bluuebunny/crossref_metadata_2025 huggingface dataset:
Menadione-induced apoptosis: roles of cytosolic Ca2+elevations and the mitochondrial permeability transition pore
Authors: Gerasimenko Julia V. , Gerasimenko Oleg V. , Palejwala Altaf , Tepikin Alexei V. , Petersen Ole H. , Watson Alastair J. M.
Published: April 2021

In normal pancreatic acinar cells, the oxidant menadione evokes repetitive cytosolic Ca2+ spikes, partial mitochondrial depolarisation,cytochrome c release and apoptosis. The physiological agonists acetylcholine and cholecystokinin also evoke cytosolic Ca2+ spikes but do not depolarise mitochondria and fail to induce apoptosis. Ca2+ spikes induced by low agonist concentrations are confined to the apical secretory pole of the cell by the buffering action of perigranular mitochondria. Menadione prevents mitochondrial Ca2+ uptake, which permits rapid spread of Ca2+ throughout the cell. Menadione-induced mitochondrial depolarisation is due to induction of the permeability transition pore. Blockade of the permeability transition pore with bongkrekic acid prevents activation of caspase 9 and 3. In contrast, the combination of antimycin A and acetylcholine does not cause apoptosis but elicits a global cytosolic Ca2+ rise and mitochondrial depolarisation without induction of the permeability transition pore. Increasing the cytosolic Ca2+buffering power by BAPTA prevents cytosolic Ca2+ spiking, blocks the menadione-elicited mitochondrial depolarisation and blocks menadione-induced apoptosis. These results suggest a twin-track model in which both intracellular release of Ca2+ and induction of the permeability transition pore are required for initiation of apoptosis.
Integrin-mediated functional polarization of Caco-2 cells through E-cadherin—actin complexes
Authors: Schreider Cyrille , Peignon Gregory , Thenet Sophie , Chambaz Jean , Pinçon-Raymond Martine
Published: April 2021

Enterocyte differentiation is a dynamic process during which reinforcement of cell-cell adhesion favours migration along the crypt-to-villus axis. Functional polarization of Caco-2 cells, the most commonly used model to study intestinal differentiation, is assessed by dome formation and tightness of the monolayer and is under the control of the extracellular matrix (ECM). Furthermore, our biochemical and confocal microscopy data demonstrate that the ECM dramatically reinforces E-cadherin targeting to the upper lateral membrane, formation of the apical actin cytoskeleton and its colocalization with E-cadherin in functional complexes. In our model, these effects were produced by native laminin-5-enriched ECM as well as by type IV collagen or laminin 2, which suggests a common pathway of induction through integrin receptors. Indeed, these effects were antagonized by blocking anti-β1-and anti-α6-integrin antibodies and directly induced by a stimulating anti-β1-integrin antibody. These results demonstrate that integrin-dependent cell to ECM adhesion reinforces E-cadherin-dependent cell-cell adhesion in Caco-2 cells and further support the notion that enterocyte differentiation is supported by a molecular crosstalk between the two adhesion systems of the cell.
The incorporation of fibrinogen into extracellular matrix is dependent on active assembly of a fibronectin matrix
Authors: Pereira Marian , Rybarczyk Brain J. , Odrljin Tatjana M. , Hocking Denise C. , Sottile Jane , Simpson-Haidaris Patricia J.
Published: April 2021

Fibrinogen is a soluble protein produced by hepatocytes and secreted into plasma, where it functions in hemostasis. During inflammation, the hepatic synthesis of fibrinogen is induced 2-10 fold. Recent studies demonstrate that after an inflammatory stimulus, fibrinogen gene expression and protein production is upregulated in lung epithelial cells, where it is secreted basolaterally and consequently deposited into the extracellular matrix in fibrils that extensively colocalize with fibronectin fibrils. In this study, we show that the deposition of fibrinogen into the matrix of fibroblasts occurred rapidly and in a Rho-dependent manner in response to serum or lysophosphatidic acid; RhoA GTPase signaling is also required for fibronectin matrix assembly. Using mouse embryonic fibronectin-null cells, we show that incorporation of exogenous fibrinogen into matrix fibrils occurred only in the presence of exogenous fibronectin, which is also assembled into matrix fibrils. Furthermore, treatment of fibroblasts and fibronectin-null cells with an antibody that inhibits fibronectin matrix assembly impaired incorporation of fibrinogen into matrix fibrils. Collectively, these data suggest that incorporation of fibrinogen into the extracellular matrix requires active fibronectin polymer elongation into matrix fibrils. From these data, we hypothesize that fibrinogen deposition rapidly changes the topology of the extracellular matrix to provide a surface for cell migration and matrix remodeling during tissue repair.
Role of cytoplasmic C-terminal amino acids of membrane proteins in ER export
Authors: Nufer Oliver , Guldbrandsen Svend , Degen Martin , Kappeler Felix , Paccaud Jean-Pierre , Tani Katsuko , Hauri Hans-Peter
Published: April 2021

