bluuebunny/crossref_metadata_2025 huggingface dataset:
A localisation signal in the 3′ untranslated region of c-myc mRNA targets c-myc mRNA and β-globin reporter sequences to the perinuclear cytoplasm and cytoskeletal-bound polysomes
Authors: Veyrune J.-L. , Campbell G. P. , Wiseman J. , Blanchard J.-M. , Hesketh J. E.
Published: April 2021

There is increasing evidence that in mammalian cells some mRNAs are localised to specific parts of the cytoplasm and a proportion of mRNAs and polyribosomes are associated with the cytoskeleton. It has been shown previously that c-myc mRNA is present in the perinuclear cytoplasm and associated with the cytoskeleton, and that this localisation is dependent upon the 3′ untranslated region of the mRNA. The present studies show that in transfected fibroblasts the c-myc 3′ untranslated region is able to localise β-globin reporter sequences to the perinuclear cytoplasm. Studies with constructs containing deletions within the 3′ untranslated region identify the region between bases 194 and 280 as critical for localisation. Transfection of cells with constructs in which this region is linked to β-globin sequences showed that it was sufficient to localise the chimaeric tran-scripts to the perinuclear cytoplasm and to cytoskeletal-bound polyribosomes. Transfection with constructs containing a mutated AUUUA sequence within the 194-280 base region showed that this conserved AUUUA is required for targeting of both c-myc mRNA and a chimaeric tran-script of β-globin transcripts linked to the c-myc 3′ untranslated region. The region between bases 194 and 280 did not induce instability of β-globin transcripts and the AUUUA mutation had little effect upon mRNA stability. We propose that this 86 nt region of the 3′ untranslated region contains a localisation signal to target c-myc mRNA so that it is retained on cytoskeletal-bound polysomes in the perinuclear cytoplasm; a conserved AUUUA sequence appears to be a critical part of this signal.
Intracellular targeting of GLUT4 in transfected insulinoma cells: evidence for association with constitutively recycling vesicles distinct from synaptophysin and insulin vesicles
Authors: Thorens Bernard , Roth Jürgen
Published: April 2021

In adipocytes and muscle cells, the GLUT4 glucose trans-porter isoform is present in intracellular vesicles which continuously recycle between an intracytoplasmic location and the plasma membrane. It is not clear whether the GLUT4-vesicles represent a specific kind of vesicle or resemble typical secretory granules or synaptic-like microvesicles. To approach this question, we expressed GLUT4 in the β cell line RINm5F and determined its intra-cellular localization by subcellular fractionation and by immunofluorescence and immunoelectron microscopy. GLUT4 was not found in insulin granules but was associated with a subpopulation of smooth-surface vesicles present in the trans-Golgi region and in vesicular structures adjacent to the plasma membrane. In the trans-Golgi region, GLUT4 did not colocalize with synaptophysin or TGN38. Incubation of the cells with horseradish peroxidase (HRP) led to colocalization of HRP and GLUT4 in some endosomal structures adjacent to the plasma membrane and in occasional trans-Golgi region vesicles. When cells were incubated in the presence of Bafilomycin A, analysis by confocal microscopy revealed GLUT4 in numerous large spots present throughout the cytoplasm, many of which costained for TGN38 and synaptophysin. By immunoelectron microscopy, numerous endosomes were observed which stained strongly for GLUT4. Together our data demonstrate that ectopic expression of GLUT4 in insulinoma cells reveals the presence of a subset of vesicular structures distinct from synaptic-like vesicles and insulin secretory granules. Furthermore, they indicate that GLUT4 constitutively recycles between the plasma membrane and its intracellular location by an endocytic route also taken by TGN38 and synaptophysin.
Syndecan-1 expression in mammary epithelial tumor cells is E-cadherin-dependent
Authors: Leppä Sirpa , Vleminckx Kris , Roy Frans Van , Jalkanen Markku
Published: April 2021

E-cadherin is a Ca2+-dependent cell-cell adhesion molecule, which is mainly expressed in epithelial cells. Recent studies have shown that E-cadherin has an important role as an invasion suppressor molecule in epithelial tumor cells. Syndecan-1 is a cell surface proteoglycan that has been implicated in a number of cellular functions including cell-cell adhesion, cell-matrix anchorage and growth factor pre-sentation for signalling receptors. Its suppression has also been shown to be associated with malignant transforma-tion of epithelial cells. In order to better understand the coordinated regulation of cell-cell and cell-matrix interac-tions during malignant transformation, we have studied the expression of syndecan-1 in malignant mammary tumor cells genetically manipulated for E-cadherin expression. In invasive NM-e-ras-MAC1 cells, where E-cadherin was partially downregulated by specific antisense RNA, syndecan-1 expression was suppressed. Furthermore, transfection of E-cadherin cDNA into invasive NM-f-ras-TD cells resulted in the upregulation of syndecan-1 expression in association with decreased invasiveness. In both cases, regulation of syndecan-1 occurred post-tran-scriptionally, since syndecan-1 mRNA levels remained unchanged. Instead, a translational regulation is suggested, since syndecan-1 core protein synthesis was E-cadherin dependent. Another cell adhesion protein, β1-integrin was not affected by E-cadherin expression. The data provide an example of coordinated changes in the expression of two cell adhesion molecules, syndecan-1 and E-cadherin during epithelial cell transformation.
Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts
Authors: Strausfeld Ulrich P. , Howell Mike , Descombes Patrick , Chevalier Stephane , Rempel Rachel E. , Adamczewski Jörg , Maller James L. , Hunt Tim , Blow J. Julian
Published: April 2021

