium mobilization. However, exogenous vitamin D3 did not appear to influence the calcium uptake activity of the chorioallantoic membrane (CAM), the tissue responsible for translocating eggshell calcium. On the other hand, when embryos were rendered vitamin K deficient by the administration of its antagonist, Warfarin, CAM calcium activity was significantly depressed, an effect which was remedied by vitamin K supplementation. We conclude that, during normal chick embryonic development, vitamin D is primarily involved in regulating yolk calcium mobilization whereas vitamin K is required for eggshell calcium translocation by the CAM.
Small eyes (Sey) : a homozygous lethal mutation on chromosome 2 which affects the differentiation of both lens and nasal placodes in the mouse
Authors: Hogan Brigid L. M. , Horsburgh Gwynn , Cohen James , Hetherington Colin M. , Fisher Graham , Lyon Mary F.
Published: April 2021

Small eyes (Sey) is a semidominant, homozygous lethal mutation in the mouse (Roberts, 1967). It is allelic with SeyH, a radiation-induced homozygous prenatal lethal which has been mapped on chromosome 2. The effect of the Sey mutation is apparently limited to the growth and differentiation of the presumptive lens and nasal placodes. Homozygous Sey/Sey embryos can be distinguished as early as 10·5 days post coitum (p.c.f, the optic vesicles grow out, but the ectoderm does not give rise to a lens and nasal pits never form. Immunohistochemical studies show that the distribution of the extracellular matrix glycoprotein laminin is not significantly different in the cephalic region of Sey/Sey versus Sey/+ or +/+ embryos. Sey/Sey embryos develop to term but without eyes or nose, and die soon after birth. Further analysis of Sey/Sey embryos may throw light on the mechanisms underlying morphogenesis of craniofacial structures in mammals.
Development of erythroid colony-forming cells in rat fetal spleen: apparent lack of sensitivity to anin vivocorticosteroid excess as compared to fetal liver
Authors: Nagel M.-D. , Nagel J.
Published: April 2021

The development of splenic erythroid colony-forming cells from rat embryos in the last 4 days of intrauterine life was examined after 2 and 7 days in a methylcellulose culture system. The number of 2- and 7-day erythroid colonies decreased sharply between, respectively, days 20 and 21 of gestation and days 19 and 20. Concomitantly, a maturation of proerythro-blasts and basophilic erythroblasts to mature erythroblasts was detected on smears of splenic cellular suspensions.

The effect of a corticosteroid excess induced by a maternal laparotomy was tested on spleen and liver cultures from the same control or experimental fetuses. The ratio of the number of 2-day to the number of 7-day erythroid colonies did not differ in experimental and control splenic cultures, but in liver cultures was significantly lower at days 19 and 20 in experimental than in control cultures.
A method for determining the periodicity of a troponin component in isolated insect flight muscle thin filaments by gold/Fab labelling
Authors: Newman R. , Butcher G. W. , Bullard B. , Leonard K. R.
Published: April 2021

Insect flight muscle has a large component (Tn-H) in the tropomyosin-troponin complex that is not present in vertebrate striated muscle thin filaments. Tn-H is shown by gold/Fab labelling to be present at regular intervals in insect flight muscle thin filaments. The Fab fragment of a monoclonal antibody to Tn-H was conjugated directly with colloidal gold and this probe used to label isolated thin filaments from the flight muscle of Lethocerus indicus (water bug). The distribution of gold particles seen in electron microscope images of negatively stained thin filaments was analysed to show that the probe bound to sites having a periodicity of approximately 40 nm, which is the expected value for the tropomyosintroponin repeat. Conjugates of Fab with colloidal gold particles of 3 nm diameter labelled almost all sites. Conjugates with gold particles of 5 nm and 10 nm diameter labelled less efficiently (70% and 30%, respectively) but analysis of the distribution of inter-particle intervals among a number of filaments again gave the same fundamental spacing of 40 nm. The error in the measurements (standard deviation approximately ±4.2 for 5 nm gold/Fab) is less than earlier estimates for the size of the gold/Fab complex. Measurements on gold/Fab in negative stain suggest that the bound Fab contributes a shell about 2 nm in thickness around the gold particle. The radius of the probe (about 4.5 nm for 5 nm gold/Fab) would then be consistent with the value of error found. The size of the probe suggests that the gold particle binds to the side of the Fab molecule, rather close to the antibody combining site. The potential resolution of the technique may thus be better than originally expected.
Mutants of the microtubule motor protein, nonclaret disjunctional, affect spindle structure and chromosome movement in meiosis and mitosis
Authors: Hatsumi Machiko , Endow Sharyn A.
Published: April 2021