Export of membrane proteins from the ER is believed to be selective and require transport signals, but the identity of such signals has remained elusive. The recycling type I membrane protein ERGIC-53 carries a C-terminal diphenylalanine motif that is required for efficient ER export. Here we show that this motif can be functionally substituted by a single phenylalanine or tyrosine at position -2, two leucines or isoleucines at position -1 and -2 or a single valine at position -1. These motifs are common among mammalian type I membrane proteins. A single C-terminal valine, but none of the other motifs,accelerates transport of inefficiently exported reporter constructs and hence operates as an export signal. The valine signal is position, but not context,dependent. All transport motifs mediate COPII binding in vitro with distinct preferences for the COPII subunits Sec23p, Sec24Bp, Sec24Cp and p125. These results suggest that cytoplasmic C-terminal amino-acid motifs, either alone or in conjunction with other transport determinants, accelerate ER export of numerous type I and probably polytopic membrane proteins by mediating interaction with COPII coat components.
Biological roles and mechanistic actions of co-repressor complexes
Authors: Jepsen Kristen , Rosenfeld Michael G.
Published: April 2021

Transcriptional repression, which plays a crucial role in diverse biological processes, is mediated in part by non-DNA-binding co-repressors. The closely related co-repressor proteins N-CoR and SMRT, although originally identified on the basis of their ability to associate with and confer transcriptional repression through nuclear receptors, have been shown to be recruited to many classes of transcription factor and are in fact components of multiple protein complexes containing histone deacetylase proteins. This association with histone deacetylase activity provides an important component of the mechanism that allows DNA-binding proteins interacting with N-CoR or SMRT to repress transcription of specific target genes. Both N-CoR and SMRT are important targets for cell signaling pathways, which influence their expression levels, subcellular localization and association with other proteins. Recently, the biological importance of these proteins has been revealed by studies of genetically engineered mice and human diseases such as acute promyelocytic leukemia (APL) and resistance to thyroid hormone(RTH).
N-WASP activation by a β1-integrin-dependent mechanism supports PI3K-independent chemotaxis stimulated by urokinase-type plasminogen activator
Authors: Sturge Justin , Hamelin Jocelyne , Jones Gareth E.
Published: April 2021

Urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR) and epidermal growth factor (EGF)-EGF receptor (EGFR) expression is highly correlated with breast cancer metastasis. Phosphoinositide 3-kinase (PI3K),small Rho GTPases, such as Cdc42 and Rac1, and neuronal Wiskott Aldrich syndrome protein (N-WASP) are key effectors that regulate dynamic changes in the actin cytoskeleton and cell migration. uPA- and EGF-stimulated chemotaxis,cytoskeletal rearrangements and activation of Cdc42, Rac1 and N-WASP were studied in the highly metastatic human breast cancer cell line MDA MB 231. These studies reveal that divergent signalling occurs downstream of PI3K. The activity of PI3K was not necessary for uPA-induced chemotactic responses, but those induced by EGF were entirely dependent upon PI3K. Furthermore,PI3K-independent chemotactic signalling by uPA was shown to involve disruption of an interaction between β1-integrins and N-WASP and translocation of N-WASP to the actin cytoskeleton.
Vimentin affects localization and activity of sodium-glucose cotransporter SGLT1 in membrane rafts
Authors: Runembert Isabelle , Queffeulou Guillaume , Federici Pierre , Vrtovsnik François , Colucci-Guyon Emma , Babinet Charles , Briand Pascale , Trugnan Germain , Friedlander Gérard , Terzi Fabiola
Published: April 2021

It has been reported that vimentin, a cytoskeleton filament that is expressed only in mesenchymal cells after birth, is re-expressed in epithelial cells in vivo under pathological conditions and in vitro in primary culture. Whether vimentin re-expression is only a marker of cellular dedifferentiation or is instrumental in the maintenance of cell structure and/or function is a matter of debate. To address this issue, we used renal proximal tubular cells in primary culture from vimentin-null mice (Vim-/-) and from wild-type littermates (Vim+/+). The absence of vimentin did not affect cell morphology, proliferation and activity of hydrolases, but dramatically decreased Na-glucose cotransport activity. This phenotype was associated with a specific reduction of SGLT1 protein in the detergent-resistant membrane microdomains (DRM). In Vim+/+cells, disruption of these microdomains by methyl-β-cyclodextrin decreased SGLT1 protein abundance in DRM, a change that was paralleled by a decrease of Na-glucose transport activity. Importantly, we showed that vimentin is located to DRM, but it disappeared after methyl-β-cyclodextrin treatment. In Vim-/- cells,supplementation of cholesterol with cholesterol-methyl-β-cyclodextrin complexes completely restored Na-glucose transport activity. Interestingly,neither cholesterol content nor cholesterol metabolism changed in Vim-/- cells. Our results are consistent with the view that re-expression of vimentin in epithelial cells could be instrumental to maintain the physical state of rafts and, thus, the function of DRM-associated proteins.
Bone morphogenetic proteins promote development of fetal pancreas epithelial colonies containing insulin-positive cells
Authors: Jiang Fang-Xu , Stanley Edouard G. , Gonez L. Jorge , Harrison Leonard C.
Published: April 2021