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define ‘S-phase promoting factor’ (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclu-sively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cdc2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.
The SAC3 gene encodes a nuclear protein required for normal progression of mitosis
Authors: Bauer Andreas , Kölling Ralf
Published: April 2021

The SAC3 gene of Saccharomyces cerevisiae has been implicated in actin function by genetic experiments showing that a temperature sensitive mutation in the essential actin gene (act1-1) can be suppressed by mutations in SAC3. An involvement of SAC3 in actin function is further suggested by the observation that the actin cytoskeleton is altered in SAC3 mutants. Our fractionation experiments, however, point to a nuclear localization of Sac3p. On sucrose density gradients Sac3p co-fractionated with the nuclear proteins Ssb1p, Nop1p and Nop2p but not with the other organelle markers examined. Furthermore, Sac3p was enriched 10-fold in a nuclei preparation along with the nuclear protein Nop1p. In this report we further show that SAC3 function is required for normal progression of mitosis. SAC3 mutants showed a higher fraction of large-budded cells in culture, indicative of a cell cycle delay. The predominant population among the large-budded sac3 cells were cells with a single nucleus at the bud-neck and a short intranuclear spindle. This suggests that a cell cycle delay occurs in mitosis prior to anaphase. The observation that SAC3 mutants lose chromosomes with higher frequency than wild-type is another indication for a mitotic defect in SAC3 mutants. We further noticed that SAC3 mutants are more resistant against the microtubule destabilizing drug benomyl. This finding suggests that SAC3 is involved, directly or indirectly, in microtubule function. In summary, our data indicate that SAC3 is involved in a process which affects both the actin cytoskeleton and mitosis.
The kinetics of the B cyclin p56cdc13 and the phosphatase p80cdc25 during the cell cycle of the fission yeast Schizosaccharomyces pombe
Authors: Creanor J. , Mitchison J. M.
Published: April 2021

The levels of the B cyclin p56cdc13 and the phosphatase p80cdc25 have been followed in selection-synchronised cultures of Schizosaccharomyces pombe wild-type and wee1 mutant cells. p56cdc13 has also been followed in induction-synchronised cells of the mutant cdc2-33. The main con-clusions are: (1) cdc13 levels in wild-type cells start to rise from base line at about mid-G2, reach a peak before mitosis and then fall slowly through G1. Cells exit mitosis with appreciable levels of cdc13. (2) cdc13 levels in wee1 cells fall to zero in interphase. They also start to rise at the beginning of G2, which may be related to the absence of a mitotic size control. (3) cdc25 starts to rise later and reaches a peak after mitosis. This is not what would be expected from a simple mitotic inducer and suggests that cdc25 has an important function at the end of mitosis. (4)

An upper (heavier) band of cdc25 peaks at the same time as the main band but rises and falls more rapidly. If this is a hyperphosphorylated form, its timing shows that it is most unlikely to function in the ways shown for such a form in eggs and mammalian cells. (5) Experiments with the mutant cdc10-129 and with hydroxyurea show that the initial signal to begin synthesis of cdc13 originates at Start. (6) In induction synchrony, where G2 spans across cell division, there is evidence that some events in one cycle cannot start in the previous one. (7) Revised timings are given for the times of mitosis in these cultures.
Mutational analysis of Hsp90α dimerization and subcellular localization: dimer disruption does not impede ‘in vivo’ interaction with estrogen receptor
Authors: Meng Xia , Devin Jocelyne , Sullivan William P. , Toft David , Baulieu Etienne-Emile , Catelli Maria-Grazia
Published: April 2021

The molecular chaperone Hsp90 has been found ubiquitously as a predominantly cytoplasmic dimer. By interacting with cytoplasmic or nuclear proteins such as pp60v-src or steroid receptors, Hsp90 helps its targets to become competent for full biological activity.

Mutational deletion analysis of some properties of chicken Hsp90α was undertaken after transient transfection of the constructs in COS7 cells. First, Hsp90 mutants were analyzed for their ability to behave as cytosolic dimers. We confirmed that the C-terminal Hsp90 region (amino acids 446-728) was sufficient for dimerization, and found that deletion of three small subregions in the 200 C-terminal residues precluded Hsp90 dimer formation. Moreover, we demonstrated that the N-terminal region of the protein (1-442) was not involved in dimerization. Second, the subcellular localization of the wild-type (WT) protein and mutants was analyzed by specific immunodetection and confocal microscopy. Most of the mutants were cytoplasmic like Hsp90WT, a nuclear localization being barely detectable in the WT protein or in mutants with a C-terminal truncation equal to or shorter than 286 residues. Surprisingly a mutant encoding the N-terminal region (1-285) was nuclear localized.

In addition, the in vivo interaction between the cytoplasmic Hsp90 and the nuclear ER was documented after coexpression of both proteins in the same cells: some Hsp90 was shifted into the nucleus via its interaction with ER. From an analysis of dimeric or monomeric cytoplasmic Hsp90 mutants, we found that disruption of Hsp90 dimer did not systematically impede its interaction with ER.