The Drosophila microtubule motor protein, nonclaret disjunctional (ned), is required for proper chromosome distribution in meiosis and mitosis. We have examined the meiotic and mitotic divisions in wild-type Drosophila oocytes and early embryos, and the effects of three ncdmutants (cand, ncd and ncdD) on spindle structure and chromosome movement. The ncdmutants cause abnormalities in spindle structure early in meiosis I, and abnormal chromosome configurations throughout meiosis I and n. Defective divisions continue in early embryos of the motor null mutant, cand, with abnormal early mitotic spindles. The effects of mutants on spindle structure suggest that ncdis required for proper meiotic spindle assembly, and may play a role in forming or maintaining spindle poles in meiosis. The disruption of normal meiotic and mitotic chromosome distribution by ncdmutants can be attributed to its role as a spindle motor, although a role for ncdas a chromosome-associated motor protein is not excluded. The ncd motor protein functions not only in meiosis, but also performs an active role in the early mitotic divisions of the embryo.
Screening and identification of a gene, PSE-1, that affects protein secretion in Saccharomyces cerevisiae
Authors: Chow Terry Y.-K. , Ash Josee J. , Dignard Daniel , Thomas David Y.
Published: April 2021

Utilizing a screening method designed for the identification of genes involved with enhanced protein secretion in Saccharomyces cerevisiae we identified a gene, which we named PSE-1 (Protein Secretion Enhancer). Overexpression of PSE-1 in a multi-copy plasmid, as shown by Northern hybridization, gave a fourfold enhancement in total protein secretion. The repertoire of proteins that are found to be secreted in greater quantities include three known biologically active proteins: k 1 killer toxin, a-factor, and acid phosphatase. The PSE-1 gene is located on chromosome XII of the yeast genome and codes for a hydrophobic protein containing 1089 amino acids. Haploid yeast cells that contained a LEU2 insertion mutation in PSE-1 grow very poorly, a phenotype similar to other conditional SEC mutants at restrictive temperature.
The Bacillus thuringiensis Cry1Ac toxin-induced permeability change in Manduca sexta midgut brush border membrane vesicles proceeds by more than one mechanism
Authors: Carroll Joe , Wolfersberger Michael G. , Ellar David J.
Published: April 2021

Aminopeptidase N purified from whole Manduca sexta midgut binds the Cry1Ac insecticidal toxin from Bacillus thuringiensis and this binding is inhibited by N-acetyl-galactosamine (GalNAc). We have examined the membrane permeabilising activity of the Cry1Ac toxin using brush border membrane vesicles (BBMV) prepared from the anterior (A-BBMV) and posterior (P-BBMV) subregions of the M. sexta midgut. A toxin mixing assay demonstrated a faster rate of toxin activity on P-BBMV than on A-BBMV. In the presence of GalNAc this rapid activity on P-BBMV was reduced to the rate seen with A-BBMV. GalNAc had no effect on the rate of A-BBMV permeabilisation by Cry1Ac. Aminopeptidase N assays of A- and P-BBMV demonstrated that this Cry1Ac binding protein is concentrated in the posterior midgut region of M. sexta. It therefore appears that there are two mechanisms by which Cry1Ac permeabilises the M. sexta midgut membrane: a GalNAc-sensitive mechanism restricted to the posterior midgut region, probably involving aminopeptidase N binding, and a previously undetected mechanism found in both the posterior and anterior regions.
Two kinds of BIR-containing protein - inhibitors of apoptosis, or required for mitosis
Authors: Silke John , Vaux David L.
Published: April 2021