Extracellular signals that guide pancreas cell development are not well characterized. In an in vitro culture system of dissociated pancreas cells from the E15.5 mouse fetus we show that, in the presence of the extracellular matrix protein laminin-1, bone morphogenetic proteins (BMPs-4, -5 and -6)promote the development of cystic epithelial colonies. Transforming growth factor β1 (TGF-β1) and activin A antagonise this effect of BMP-6 and inhibit colony formation. Histological analysis revealed that the colonies are composed of E-cadherin-positive epithelial cells, which in localised areas are insulin positive. The colonies also contain occasional glucagon-positive cells, but no somatostatin- or α-amylase-positive cells. These findings indicate that members of the TGF-β superfamily regulate pancreas epithelial cell development and can promote the formation of islet-like structures in vitro.
Identification and characterization of a novel human plant pathogenesis-related protein that localizes to lipid-enriched microdomains in the Golgi complex
Authors: Eberle Heike B. , Serrano Ramon L. , Füllekrug Joachim , Schlosser Andreas , Lehmann Wolf D. , Lottspeich Friedrich , Kaloyanova Dora , Wieland Felix T. , Helms J. Bernd
Published: April 2021

Group 1 of plant pathogenesis-related proteins (PR-1) and a variety of related mammalian proteins constitute a superfamily of proteins that share structural similarities. Little is known about their function, but all the family members identified to date are co-translationally translocated to the lumen of the endoplasmic reticulum and are secreted as soluble proteins or are targeted to vacuoles. Here we report the identification of a novel family member that localizes to the cytosolic site of the endomembrane system in mammalian cells. After detergent solubilization of isolated Golgi membranes, a 17 kDa protein was found associated with a low-density detergent-insoluble fraction. The amino-acid sequence, determined by microsequencing and molecular cloning, revealed a significant homology with the superfamily of PR-1 proteins. Golgi-associated PR-1 protein (GAPR-1) showed a brefeldin-A-sensitive Golgi localization in immunofluorescence. Interestingly,the protein remained associated with the microdomain fraction in the presence of Brefeldin A.

By mass spectrometry, GAPR-1 was shown to be myristoylated. Immunoprecipitation of GAPR- 1 from Golgi membranes resulted in the coimmunoprecipitation of caveolin-1, indicating a direct interaction between these two proteins. Myristoylation, together with protein-protein or electrostatic interactions at physiological pH owing to the highly basic pI of GAPR-1 (pI 9.4) could explain the strong membrane association of GAPR-1.

Tissue screening revealed that GAPR-1 is not detectably expressed in liver,heart or adrenal glands. High expression was found in monocytes, leukocytes,lung, spleen and embryonic tissue. Consistent with the involvement of PR-1 proteins in the plant immune system, these data could indicate that GAPR-1 is involved in the immune system.
Disruption of microtubules uncouples budding and nuclear division inToxoplasma gondii
Authors: Morrissette Naomi S. , Sibley L. David
Published: April 2021

The tachyzoite stage of the protozoan parasite Toxoplasma gondiihas two populations of microtubules: spindle microtubules and subpellicular microtubules. To determine how these two microtubule populations are regulated, we investigated microtubule behavior during the cell cycle following treatment with microtubule-disrupting drugs. Previous work had established that the microtubule populations are individually nucleated by two distinct microtubule-organizing centers (MTOCs): the apical polar ring for the subpellicular microtubules and spindle pole plaques/centrioles for the spindle microtubules. When replicating tachyzoites were treated with 0.5 μM oryzalin or 1.0 mM colchicine they retained the capacity to form a spindle and undergo nuclear division. Although these parasites could complete budding,they lost the bulk of their subpellicular microtubules and the ability to reinvade host cells. Both nascent spindle and subpellicular microtubules were disrupted in 2.5 μM oryzalin or 5.0 mM colchicine. Under these conditions,parasites grew in size and replicated their genome but were incapable of nuclear division. After removal from 0.5 μM oryzalin, Toxoplasmatachyzoites were able to restore normal subpellicular microtubules and a fully invasive phenotype. When oryzalin was removed from Toxoplasmatachyzoites treated with 2.5 μM drug, the parasites attempted to bud as crescent-shaped tachyzoites. Because the polyploid nuclear mass could not be correctly segregated, many daughter parasites lacked nuclei altogether although budding and scission from the maternal mass was able to be completed. Multiple MTOCs permit Toxoplasma tachyzoites to control nuclear division independently from cell polarity and cytokinesis. This unusual situation grants greater cell cycle flexibility to these parasites but abolishes the checks for coregulation of nuclear division and cytokinesis found in other eukaryotes.
Repairing a torn cell surface: make way, lysosomes to the rescue
Authors: McNeil Paul L.
Published: April 2021

Biological membranes are often described as `self-sealing' structures. If indeed membranes do have an inherent capacity for repair, does this explain how a cell can rapidly reseal a very large (1-1000 μm2)disruption in its plasma membrane? It is becoming increasingly clear that, in nucleated animal cells, the cytoplasm plays an active and essential role in resealing. A rapid and apparently chaotic membrane fusion response is initiated locally in the cytoplasm by the Ca2+ that floods in through a disruption: cytoplasmic vesicles are thereby joined with one another(homotypically) and with the surrounding plasma membrane (exocytotically). As a consequence, internal membrane is added to cell surface membrane at the disruption site. In the case of large disruptions, this addition is hypothesized to function as a `patch'. In sea urchin eggs, the internal compartment used is the yolk granule. Several recent studies have significantly advanced our understanding of how cells survive disruption-inducing injuries. In fibroblasts, the lysosome has been identified as a key organelle in resealing. Protein markers of the lysosome membrane appear on the surface of fibroblasts at sites of disruption. Antibodies against lysosome-specific proteins, introduced into the living fibroblast,inhibit its resealing response. In gastric eptithelial cells, local depolymerization of filamentous actin has been identified as a crucial step in resealing: it may function to remove a barrier to lysosome-plasma membrane contact leading to exocytotic fusion. Plasma membrane disruption in epithelial cells induces depolymerization of cortical filamentous actin and, if this depolymerization response is inhibited, resealing is blocked. In the Xenopus egg, the cortical cytoskeleton has been identified as an active participant in post-resealing repair of disruption-related damage to underlying cell cortex. A striking, highly localized actin polymerization response is observable around the margin of cortical defects. A myosin powered contraction occurring within this newly formed zone of F-actin then drives closure of the defect in a purse-string fashion.
F-actin ring formation and the role of F-actin cables in the fission yeastSchizosaccharomyces pombe
Authors: Arai Ritsuko , Mabuchi Issei
Published: April 2021