Finally, Hsp90WT and cytoplasmic mutants were tested for their ability to rescue from lethality a yeast strain deleted of both Hsp90 genes. Interestingly, the Δ661-677 mutant that showed an impaired dimerization but interacted with ER was able to confer viability, while the mutant deleted of the 30 C-terminal residues (NC6) was monomeric, did not confer viability and did not interact with ER. We therefore suggest that Hsp90 properties analyzed here are not necessarily interdependent.
Nuclear localization of DP and E2F transcription factors by heterodimeric partners and retinoblastoma protein family members
Authors: Magae Junji , Wu Chin-Lee , Illenye Sharon , Harlow Ed , Heintz Nicholas H.
Published: April 2021

E2F is a family of transcription factors implicated in the regulation of genes required for progression through G1 and entry into the S phase. The transcriptionally active forms of E2F are heterodimers composed of one polypeptide encoded by the E2F gene family and one polypeptide encoded by the DP gene family. The transcriptional activity of E2F/DP heterodimers is influenced by association with the members of the retinoblastoma tumor suppressor protein family (pRb, p107, and p130). Here the intracellular distribution of E2F and DP proteins was investigated in transiently transfected Chinese hamster and human cells. In transfected cells, DP-1 did not accumulate in the nucleus unless it was coexpressed with the heterodimeric partners E2F-1, E2F-2, or E2F-3. Domain mapping experiments showed that regions of E2F-1 and DP-1 that are required for stable association of the two proteins were also required for nuclear localization of DP-1. Unlike E2F-1, -2, and -3, E2F-4 did not accumulate in the nucleus unless it was coexpressed with DP-2. p107 and p130, but not pRb, stimulated nuclear localization of E2F-4, either alone or in combination with DP-2. These results indicate that DP proteins preferentially associate with specific E2F partners, and suggest that the ability of specific E2F/DP heterodimers to localize in the nucleus contributes to the regulation of E2F activity.
Vaccinia virus: a model system for actin-membrane interactions
Authors: Cudmore Sally , Reckmann Inge , Griffiths Gareth , Way Michael
Published: April 2021

Our understanding of the interactions between the actin cytoskeleton and cellular membranes at the molecular level is rudimentary. One system that offers an opportunity to examine these interactions in greater detail is provided by vaccinia virus, which induces the nucleation of actin tails from the outer membrane surrounding the virion. To further understand the mechanism of their formation and how they generate motility, we have examined the structure of these actin tails in detail. Actin filaments in vaccinia tails are organized so they splay out at up to 45° from the centre of the tail and are up to 0.74 μm in length, which is considerably longer than those reported in the Listeria system. Actin filaments show unidirectional polarity with their barbed filament ends pointing towards the surface of the virus particle. Rhodamine-actin incorporation experiments show that the first stage of tail assembly involves a polarized recruitment of G-actin, and not pre-formed actin filaments, to the membrane surrounding the virion. Incorporation of actin into the tail only occurs by nucleation from the viral surface, suggesting filament ends in the tail are blocked against further actin addition. As virus particles fuse with the plasma membrane during the extention of projections, actin nucleation sites previously in the viral membrane become localized to the plasma membrane, where they are able to nucleate actin polymerization in a manner analogous to the leading edge of motile cells.
Paradoxical accumulation of the cyclin-dependent kinase inhibitor p27kip1 during the cAMP-dependent mitogenic stimulation of thyroid epithelial cells
Authors: Depoortere Fabienne , Dumont Jacques E. , Roger Pierre P.
Published: April 2021

In different systems, cAMP either blocks or promotes cell cycle progression in mid to late G1 phase. Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S pre-replicative phase progression by cyclic AMP (cAMP) as a second messenger for thyrotropin (TSH). We report here that TSH markedly increases the expression of p27kip1, the inhibitor of the cell cycle and cyclin-dependent kinases. This effect was prevented by the concomitant administration of the cAMP-independent mitogens, epidermal growth factor (EGF)+serum. EGF+serum also slightly inhibited the weak basal accumulation of p27kip1. Nevertheless, in the case of stimulation by TSH alone, the cAMP-dependent cell cycle progression was fully compatible with the enhanced expression of p27kip1. This observation is paradoxical since a decrease of p27kip1 is generally associated with growth stimulation in other systems, and since a similar cAMP-dependent increase of p27kip1 in macrophages has been found responsible for mid-G1 cell cycle arrest. The opposite regulation of p27kip1 in response to TSH or EGF+serum in dog thyroid epithelial cells suggests a major difference at mid to late G1 stages between cAMP-dependent and cAMP-independent mitogenic pathways.
Characterization and quantitation of three B-type lamins in Xenopus oocytes and eggs: increase of lamin LI protein synthesis during meiotic maturation
Authors: Lourim David , Kempf Anja , Krohne Georg
Published: April 2021

We have previously shown that Xenopus oocytes, eggs, and early embryos contain lamins LII and LIII, and that portions of each are associated with distinct egg vesicle populations. We now report that a lamin similar or identical to the B-type lamin LI is also present in oocyte nuclei and in egg extracts. We have quantitated the three B-type lamins per oocyte nucleus, and have calculated relative ratios of LI:LIII = 1:100, and LII:LIII = 1:10. Similar to lamin LII, 5-15% of lamin LI is associated with egg membranes in a biochemically stable manner. Egg vesicles absorbed with lamin isoform-specific antibodies to magnetic beads indicate that lamin LI-associated egg membranes are of heterogenous morphology, and are independent from the lamin LII and LIII vesicle populations.

