bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of cytoplasmic and axonemal dyneins in rat tissues
Authors: Yoshida Toshimichi , Takanari Hideki , Izutsu Kosaku
Published: April 2021

Microtubule-associated protein IC (MAP 1C) is now defined as brain cytoplasmic dynein. Recent studies have suggested that cytoplasmic dynein is a motor protein responsible for the intracellular microtubule-based motility in neuronal and non-neuronal cells. We have prepared an antibody against bovine brain MAP 1C and have examined the localizations of cytoplasmic dynein in rat tissues. Immunoblots of extracts from the tissues showed that the dynein was present in brain, testis, liver, kidney and lung. Immunohistochemical experiments have demonstrated that dynein is localized in Purkinje cells of cerebellum and axons of central and peripheral nervous systems. In non-neuronal tissues, the antibody staining was intense in many types of cells, such as hepatocytes, epithelia of renal convoluted tubules, secretory cells of adrenal medulla and spermatids. Glomeruli of kidney, bronchial epithelia and type II pneumocytes of lung, pancreatic islets and acini, adrenal cortex and Sertoli cells were moderately positive upon exposure to the cytoplasmic dynein antibody. On the other hand, the localization of axonemal dynein was examined using antibodies against flagellar dynein of sea urchin spermatozoa. Anti-axonemal dynein labeled cilia and flagella in rat tissues whereas anti-MAP 1C did not stain axonemes. We also tested for immunological crossreactivity between cytoplasmic and axonemal dyneins to probe for molecular similarities. Anti-axonemal dynein reacted with MAP 1C weakly. These results have confirmed that cytoplasmic dyneins are distributed widely among rat organs, not only in neuronal but also in non-neuronal tissues. There is no similarity in the localization of cytoplasmic and axonemal dyneins but there is some similarity in molecular antigenicity.
Recognition of collagen by fibroblasts through cell surface glycoproteins reactive with Phaseolus vulgaris agglutinin
Authors: Asaga Hiroaki , Yoshizato Katsutoshi
Published: April 2021

The role of glycochains of cell surface glycoproteins in the cell to collagen interaction was examined by studying the effect of lectins on the fibroblast-mediated collagen gel contraction. Lectins of Phaseolus vulgaris agglutinin (PHA), concanavalin A (ConA), lentil seed agglutinin (LCA), pea agglutinin (PSA), Ricinus communis ag-glutinin-60 (RCA), and wheat germ agglutinin (WGA) dose-dependently inhibited gel contraction, while lectins of mushroom agglutinin (ABA), peanut agglutinin (PNA), pokeweed mitogen (PWM), and soybean agglutinin (SBA) did not. Of these lectins, PHA seemed to be worthy of further analysis, because PHA, but not other lectins, inhibited spreading of fibroblasts on collagen fibrils but not on plastic or gelatin, suggesting that cell-surface glycoproteins responsive to the lectin are involved in the specific binding of fibroblasts to native collagen fibrils. The inhibitory effect of PHA-E4, an isolectin of PHA, was more intense than that of PHA-L4, another isolectin of PHA. The collagen gel contraction was also inhibited by tunicamycin and monensin in a concentration-dependent and reversible manner. These results strongly suggest that PHA-E4-reactive glycoproteins of the fibroblast surface play an important role in cell to collagen binding during the gel contraction. Five membrane proteins including β1 subunits of the integrin family were obtained by affinity chromatography with PHA-E4.
The extended tubulin superfamily
Authors: McKean Paul G. , Vaughan Sue , Gull Keith
Published: April 2021

Although most eukaryotic cells can express multiple isotypes of αβ-tubulin, the significance of this diversity has not always been apparent. Recent data indicate that particular αβ-tubulin isotypes, both genome encoded and those derived by post-translational modification, can directly influence microtubule structure and function — thus validating ideas originally proposed in the multitubulin hypothesis over 25 years ago.

It has also become increasingly evident over the past year that some (but intriguingly not all) eukaryotes encode several other tubulin proteins, and to date five further members of the tubulin superfamily, γ, δ, ϵ, 𝛇 and η, have been identified. Although the role of γ-tubulin in the nucleation of microtubule assembly is now well established, far less is known about the functions of δ-, ϵ-, 𝛇- and η-tubulin. Recent work has expanded our knowledge of the functions and localisation of these newer members of the tubulin superfamily, and the emerging data suggesting a restricted evolutionary distribution of these `new' tubulin proteins, conforms to established knowledge of microtubule cell biology. On the basis of current evidence, we predict that δ-, ϵ-, 𝛇- and η-tubulin all have functions associated with the centriole or basal body of eukaryotic cells and organisms.
Phosphorylation of MAP4 affects microtubule properties and cell cycle progression
Authors: Chang Winston , Gruber Dorota , Chari Sripriya , Kitazawa Hidefumi , Hamazumi Yuko , Hisanaga Shin-ichi , Bulinski J. Chloë
Published: April 2021