Cells of the fission yeast Schizosaccharomyces pombe divide by the contraction of the F-actin ring formed at the medial region of the cell. We investigated the process of F-actin ring formation in detail using optical sectioning and three-dimensional reconstruction fluorescence microscopy. In wild-type cells, formation of an aster-like structure composed of F-actin cables and accumulation of F-actin cables were recognized at the medial cortex of the cell during prophase to metaphase. The formation of the aster-like structure seemed to initiate from branching of the longitudinal F-actin cables at a site near the spindle pole bodies, which had been duplicated but not yet separated. A single cable extended from the aster and encircled the cell at the equator to form a primary F-actin ring during metaphase. During anaphase,the accumulated F-actin cables were linked to the primary F-actin ring, and then all of these structures seemed to be packed to form the F-actin ring. These observations suggest that formation of the aster-like structure and the accumulation of the F-actin cables at the medial region of the cell during metaphase may be required to initiate the F-actin ring formation. In the nda3 mutant, which has a mutation in ß-tubulin and has been thought to be arrested at prophase, an F-actin ring with accumulated F-actin cables similar to that of anaphase wild-type cells was formed at a restrictive temperature. Immediately after shifting to a permissive temperature, this structure changed into a tightly packed ring. This suggests that the F-actin ring formation progresses beyond prophase in the nda3 cells once the cells enter prophase. We further examined F-actin structures in both cdc12 and cdc15 early cytokinesis mutants. As a result,Cdc12 seemed to be required for the primary F-actin ring formation during prophase, whereas Cdc15 may be involved in both packing the F-actin cables to form the F-actin ring and rearrangement of the F-actin after anaphase. In spg1, cdc7 and sid2 septum initiation mutants, the F-actin ring seemed to be formed in order.
Perinuclear localization of huntingtin as a consequence of its binding to microtubules through an interaction with β-tubulin: relevance to Huntington's disease
Authors: Hoffner Guylaine , Kahlem Pascal , Djian Philippe
Published: April 2021

Huntington's disease results from an expansion of a series of glutamine repeats in the protein huntingtin. We have discovered from immunopurification studies that huntingtin combines specifically with the β subunit of tubulin. This binding explains why huntingtin can be shown on assembled microtubules by electron microscopy. Immunostaining shows that most of the huntingtin in the cytoplasm is associated with microtubules. Huntingtin is particularly abundant in the perinuclear region, where it is also associated with microtubules and in the centrosomal region, where it co-localizes withγ-tubulin. In Huntington's disease, inclusions are often nuclear or perinuclear. Since the perinuclear concentration of huntingtin does not depend on the number of its glutamine repeats, we propose that inclusions are found in perinuclear and intranuclear locations because the β-tubulin binding property of huntingtin brings it to the perinuclear region, from which it readily gains access to the nucleus. The mutational glutamine expansion then promotes insolubility and results in an inclusion.
Phenotypic characterization ofDrosophila idamutants: defining the role of APC5 in cell cycle progression
Authors: Bentley A. M. , Williams Byron C. , Goldberg Michael L. , Andres Andrew J.
Published: April 2021

We have cloned and characterized the ida gene that is required for proliferation of imaginal disc cells during Drosophila development. IDA is homologous to APC5, a subunit of the anaphase-promoting complex(APC/cyclosome). ida mRNA is detected in most cell types throughout development, but it accumulates to its highest levels during early embryogenesis. A maternal component of IDA is required for the production of eggs and viable embryos. Homozygous ida mutants display mitotic defects: they die during prepupal development, lack all mature imaginal disc structures, and have abnormally small optic lobes. Cytological observations show that ida mutant brains have a high mitotic index and many imaginal cells contain an aneuploid number of aberrant overcondensed chromosomes. However, cells are not stalled in metaphase, as mitotic stages in which chromosomes are orientated at the equatorial plate are never observed. Interestingly, some APC/C-target substrates such as cyclin B are not degraded in ida mutants, whereas others controlling sister-chromatid separation appear to be turned over. Taken together, these results suggest a model in which IDA/APC5 controls regulatory subfunctions of the anaphase-promoting complex.
The involvement of lipid rafts in the regulation of integrin function
Authors: Leitinger Birgit , Hogg Nancy
Published: April 2021