Compared to other nuclear envelope proteins, the synthesis of lamin LI protein is specifically elevated during meiotic maturation, resulting in a 4- to 12-fold higher amount of lamin LI in eggs than is present in oocyte nuclei. Immunofluorescence and immunoblot analysis demonstrated that lamins LI, LII, and LIII are associated with the nuclear envelope formed on demembranated sperm when added to activated egg extract. These results strongly suggest that three different lamin-associated vesicle populations are involved in the formation of a nuclear envelope in egg extracts.
Structure/function studies on the pH-dependent actin-binding protein hisactophilin in Dictyostelium mutants
Authors: Stoeckelhuber Mechthild , Noegel Angelika A. , Eckerskorn Christoph , Köhler Jana , Rieger Daniela , Schleicher Michael
Published: April 2021

Our previous studies have shown that the actin-binding protein hisactophilin from Dictyostelium discoideum is a candidate for organizing the actin cytoskeleton at the plasma membrane in a pH-dependent manner. To further characterize this interaction we isolated hisactophilin over-expression (hisII+) and hisactophilin minus (his−) mutants. D. discoideum contains two hisactophilin isoforms; both genes are independently transcribed and carry a short intron at the same position of the coding region. The deduced amino acid sequence of hisactophilin II showed a characteristic high content of 35 histidine residues out of a total 118 amino acids. After transformation of Dictyostelium AX2 wild-type cells with a genomic fragment designed to inactivate the hisactophilin I gene we obtained hisactophilin II overexpressing mutants (hisII+). Multiple integration of the vector led to strong overexpression of hisactophilin II which even outnumbered the actin concentration by a factor of two. Hisactophilin II protein showed the same biochemical properties as hisactophilin I during purification and in its pH-dependent binding to F-actin; as shown by mass spectrometry the hisactophilin II fraction was almost completely myristoylated despite of this high overexpression. The inactivation of both hisactophilin genes was achieved by gene replacement with a vector construct encompassing parts of gene I and gene II connected by a geneticin cassette. The properties of the hisII+ and his− cells with regard to growth in shaking culture and on Klebsiella plates, development, chemotaxis and morphology were not affected under normal conditions. However, the hisII+ transformants revealed a significant difference to wild-type cells and his− cells when the cytoplasmic pH was lowered by diethylstilbestrol (DES), a proton pump inhibitor. HisII+ cells were more resistant to the acidification; in contrast to AX2 wild-type cells and his− cells they did not form plasma membrane protrusions, showed an increase in F-actin content, and contained large clusters of F-actin. Lowering the internal pH caused an accumulation of hisactophilin below the plasma membrane. The fact that cells deficient in hisactophilin again lose resistance to acidification is in good agreement with the hypothesis that hisactophilin functions as a pH sensor at the plasma membrane by reversibly connecting the membrane with the actin cortical network upon local changes of the proton concentration.
Non-neural agrin codistributes with acetylcholine receptors during early differentiation of Torpedo electrocytes
Authors: Cartaud A. , Ludosky M. A. , Haasemann M. , Jung D. , Campbell K. , Cartaud J.
Published: April 2021

Agrin, an extracellular matrix protein synthesized by nerves and muscles is known to promote the clustering of acetylcholine receptors and other synaptic proteins in cultured myotubes. This observation suggests that agrin may provide at least part of the signal for synaptic specialization in vivo. The extracellular matrix components agrin, laminin and merosin bind to α-dystroglycan, a heavily glycosylated peripheral protein part of the dystrophin-glycoprotein complex, previously characterized in the sarcolemma of skeletal and cardiac muscles and at the neuromuscular junction.

In order to understand further the function of agrin and αDG in the genesis of the acetylcholine receptor-rich membrane domain, the settling of components of the dystrophin-glycoprotein complex and agrin was followed by immunofluorescence localization in developing Torpedo marmorata electrocytes. In 40-45 mm Torpedo embryos, a stage of development at which the electrocytes exhibit a definite structural polarity, dystrophin, α/β-dystroglycan and agrin accumulated concomitantly with acetylcholine receptors at the ventral pole of the cells. Among these components, agrin appeared as the most intensely concentrated and sharply localized. The scarcity of the nerve-electrocyte synaptic contacts at this stage of development, monitored by antibodies against synaptic vesicles, further indicates that before innervation, the machinery for acetylcholine receptor clustering is provided by electrocyte-derived agrin rather than by neural agrin.

These observations suggest a two-step process of acetylcholine receptor clustering involving: (i) an instructive role of electrocyte-derived agrin in the formation of a dystrophin-based membrane scaffold upon which acetylcholine receptor molecules would accumulate according to a diffusion trap model; and (ii) a maturation and/or stabilization step controlled by neural agrin. In the light of these data, the existence of more than one agrin receptor is postulated to account for the action of agrin variants at different stages of the differentiation of the postsynaptic membrane in Torpedo electrocytes.
Structural requirements for α1β1 and α2β1 integrin mediated cell adhesion to collagen V
Authors: Ruggiero Florence , Comte Jane , Cabañas Carlos , Garrone Robert
Published: April 2021

A large variety of cells adhere to and spread on specific regions within the triple helix of collagens, mainly via α1β1 and α2β1 integrins. Disruption of collagen triple helical integrity generally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the importance of the linear sequence of the constitutive α-chains versus the triple helical conformation in the recognition of collagen V binding sites.

