bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase Cα actively downregulates through caveolae-dependent traffic to an endosomal compartment
Authors: Prevostel Corinne , Alice Vallentin , Joubert Dominique , Parker Peter J.
Published: April 2021

Receptor desensitization occurs through receptor internalization and targeting to endosomes, a prerequisite for sorting and degradation. Such trafficking processes may not be restricted to membrane associated receptors but may also play an important role in the downregulation of cytoplasmic transducers such as protein kinase C (PKC). It is demonstrated here that acute TPA exposure induces the transport of activated PKC α from the plasma membrane to endosomes. This process requires PKC activity and catalytically competent PKC can even promote a similar process for a truncated regulatory domain PKC α protein. It is established that PKC α is targeted to the endosome compartment as an active kinase, where it colocalizes with annexin I, a substrate of PKC. Thus, PKC α downregulation shares features with plasma membrane associated receptor sorting and degradation. However, it is shown that PKC α delivery to the endosome compartment is not a Rab5 mediated process in contrast to the well characterised internalisation of the transferrin receptor. An alternative route for PKC α is evidenced by the finding that the cholesterol binding drugs nystatin and filipin, known to inhibit caveolae mediated trafficking, are able to block PKC α traffic and down regulation. Consistent with this, the endosomes where PKC α is found also contain caveolin. It is concluded that the initial step in desensitisation of PKC α involves active delivery to endosomes via a caveolae mediated process.
High resolution analysis of interphase chromosome domains
Authors: Visser Astrid E. , Jaunin Françoise , Fakan Stanislav , Aten Jacob A.
Published: April 2021

Chromosome territories need to be well defined at high resolution before functional aspects of chromosome organization in interphase can be explored. To visualize chromosomes by electron microscopy (EM), the DNA of Chinese hamster fibroblasts was labeled in vivo with thymidine analogue BrdU. Labeled chromosomes were then segregated during several cell cycles to obtain nuclei containing only 2 to 3 labeled chromosomes. Subsequent immunocytochemical detection of BrdU allowed analysis by EM of chromosome territories and subchromosomal domains in well preserved nuclei. Our results provide the first high resolution visualization of chromosomes in interphase nuclei. We show that chromosome domains are either separated from one another by interchromatin space or are in close contact with no or little intermingling of their DNA. This demonstrates that, while chromosomes form discrete territories, chromatin of adjacent chromosomes may be in contact in limited regions, thus implying chromosome-chromosome interactions. Chromosomes are organized as condensed chromatin with dispersed chromatin extending into the interchromatin space that is largely devoid of DNA. The interchromatin space, which is known to be involved in various nuclear functions, forms interconnecting channels running through and around chromosome territories. Functional implications of this organization are discussed.
CAF-1 and the inheritance of chromatin states: at the crossroads of DNA replication and repair
Authors: Ridgway Patricia , Almouzni Geneviève
Published: April 2021

Chromatin is no longer considered to be a static structural framework for packaging DNA within the nucleus but is instead believed to be an interactive component of DNA metabolism. The ordered assembly of chromatin produces a nucleoprotein template capable of epigenetically regulating the expression and maintenance of the genome. Factors have been isolated from cell extracts that stimulate early steps in chromatin assembly in vitro. The function of one such factor, chromatin-assembly factor 1 (CAF-1), might extend beyond simply facilitating the progression through an individual assembly reaction to its active participation in a marking system. This marking system could be exploited at the crossroads of DNA replication and repair to monitor genome integrity and to define particular epigenetic states.
Mammalian cell lines expressing functional RNA polymerase II tagged with the green fluorescent protein
Authors: Sugaya Kimihiko , Vigneron Marc , Cook Peter R.
Published: April 2021