The baculoviral IAP repeat (BIR) is a zinc-binding fold. Some BIR-containing proteins (BIRCs), including several from insect viruses, insects and vertebrates, are inhibitors of cell death and act by binding to active caspases. Their ability to do so can be antagonized by pro-apoptotic insect proteins such as Grim, HID and Reaper, or the mammalian protein Diablo/Smac. Members of one structurally distinct subgroup of BIR-containing proteins, which are present in yeasts and Caenorhabditis elegans as well as insects and vertebrates, do not act as caspase inhibitors; instead, they are required for chromosome segregation and cytokinesis, and act in concert with inner centromere protein (INCENP) homologues and aurora kinase homologues.
Rho GTPases and cell migration
Authors: Ridley Anne J.
Published: April 2021

Cell migration involves dynamic and spatially regulated changes to the cytoskeleton and cell adhesion. The Rho GTPases play key roles in coordinating the cellular responses required for cell migration. Recent research has revealed new molecular links between Rho family proteins and the actin cytoskeleton, showing that they act to regulate actin polymerization, depolymerization and the activity of actin-associated myosins. In addition, studies on integrin signalling suggest that the substratum continuously feeds signals to Rho proteins in migrating cells to influence migration rate. There is also increasing evidence that Rho proteins affect the organization of the microtubule and intermediate filament networks and that this is important for cell migration.
The AP-3-dependent targeting of the melanosomal glycoprotein QNR-71 requires a di-leucine-based sorting signal
Authors: Le Borgne Roland , Planque Nathalie , Martin Patrick , Dewitte Frédérique , Saule Simon , Hoflack Bernard
Published: April 2021

The Quail Neuroretina clone 71 gene (QNR-71) is expressed during the differentiation of retinal pigmented epithelia and the epidermis. It encodes a type I transmembrane glycoprotein that shares significant sequence homologies with several melanosomal proteins. We have studied its intracellular traffic in both pigmented and non-pigmented cells. We report that a di-leucine-based sorting signal (ExxPLL) present in the cytoplasmic domain of QNR-71 is necessary and sufficient for its proper targeting to the endosomal/premelanosomal compartments of both pigmented and non-pigmented cells. The intracellular transport of QNR-71 to these compartments is mediated by the AP-3 assembly proteins. As previously observed for the lysosomal glycoproteins LampI and LimpII, overexpression of QNR-71 increases the amount of AP-3 associated with membranes, and inhibition of AP-3 synthesis increases the routing of QNR-71 towards the cell surface. In addition, expression of QNR-71 induces a misrouting of endogenous LampI to the cell surface. Thus, the targeting of QNR-71 might be similar to that of the lysosomal integral membrane glycoproteins LampI and LimpII. This suggests that sorting to melanosomes and lysosomes requires similar sorting signals and transport machineries.
Novel non-TGF-β-binding splice variant of LTBP-4 in human cells and tissues provides means to decrease TGF-β deposition
Authors: Koli Katri , Saharinen Juha , Kärkkäinen Mira , Keski-Oja Jorma
Published: April 2021

Small latent TGF-β consists of latency associated peptide (LAP) bound to the 25 kDa TGF-β by noncovalent interactions. Small latent TGF-β is secreted from cells and deposited into the extracellular matrix as covalent complexes with its binding proteins, LTBPs. Four LTBPs have been molecularly cloned and their structures contain repetitive sequences. The 3rd 8-Cys repeats of LTBP-1, -3 and -4 are able to associate with small latent TGF-β. We analyzed by RT-PCR the expression of LTBPs 1-4 in a panel of cultured human cell lines including fibroblasts of different origin, endothelial cells and immortalized keratinocytes. LTBPs were expressed in an overlapping manner, but differences in their expression levels were detected. SV-40 transformed human embryonic lung fibroblasts contained less of the mRNAs for the LTBPs, suggesting that malignant transformation leads to decrease in LTBP expression. A novel alternatively spliced form of LTBP-4 lacking the 3rd 8-Cys repeat (LTBP-4Δ8-Cys3rd) was identified. LTBP-4Δ8-Cys3rd does not bind TGF-β and it was found to be expressed in the same tissues as the full length LTBP-4. The exon-intron structure of LTBP-4 around the 3rd 8-Cys repeat was similar to those of LTBP-2 and -3. LTBP-4Δ8-Cys3rd was produced by alternative splicing over two exons. In addition, HL-60 promyelocytic leukemia cells expressed a splice variant lacking only one exon of this region. The expression of the non-TGF-β-binding variant of LTBP-4 may be important for the regulation of TGF-β deposition in tissues. Since LTBPs are a part of the extracellular matrix microfibrils, the LTBP-4Δ8-Cys3rd protein may also be involved in various structural functions not related to TGF-β signaling.
Centrosome cohesion is regulated by a balance of kinase and phosphatase activities
Authors: Meraldi Patrick , Nigg Erich A.
Published: April 2021