To investigate this question, in vitro renaturation of the isolated α1(V) and α2(V) chains was performed according to the annealing procedure and formation of the triple helix was monitored by rotary shadowing and by mild trypsin digestion followed by electrophoretic analysis. The results indicate that the α1(V) and α2(V) homotrimeric reassociation can occur up to a full-length triple helix but intermediate forms of 50-200 nm long rod-like segments are also observed.

We have previously shown that α1β1 and α2β1 integrins, the major collagen receptors, are also involved in cell adhesion to native collagen V. Therefore we chose the following two different cell lines for this study: HT1080 (a human fibrosarcoma cell line) expressing α2β1 and HBL100 (a human mammary epithelial cell line) containing significant amounts of α1β1 and α2β1 integrins. We showed that both α1(V) and α2(V) homotrimers induced cell adhesion but refolded α2(V) chains were more efficient and promoted cell adhesion as well as native collagen V.

Thermal stability of refolded α-chains was monitored by adhesion promoting activity and showed that cell adhesion was dependent on triple helical conformation of the substrates. Adhesion in all cases was strongly Mg2+ and Mn2+-dependent and Ca2+ ions alone were ineffective. Antibodies against α2 and β1 integrin subunits completely inhibited HT1080 cell adhesion to all substrates. Moreover, addition of cyclic RGD peptides, which had been shown to interact with α2β1, dramatically affected HT1080 cell adhesion to native collagen V and to the refolded α-chains. Antibody to β1 subunits abolished HBL100 cell adhesion to all substrates. A complete inhibition of HBL100 cell adhesion to native collagen V was achieved only by simultaneous addition of function-blocking specific monoclonal antibodies against α1 and α2 integrin subunits. However, only α2β1 was engaged obviously in HBL100 cell adhesion to refolded α-chains.

These data indicate that triple helical conformation is particularly critical for α2β1- and α1β1-dependent adhesion and that the integrin α2β1 is a dominant functional receptor for refolded α-chains. We conclude that α2β1-dependent adhesion seems to involve multiple different conformational binding sites while α1β1-dependent adhesion is more restricted to the heterotrimeric native form of the molecule.
Apical β1 integrin in polarized MDCK cells mediates tubulocyst formation in response to type I collagen overlay
Authors: Zuk Anna , Matlin Karl S.
Published: April 2021

A number of epithelia form tubulocysts in vitro when overlaid with type I collagen gel. Because collagen receptors are generally believed to be expressed on the basolateral domain, the mechanism by which collagen elicits this morphogenetic response from the apical surface is unclear. To investigate the role of β1 integrins, the major receptor family for collagen, in this process, we overlaid polarized monolayers of MDCK II cells grown on permeable supports with type I collagen gel and correlated integrin polarity with the polarity of other apical and basolateral membrane markers during tubulocyst formation. Polarized monolayers of one clone of MDCK II cells, referred to as Heidelberg MDCK, initially respond to collagen overlay by stratifying; within 48 hours, lumena develop between the cell layers giving rise to tubulocysts. Tight junctions remain intact during tubulocyst formation because transepithelial electrical resistance does not significantly change. Major alterations are observed, however, in the expression and localization of apical and basolateral membrane markers. β1 integrins are necessary for tubulocyst morphogenesis because a function-blocking antibody administered to the apical pole of the cells completely inhibits the formation of these structures. To determine how apical-cell collagen interactions elicit tubulocyst formation, we examined whether β1 integrins are mobilized to apical plasma membranes in response to collagen overlay. We found that in the absence of collagen, polarized monolayers of Heidelberg MDCK cells endogenously express on apical plasma membranes a small pool of the β1 family, including α2β1 and α3β1. Collagen overlay does not mobilize additional β1 integrins to apical domains. If β1 integrins are not already apically expressed, as in the C6 MDCK cell line (Schoenenberger et al. (1994)J. Cell Biol. 107, 527-541), β1 integrins are not directed apically and tubulocysts do not develop in response to collagen. Thus, interaction of β1 integrins pre-existing on apical plasma membranes of polarized epithelia with type I collagen gel is the mechanism by which apical application of collagen elicits the formation of tubulocysts. Depolarized integrins on apical plasma membranes of polarized epithelia may be relevant to the pathogenesis of disease and injury.
Functional compartmentalization of the nucleus
Authors: Strouboulis John , Wolffe Alan P.
Published: April 2021

Recent applications of cell biology and molecular genetics have built an image of nuclear organization in which the molecular machines involved in transcription, RNA processing and replication assemble morphologically distinct nuclear organelles with defined functional properties. These observations indicate a very high level of structural organization for the various metabolic activities occurring within the nucleus. We discuss the possible existence of novel regulatory functions inherent to nuclear architecture itself.
Granular cells are required for encapsulation of foreign targets by insect haemocytes
Authors: Pech Louis L. , Strand Michael R.
Published: April 2021