RNA polymerase II is a multi-subunit enzyme responsible for transcription of most eukaryotic genes. It associates with other complexes to form enormous multifunctional ‘holoenzymes’ involved in splicing and polyadenylation. We wished to study these different complexes in living cells, so we generated cell lines expressing the largest, catalytic, subunit of the polymerase tagged with the green fluorescent protein. The tagged enzyme complements a deficiency in tsTM4 cells that have a temperature-sensitive mutation in the largest subunit. Some of the tagged subunit is incorporated into engaged transcription complexes like the wild-type protein; it both resists extraction with sarkosyl and is hyperphosphorylated at its C terminus. Remarkably, subunits bearing such a tag can be incorporated into the active enzyme, despite the size and complexity of the polymerizing complex. Therefore, these cells should prove useful in the analysis of the dynamics of transcription in living cells.
Goosecoid suppresses cell growth and enhances neuronal differentiation of PC12 cells
Authors: Sawada Ken , Konishi Yuko , Tominaga Mari , Watanabe Yoshiya , Hirano Jun , Inoue Shimpei , Kageyama Ryoichiro , Blum Martin , Tominaga Akira
Published: April 2021

In all vertebrate species, the homeobox gene goosecoid serves as a marker of the Spemann organizer tissue. One function of the organizer is the induction of neural tissue. To investigate the role of goosecoid in neuronal differentiation of mammalian cells, we have introduced goosecoid into PC12 cells. Expression of goosecoid resulted in reduced cell proliferation and enhanced neurite outgrowth in response to NGF. Expression of goosecoid led to a decrease in the percentage of S-phase cells and to upregulation of the expression of the neuron-specific markers MAP-1b and neurofilament-L. Analysis of goosecoid mutants revealed that these effects were independent of either DNA binding or homodimerization of Goosecoid. Coexpression of the N-terminal portion of the ets transcription factor PU.1, a protein that can bind to Goosecoid, repressed neurite outgrowth and rescued the proliferation of PC12 cultures. In contrast, expression of the bHLH transcription factor HES-1 repressed goosecoid- mediated neurite outgrowth without changing the proportion of S-phase cells. These results suggest that goosecoid is involved in neuronal differentiation in two ways, by slowing the cell cycle and stimulating neurite outgrowth, and that these two events are separately regulated.
Role of the cytoskeleton in rapid activation of CD11b/CD18 function and its subsequent downregulation in neutrophils
Authors: Anderson Stephen I. , Hotchin Neil A. , Nash Gerard B.
Published: April 2021

When rolling adherent neutrophils are stimulated, they rapidly immobilise through activation of integrin CD11b/CD18, and then modulate attachment through this integrin to allow migration. We investigated links between cytoskeletal rearrangement and changes in function of integrin CD11b/CD18 in neutrophils stimulated with formyl peptide (fMLP). Neutrophils treated with the actin- polymerising agent jasplakinolide became rolling adherent on monolayers of activated platelets, but could not use CD11b/CD18 to become immobilised when fMLP was perfused over them. If treated with jasplakinolide after fMLP, the cells stopped migrating but could not detach when fMLP was removed. Jasplakinolide did not inhibit changes in intracellular Ca2+ seen after fMLP treatment, or inhibit neutrophil immobilisation induced by externally added Mn2+. Thus cytoskeletal rearrangement was directly implicated in upregulation and, later, downregulation of CD11b/CD18 binding. Inhibition of RhoA with C3- transferase caused a dose-dependent reduction of initial rolling adhesion of neutrophils, and reduced the rate of migration after stimulation; however, neither the conversion of rolling to stationary adhesion, nor the ability of neutrophils to detach on removal of the stimulus, were inhibited. Thus, Rho may regulate actin polymerisation and motility in neutrophils, but did not appear to control integrin-mediated adhesion itself. Integrin binding may be promoted by disruption of links to the cytoskeleton, effected through depolymerisation of actin or cleavage of linking protein talin by calpain. Disruption of actin filaments with cytochalasin D did not, however, cause integrin-mediated immobilisation of rolling neutrophils. Although the calpain inhibitor calpeptin did inhibit the adhesion response to fMLP, this was only at doses where actin polymerisation was also ablated. We suggest that the cytoskeleton actively regulates binding conformation of CD11b/CD18 as well as its mobility in the membrane.
Trafficking and signaling through the cytoskeleton: a specific mechanism
Authors: Shafrir Yinon , ben-Avraham Daniel , Forgacs Gabor
Published: April 2021