Centrosome cohesion and separation are regulated throughout the cell cycle, but the underlying mechanisms are not well understood. Since overexpression of a protein kinase, Nek2, is able to trigger centrosome splitting (the separation of parental centrioles), we have surveyed a panel of centrosome-associated kinases for their ability to induce a similar phenotype. Cdk2, in association with either cyclin A or E, was as effective as Nek2, but several other kinases tested did not significantly interfere with centrosome cohesion. Centrosome splitting could also be triggered by inhibition of phosphatases, and protein phosphatase 1α (PP1α) was identified as a likely physiological antagonist of Nek2. Furthermore, we have revisited the role of the microtubule network in the control of centrosome cohesion. We could confirm that microtubule depolymerization by nocodazole causes centrosome splitting. Surprisingly, however, this drug-induced splitting also required kinase activity and could specifically be suppressed by a dominant-negative mutant of Nek2. These studies highlight the importance of protein phosphorylation in the control of centrosome cohesion, and they point to Nek2 and PP1α as critical regulators of centrosome structure.
Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells
Authors: Trinkle-Mulcahy Laura , Sleeman Judith E. , Lamond Angus I.
Published: April 2021

Protein phosphatase 1 (PP1) is expressed in mammalian cells as three closely related isoforms, α, β/δ and γ1, which are encoded by separate genes. It has yet to be determined whether the separate isoforms behave in a similar fashion or play distinct roles in vivo. We report here on analyses by fluorescence microscopy of functional and fluorescently tagged PP1 isoforms in live cells. PP1α and PP1γ fluorescent protein fusions show largely complimentary localization patterns, particularly within the nucleus where tagged PP1γ accumulates in the nucleolus, whereas tagged PP1α is primarily found in the nucleoplasm. Overexpression of NIPP1 (nuclear inhibitor of PP1), a PP1 targeting subunit that accumulates at interchromatin granule clusters in the nucleoplasm, results in a retargeting of both isoforms to these structures, indicating that steady-state localization is based, at least in part, on relative affinities for various targeting subunits. Photobleaching analyses show that PP1γ is rapidly exchanging between the nucleolar, nucleoplasmic and cytoplasmic compartments. Fluorescence resonance energy transfer (FRET) analyses indicate that the direct interaction of the two proteins predominantly occurs at or near interchromatin granule clusters. These data indicate that PP1 isoforms are highly mobile in cells and can be dynamically (re)localized through direct interaction with targeting subunits.
β1PIX, the PAK-interacting exchange factor, requires localization via a coiled-coil region to promote microvillus-like structures and membrane ruffles
Authors: Koh Cheng-Gee , Manser Ed , Zhao Zhou-Shen , Ng Chee-Peng , Lim Louis
Published: April 2021