Haemocytes play an essential role in defending invertebrates against pathogens and parasites that enter their haemocoel. A primary defense response is encapsulation; a process in which haemocytes attach to the foreign organism and kill it. Whether encapsulation requires cooperation between specific subpopulations of haemocytes is unknown. Using purified subpopulations of haemocytes and an in vitro encapsulation assay, we investigated the process of capsule formation in the insect Pseudoplusia includens. Immunocytochemical staining revealed that capsule formation involves a three step process. Encapsulation began when granular cells attached to the foreign target. This was followed by attachment of multiple layers of plasmatocytes. Termination of capsule formation occurred when a subpopulation of granular cells formed a monolayer around the periphery of the capsule. Neither granular cells nor plasmatocytes were capable of forming a capsule independently. However, plasmatocytes encapsulated targets if granular cells were present or if targets were preincubated in medium conditioned by granular cells. The effect of granular cell-conditioned medium could be blocked by the addition of the cell adhesion recognition sequence, RGDS, but not by RGES. These results demonstrate experimentally that granular cells are required for encapsulation of foreign targets by plasmatocytes in vitro, and that the role of granular cells in this process involves an RGD-dependent cell adhesion mechanism.
The intracompartmental sorting of myosin alkali light chain isoproteins reflects the sequence of developmental expression as determined by double epitope-tagging competition
Authors: Komiyama Masatoshi , Soldati Thierry , Arx Pierre von , Perriard Jean-Claude
Published: April 2021

In order to compare within the same cell the various degrees of specificity of myosin alkali light chain (MLC) isoproteins sorting to sarcomeres, a competition assay was established using double epitope tagging. Various combinations of two different MLC isoform cDNAs tagged with either a vesicular stomatitis virus VSV-G (VSV) or a medium T (mT) protein epitope were co-expressed in cultured cardiomyocytes from adult and neonatal rat ventricles. Expressed isoproteins were detected by means of anti-VSV and anti-mT antibodies and their sorting patterns were analyzed by confocal microscopy. The sorting specificity of MLC isoforms to sarcomeric sites was shown to increase in the order MLC3nm, to ML1sa, to MLC1sb, to MLC1f and MLC3f following the sequence of developmental expression. Expressed fast skeletal muscle isoforms (MLC1f and MLC3f) were always localized at the A-bands of myofibrils, while nonmuscle type (MLC3nm) was distributed throughout the cytoplasm. The slow skeletal muscle type (MLC1sa) showed a weak sarcomeric pattern if it was co-expressed with MLC3nm, but it was distributed throughout the cytoplasm when expressed in combination with MLC1f, MLC3f or the slow skeletal/ventricular muscle isoform (MLC1sb). The MLC1sb was localized at the A-bands when it was co-expressed with MLC3nm or MLC1sa, while it was also distributed to the cytoplasm if co-expressed with MLC1f or MLC3f. Further, expression of chimeric cDNAs revealed that the N-terminal lobe of each isoprotein is responsible for the isoform-specific sorting pattern.
Desmoplakin expression and organization at human umbilical vein endothelial cell-to-cell junctions
Authors: Valiron Odile , Chevrier Véronique , Usson Yves , Breviario Ferruccio , Job Didier , Dejana Elisabetta
Published: April 2021

Desmoplakin is an intracellular component of desmosomes which plays a role in the anchorage of intermediate filaments to these structures. We report here that, despite the absence of desmosomes, cultured endothelial cells from human umbilical vein express desmoplakin I and II both at mRNA and protein level. Desmoplakin I/II are found only in the detergent insoluble fraction suggesting that most of the protein is linked to the cytoskeleton. Desmoplakin I/II could be detected by western blot only in long confluent cells even if desmoplakin mRNA levels are unchanged by cell confluency. This suggests that desmoplakin might be stabilized at protein level by its association with junctional components. Immunofluorescence confocal microscopy showed that desmoplakin codistributes with VE-cadherin and plakoglobin along the lateral cell membrane. In contrast, desmoplakin localization was distinct from that of PECAM, an endothelial specific junctional protein localized outside adherence junctions. Endothelial cells do not have keratins but they express vimentin. In confluent cells vimentin forms peripheral filaments which attach to the cell membrane in areas at desmoplakin localization. These data suggest that desmoplakin may participate in the molecular organization of interendothelial junctions by interacting with VE-cadherin and promoting vimentin anchorage. This new type of intercellular junction seems to correspond to the ‘complexus adhaerentes’ described in vivo in lymphatic endothelium.
Dot-like focal contacts in adherent eosinophils, their redistribution into peripheral belts, and correlated effects on cell migration and protected zone formation
Authors: Tourkin Andre , Bonner Michael , Mantrova Ekaterina , LeRoy E. Carwile , Hoffman Stanley
Published: April 2021

We have examined the organization of F-actin and focal contacts in eosinophils and the functional consequences correlated with their redistribution. In activated eosinophils adherent to laminin, F-actin is localized in large, uniformly distributed, dot-like structures. Colocalized with the F-actin are a variety of typical components of focal contacts including: the α6 and β1 integrins subunits (which mediate eosinophil adhesion to laminin), vinculin, talin, pp125FAK, paxillin, and tyrosine phosphorylated proteins, but not the β2 integrin subunit which is not involved in eosinophil adhesion to laminin. In unactivated eosinophils on laminin (which do not adhere well) and in activated eosinophils on non-adhesive surfaces (human serum albumin, tenascin, or a mixture of tenascin and laminin), dot-like accumulations of F-actin do not form.