In human cells, MAP4, a microtubule-associated protein ubiquitously expressed in proliferating cells, has been shown to undergo in vivo phosphorylation. Two phosphorylation sites, serines 696 and 787, lie within the proline-rich region of its microtubule-binding domain. To test the hypothesis that phosphorylation at these sites influences microtubule properties or cell cycle progression, we prepared stable cell lines that inducibly express versions of MAP4 in which phosphorylation of these two serines was prevented by their replacement with alanine, lysine, or glutamate residues (AA-, KK-, or EE-MAP4). All non-phosphorylatable mutant forms of MAP4 expressed in mouse Ltk- cells were localized to MT arrays that were unremarkable in appearance. Expression of non-phosphorylatable mutants of MAP4 did not affect cell doubling time; however, expression of some mutants altered progression into or through cell division. Interactions of mutant MAP4 with MTs were examined in vitro. KK mutant MAP4 bound MTs more avidly than its wild-type counterpart, WT-MAP4. In vivo MT polymer also differed among the mutants: MTs in cells expressing the KK- and AA-MAP4 forms were more resistant to nocodazole depolymerization than those in cells expressing EE- or WT-MAP4 forms. Our results demonstrate that phosphorylation alters MAP4 properties and suggest a raison d'être for phosphorylation of the MAP4 microtubule-binding domain during cell cycle progression.
Roles of the cytoplasmic and transmembrane domains of syntaxins in intracellular localization and trafficking
Authors: Kasai Kazuo , Akagawa Kimio
Published: April 2021

Syntaxins are target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) involved in docking and fusion of vesicles in exocytosis and endocytosis. Many syntaxin isoforms have been isolated, and each one displays a distinct intracellular localization pattern. However, the signals that drive the specific intracellular localization of syntaxins are poorly understood. In this study, we used indirect immunofluorescence analysis to examine the localization of syntaxin chimeras, each containing a syntaxin transmembrane domain fused to a cytoplasmic domain derived from a different syntaxin. We show that the cytoplasmic domains of syntaxins 5, 6, 7 and 8 have important effects on intracellular localization. We also demonstrate that the transmembrane domain of syntaxin 5 is sufficient to localize the chimera to the compartment expected for wild-type syntaxin 5. Additionally, we find that syntaxins 6, 7 and 8, but not syntaxin 5, are present at the plasma membrane, and that these syntaxins cycle through the plasma membrane by virtue of their cytoplasmic domains. Finally, we find that di-leucine-based motifs in the cytoplasmic domains of syntaxins 7 and 8 are necessary for their intracellular localization and trafficking via distinct transport pathways. Combined, these results suggest that both the cytoplasmic and the transmembrane domains play important roles in intracellular localization and trafficking of syntaxins.
Dynamics of integrin clustering at focal contacts of endothelial cells studied by multimode imaging microscopy
Authors: Kawakami Keisuke , Tatsumi Hitoshi , Sokabe Masahiro
Published: April 2021

Human umbilical vein endothelial cells were stained with FITC-labeled anti-β1 integrin antibody and plated on a glass cover slip to elucidate the mechanism of integrin clustering during focal contact formation. The process of integrin clustering was observed by time-lapse total-internal-reflection fluorescence microscopy, which can selectively visualize the labeled integrins at the basal surface of living cells. The clustering of integrins at focal contacts started at 1 hour after plating and individual clusters kept growing for ∼6 hours. Most integrin clusters (∼80%) elongated towards the cell center or along the cell margin at a rate of 0.29±0.24 μm minute−1. Photobleaching and recovery experiments with evanescent illumination revealed that the integrins at the extending tip of the clusters were supplied from the intracellular space. Simultaneous time-lapse imaging of exocytosis of integrin-containing vesicles and elongating focal contacts showed that most exocytosis occurred at or near the focal contacts followed by their elongation. Double staining of F-actins and integrins demonstrated that stress fibers were located near the integrin clusters and that intracellular punctate integrins were associated with these stress fibers. These results suggest that the clustering of integrins is mediated by actin-fiber-dependent translocation of integrins to the extending tip of focal contacts.
αT-Catenin: a novel tissue-specific β-catenin-binding protein mediating strong cell-cell adhesion
Authors: Janssens Barbara , Goossens Steven , Staes Katrien , Gilbert Barbara , van Hengel Jolanda , Colpaert Cecile , Bruyneel Erik , Mareel Marc , van Roy Frans
Published: April 2021

Cadherins are major cell-cell adhesion proteins whose cytoplasmic domains bind to catenin proteins. Strong intercellular adhesion depends on linkage of the cadherin/catenin complex to the actin cytoskeleton via α-catenin. To date, it is not clear how different cell types achieve the variable strength of cell-cell adhesion clearly needed in a multicellular organism. Here, we report the cloning and molecular characterization of αT(testis)-catenin, a novel human cDNA encoding a protein with homology to both human αE(epithelial)-catenin and αN(neural)-catenin. Although originally discovered in testis, αT-catenin is expressed in other tissues, the highest levels being observed in heart. Immunohistochemical analysis showed human αT-catenin localization at intercalated discs of cardiomyocytes and in peritubular myoid cells of testis. In cells transfected with αT-catenin cDNA, interaction with β-catenin was demonstrated by co-immunoprecipitation. Transfection of α-catenin-deficient colon carcinoma cells recruited E-cadherin and β-catenin to cell-cell contacts and functional cadherin-mediated cell-cell adhesion was restored in this way. Moreover, compaction of these cells was at least as prominent as in the case of cells expressing endogenous αE-catenin. We propose that αT-catenin is necessary for the formation of stretch-resistant cell-cell adhesion complexes, in particular, muscle cells.
Alterations in an IRE1-RNA complex in the mammalian unfolded protein response
Authors: Bertolotti Anne , Ron David
Published: April 2021