Integrin activity on cells such as T lymphocytes is tightly controlled. Here we demonstrate a key role for lipid rafts in regulating integrin function. Without stimulation integrin LFA-1 is excluded from lipid rafts, but following activation LFA-1 is mobilised to the lipid raft compartment. An LFA-1 construct from which the I domain has been deleted mimics activated integrin and is constitutively found in lipid rafts. This correlation between integrin activation and raft localisation extends to a second integrin,α4β1, and the clustering of α4β1 is also raft dependent. Both LFA-1 and α4β1-mediated adhesion is dependent upon intact lipid rafts providing proof of the functional relevance of the lipid raft localisation. Finally we find that non-raft integrins are excluded from the rafts by cytoskeletal constraints. The presence of integrin in lipid rafts under stimulating conditions that activate these receptors strongly indicates that the rafts have a key role in positively regulating integrin activity.
The CDK inhibitor NtKIS1a is involved in plant development,endoreduplication and restores normal development of cyclin D3;1-overexpressing plants
Authors: Jasinski Sophie , Riou-Khamlichi Catherine , Roche Odile , Perennes Claudette , Bergounioux Catherine , Glab Nathalie
Published: April 2021

Plant development requires stringent controls between cell proliferation and cell differentiation. Proliferation is positively regulated by cyclin dependent kinases (CDKs). Acting in opposition to CDKs are CDK inhibitors(CKIs). The first tobacco CKI (NtKIS1a) identified was shown to inhibit in vitro the kinase activity of CDK/cyclin complexes and to interact with CDK and D-cyclins. However, these features, which are common to other plant and animal CKIs already characterised, did not provide information about the function of NtKIS1a in plants. Thus, to gain insight into the role of NtKIS1a and especially its involvement in cell proliferation during plant development, we generated transgenic Arabidopsis thaliana plants that overexpress NtKIS1a. These plants showed reduced growth with smaller organs that contained larger cells. Moreover, these plants displayed modifications in plant morphology. These results demonstrated that plant organ size and shape,as well as organ cell number and cell size, might be controlled by modulation of the single NtKIS1a gene activity. Since in mammals, D-cyclins control cell cycle progression in a CDK-dependent manner but also play a CDK independent role by sequestering the CKIs p27Kip1 and p21Cip1, we tested the significance of cyclin D-CKI interaction within a living plant. With this aim, NtKIS1a and AtCycD3;1 were overexpressed simultaneously in plants by two different methods. Our results demonstrated that overexpression of the CKI NtKIS1a restores essentially normal development in plants overexpressing AtCycD3;1, providing the first evidence of cyclin D-CKI co-operation within the context of a living plant.
Human myosin-Vc is a novel class V myosin expressed in epithelial cells
Authors: Rodriguez Olga C. , Cheney Richard E.
Published: April 2021

Class V myosins are one of the most ancient and widely distributed groups of the myosin superfamily and are hypothesized to function as motors for actin-dependent organelle transport. We report the discovery and initial characterization of a novel member of this family, human myosin-Vc (Myo5c). The Myo5c protein sequence shares ∼50% overall identity with the two other class V myosins in vertebrates, myosin-Va (Myo5a) and myosin-Vb (Myo5b). Systematic analysis of the mRNA and protein distribution of these myosins indicates that Myo5a is most abundant in brain, whereas Myo5b and Myo5c are expressed chiefly in non-neuronal tissues. Myo5c is particularly abundant in epithelial and glandular tissues including pancreas, prostate, mammary,stomach, colon and lung. Immunolocalization in colon and exocrine pancreas indicates that Myo5c is expressed chiefly in epithelial cells. A dominant negative approach using a GFP-Myo5c tail construct in HeLa cells reveals that the Myo5c tail selectively colocalizes with and perturbs a membrane compartment containing the transferrin receptor and rab8. Transferrin also accumulates in this compartment, suggesting that Myo5c is involved in transferrin trafficking. As a class V myosin of epithelial cells, Myo5c is likely to power actin-based membrane trafficking in many physiologically crucial tissues of the human body.
GIT1 functions in a motile, multi-molecular signaling complex that regulates protrusive activity and cell migration
Authors: Manabe Ri-ichiroh , Kovalenko Mykola , Webb Donna J. , Horwitz Alan Rick
Published: April 2021

GIT1 is a multidomain protein that is thought to function as an integrator of signaling pathways controlling vesicle trafficking, adhesion and cytoskeletal organization. It regulates ARF GTPases and has binding domains for paxillin and PIX, which is a PAK-binding protein and an exchange factor for Rac. We show that GIT1 cycles between at least three distinct subcellular compartments, including adhesion-like structures, the leading edge and cytoplasmic complexes. The cytoplasmic structures, which also contain paxillin, PAK and PIX, do not detectably co-localize with endosomal Golgi or membrane markers, suggesting that they represent a novel supramolecular complex. The GIT1 cytoplasmic complexes are motile and tended to move toward the cell periphery where they joined existing adhesions. In retracting regions of the cells, the GIT1 complexes moved away from the disassembling adhesions toward the cell body. Using deletion mutants, we have identified domains that target GIT1 to each of the compartments. Localization to adhesions and the leading edge requires the paxillin-binding domain, which comprises the C-terminal 140 residues (cGIT1), whereas targeting to the cytoplasmic complexes requires the central region that contains ankyrin repeats and the PIX-binding domain. Expression of GIT1 or cGIT, but not nGIT1 in which the paxillin-binding domain is deleted, increases the rate of migration and the size and number of protrusions. The latter are inhibited when GIT1 is co-expressed with a kinase-dead PAK, suggesting that the GIT1 interaction with PAK is required for enhanced migration and protrusive activity. Furthermore,GIT1 targets constitutively activated PAK to adhesions and the leading edge via its interaction with paxillin. Since expression of cGIT targets endogenous GIT1 to the leading edge, it appears that the leading edge is the location of GIT1 responsible for these activities. Thus, GIT1 is a component of a motile,multimolecular complex that traffics a set of signaling components to specific locations in the cell where they regulate localized activities.
DNA ligase I null mouse cells show normal DNA repair activity but altered DNA replication and reduced genome stability
Authors: Bentley Darren J. , Harrison Caroline , Ketchen Ann-Marie , Redhead Nicola J. , Samuel Kay , Waterfall Martin , Ansell John D. , Melton David W.
Published: April 2021