When activated eosinophils are incubated on laminin in the presence of low concentrations of 1,1’-ethylidenebis(L-tryptophan), a remarkable rearrangement occurs. Both conventional and confocal microscopy suggest that the dotlike structures rearrange from a uniform distribution into thick, peripheral belts surrounding each cell with few dotlike structures internal to the belt. Functional consequences occur as a result of this rearrangement of focal contacts and the cytoskeleton. The belt of focal contacts acts as a size-dependent permeability barrier; fluorescent 8 kDa polyethylene glycol does not label the substrate underneath the cells whereas 3.5 kDa polyethylene glycol labels the substrate uniformly. The formation of belts of focal contacts also blocks the ability of eosinophils to migrate on laminin in response to a chemotactic gradient. While the mechanism of 1,1’-ethylidenebis(L-tryptophan) action remains to be determined, experiments indicate that the function of 1,1’-ethylidenebis(L-tryptophan) is distinct from the function of drugs that alter tyrosine kinase and serine/threonine kinase activities. Our observations reveal a novel mechanism for regulating cell behavior in invasive cells in which the rearrangement of focal contacts and the associated cytoskeleton may control cell migration and the sequestration of secreted cytotoxic and degradatory molecules. We propose that 1,1’-ethylidenebis(L-tryptophan) may mimic the function of an endogenous regulator of eosinophil morphology and behavior.
A different approach to tumour suppression: The Alexandra Kefalides Memorial Lecture
Authors: Harris Henry , Rawlins Joy , Sharps Jane
Published: April 2021

When tumour cells are fused with normal ones, malignancy is suppressed. It has been shown that this suppression is associated with the imposition on the hybrid cell of the terminal differentiation programme of the normal parent cell. We report here the consequences of imposing the synthesis of keratin 1 and keratin 10, markers of terminal differentiation in the epidermal keratinocyte, on malignant cells of keratinocyte and non-keratinocyte lineage. We find that there is extreme selection in vivo against cells making keratin 1: tumours arising from inocula of such cells are invariably produced by the selective overgrowth of cells in which keratin 1 synthesis has been drastically reduced, usually to trace levels. No such selection operates against keratin 10. It is possible that if substantial synthesis of keratin 1 could be induced in malignant cells in a clinical context, some therapeutic benefit might accrue.
Epigenetic control of mammalian centromere protein binding: Does DNA methylation have a role?
Authors: Mitchell Arthur R. , Jeppesen Peter , Nicol Linda , Morrison Harris , Kipling David
Published: April 2021

Chromosome 1 of the inbred mouse strain DBA/2 has a polymorphism associated with the minor satellite DNA at its centromere. The more terminal block of satellite DNA sequences on this chromosome acts as the centromere as shown by the binding of CREST ACA serum, anti-CENP-B and anti-CENP-E polyclonal sera. Demethylation of the minor satellite DNA sequences accomplished by growing cells in the presence of the drug 5-aza-2’-deoxycytidine results in a redistribution of the CENP-B protein. This protein now binds to an enlarged area on the more terminal block and in addition it now binds to the more internal block of minor satellite DNA sequences on chromosome 1. The binding of the CENP-E protein does not appear to be affected by demethylation of the minor satellite sequences. We present a model to explain these observations. This model may also indicate the mechanism by which the CENP-B protein recognises specific sites within the arrays of minor satellite DNA on mouse chromosomes.
N-tropomodulin: A novel isoform of tropomodulin identified as the major binding protein to brain tropomyosin
Authors: Watakabe Akiya , Kobayashi Ryuji , Helfman David M.
Published: April 2021

We have identified and characterized two proteins in rat brain that bind to the neuron-specific tropomyosin isoform, TMBr3. The two proteins were identified by blot overlay assay, in which the proteins immobilized on the membrane were probed by epitope-tagged TMBr3, followed by detection with anti-epitope antibody. We have purified these proteins using a TMBr3 affinity column. Peptide sequencing as well as immunoblotting showed that one of the two proteins is identical to tropomodulin, a tropomyosin-binding protein originally identified in erythrocytes. The cDNA for the other protein was cloned from an adult rat brain cDNA library using degenerate oligonucleotides that we designed based on the peptide sequences. Sequence analysis of the cDNA clone revealed this protein to be a novel isoform of tropomodulin which is the product of a distinct gene, and is herein referred to as N-tropo-modulin. Recombinant N-tropomodulin bound to TMBr3 as well as to other low molecular mass tropomyosins (TM5a or TM5), but not to high molecular mass tropomyosins (TM2 or TMBr1). Northern blotting and RNase protection assays as well as immunoblotting showed that N-tropo-modulin is expressed predominantly in brain. Furthermore, RNase protection assays revealed no alternatively spliced regions within the coding sequence. Developmentally, N-tropomodulin was detected in rat brain as early as embryonic day 14 and reaches the adult level before birth. Immunofluorescence of primary frontal cortex cell cultures showed that N-tropomodulin is specifically expressed in neurons. The neuron-specific expression of N-tropomod-ulin strongly suggests specialized roles of this TM-binding protein in neurons.
C-Met signalling in an HGF/SF-insensitive variant MDCK cell line with constitutive motile/invasive behaviour
Authors: Webb C. P. , Lane K. , Dawson A. P. , Woude G. F. Vande , Warn R. M.
Published: April 2021