IRE1 proteins mediate cellular responses to accumulation of malfolded proteins in the endoplasmic reticulum in the yeast and mammalian unfolded protein responses. A sensitive in vivo u.v. crosslinking assay showed that IRE1 proteins are intimately associated with RNA in mammalian cells. The IRE1-associated RNA fragments recovered by this assay were different in stressed and unstressed cells. The amount of RNA associated with IRE1 that could be revealed by end-labeling with T4 kinase was greater in IRE1-containing complexes isolated from stressed cells. Furthermore, the RNA fragments recovered from complexes found in stressed cells were shorter than those from unstressed cells, revealing a dynamic change in the IRE1-RNA complex during the UPR. Formation of the complex between IRE1 and RNA was dependent on both the kinase and endonuclease domains of IRE1, and involved pre-existing RNA species. When viewed in the context of the known importance of Ire1p-HAC1 mRNA interactions to the yeast unfolded protein response, these findings suggest that full-length mammalian IRE1s also engage RNA molecules as downstream effectors.
Functions of BRCA1 and BRCA2 in the biological response to DNA damage
Authors: Venkitaraman Ashok R.
Published: April 2021

Inheritance of one defective copy of either of the two breast-cancer-susceptibility genes, BRCA1 and BRCA2, predisposes individuals to breast, ovarian and other cancers. Both genes encode very large protein products; these bear little resemblance to one another or to other known proteins, and their precise biological functions remain uncertain. Recent studies reveal that the BRCA proteins are required for maintenance of chromosomal stability in mammalian cells and function in the biological response to DNA damage. The new work suggests that, although the phenotypic consequences of their disruption are similar, BRCA1 and BRCA2 play distinct roles in the mechanisms that lead to the repair of DNA double-strand breaks.
PML bodies associate specifically with the MHC gene cluster in interphase nuclei
Authors: Shiels Carol , Islam Suhail A. , Vatcheva Radost , Sasieni Peter , Sternberg Michael J. E. , Freemont Paul S. , Sheer Denise
Published: April 2021

Promyelocytic leukemia (PML) bodies are nuclear multi-protein domains. The observations that viruses transcribe their genomes adjacent to PML bodies and that nascent RNA accumulates at their periphery suggest that PML bodies function in transcription. We have used immuno-FISH in primary human fibroblasts to determine the 3D spatial organisation of gene-rich and gene-poor chromosomal regions relative to PML bodies. We find a highly non-random association of the gene-rich major histocompatibilty complex (MHC) on chromosome 6 with PML bodies. This association is specific for the centromeric end of the MHC and extends over a genomic region of at least 1.6 megabases. We also show that PML association is maintained when a subsection of this region is integrated into another chromosomal location. This is the first demonstration that PML bodies have specific chromosomal associations and supports a model for PML bodies as part of a functional nuclear compartment.
Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels
Authors: Martin Patricia E. M. , Blundell Geraldine , Ahmad Shoeb , Errington Rachel J. , Evans W. Howard
Published: April 2021

The assembly of gap junctions was investigated in mammalian cells expressing connexin (Cx) 26, 32 and 43 fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Targeting of Cx32-CFP and 43-GFP to gap junctions and gap junctional communication was inhibited in cells treated with Brefeldin A, a drug that disassembles the Golgi. However gap junctions constructed of Cx26-GFP were only minimally affected by Brefeldin A. Nocodazole, a microtubule disruptor, had little effect on the assembly of Cx43-GFP gap junctions, but perturbed assembly of Cx26-GFP gap junctions. Co-expression of Cx26-YFP and Cx32-CFP in cells treated with Brefeldin A resulted in assembly of gap junctions constructed of Cx26-YFP. Two amino acids that distinguish Cx26 from Cx32 in transmembrane domains were mutated in Cx32 to investigate underlying mechanisms determining trafficking routes to gap junctions. One mutation, Cx32I28L, conferred on it partial Cx26-like trafficking properties as well the post-translational membrane insertion characteristics of Cx26, suggesting that a key determinant regulating trafficking was present in the first transmembrane domain. The results provide a protein trafficking basis for specifying and regulating connexin composition of gap junctions and thus selectivity of intercellular signaling, with Cx32 and 43 trafficking through the secretory pathway and Cx26 also following an alternative pathway.
Identification and isolation of human prostate epithelial stem cells based on α2β1-integrin expression
Authors: Collins Anne T. , Habib Fouad K. , Maitland Norman J. , Neal David E.
Published: April 2021

A major impediment to our understanding of the biology of stem cells is the inability to distinguish them from their differentiating progeny. We made use of the known association of stem cells with basement membranes to isolate prostate epithelial stem cells. We show that, in vivo, putative stem cells express higher levels of the α2-integrin subunit than other cells within the basal layer. Approximately 1% of basal cells examined by confocal microscopy were integrin ‘bright’, and these cells can be selected directly from the tissue on the basis of rapid adhesion to type I collagen. This selected population has a basal phenotype, as determined by expression of CK5 and CK14 and lack of expression of the differentiation-specific markers prostate specific antigen (PSA) and prostatic acid phosphatase (PAP), and has a fourfold greater ability to form colonies in vitro than the total basal population. These putative stem cells are distinguished from other basal cells by their ability to generate prostate-like glands in vivo with morphologic and immuno-histochemical evidence of prostate-specific differentiation. These properties are consistent with a stem cell origin. Furthermore, the presence of surface integrins on prostate stem cells suggests that these cells share common pathways with stem cells in other tissues.
Cell biology beyond the diffraction limit: near-field scanning optical microscopy
Authors: de Lange Frank , Cambi Alessandra , Huijbens Richard , de Bakker Bärbel , Rensen Wouter , Garcia-Parajo Maria , van Hulst Niek , Figdor Carl G.
Published: April 2021