A specific mechanism for the intracellular translocation of nonvesicle-associated proteins is proposed. This movement machinery is based on the assumption that the cytoskeleton represents an interconnected network of filamentous macromolecules, which extends over the entire cytoplasm. Diffusion along the filaments provides an efficient way for movement and with this, for signal transduction, between various intracellular compartments. We calculate the First Passage Time (FPT), the average time it takes a signaling molecule, diffusing along the cytoskeleton, to arrive from the cell surface to the nucleus for the first time. We compare our results with the FPT of free diffusion and of diffusion in the permeating cytoplasm. The latter is hindered by intracellular organelles and the cytoskeleton itself. We find that for filament concentrations even below physiological values, the FPT along cytoskeletal filaments converges to that for free diffusion. When filaments are considered as obstacles, the FPT grows steadily with filament concentration. At realistic filament concentrations the FPT is insensitive to local modifications in the cytoskeletal network, including bundle formation. We conclude that diffusion along cytoskeletal tracks is a reliable alternative to other established ways of intracellular trafficking and signaling, and therefore provides an additional level of cell function regulation.
SOCS: physiological suppressors of cytokine signaling
Authors: Krebs Danielle L. , Hilton Douglas J.
Published: April 2021

Cytokines regulate cellular behavior by interacting with receptors on the plasma membrane of target cells and activating intracellular signal transduction cascades such as the JAK-STAT pathway. Suppressors of cytokine signaling (SOCS) proteins negatively regulate cytokine signaling. The SOCS family consists of eight proteins: SOCS1-SOCS7 and CIS, each of which contains a central Src-homology 2 (SH2) domain and a C-terminal SOCS box. The expression of CIS, SOCS1, SOCS2 and SOCS3 is induced in response to stimulation by a wide variety of cytokines, and overexpression of these proteins in cell lines results in inhibition of cytokine signaling. Thus, SOCS roteins appear to form part of a classical negative feedback loop. The analysis of mice lacking SOCS1 has revealed that it is critical in the negative regulation of IFNγ signaling and in the differentiation of T cells. Additionally, the analysis of mouse embryos lacking SOCS3 suggests that SOCS3 negatively regulates fetal liver erythropoiesis, probably through its ability to modulate erythropoietin (Epo) signaling. Thus, the use of gene targeting has confirmed that SOCS proteins regulate cytokine signaling in a physiological setting.
RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis
Authors: Jullien-Flores Viviana , Mahé Yannick , Mirey Gladys , Leprince Corinne , Meunier-Bisceuil Brigitte , Sorkin Alexander , Camonis Jacques H.
Published: April 2021

RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral- RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. μ2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates μ2 from μ1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.
Laminin and α7β1 integrin regulate agrin-induced clustering of acetylcholine receptors
Authors: Burkin Dean J. , Kim Jae Eun , Gu Maojian , Kaufman Stephen J.
Published: April 2021

The clustering of acetylcholine receptors (AChRs) in the post-synaptic membrane of skeletal muscle is an early developmental event in the formation of the neuromuscular junction. Several studies show that laminin, as well as neural agrin, can induce AChR clustering in C2C12 myofibers. We recently showed that specific isoforms of the α7β1 integrin (a receptor normally found at neuromuscular junctions) colocalize and physically interact with AChR clusters in a laminin-dependent fashion. In contrast, induction with agrin alone fails to promote localization of the integrin with AChR clusters. Together both agrin and laminin enhance the interaction of the integrin with AChRs and their aggregation into clusters. To further understand this mechanism we investigated cluster formation and the association of the α7β1 integrin and AChR over time following induction with laminin and/or agrin. Our results show that the α7β1 integrin associates with AChRs early during the formation of the post-synaptic membrane and that laminin modulates this recruitment. Laminin induces a rapid stable association of the integrin and AChRs and this association is independent of clustering. In addition to laminin-1, merosin (laminin-2/4) is present both before and after formation of neuromuscular junctions and also promotes AChR clustering and colocalization with the integrin as well as synergism with agrin. Using site directed mutagenesis we demonstrate that a tyrosine residue in the cytoplasmic domain of both α7A and α7B chains regulates the localization of the integrin with AChR clusters. We also provide evidence that laminin, through its association with the α7β1 integrin, reduces by 20-fold the concentration of agrin required to promote AChR clustering and accelerates the formation of clusters. Thus laminin, agrin and the α7β1 integrin act in a concerted manner early in the development of the post-synaptic membrane, with laminin priming newly formed myofibers to rapidly and vigorously respond to low concentrations of neural agrin produced by innervating motor neurons.
Somatic linker histone H1 is present throughout mouse embryogenesis and is not replaced by variant H1°
Authors: Adenot Pierre G. , Campion Evelyne , Legouy Edith , Allis C. David , Dimitrov Stefan , Renard Jean-Paul , Thompson Eric M.
Published: April 2021