DNA ligase I is the key ligase for DNA replication in mammalian cells and has also been reported to be involved in a number of recombination and repair processes. Our previous finding that Lig1 knockout mouse embryos developed normally to mid-term before succumbing to a specific haematopoietic defect was difficult to reconcile with a report that DNA ligase I is essential for the viability of cultured mammalian cells. To address this issue, we generated a second Lig1 targeted allele and found that the phenotypes of our two Lig1 mutant mouse lines are identical. Widely different levels of Lig1 fusion transcripts were detected from the two targeted alleles, but we could not detect any DNA ligase I protein, and we believe both are effective Lig1 null alleles. Using foetal liver cells to repopulate the haematopoietic system of lethally irradiated adult mice, we demonstrate that the haematopoietic defect in DNA-ligase-I-deficient embryos is a quantitative deficiency relating to reduced proliferation rather than a qualitative block in any haematopoietic lineage. DNA ligase I null fibroblasts from Lig1 mutant embryos showed an accumulation of DNA replication intermediates and increased genome instability. In the absence of a demonstrable deficiency in DNA repair we postulate that, unusually, genome instability may result directly from the DNA replication defect. Lig1null mouse cells performed better in the survival and replication assays than a human LIG1 point mutant, and we suggest that the complete absence of DNA ligase I may make it easier for another ligase to compensate for DNA ligase I deficiency.
Dissecting interactions between EB1, microtubules and APC in cortical clusters at the plasma membrane
Authors: Barth Angela I. M. , Siemers Kathleen A. , Nelson W. James
Published: April 2021

End-binding protein (EB) 1 binds to the C-terminus of adenomatous polyposis coli (APC) protein and to the plus ends of microtubules (MT) and has been implicated in the regulation of APC accumulation in cortical clusters at the tip of extending membranes. We investigated which APC domains are involved in cluster localization and whether binding to EB1 or MTs is essential for APC cluster localization. Armadillo repeats of APC that lack EB1- and MT-binding domains are necessary and sufficient for APC localization in cortical clusters; an APC fragment lacking the armadillo repeats, but containing MT-and EB1-binding domains, does not localize to the cortical clusters but instead co-aligns with MTs throughout the cell. Significantly, analysis of endogenous proteins reveals that EB1 does not accumulate in the APC clusters. However, overexpressed EB1 does accumulate in APC clusters; the APC-binding domain in EB1 is located in the C-terminal region of EB1 between amino acids 134 and 268. Overexpressed APC- or MT-binding domains of EB1 localize to APC cortical clusters and MT, respectively, without affecting APC cluster formation itself. These results show that localization of APC in cortical clusters is different from that of EB1 at MT plus ends and appears to be independent of EB1.
An ultrastructural and radioautographic investigation of the nucleolonemal component of plant interphase nucleoli
Authors: Lafontaine J.-G. , Lord A.
Published: April 2021

The interphase nucleolus in Allhim porrum meristematic cells is characterized by the presence of 1–4 dense fibrillar zones of rather complex organization. Each such zone appears to consist essentially of a convoluted, evacuolated, filamentous structure approximately 1·5 μm in diameter. At the ultrastructural level, these structures exhibit an intricate array of lacunar spaces each of which is surrounded by a dense coating. These lacunae are filled with a loose fibrillar material and the largest ones sometimes also show a dense central core. In appropriate preparations, certain of the peripherally located lacunae are found to be continuous with segments of chromosomes. High-resolution radioautography reveals, moreover, that DNA is present within both the dense and lighter portions of the nucleolar loops. These observations add further support to the hypothesis that the convoluted filamentous structures in question correspond to loops of chromosomal origin and are thus related to the nucleolar organizer.
Ultrastructural aspects of the self-incompatibility mechanism in Lycopersicum Peruvianum mill
Authors: Nettancourt D. De , Devreux M. , Bozzini A. , Cresti M. , Pacini E. , Sarfatti G.
Published: April 2021

The experimental results obtained show that the tip of the incompatible pollen tube bursts open after the outer-wall has considerably expanded in the intercellular spaces of the conducting tissue and the inner-wall has disappeared and numerous particles have accumulated in the tube cytoplasm. These particles, which measure approximately 0·2 μm in diameter and give a weak reaction to the test of Thiéry, differ in many respects from the vesicles normally present in compatible pollen tubes growing through the style; they appear to resemble, in some cases, the spheres which are discharged by the compatible pollen tubes after they have reached the embryo-sac.