Throughout the years, fluorescence microscopy has proven to be an extremely versatile tool for cell biologists to study live cells. Its high sensitivity and non-invasiveness, together with the ever-growing spectrum of sophisticated fluorescent indicators, ensure that it will continue to have a prominent role in the future. A drawback of light microscopy is the fundamental limit of the attainable spatial resolution – ∼250 nm – dictated by the laws of diffraction. The challenge to break this diffraction limit has led to the development of several novel imaging techniques. One of them, near-field scanning optical microscopy (NSOM), allows fluorescence imaging at a resolution of only a few tens of nanometers and, because of the extremely small near-field excitation volume, reduces background fluorescence from the cytoplasm to the extent that single-molecule detection sensitivity becomes within reach. NSOM allows detection of individual fluorescent proteins as part of multimolecular complexes on the surface of fixed cells, and similar results should be achievable under physiological conditions in the near future.
Growth factors but not gap junctions play a role in injury-induced Ca2+ waves in epithelial cells
Authors: Klepeis Veronica E. , Cornell-Bell Ann , Trinkaus-Randall Vickery
Published: April 2021

This paper characterizes the early responses of epithelial cells to injury. Ca2+ is an important early messenger that transiently increases in the cytoplasm of cells in response to external stimuli. Its elevation leads to the regulation of signaling pathways responsible for the downstream events important for wound repair, such as cell migration and proliferation. Live cell imaging in combination with confocal laser scanning microscopy of fluo-3 AM loaded cells was performed. We found that mechanical injury in a confluent region of cells creates an elevation in Ca2+ that is immediately initiated at the wound edge and travels as a wave to neighboring cells, with [Ca2+]i returning to background levels within two minutes. Addition of epidermal growth factor (EGF), but not platelet-derived growth factor-BB, resulted in increased [Ca2+]i, and EGF specifically enhanced the amplitude and duration of the injury-induced Ca2+ wave. Propagation of the Ca2+ wave was dependent on intracellular Ca2+ stores, as was demonstrated using both thapsigargin and Ca2+ chelators (EGTA and BAPTA/AM). Injury-induced Ca2+ waves were not mediated via gap junctions, as the gap-junction inhibitors 1-heptanol and 18α-glycyrrhetinic acid did not alter wave propagation, nor did the cells recover in photobleaching experiments. Additional studies also demonstrated that the wave could propagate across an acellular region. The propagation of the injury-induced Ca2+ wave occurs via diffusion of an extracellular mediator, most probably via a nucleotide such as ATP or UTP, that is released upon cell damage.

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Targeting and functional role of N-RAP, a nebulin-related LIM protein, during myofibril assembly in cultured chick cardiomyocytes
Authors: Carroll Stefanie L. , Herrera Amy H. , Horowits Robert
Published: April 2021

Targeting and functional effects of N-RAP domains were studied by expression as GFP-tagged fusion proteins in cultured embryonic chick cardiomyocytes. GFP-tagged N-RAP was targeted to myofibril precursors, myofibril ends and cell contacts, expression patterns that are similar to endogenous N-RAP. The GFP-tagged N-RAP LIM domain (GFP-N-RAP-LIM) was targeted to the membrane in cells with myofibril precursors and cell-cell contacts. The GFP-tagged super repeats (N-RAP-SR) and the GFP-tagged domain normally found in between the super repeats and the LIM domain (N-RAP-IB) were each observed at sites of myofibril assembly, incorporating into myofibril precursors in a manner similar to full length N-RAP. However, unlike full-length N-RAP, N-RAP-SR and N-RAP-IB were also found in mature myofibrils, associating with the sarcomeric actin filaments and the Z-lines, respectively. N-RAP-IB was also colocalized with α-actinin at cell contacts. Each of the N-RAP constructs could inhibit the formation of mature myofibrils in cultured cardiomyocytes, with the effects of N-RAP-SR and N-RAP-IB depending on the time of transfection. The results show that each region of N-RAP is crucial for myofibril assembly. Combining the targeting and functional effects of N-RAP domains with information in the literature, we propose a new model for initiation of myofibrillogenesis.
Cell-cell adhesion and signal transduction duringDictyosteliumdevelopment
Authors: Coates Juliet C. , Harwood Adrian J.
Published: April 2021

The development of the non-metazoan eukaryote Dictyostelium discoideum displays many of the features of animal embryogenesis, including regulated cell-cell adhesion. During early development, two proteins, DdCAD-1 and csA, mediate cell-cell adhesion between amoebae as they form a loosely packed multicellular mass. The mechanism governing this process is similar to epithelial sheet sealing in animals. Although cell differentiation can occur in the absence of cell contact, regulated cell-cell adhesion is an important component of Dictyostelium morphogenesis, and a third adhesion molecule, gp150, is required for multicellular development past the aggregation stage.

Cell-cell junctions that appear to be adherens junctions form during the late stages of Dictyostelium development. Although they are not essential to establish the basic multicellular body plan, these junctions are required to maintain the structural integrity of the fruiting body. The Dictyostelium β-catenin homologue Aardvark (Aar) is present in adherens junctions, which are lost in its absence. As in the case of its metazoan counterparts, Aar also has a function in cell signalling and regulates expression of the pre-spore gene psA.