A striking feature of early embryogenesis in a number of organisms is the use of embryonic linker histones or high mobility group proteins in place of somatic histone H1. The transition in chromatin composition towards somatic H1 appears to be correlated with a major increase in transcription at the activation of the zygotic genome. Previous studies have supported the idea that the mouse embryo essentially follows this pattern, with the significant difference that the substitute linker histone might be the differentiation variant H1°, rather than an embryonic variant. We show that histone H1° is not a major linker histone during early mouse development. Instead, somatic H1 was present throughout this period. Though present in mature oocytes, somatic H1 was not found on maternal metaphase II chromatin. Upon formation of pronuclear envelopes, somatic H1 was rapidly incorporated onto maternal and paternal chromatin, and the amount of somatic H1 steadily increased on embryonic chromatin through to the 8-cell stage. Microinjection of somatic H1 into oocytes, and nuclear transfer experiments, demonstrated that factors in the oocyte cytoplasm and the nuclear envelope, played central roles in regulating the loading of H1 onto chromatin. Exchange of H1 from transferred nuclei onto maternal chromatin required breakdown of the nuclear envelope and the extent of exchange was inversely correlated with the developmental advancement of the donor nucleus.
The C-terminal tail domain of neurofilament protein-H (NF-H) forms the crossbridges and regulates neurofilament bundle formation
Authors: Chen Jianguo , Nakata Takao , Zhang Zhizeng , Hirokawa Nobutaka
Published: April 2021

In order to study the role of NF-H in a neurofilament network formation in neurons, we coexpressed NF-H with neurofilament protein-L (NF-L) in Sf9 cells using the baculovirus expression system. Electron microscopy observations revealed that parallel arrays of 10 nm filaments with frequent crossbridges between adjacent filaments were formed in the cytoplasm of Sf9 cells infected with the recombinant virus that co-expressed NF-L and NF-H. To explore the function of the C-terminal tail domain of NF-H, various deletion mutants lacking portions of the tail domain were constructed, and each of them was coexpressed with NF-L. The results show that the tail domain of NF-H is a structural component of crossbridges and is involved in parallel bundle formation of neurofilaments, as core filaments of the axon. The last 191 amino acids of the C-terminal tail domain of NF-H play a key role in crossbridge formation.
Expression-site-associated gene 8 (ESAG8) of Trypanosoma brucei is apparently essential and accumulates in the nucleolus
Authors: Hoek Maarten , Engstler Markus , Cross George A. M.
Published: April 2021

Trypanosoma brucei variant surface glycoprotein expression sites are interesting examples of genomic loci under complex epigenetic control. In the infectious bloodstream stage, only one of about 20 expression sites is actively transcribed. In the Tsetse midgut (procyclic) stage, chromatin remodeling silences all expression sites. We have begun to explore the function of one of the expression-site-associated genes, ESAG8. Gene knockout experiments implied that ESAG8 is essential. ESAG8 is present at a very low level and apparently accumulates in the nucleolus. A 32-amino-acid domain, which contains a putative bipartite nuclear localization signal (NLS), is both necessary and sufficient to target fusions of ESAG8, with Aequorea victoria green fluorescent protein, to the trypanosome nucleolus. This same sequence functioned only as an NLS in mammalian cells, supporting the idea that nucleolar accumulation requires specific interactions. These results have implications for models of ESAG8 function.
High dosage expression of a zinc finger protein, Grt1, suppresses a mutant of fission yeast slp1+, a homolog of CDC20/p55CDC/Fizzy
Authors: Yamada Hiroshi Y. , Matsumoto Sanae , Matsumoto Tomohiro
Published: April 2021