PIX is a Rho-family guanine nucleotide exchange factor that binds PAK. We previously described two isoforms of PIX that differ in their N termini. Here, we report the identification of a new splice variant of βPIX, designated β2PIX, that is the dominant species in brain and that lacks the region of ∼120 residues with predicted coiled-coil structure at the C terminus of β1PIX. Instead, β2PIX contains a serine-rich C terminus. To determine whether these splice variants differ in their cellular function, we studied the effect of expressing these proteins in HeLa cells. We found that the coiled-coil region plays a key role in the localization of β1PIX to the cell periphery and is also responsible for PIX dimerization. Overexpression of β1, but not β2PIX, drives formation of membrane ruffles and microvillus-like structures (via activation of Rac1 and Cdc42, respectively), indicating that its function requires localized activation of these GTPases. Thus, β1PIX, like other RhoGEFs, exerts specific morphological functions that are dependent on its intracellular location and are mediated by its C-terminal dimerization domain.
μ1A deficiency induces a profound increase in MPR300/IGF-II receptor internalization rate
Authors: Meyer Christoph , Eskelinen Eeva-Liisa , Guruprasad Medigeshi Ramarao , von Figura Kurt , Schu Peter
Published: April 2021

The mannose-6-phosphate/IGF-II receptor MPR300 mediates sorting of lysosomal enzymes from the trans-Golgi network to endosomes and endocytosis of hormones, for example, of IGF-II. We analyzed transport of MPR300 in μ1A-adaptin-deficient fibroblasts, which lack a functional AP-1 clathrin adaptor complex. In μ1A-adaptin-deficient fibroblasts, the homologous MPR46 accumulates in endosomes due to a block in retrograde transport to the trans-Golgi network. The MPR300-mediated endocytosis is markedly enhanced. We demonstrate that the seven-fold increase in endocytosis is not associated with an increased steady-state concentration of receptors at the plasma membrane, but with an increased internalization rate of MPR300. Internalization of other receptors that are also endocytosed by AP-2 is not affected. More MPR300 receptors are found in clathrin-coated pits of the plasma membrane, whereas outside coated-areas, more MPR300 are concentrated in clusters and all intracellular receptors reside in endosomes, which are in equilibrium with the plasma membrane. Thus AP-1-mediated transport of MPR300 from endosomes to the TGN controls indirectly the recycling rate of the receptor between the plasma membrane and endosomes.
Lysosome proteins are redistributed during expression of a GTP-hydrolysis-defective rab5a
Authors: Rosenfeld Jennifer L. , Moore Robert H. , Zimmer K.-Peter , Alpizar-Foster Estrella , Dai Wenping , Zarka M. Nader , Knoll Brian J.
Published: April 2021

The functioning of the endocytic pathway is influenced by a distinct set of rab GTPases, including rab5a, which regulates homotypic fusion of early endosomes. Expression of a dominant active, GTPase-defective rab5a accelerates endosome fusion, causing the formation of a greatly enlarged endocytic compartment. Here we present evidence that rab5a also regulates trafficking between endosomes and lysosomes and may play a role in lysosome biogenesis. The GTPase defective rab5aQ79L mutant was inducibly expressed as an EGFP fusion in HEK293 cells, and the distribution of lysosome proteins and endocytic markers then assessed by deconvolution fluorescence microscopy. During expression of EGFP-rab5aQ79L, the lysosome proteins LAMP-1, LAMP-2 and cathepsin D were found in dilated EGFP-rab5aQ79L-positive vesicles, which also rapidly labeled with transferrin Texas Red. Exogenous tracers that normally traffic to lysosomes after prolonged chase (dextran Texas Red and DiI-LDL) also accumulated in these vesicles. Dextran Texas Red preloaded into lysosomes localized with subsequently expressed EGFP-rab5a Q79L, suggesting the existence of lysosome to endosome traffic. Cells expressing EGFP-rab5a wt or the dominant negative EGFP-rab5aS34N did not exhibit these abnormalities. Despite the dramatic alterations in lysosome protein distribution caused by expression of EGFP-rab5a Q79L, there was little change in the endocytosis or recycling of a cell-surface receptor (β2-adrenergic receptor). However, there was a deficiency of dense β-hexosaminidase-containing lysosomes in cells expressing EGFP-rab5aQ79L, as assessed by Percoll gradient fractionation. These results suggest that expression of a GTPase-defective rab5a affects lysosome biogenesis by alteration of traffic between lysosomes and endosomes.
BAF is required for emerin assembly into the reforming nuclear envelope
Authors: Haraguchi Tokuko , Koujin Takako , Segura-Totten Miriam , Lee Kenneth K. , Matsuoka Yosuke , Yoneda Yoshihiro , Wilson Katherine L. , Hiraoka Yasushi
Published: April 2021