It is becoming clear that cell-cell adhesion is an integral part of Dictyostelium development. As in animals, cell adhesion molecules have a mechanical function and may also interact with the signal-transduction processes governing morphogenesis.
Alteration of nuclear architecture in male germ cells of chromosomally derived subfertile mice
Authors: Garagna Silvia , Zuccotti Maurizio , Thornhill Alan , Fernandez-Donoso Raul , Berrios Soledad , Capanna Ernesto , Redi Carlo Alberto
Published: April 2021

The mammalian cell nucleus consists of numerous compartments involved in the regular unfolding of processes such as DNA replication and transcription, RNA maturation, protein synthesis and cell division. Knowledge is increasing of the relationships between high-order levels of chromatin organization and its spatial organization, and of how these relationships contribute to the various functions carried out in the nucleus. We have studied the spatial arrangement of mouse telocentric chromosomes 5, 11, 13, 15, 16 and 17, some of their metacentric Robertsonian derivatives, and X and Y chromosomes by whole chromosome painting in male germ (spermatogonia, pachytene spermatocytes and spermatids) and Sertoli cells of homozygous and heterozygous individuals. Using dual-colour fluorescence in situ hybridization we found that these chromosomes occupy specific nuclear territories in each cell type analysed. When chromosomes are present as Robertsonian metacentrics in the heterozygous state, that is, as Robertsonian metacentrics and their homologous telocentrics, differences in their nuclear positions are detectable: heterozygosity regularly produces a change in the nuclear position of one of the two homologous telocentrics in all the cell types studied. In the Robertsonian heterozygotes, the vast majority of the Sertoli cells show the sex chromosomes in a condensed state, whereas they appear decondensed in the Robertsonian homozygotes. As the Robertsonian heterozygosities we studied produce a chromosomally derived impairment of male germ-cell differentiation, we discuss the possibility that changes in chromosome spatial territories may alter some nuclear machinery (e.g., synapsis, differential gene expression) important for the correct unfolding of the meiotic process and for the proper functioning of Sertoli cells.
Molecular mechanisms regulating the subcellular localization of p95-APP1 between the endosomal recycling compartment and sites of actin organization at the cell surface
Authors: Matafora Vittoria , Paris Simona , Dariozzi Simona , Curtis Ivan de
Published: April 2021

Cell migration requires coordination between adhesion, actin organization and membrane traffic. Rac and ARF6 have been shown to cooperate for the organization of actin at the cell surface. Recently, the GIT family of ARF-GAPs has been identified, which includes proteins that can functionally interact with both ARF and Rac GTPases. The p95-APP1 protein is a member of this family, isolated as part of a multi-molecular complex interacting with GTP-Rac. Our previous work has indicated that this protein may be part of the machinery redirecting membrane recycling towards sites of protrusion during locomotion. By analyzing the distribution and the effects of truncated forms of p95-APP1, we show here that the lack of the ARF-GAP domain of p95-APP1 dramatically shifts its localization to large vesicles. The use of several markers of the endocytic pathway has revealed that the truncated p95-APP1 localizes specifically to a Rab11-, transferrin receptor-positive compartment. Other markers are excluded from the p95-APP1-positive vesicles, while known components of the multi-molecular complex colocalize with truncated p95-APP1 in this compartment. Coexpression of a constitutively active form of Rac induces the redistribution of the truncated constructs and of the associated PIX, PAK, and paxillin to peripheral sites of Rac-mediated actin organization, and the disassembly of the large Rab11-positive vesicles. Together, the data presented indicate that p95-APP1 is part of a complex that shuttles between the plasma membrane and the endocytic recycling compartment, and suggest that the dynamic redistribution of the p95-APP1-containing complex is mediated both by the ARF-GAP domain, and by the recruitment of the complex at the cell surface at sites of Rac activation.
Enhancement of branching efficiency by the actin filament-binding activity of N-WASP/WAVE2
Authors: Suetsugu Shiro , Miki Hiroaki , Yamaguchi Hideki , Obinata Takeshi , Takenawa Tadaomi
Published: April 2021

The actin-related protein (Arp) 2/3 complex is an essential regulator of de novo actin filament formation. Arp2/3 nucleates the polymerization of actin and creates branched actin filaments when activated by Arp2/3-complex activating domain (VCA) of Wiskott-Aldrich syndrome proteins (WASP family proteins). We found that the branching of actin filaments on pre-existing ADP filaments mediated by the Arp2/3 complex is twice as efficient when Arp2/3 was activated by wild-type neural WASP (N-WASP) or WASP-family verprolin-homologous protein (WAVE) 2 than when activated by the VCA domain alone. By contrast, there was no difference between wild-type N-WASP or WAVE2 and VCA in the branching efficiency on de novo filaments, which are thought to consist mainly of ADP-phosphate filaments. This increased branching efficiency on ADP filaments is due to the basic region located in the center of N-WASP and WAVE2, which was found to associate with ADP actin filaments. Actin filaments and phosphatidylinositol bisphosphate (PIP2) associate with N-WASP at different sites. This association of N-WASP and WAVE2 with actin filaments enhanced recruitment of Arp2/3 to the pre-existing filaments, presumably leading to efficient nucleation and branch formation on pre-existing filaments. These data together suggest that the actin filament binding activity of N-WASP and WAVE2 in the basic region increases the number of barbed ends created on pre-existing filaments. Efficient branching on ADP filaments may be important for initiation of actin-based motility.
Relationship between the function and the location of G1 cyclins inS. cerevisiae
Authors: Edgington Nicholas P. , Futcher Bruce
Published: April 2021