Selective proteolysis at and after the onset of anaphase is a key cell cycle event required for sister chromatid separation as well as for exit from mitosis. It requires ubiquitination of substrates by Anaphase Promoting Complex(APC)/ Cyclosome. Slp1, a WD-repeat protein, is a putative activator for APC in fission yeast. With another WD-repeat protein, Ste9/Srw1, it is thought to promote the proteolysis in a substrate-specific manner. We report here characterization of a temperature-sensitive (ts) slp1 mutant and its high-dosage suppressor, grt1+. In cells arrested in metaphase, wild-type Slp1 was preferentially found in a complex with hyperphosphorylated Cut9 (subunit of APC), whereas the ts Slp1 protein, lacking the last 113 amino acids, failed to interact with Cut9. The temperature sensitivity was suppressed by high dosage expression of a zinc finger protein, Grt1. The ts slp1 mutant was unable to maintain the normal level of Grt1 protein. The reduction in the Grt1 level may be a primary defect since high dosage expression of grt1+rescues the slp1 mutant. The grt1-suppression had an additive effect to Δste9 and wee1-50, both of which partially suppress the ts slp1 mutant. Therefore, grt1+ would define an independent pathway that facilitates the function of Slp1.
Connexin-specific distribution within gap junctions revealed in living cells
Authors: Falk Matthias M.
Published: April 2021

To study the organization of gap junctions in living cells, the connexin isotypes α1(Cx43), β1(Cx32) and β2(Cx26) were tagged with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. The cellular fate of the tagged connexins was followed by high-resolution fluorescence deconvolution microscopy and time-lapse imaging. Comprehensive analyses demonstrated that the tagged channels were functional as monitored by dye transfer, even under conditions where the channels were assembled solely from tagged connexins. High-resolution images revealed a detailed structural organization, and volume reconstructions provided a three-dimensional view of entire gap junction plaques. Specifically, deconvolved dual-color images of gap junction plaques assembled from CFP- and YFP-tagged connexins revealed that different connexin isotypes gathered within the same plaques. Connexins either codistributed homogeneously throughout the plaque, or each connexin isotype segregated into well-separated domains. The studies demonstrate that the mode of channel distribution strictly depends on the connexin isotypes. Based on previous studies on the synthesis and assembly of connexins I suggest that channel distribution is regulated by intrinsic connexin isotype specific signals.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs1735.html, Movie 2, Movie 3, Movie 4, Movie 5
Book Reviews
Authors: Witkowski J.
Published: April 2021

If you are anywhere on the spectrum from frequent Cold Spring Harbor visitor to someone who barely knows that Symposia of that name were until recently published in maroon covers, and if you want to learn more of the history of this remarkable research centre, then this book is for you. At first sight, Illuminating Life looks like a coffee table book, but it is much more than that. Jan Witkowski has assembled a history of the Cold Spring Harbor Laboratories from their inception in 1890 through to 1968, illustrated by a selection of research papers from 1903 to 1969. Each one or two papers is preceded by an interpretative essay and a biographical note on the principal author(s), and the whole is introduced by an informative historical preface. At the end are three obituaries from the literature summarizing the lives of three key players, Davenport, Harris and Demerec.
The FHA domain mediates phosphoprotein interactions
Authors: Li Jia , Lee Gui-in , Doren Steven R. Van , Walker John C.
Published: April 2021

The forkhead-associated (FHA) domain is a phosphopeptide-binding domain first identified in a group of forkhead transcription factors but is present in a wide variety of proteins from both prokaryotes and eukaryotes. In yeast and human, many proteins containing an FHA domain are found in the nucleus and involved in DNA repair, cell cycle arrest, or pre-mRNA processing. In plants, the FHA domain is part of a protein that is localized to the plasma membrane and participates in the regulation of receptor-like protein kinase signaling pathways. Recent studies show that a functional FHA domain consists of 120-140 amino acid residues, which is significantly larger than the sequence motif first described. Although FHA domains do not exhibit extensive sequence similarity, they share similar secondary and tertiary structures, featuring a sandwich of two anti-parallel β-sheets. One intriguing finding is that FHA domains may bind phosphothreonine, phosphoserine and sometimes phosphotyrosine, distinguishing them from other well-studied phosphoprotein-binding domains. The diversity of proteins containing FHA domains and potential differences in binding specificities suggest the FHA domain is involved in coordinating diverse cellular processes.
Live analysis of lagging chromosomes during anaphase and their effect on spindle elongation rate in fission yeast
Authors: Pidoux Alison L. , Uzawa Satoru , Perry Paul E. , Cande W. Zacheus , Allshire Robin C.
Published: April 2021