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envelope assembly in living HeLa cells. During telophase, emerin accumulates briefly at the ‘core’ region of telophase chromosomes, and later distributes over the entire nuclear rim. Barrier-to-autointegration factor (BAF), a protein that binds nonspecifically to double-stranded DNA in vitro, co-localized with emerin at the ‘core’ region of chromosomes during telophase. An emerin mutant defective for binding to BAF in vitro failed to localize at the ‘core’ in vivo, and subsequently failed to localize at the reformed NE. In HeLa cells that expressed BAF mutant G25E, which did not show ‘core’ localization, the endogenous emerin proteins failed to localize at the ‘core’ region during telophase, and did not assemble into the NE during the subsequent interphase. BAF mutant G25E also dominantly dislocalized LAP2β and lamin A from the NE, but had no effect on the localization of lamin B. We conclude that BAF is required for the assembly of emerin and A-type lamins at the reforming NE during telophase, and may mediate their stability in the subsequent interphase.
How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane
Authors: Tilney Lewis G. , Harb Omar S. , Connelly Patricia S. , Robinson Camenzind G. , Roy Craig R.
Published: April 2021

Within five minutes of macrophage infection by Legionella pneumophila, the bacterium responsible for Legionnaires’ disease, elements of the rough endoplasmic reticulum (RER) and mitochondria attach to the surface of the bacteria-enclosed phagosome. Connecting these abutting membranes are tiny hairs, which are frequently periodic like the rungs of a ladder. These connections are stable and of high affinity - phagosomes from infected macrophages remain connected to the ER and mitochondria (as they were in situ) even after infected macrophages are homogenized. Thin sections through the plasma and phagosomal membranes show that the phagosomal membrane is thicker (72±2 Å) than the ER and mitochondrial membranes (60±2 Å), presumably owing to the lack of cholesterol, sphingolipids and glycolipids in the ER. Interestingly, within 15 minutes of infection, the phagosomal membrane changes thickness to resemble that of the attached ER vesicles. Only later (e.g. after six hours) does the ER-phagosome association become less frequent. Instead ribosomes stud the former phagosomal membrane and L. pneumophila reside directly in the rough ER. Examination of phagosomes of various L. pneumophila mutants suggests that this membrane conversion is a four-stage process used by L. pneumophila to establish itself in the RER and to survive intracellularly. But what is particularly interesting is that L. pneumophila is exploiting a poorly characterized naturally occuring cellular process.
The effect of inhalational anaesthetics on the swimming velocity of Tetrahymena pyriformis
Authors: Nunn J. F. , Sturrock Jean E. , J-Wills E. , Richmond Joan E. , Mcpherson C. K.
Published: April 2021

The effect of 6 inhalational anaesthetics on speed of swimming (produced by ciliary movement) has been studied in Tetrahymena pyriformis. There was no evidence of stimulation at low dose levels and higher levels caused rapid, reversible, dose-dependent reduction in swimming velocity. The concentrations of anaesthetics which depressed motility by 50 % were of the same order as those required for anaesthesia in mammals, except in the case of cyclopropane, for which the required level was 4 times higher than the anaesthetic level. Correlation with lipid solubility was not as close as is the case for narcotic concentration. Oxygen consumption was reduced with increasing amounts of halothane in parallel with the reduction in swimming velocity. Halothane produced deciliation of Tetrahymena at about 10 times the anaesthetic dose for man; regrowth of cilia took place within 4 h of withdrawal of the drug. There were no changes in the ultrastructure of the cilia, basal bodies and associated microtubular systems at levels of halothane sufficient to stop cilial beat. At higher concentrations deciliation occurred immediately distal to the axosome and there was variable swelling of the mitochondria.
Erythrophagocytosis by the epithelial cells of the bladder
Authors: Wakefield J. ST J. , Hicks R. M.
Published: April 2021