The Saccharomyces cerevisiae cyclin-dependent kinase Cdc28 forms complexes with nine different cyclins to promote cell division. These nine cyclin-Cdc28 complexes have different roles, but share the same catalytic subunit; thus, it is not clear how substrate specificity is achieved. One possible mechanism is specific sub-cellular localization of specific complexes. We investigated the location of two G1 cyclins using fractionation and microscopy. In addition, we developed ‘forced localization’ cassettes, which direct proteins to particular locations, to test the importance of localization. Cln2 was found in both nucleus and cytoplasm. A substrate of Cln2, Sic1, was also in both compartments. Cytoplasmic Cln2 was concentrated at sites of polarized growth. Forced localization showed that some functions of Cln2 required a cytoplasmic location, while other functions required a nuclear location. In addition, one function apparently required shuttling between the two compartments. The G1 cyclin Cln3 required nuclear localization. An autonomous, nuclear localization sequence was found near the C-terminus of Cln3. Our data supports the hypothesis that Cln2 and Cln3 have distinct functions and locations, and the specificity of cyclin-dependent kinases is mediated in part by subcellular location.
The factor affecting the spreading of chondrocytes upon inorganic substrate
Authors: Takeichi M.
Published: April 2021

The effect of conditioned medium (CM) prepared from mass-cultures of chick embryonic cells was studied on the spreading behaviour of chondrocyte derived from sterna of 16-day-old chick embryos.

Freshly dissociated chondrocytes exhibited a quite rounded form, and this shape did not change when they were cultured with fresh medium (Eagle’s MEM + 6 % foetal calf serum) in vitro for several days. Non-dialysable material(s) in CM added into the fresh medium stimulated the formation of pseudopods of chondrocytes, without primarily affecting the synthesis of chondroitin sulphates. Such an activity of CM was not lost after boiling, but it was lost following treatment with proteases. The chondrocytes covered with newly deposited acsidmucopoly saccharides were insensitive to the effect of CM, but they became sensitive to form pseudopods after treatment of the cells with chondroitinase.

These results suggest that CM contains a macromolecular material(s) to enhance the motility or adhesiveness of chondrocytes.
A quantitative analysis of the effects of the intraneural injection of lysophosphatidyl choline
Authors: Gregson N. A. , Hall Susan M.
Published: April 2021

Some quantitative aspects of the demyelinating lesion, and its progress, produced by the in vivo injection of lysophosphatidyl choline (LPC) into the myelinated peripheral nerves of adult mice have been investigated. The extent of the lesion has been shown to be highly localized to the injection site and dependent on the concentration of LPC. The length of the demyelinating zone appears to be determined within the first hour following the injection. The resulting demyelination occurs in units of whole internodes and over 40 % of the fibres involved show the loss of only one internode. The length of the demyelinated zone does not show any obvious dependency on axon diameter. Remyelination has started by day 14 and involves on average the replacement of each original internode by 2 new segments. There is a cellular proliferation in response to the injection, starting within the first week and achieving a maximum between 2–3 weeks. Of the extra cells 70—80 % are not axon-associated and of these 50% are supemumary Schwann cells. The use of 14C-oleate and 3H-stearyl LPC indicates that the injected LPC can be converted into lecithin, but also that the fatty acid moiety of LPC becomes distributed amongst the other lipid classes. Pretreatment of the nerve with small amounts of KCN blocks the metabolism of the injected LPC and renders all of the cells subject to lysis.
Ultrastructure of an unusual contractile vacuole in several chrysomonad phytoflagellates
Authors: Aaronson S. , Behrens U.
Published: April 2021

The ultrastructure of the contractile vacuole of Ochromonas and Poterioochromonas species is described. It appears to be a permanent structure in the anterior end of the phytoflagellate, consisting of a central cavity with a unit-membrane lining from which flattened saccules protrude in all directions.

The contracted saccules are about 40 nm in diameter and are made up of a single trilammellar unit membrane about 9 nm thick and continuous with the central cavity membrane. In their contracted state the saccules contain transverse fibrils which may be involved in the contraction of the saccules ; these fibrils are no longer seen when the central cavity and the saccules swell. The saccules seem to merge and become continuous with the central cavity during the swelling of the contractile vacuole.
Nuclear sheets in the egg of a fern, Dryopteris filix-mas
Authors: Bell P. R.
Published: April 2021

In the maturing egg of Dryopteris filix-mas the nucleus is shown to produce sheet-like extensions into the cytoplasm, resembling those described in certain somatic cells of animals. In these sheets the distance between the 2 inner membranes of the envelope is regularly of the order of 45-50 nm, and the nuclear material is organized into a monolayer of electron-opaque aggregates 35-40 nm in diameter, occasionally with a central space.