The fission yeast Schizosaccharomyces pombe is widely used as a model system for studies of the cell cycle and chromosome biology. To enhance these studies we have fused GFP to the chromodomain protein Swi6p, thus allowing nuclear and chromosome behaviour to be followed in living cells using time-lapse fluorescence microscopy. Like endogenous Swi6p, GFP-Swi6p localises to the nucleus and is concentrated at the heterochromatic centromeres and telomeres. The nucleus is highly dynamic during interphase: the clustered centromeres, in particular, are highly mobile. By expressing GFP-α2-tubulin and GFP-Swi6p in the same cells we observe that the clustered centromeres move in concert with the cytoplasmic microtubules, which is likely to reflect their association with the spindle pole body. Drug treatment indicates that this movement is dependent on intact cytoplasmic microtubules. We have also used GFP-Swi6p to investigate the properties of lagging chromosomes observed in mutants with defects in chromosome segregation. Lagging chromosomes display a variety of behaviours on anaphase spindles, most surprisingly, chromosomes appear to initiate microtubule interactions and move to the poles late in anaphase B. Interestingly, in cells displaying lagging chromosomes, the rate of spindle elongation is slowed by a factor of two. This suggests that cells are able to sense the presence of a lagging chromosome and slow anaphase B in order to allow it extra time to reach the pole. However, this mechanism is not dependent on the spindle checkpoint proteins Bub1p or Dma1p, raising the possibility that a novel checkpoint mechanism operates to retard spindle elongation if lagging chromosomes are detected. An alternative model is also discussed in which single defective kinetochores on lagging chromatids are able to interact simultaneously with microtubules emanating from both poles and affect spindle dynamics by counteracting the spindle elongation force.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs1686.html, Movie FIG2A, Movie FIG2B, Movie FIG2C, Movie FIG2D, Movie FIG3A, Movie FIG3B, Movie FIG3C, Movie FIG5A, Movie FIG5B, Movie FIG5C, Movie FIG5D, Movie FIG5E, Movie FIG5F, Movie FIG5G, Movie FIG5H, Movie FIG5I, Movie FIG5J, Movie FIG8CSP10-2
Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation
Authors: Meerson Nirina Rajho , Bello Valérie , Delaunay Jean-Louis , Aït Slimane Tounsia , Delautier Danièle , Lenoir Christelle , Trugnan Germain , Maurice Michèle
Published: April 2021

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic 35S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.
p125 focal adhesion kinase promotes malignant astrocytoma cell proliferation in vivo
Authors: Wang Dongyan , Grammer J. Robert , Cobbs Charles S. , Stewart Jerry E. , Liu Zhiyong , Rhoden Rosaline , Hecker Timothy P. , Ding Qiang , Gladson Candece L.
Published: April 2021

p125 focal adhesion kinase (p125FAK) is a cytoplasmic tyrosine kinase that is activated upon engagement of integrin cell adhesion receptors, and initiates several signaling events that modulate cell function in vitro. To determine the biologic role of p125FAK in malignant astrocytic tumor cells, U-251MG human malignant astrocytoma cells were stably transfected with p125FAK cDNA using the TET-ON system, and stable clones isolated that exhibited an estimated 5-or 20-fold increase in p125FAK expression on administration of 0.1 or 2.0 μg/ml doxycycline, respectively. In vitro studies demonstrated that induction of p125FAK resulted in a 2-to 3-fold increase in cell migration, increased p130CAS phosphorylation, localization of exogenous p125FAK to focal adhesions, and a 2-fold increase in soft agar growth. To determine the role of p125FAK in vivo, clones were injected stereotactically into the brains of scid mice. A 4.5-fold estimated increase in p125FAK expression was induced by administration of doxycycline in the drinking water. Analysis of xenograft brains demonstrated that, upon induction of p125FAK, there was a 1.6-to 2.8-fold increase in tumor cell number, and an increase in mAb PCNA-labeling of tumor cells in the absence of a change in the apoptotic index. Compared to normal brain, the expression of p125FAK was elevated in malignant astrocytic tumor biopsies from patient samples. These data demonstrate for the first time that p125FAK promotes tumor cell proliferation in vivo, and that the underlying mechanism is not associated with a reduction in apoptosis.
Contractile apparatus of the normal and abortive cytokinetic cells during mouse male meiosis
Authors: Manandhar G. , Moreno R. D. , Simerly C. , Toshimori K. , Schatten G.
Published: April 2021