It is considered that these observations support the current belief that the tube wall is the site of action for the incompatibility proteins and suggest that self-incompatibility is not a passive process resulting from lack of growth stimulation but an active event which leads to the destruction of the incompatible pollen tubes. The degradation mechanism involved appears similar to the one which enables the compatible pollen tube to release its contents in the degenerated synergid and presents some analogies with the lytic process taking place in virus-infected cells.

The general hypothesis is presented that the particles observed in the cytoplasm of self-incompatible pollen tubes consist of a mixture of incompatibility proteins and of basic constituents of the tube wall.
Pollen wall proteins: Pollen-Stigma interactions in ragweed and Cosmos (compositae)
Authors: Knox R. B.
Published: April 2021

The release of wall-held materials from the pollen of ragweed (Ambrosia tenuifolia) and Cosmos bipimuxtus on to the stigma surface has been followed. When fresh stigmas were viewed by scanning electron microscopy, a fluid material was observed coating pollen grains, pollen tubes and adjacent stigmatic papillae. This fluid contained pollen-wall antigens, including the allergen Antigen E, detected by immunofluorescence, and proteins, lipids and carbohydrates detected by cytochemical methods.

In Cosmos, the fate of the antigens was much the same after both compatible and incompatible matings. In incompatible matings, pollen tubes were blocked with a polysaccharide considered to be callose. Callose particles appeared on neighbouring stigmatic papillae, and adjacent stigmatic hairs accumulated callose internally. This reaction may be an important one for the incompatibility response.
Complementary plasma membrane fracture faces in freeze-etch replicas
Authors: Flower N. E.
Published: April 2021

Recent studies have all shown that membranes fracture internally during freeze-etching or freeze-cleaving, confirming that 2 internal membrane faces are produced. That these faces are not complementary is one of the main features of freeze-etch replicas still requiring explanation. In the present investigation certain plasma membranes in light organs of the New Zealand glow worm are found to be an exception to this general rule of non-complementarity, as there is a sufficient number of holes present in one membrane face to match the number of particles on the other. This finding suggests that most of the theories put forward to explain non-complementarity do not apply and that holes are probably lost in most membrane faces by distortion during fracturing.
Erythropoiesis in the newt, Triturus Cristatus laur. : II. characteristics of the erythropoietic process
Authors: Grasso J. A.
Published: April 2021

In splenectomized newts (Triturus cristatus Laur.) rendered anaemic by acetylphenylhydrazine (APH), an erythropoietic response is delayed so that the animals are completely devoid of the erythron, including erythrocytes. At 11–14 days after APH, ‘erythroid precursor cells’ (EPC) in the blood signal the occurrence of an erythropoietic response. Ultrastructural studies have shown few or no ribosomes in EPCs, but well developed nucleoli and intense RNA synthesis are seen in these cells. Correlated morphological and cytochemical data indicate the production of ribosomes in EPCs, a process culminating in the formation of basophilic erythroblasts (BE). Microphotometric studies show the accumulation of haem during this interval. Thus, in EPCs and BEs, both ribosomal (rRNA) and messenger (m) RNA are synthesized, making possible the early synthesis of haemoglobin. In subsequent stages, nucleoli exhibit a size decrease, most evident in the particulate component, while all RNA synthesis ceases during the mid-poly chromatophilic erythroblast (MPE) stage. Coupled with the gradual loss of ribosomes characteristic of this developmental period, the results suggest that rRNA synthesis occurs in EPCs, BEs, and in early polychromatophilic erythroblasts, where it is completed. Haemoglobin mRNA is also formed in these stages since ultrastructural and microphotometric data show the accumulation of haemoglobin. Beyond the MPE, haemoglobin production is dependent upon stable messenger RNA since no RNA synthesis is detected in this period. Ultrastructural studies demonstrate that autophagy may play an important role in the loss of cytoplasmic organelles characteristic of the erythropoietic process. The occurrence of swollen cristae and outer compartment in mitochondria coupled with the presence of myelin-like membrane whorls in association with mitochondria is seen in all erythroid cells except EPCs. Membrane profiles derived from endoplasmic reticulum are frequently encountered in the vicinity of mitochondria and appear to encircle these organelles and adjacent cytoplasm to form autophagic vacuoles. In the process, the limiting membranes assume a dense laminar appearance. Elements of the Golgi complex also display morphological alterations suggestive of degeneration. Cytoplasmic bodies of varying appearance and content, some of which are similar to lysosomes in other cell types, are seen in all erythroid cells. Ferritin is seen in some bodies, in vacuoles, or in aggre gates, but does not appear in micropinocytotic vesicles.
The effect of vincristine sulphate on the axoplasmic flow of proteins in cultured sympathetic neurons
Authors: England J. M. , Kadin M. E. , Goldstein M. N.
Published: April 2021

The effect of vincristine sulphate on the axoplasmic flow of labelled proteins in neurites of chick embryo sympathetic neurons growing in tissue culture was studied by autoradiography. In control neurons most of the 3H-proteins synthesized during a 90-min pulse with a 3H-amino acid were localized in cell bodies. There was a diminishing gradient of labelled proteins in the neurites which was highest in portions adjacent to the cell bodies and lowest at the periphery. During a physiological chase there was a gradual increase in the amount of label in the neurites, so that after a 15-h chase even the most peripheral portions were well labelled. This indicates that a portion of the labelled proteins synthesized in the cell bodies are transported peripherally into the neurites.