Much of the osmiophilic material of the aggregates is removed by pronase and trypsin, leaving granular osmiophilic material, but no fibrils. Ribonuclease and deoxyribonuclease produced little effect. Tritiated thymidine, fed for 6 h before fixation to maturing eggs, could not be shown to be incorporated into the aggregates, despite marked incorporation into plastids and mitochondria. In the absence of compelling evidence that the electron-opaque material is chromatin, it is proposed that the aggregates represent enzyme complexes concerned with membrane growth, believed to be active in the nuclear evaginations. These complexes are not considered to be confined to the sheet-like parts of evaginations, but to be brought into an aggregated form in the sheets as a result of compression, as suggested for the similar organization of chromatin in the nuclear sheets of animal cells.
Variation in eyespot ultrastructure in Chlamydomonas reinhardi (ac-31)
Authors: Gruber Helen E. , Rosario Benjamin
Published: April 2021

Several morphological variations in eyespot complex fine structure were exhibited in some cells of the pale-green mutant strain ac-31 of Chlamydomonas reinhardi. The cells were grown in minimal medium supplemented with 0·2 % sodium acetate and were harvested by centrifugation and prepared for electron-microscopic examination.

Microtubules were seen near the flagella, confirming previous observations. Microtubules were also seen near the eyespot complex. Although a direct connexion between the microtubules of the flagellar and eyespot region was never observed, this does not exclude the possibility that it exists and thus provides a structural and functional connexion between the two organelles.

Occasionally irregular curved bodies intimately associated with the eyespot complex of some cells appeared to displace the chloroplast and cell membranes. These bodies often appeared to be nearly covered by a limiting membrane and were found near empty ‘cavities’ in the eyespot plate. Crystalline arrays of dense bodies were observed in some sections. The significance of these bodies is discussed in relation to the functional state of the carotenoid pigments making up the eyespot granules. An hypothesis for the formation of the rod-like structures is presented, based upon the observation of granules which had fused together to form a helix.
On the mechanism of the increased aggregation by neuraminidase of 16 c malignant rat dermal fibroblasts in vitro
Authors: Lloyd C. W. , Cook G. M. W.
Published: April 2021

The aggregation of briefly trypsinized 16 C malignant rat dermal fibroblasts as measured by gyratory shaker/electronic particle counting technique is increased by neuraminidase. Enzyme treatment also exposes soybean and Ricinus communis agglutinin-binding sites as judged by cell agglutination. Desialysed bovine submaxillary mucin and desialysed, agalacto fetuin reversed the neuraminidase-stimulated increase in aggregation, whereas desialysed fetuin and ovalbumin did not. The effective glycoproteins also acted as acceptors in cellular sialyl- and galactosyl-transferase reactions, and these activities are detected in plasma membrane fractions.

The possibility that neuraminidase increases cellular aggregation by generating acceptor sites for interaction with cell surface glycosyltransferases, is discussed.
Cytokinesis in the heliozoan Actinophrys sol
Authors: Ockleford C. D.
Published: April 2021

A study of cytokinesis in the heliozoan Actinophrys sol has been made using low-temperature treatments, antimitotic drugs, cytochalasin B, light microscopy and electron microscopy. It reveals that microtubular axopodia remain extended during cell division and play a major role in it.

Data indicate that when organisms are attached to the bases of culture dishes the normal locomotive mechanism of the presumptive daughter cells pulls them apart. However, when Actinophrys are floating freely in their culture medium, they are still able to undertake division. In this situation interactions between axopodia from opposite daughter cell bodies appear to facilitate the movement apart of the prospective daughter cells.

The present study and other published accounts indicate that a type of cytokinesis exists which is not explicable in terms of the ‘contractile ring’ or ‘fusing vesicle’ theories. To account for these observations a ‘barge pole’ model of division is suggested.
The localization of enzymes in fixed sections of soybean root nodules by electron microscopy
Authors: Marks I. , Sprent Janet I.
Published: April 2021

Attempts were made to localize the following enzymes within bacteroids and host tissue: reduced-NAD oxidoreductase, succinate oxidoreductase, adenosinetriphosphate phosphorylase, diaminobenzidineoxidase, and xylylene diamine oxidase. Diaminobenzidine (DAB) staining was localized within the mitochondrial membranes and within the bacteroid cytomembrane and associated vesicles, whereas dehydrogenase enzymes within the bacteroids were for the greater part mesosomal. Peroxidative reduction of DAB and xylylene diamine within the bacteroid nuclear regions occurred in the presence of catalase, to which the cytomembrane appeared to be impermeable. A cyanide-insensitive oxidase localized within the bacteroid mesosomes and nuclear regions was not affected by the addition of catalase, aminotriazole or pyruvate. Ca2+-stimulated ATP-phosphorylase was localized within the bacteroid cytomembrane, whereas a Mg2+-stimulated ATP-phosphorylase was localized within the host cell plasma membrane.
Ultrastructural and biochemical observations on interphase nuclei isolated from chicken erythrocytes
Authors: Walmsley M. E. , Davies H. G.
Published: April 2021

Adult hen erythrocyte nuclei are isolated from cells or haemolysed in situ by acting on the plasma membrane with rotating knives or with non-ionic detergents. When the isolation medium contains magnesium 10ns (1 mM), sucrose (0·4 M) and Tris buffer (0· 01 M, pH 7· 5) called SMTOG (see text), the ultrastructure in thin sections through the condensed chromatin bodies, after staining with either uranyl-lead or phosphotungstic acid (PTA), is similar to that found in the intact cell. Hence it can be concluded that the 2 phases which comprise chromatin, the o- and the "-phase, survive nuclear isolation. These are so called because the structural units in chromatin are arranged at the surface of the nucleus into one or more layers and give rise to oddly (o) and evenly (e) numbered bands. The o-phase is also largely retained after extensive washing in 0 ·07 M NaCl as shown by electron microscopy and biochemical measurements; only 6% of the total nuclear protein is removed, a value small compared with the fractional amount of the chromatin protein calculated to lie in the o-phase, about 70%. After extensive washing in saline-EDTA there are structural changes in chromatin, but biochemical data show that the molecules in the o-phase are also largely retained; loss of protein amounts to between 5 and 11%.