Mouse male meiotic cytokinesis was studied using immunofluorescent probes against various elements of cytokinetic apparatus and electron microscopy. In normal mice, some spermatocytes fail to undergo cytokinesis after meiotic I or II nuclear divisions, forming syncytial secondary spermatocytes and spermatids. Abnormal cytokinetic cells develop sparse and dispersed midzone spindles during the early stage. However, during late stages, single and compact midzone spindles are formed as in normal cells, but localize asymmetrically and attach to the cortex. Myosin and f-actin were observed in the midzone spindle and midbody regions of normally cleaving cells as well as in those cells that failed to develop a cytokinetic furrow, implying that cytokinetic failure is unlikely to be due to defect in myosin or actin assembly. Depolymerization of microtubules by nocodazole resulted in the loss of the midbody-associated f-actin and myosin. These observations suggest that actin-myosin localization in the midbody could be a microtubule-dependent process that may not play a direct role in cytokinetic furrowing. Anti-centrin antibody labels the putative centrioles while anti-γ-tubulin antibody labels the minus-ends of the midzone spindles of late-stage normal and abnormal cytokinetic cells, suggesting that the centrosome and midzone spindle nucleation in abnormal cytokinetic cells is not different from those of normally cleaving cells. Possible use of mouse male meiotic cells as a model system to study cytokinesis has been discussed.
The novel protein Ccz1p required for vacuolar assembly in Saccharomyces cerevisiae functions in the same transport pathway as Ypt7p
Authors: Kucharczyk Róza , Dupre Sophie , Avaro Sandrine , Haguenauer-Tsapis Rosine , Słonimski Piotr P. , Rytka Joanna
Published: April 2021

CCZ1 was previously identified by the sensitivity of ccz1Δ mutants to high concentrations of Caffeine and the divalent ions Ca2+ and Zn2+. In this paper we show that deletion of CCZ1 leads to aberrant vacuole morphology, similar to the one reported for the family of vacuolar protein sorting (vps) mutants of class B. The ccz1Δ cells display severe vacuolar protein sorting defects for both the soluble carboxipeptidase Y and the membrane-bound alkaline phosphatase, which are delivered to the vacuole by distinct routes. Ccz1p is a membranous protein and the vast majority of Ccz1p resides in late endosomes. These results, along with a functional linkage found between the CCZ1 and YPT7 genes, indicate that the site of Ccz1p function is at the last step of fusion of multiple transport intermediates with the vacuole.
Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruce
Authors: Filonova Lada H. , Bozhkov Peter V. , Brukhin Vladimir B. , Daniel Geoffrey , Zhivotovsky Boris , Arnold Sara von
Published: April 2021

In the animal life cycle, the earliest manifestations of programmed cell death (PCD) can already be seen during embryogenesis. The aim of this work was to determine if PCD is also involved in the elimination of certain cells during plant embryogenesis. We used a model system of Norway spruce somatic embryogenesis, which represents a multistep developmental pathway with two broad phases. The first phase is represented by proliferating proembryogenic masses (PEMs). The second phase encompasses development of somatic embryos, which arise from PEMs and proceed through the same sequence of stages as described for their zygotic counterparts.

Here we demonstrate two successive waves of PCD, which are implicated in the transition from PEMs to somatic embryos and in correct embryonic pattern formation, respectively. The first wave of PCD is responsible for the degradation of PEMs when they give rise to somatic embryos. We show that PCD in PEM cells and embryo formation are closely interlinked processes, both stimulated upon withdrawal or partial depletion of auxins and cytokinins. The second wave of PCD eliminates terminally differentiated embryo-suspensor cells during early embryogeny.