The Met protein is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional growth factor with mitogenic, motogenic and morphogenic properties. A morphologically altered variant of the MDCK cell line, MDCK-1, spontaneously exhibits a number of features associated with a partial HGF/SF-Met induced phenotype (less adhesive colonies in culture, enhanced invasion and motility, nascent tubule formation), but paradoxically does not respond to HGF/SF treatment. Although the overall cell surface expression and distribution of Met were found to be similar in parental MDCK cells and the MDCK-1 cell line, p145met autophosphorylation (± HGF/SF) was significantly reduced in MDCK-1 cells in vitro and in vivo when compared with parental MDCK cells. In contrast, EGF induced cell proliferation and EGF receptor autophosphorylation to similar levels in both cell lines. The basal levels of protein tyrosine phosphorylation were higher in MDCK-1 cells when compared with parental MDCK cells, including that of two prominent proteins with molecular masses of ∼185 kDa and 220 kDa. Moreover, both p185 and p220 are present and tyrosine phosphorylated in Met immunoprecipitates from MDCK-1 cells (±HGF/SF), but not parental MDCK cells. In addition, Met immunocomplexes from MDCK-1 cells exhibited an approximately 3-fold increased tyrosine kinase activity in vitro when compared with MDCK cells, correlating with the higher basal levels of total phosphotyrosine. Treatment of MDCK-1 cells with the tyrosine kinase inhibitor herbimycin A reverted the cell phenotype to a more MDCK-like morphology in culture, with a concomitant reduction in the tyrosine phosphorylation predominantly of p220. Taken together these data suggest that aberrations in Met activity and associated signalling render MDCK-1 cells insensitive to HGF/SF, and may also mediate alterations in MDCK-1 cell behaviour.
Patch-clamp capacitance analysis of the effects of α-SNAP on exocytosis in adrenal chromaffin cells
Authors: Kibble A.Victoria , Barnard Richard J. O. , Burgoyne Robert D.
Published: April 2021

We have examined the effect of α-SNAP on exocytosis in adrenal chromaffin cells by direct assay of exocytosis using patch-clamp capacitance analysis. Cells were recorded using the whole cell patch-clamp configuration and the cells dialysed with control pipette solution or with a pipette solution containing α-SNAP or the deletion mutant α-SNAP(41-295). The deletion mutant was found to be unable to bind to syntaxin allowing a test of the requirement for syntaxin-binding for any effect of α-SNAP on exocytosis. Following cell dialysis for 10 minutes, cells were depo-larised five times at 2 minute intervals. At each depolari-sation step cells dialysed with α-SNAP showed a significant increase in both the initial rate and extent of exocytosis which was seen as a rise in membrane capacitance. This increase in exocytosis was not observed with α-SNAP(41-295) which instead produced some inhibition of the extent but had no effect on the initial rate of exocytosis. These results show directly that α-SNAP has a specific and marked stimulatory effect on exocytosis in chromaffin cells.
On the existence of a guanine nucleotide trap, the role of adenosine kinase and a possible cause of excessive purine production in mammalian cells
Authors: Green H. , Ishii K.
Published: April 2021

It is usually assumed, in part from studies of bacteria, that there is free interconversion of adenosine 5′-monophosphate (AMP) and guanosine s′-monophosphate (GMP), and that the balance between the 2 nucleotides is maintained exactly by allosteric controls on the activity of the enzymes involved in the interconversion. However, there are good reasons for believing that in most mammalian cells the conversion of GMP to AMP is not an effective process. Furthermore, the activity of certain other enzymes of the purine scavenger pathways will tend to induce imbalance between AMP and GMP. This imbalance will be compensated, but the compensation mechanism will involve increased production and excretion of purines. It is suggested that the operation of this mechanism in the human would result in gout.
An ultrastructural study of the membranes of keratinizing wool follicle cells
Authors: Orwin D.F.G. , Thomson R.W.
Published: April 2021

Measurements of the widths of apposed plasma membranes and of the spaces between them were made at different stages of differentiation of keratinizing and hardening cells of the wool follicle. In contrast to findings in the hair follicle, no changes were detected for apposed cortex, cortex/fibre cuticle and fibre cuticle/fibre cuticle cells until keratinization had taken place. The trilaminar appearance of the plasma membranes was then lost and the intercellular material decreased in width.

However, the inner root sheath cells developed a ‘membrane complex’ in enlarged inter-cellular spaces prior to hardening. Desmosomes are apparently retained in the hardened ‘membrane complexes’. A band of cytoplasmic material was also formed adjacent to the inner lamellae of the plasma membranes immediately before hardening of the cells.

The presence of gap and tight junctions in differentiating cell lines of the wool follicle was noted.
Real and reflected images of cells in profile: I. a method for the study of cell movement and adhesion
Authors: Hlinka J. , Sanders F. K.
Published: April 2021

A new way of illuminating and photographing cells in profile has been developed, showing at the same time their images reflected in a glass substrate. Through its use in cinemicrography, the kinematics of cells and their adhesions can be observed with certainty, thus permitting a better understanding of their mode of locomotion.