The centrifugal movement of labelled proteins in neurites was markedly decreased when cells were grown in medium containing 10 μg/ml vincristine sulphate. After a 15-h chase in the presence of drug only a small amount of label was in the peripheral portion of the neurites. Treatment with vincristine did not decrease the rate of amino acid incorporation or alter the rate of protein turnover during the course of the experiment. Thus an explanation of the results based on an altered rate of total cell protein synthesis or degradation is unlikely. The capacity of sympathetic neurons to take up and concentrate exogenous [3H]norepinephrine in their neurites was only slightly reduced by vincristine. This indicates that at least some cellular activities requiring metabolic energy are relatively unaffected by the interruption in axoplasmic flow caused by vincristine and that the mechanism by which vincristine interferes with axoplasmic flow does not involve general cellular toxicity.

The major morphological differences between control and vincristine-treated neurons were the absence of microtubules and the presence of crystal-like structures within the cells. The relationship between the effect of vincristine on the axoplasmic flow of proteins and the arrangement of the microtubule system is discussed.
Intestinal goblet cell mucus: Isolation and identification by immuno-fluorescence of a goblet cell glycoprotein
Authors: Forstner J. , Taichman N. , Kalnins V. , Forstner G.
Published: April 2021

A high-molecular-weight glycoprotein (HMG), representing the majority of the soluble glycoprotein hexosamine and hexose in the intestine, was isolated by Sepharose 4B chromatography from high-speed supernatants of rat small intestinal homogenate. Fluorescein-labelled globulin, from rabbit antiserum produced against HMG, specifically stained supra-nuclear mucus vesicles of goblet cells in small intestine and colon as well as gastric pit cells in the body of the stomach and mucus-producing cells in the sublingual salivary gland. A single precipitin line was found when HMG was tested against its antibody by agar immunodiffusion and immunoelectrophoresis. No cross-reactivity was observed between antibody and rat serum or extracts from microvillus membrane, human colon and pig intestine. Precipitin lines which fused with the HMG precipitin arc in apparent identity were observed with antigens in intestinal lumen washings, and in small-molecular-weight fractions from intestinal cell sap. When studied by cellulose acetate electrophoresis, cetyl trimethylammonium bromide precipitation and precursor labelling with [1-14C]glucosamine, HMG behaved as a single homogeneous glycoprotein free of detectable protein contamination. These results imply that HMG is a major component of goblet-cell mucus in the small intestine, and suggest that it is similar to mucin produced throughout the gastrointestinal tract. HMG is the first glycoprotein, isolated without the aid of proteolytic agents, which has been specifically identified as a product of the goblet cell.
Association of the two glycosyl transferase activities of glycoprotein synthesis with low equilibrium density smooth microsomes
Authors: Wagner R. R. , Pettersson Elisabeth , Dallner G.
Published: April 2021

Smooth microsomes of rat liver were subfractionated on a continuous sucrose gradient to isopyenic equilibrium density. All the microsomal electron-transport enzymes and phosphatases exhibit a broad distribution in the various microsomal subtractions. On the other hand, the N-acetylglucosaminyl and galactosyl transferase activities, which are known to participate in the synthesis of the oligosaccharide chain of the plasma glycoproteins, exhibit a markedly different distribution pattern. They are concentrated to a small fraction having an isopyenic equilibrium density of 1·10. These membranes represent a separate entity of the smooth microsomal fraction, the exact cellular origin of which remains to be clarified.
Some effects of actinomycin D, cycloheximide and puromycin on cell adhesion
Authors: Weiss L. , Chang M. K.
Published: April 2021

The metabolic inhibitors cycloheximide, puromycin and actinomycin D, increased the rate of adhesion of Ehrlich ascites tumour cells to coverslips and plastic surfaces, over periods of up to 2 h in vitro. Among a number of possible explanations of this effect is a decreased production of hypothetical anti-adhesion factor(s). It is suggested that the difference between the increased rate of adhesion observed by us, and the decrease or lack of effect of the drugs observed by others, is related to prior trypsin-treatment of cells by the other observers.
A possible alteration in the permeability of ascites cell membranes after exposure to acoustic microstreaming
Authors: Williams A. R.
Published: April 2021

Single-cell suspensions of mouse Ehrlich ascites cells in isotonic saline were subjected to a small-scale acoustic microstreaming field generated around a transversely oscillating wire. Cells sheared at wire displacement amplitudes below the threshold for membrane rupture contained more fluid than their controls. Evidence is presented which suggests that a slight change has occurred in the permeability of the plasma membranes of the intact sheared cells.
Acetylcholinesterase in human erythroid cells
Authors: Skaer R. J.
Published: April 2021

Acetylcholinesterase is present in human red cells but cannot be demonstrated by the copper thiocholine test. The enzyme is revealed, however, in the perinuclear cisterna, endoplasmic reticulum and Golgi apparatus of red cell precursors. It is suggested that 2 forms of the enzyme are present, one of which can be demonstrated by the copper thiocholine test, the other cannot; one form may be the precursor of the other. These observations may cast light on the kinetics of red cell replacement and on the interpretation of the results from the copper thiocholine test on other tissues such as the nervous system.