These data suggest that the o-phase is a structural component of the chromatin bodies. They support the hypothesis that condensed chromatin is formed by folding superunit threads. These units consist of a central thread-like element about 17 nm diameter which stains preferentially with uranyl-lead and forms the e-phase, with an outer cylindrical shell forming the o-phase of total diameter about 28 nm.

The 5–10% proteins removed by salt washes are located exclusively in a particulate component, quite likely the chromatin. They have been examined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. There are about 10 or more protein species, ranging in molecular weight from 21000 upwards. The groups of large granules previously found in the nuclear sap of intact erythrocytes are shown to be associated with an amorphous or finely fibrillar body. During nuclear isolation and subsequent washes, we observed the fate of this large granule complex as well as that of the nuclear envelope and the filled cavities in chromatin.
Effects of cytochalasin b on the aggregation, electrophoretic mobility and surface morphology of chick neural retina cells
Authors: Jones G. E.
Published: April 2021

Over a concentration range of 0 · 5–10 μg/cm3, cytochalasin B caused a biphasic change in the electrophoretic mobility of disaggregated neural retina cells. An initial rise in anodal mobility at low concentrations of the drug was transformed into a reduction in the mobility below that of the control at a concentration of 10 μg/cm3.

The effect of cytochalasin B was found to be reversible by washing treated cells in cytochalasin B-free media. This was investigated at a concentration of cytochalasin at which the greatest difference existed between the mobilities of the control and experimental cell suspensions.

Reaggregation of cell dispersions failed to show any significant difference in the rate of aggregation between cytochalasin B-treated cells and the control. Scanning electron microscopy of cells fixed while in suspension also showed little significant change in the surface morphology upon application of cytochalasin B. In high concentrations of the drug cells appeared somewhat smoother in outline, but no correlation was found between changes in surface morphology and the variations in cell electrophoretic mobility.

It is concluded that the observed changes in electrophoretic mobility may be attributed to a binding of cytochalasin B to the cell membrane. This lends support to the hypothesis that the primary site of action of cytochalasin B may be the plasma membrane.
Cytoplasmic induction of changes in the ultrastructure of the Acetabularia nucleus and perinuclear cytoplasm
Authors: Berger S. , Schweiger H. G.
Published: April 2021

The ultrastructure of the cell nucleus and perinuclear cytoplasm in Acetabularia is quite different in young and old cells. When a nucleus of an old cell was implanted into the cytoplasm of a young cell the nucleus assumed the morphology typical of a young cell within less than 10 days. The cytoplasm of an old cell was able to induce the reverse change in an implanted nucleus from a young cell. This cytoplasmic induction of nuclear ageing appeared to proceed more rapidly than rejuvenation.
Inability to detect Chlamydomonas microtubule assembly in vitro: Possible implications to the in vivo regulation of microtubule assembly
Authors: Farrell K. W. , Burns R. G.
Published: April 2021

It has been demonstrated that the in vitro assembly of microtubules from Chlamydomonas preparations does not occur under a wide range of conditions, including those efficacious for mammalian brain tubulin. This incompetence of Chlamydomonas extracts to form microtubules is independent of the tubulin concentration, the presence of added nucleotides or an added seed, temperature, or the concentration of divalent cations. However, an amorphous aggregate was observed under certain conditions, whose composition was mainly tubulin.

The in vitro reassembly of microtubules in gerbil brain extracts is inhibited by Chlamydomonas preparations. Fractionation of the Chlamydomonas extracts by column chromatography suggests that the inhibitory component is Chlamydomonas tubulin itself. The mechanism of this inhibition is unknown, but reassembly experiments indicate that the 2 types of tubulins cannot copolymerize. We suggest that the Chlamydomonas tubulin, derived from a cytoplasmic pool, requires to be activated prior to its in vivo polymerization into microtubules.
Ultrastructural characteristics of the non-immune rosette-forming cell
Authors: Levy J. D. , Knieser M. R. , Briggs W. A.
Published: April 2021

Non-immune rosette formation, where a non-sensitized lymphocyte is surrounded by three or more sheep red blood cells, is an in vitro technique which is thought to be specific in the identification of thymus-dependent (T) lymphocytes. Using the electron microscope and tissue culture techniques, we have studied the ultrastructure of the rosette-forming lymphocyte which has been stimulated by various cellular mitogens. Non-sensitized but stimulated lymphocytes, which form rosettes, have a morphology similar to that of previously identified T cells, adding further credence to the concept that non-sensitized rosette-forming cells represent a population of T cells. Changes in the binding pattern of the sheep red cell membrane to the lymphocyte have been identified which may represent an early phase of cell ‘activation.’ This marker of activation offers a potential method of studying membrane responses in attempts to localize cellular defects involving the T lymphocyte. In addition, preliminary studies using fluorescein-labelled sheep red blood cell fragments suggest specific attachment of labelled membrane to the T lymphocyte, allowing rapid identification of T cells in smears or tissue preparations.