N-methyl-W-nitrosourea (MNU), methyl methane sulphonate (MMS), ethyl methane sulphonate (EMS) and cyclophosphamide (CP) are all cytotoxic to the transitional epithelium lining the urinary bladder, and also cause haemorrhage from the subepithelial blood capillaries. Erythrocytes enter the epithelium and are phagocytosed by the epithelial cells where they are subsequently digested. Two other compounds, dibutyl nitrosamine (DBN) and N-(4-(5-nitro-2-furyl)-2-thiazolyl)-formamide (NFTF) are only mildly cytotoxic but produce bladder epithelial tumours. Where haemorrhage occurs in these tumours, the erythrocytes are phago-cytosed and digested by the neoplastic epithelial cells.

The ingested red cells appear to lie free in the epithelial cell cytoplasm, surrounded by only a single unit membrane. Densitometrie tracings, however, show that this membrane is derived from the epithelial cells and that the red cells lie within a phagocytic vacuole. Careful inspection reveals the red cell membrane within the dense content of the vacuole, indicating that the permeability of the red cell membrane changes rapidly as soon as it is engulfed, so that haemoglobin is released into the phagocytic vacuole. The contents of the vacuole disintegrate and myelin figures and small amounts of residual dense material appear, but at no time was it possible to demonstrate acid phosphatase activity associated with the vacuole.
Descriptive and quantitative study of ultrastructural changes in the apical meristem of mustard in transition to flowering: I. the cell and nucleus
Authors: Havelange A. , Bernier G.
Published: April 2021

Transition to flowering was induced in the shoot apical meristem of Sinapis alba (mustard), a long-day species, by subjecting vegetative plants to a single 22-h long day. The ultrastructural changes occurring in the meristematic cells during the complete morphogenetic switch were quantitatively investigated using both the planimetric and the point-counting stereological methods. The 2 methods yield very similar results and are thus equally appropriate for the study.

The cell, nucleus, and dispersed chromatin sizes are greater in meristems of plants induced to flower (evoked meristems) than in meristems of control vegetative plants. A first size maximum is reached at 26 h after the start of the inductive long day and a second at 54 h. These 2 maxima occur just prior to 2 mitotic waves culminating respectively at 26–30 and 62 h.

Neither the condensed chromatin size nor the number of chromocentre profiles per nucleus section change. Consequently the dispersed chromatin: condensed chromatin ratio increases in evoked meristems. This change is discussed in relation to current views on the differential genetic activity of the 2 kinds of chromatin.

There is a late but large increase in size of the nucleolus. Dramatic changes in texture are associated with the enlargement of this organelle. These changes are the disappearance of the segregation of fibrillar and granular components and a loss of compactness due to marked vacuolation. All these changes in size and structure are interpreted as indicating a late increase in nucleolar synthesis of ribosomes in the evoked meristems.
Avian sarcoma virus transformed hamster cells made resistant to ethidium bromide
Authors: Altaner C. , Matoska F.
Published: April 2021

Hamster cells transformed with the Schmidt-Ruppin strain of avian sarcoma virus were selected for resistance to ethidium bromide (EB). The resistant cell lines proliferated in the presence of up to 30 μg/ml EB.

From avian sarcoma virus-transformed hamster cells already resistant to bromodeoxyuridinu (BrdU), ethidium bromide-resistant cells which were able to grow in 10μg/ml EB were also prepared. These cells remain deficient in thymidine kinase activity and are suitable for selective preparation of hybrid cells.

The EB resistance was genetically stable. The EB-resistant cell lines, and doubly resistant cells (BrdU, EB) showed no differences in mitochondrial ultrastructure compared with the original cell lines. Thymidine incorporation into mitochondrial DNA was not influenced by EB resistance.

All resistant cell lines, including the doubly resistant cell line, contained the avian sarcoma virus genome. The number of cells needed for positive rescue experiments for avian sarcoma virus genome by cell fusion with permissive chicken embryo cells was the same as with the original cell lines. The single EB-resistant cell lines contained R-type virus-like particles, while in BrdU-rcsistant and doubly resistant cells the R-type particles were absent.

The possible nature of EB resistance is discussed.