During the dismantling phase of PCD, PEM and embryo-suspensor cells exhibit progressive autolysis, resulting in the formation of a large central vacuole. Autolytic degradation of the cytoplasm is accompanied by lobing and budding-like segmentation of the nucleus. Nuclear DNA undergoes fragmentation into both large fragments of about 50 kb and multiples of approximately 180 bp. The tonoplast rupture is delayed until lysis of the cytoplasm and organelles, including the nucleus, is almost complete. The protoplasm then disappears, leaving a cellular corpse represented by only the cell wall. This pathway of cell dismantling suggests overlapping of apoptotic and autophagic types of PCD during somatic embryogenesis in Norway spruce.
Human zonulin, a potential modulator of intestinal tight junctions
Authors: Wang Wenle , Uzzau Sergio , Goldblum Simeon E. , Fasano Alessio
Published: April 2021

Intercellular tight junctions are dynamic structures involved in vectorial transport of water and electrolytes across the intestinal epithelium. Zonula occludens toxin derived from Vibrio cholerae interacts with a specific intestinal epithelial surface receptor, with subsequent activation of a complex intracellular cascade of events that regulate tight junction permeability. We postulated that this toxin may mimic the effect of a functionally and immunologically related endogenous modulator of intestinal tight junctions. Affinity-purified anti-zonula occludens toxin antibodies and the Ussing chamber assay were used to screen for one or more mammalian zonula occludens toxin analogues in both fetal and adult human intestine. A novel protein, zonulin, was identified that induces tight junction disassembly in non-human primate intestinal epithelia mounted in Ussing chambers. Comparison of amino acids in the active zonula occludens toxin fragment and zonulin permitted the identification of the putative receptor binding domain within the N-terminal region of the two proteins. Zonulin likely plays a pivotal role in tight junction regulation during developmental, physiological, and pathological processes, including tissue morphogenesis, movement of fluid, macromolecules and leukocytes between the intestinal lumen and the interstitium, and inflammatory/autoimmune disorders.
Ectodomain shedding, translocation and synthesis of SorLA are stimulated by its ligand head activator
Authors: Hampe Wolfgang , Björn Riedel I. , Lintzel Julia , Bader Christian O. , Franke Inga , Schaller H. Chica
Published: April 2021

The single transmembrane receptor SorLA is the mammalian orthologue of the head activator-binding protein, HAB, from hydra. The human neuronal precursor cell line NT2 and the neuroendocrine cell line BON produce head activator (HA) and respond to HA by entry into mitosis and cell proliferation. They express SorLA, and bind HA with nanomolar affinity. HA coupled to Sepharose is able to precipitate SorLA specifically proving that SorLA binds HA. Using antisera directed against extra- and intracellular epitopes we find SorLA as membrane receptor and as soluble protein released from cells into the culture medium. Cell lines differ strongly in processing of SorLA, with NT2 cells expressing SorLA mainly as membrane receptor, whereas release predominates in BON cells. Soluble SorLA lacks the intracellular domain and is shed from the transmembrane protein by a metalloprotease. Release from cells and brain slices is stimulated by HA and by phorbol ester, and it is blocked by a metalloprotease inhibitor and by lowering the temperature to 20°C. Blockade of SorLA shedding and treatment of cells with SorLA antisense oligonucleotides lead to a decrease in the rate of cell proliferation. From this we conclude that SorLA is necessary to mediate the mitogenic effect of endogenous HA. HA enhances the translocation of SorLA from internal membranes to the cell surface and its internalization. In addition, HA stimulates SorLA synthesis hinting at an autocatalytic feedback loop in which the ligand activates production, processing, and translocation of its receptor.
Cathepsin K in thyroid epithelial cells: sequence, localization and possible function in extracellular proteolysis of thyroglobulin
Authors: Tepel Carmen , Brömme Dieter , Herzog Volker , Brix Klaudia
Published: April 2021

Extracellular proteolysis of thyroglobulin at the apical surface of thyroid epithelial cells results in liberation of thyroxine, and is mediated by lysosomal cysteine proteases such as cathepsins B and L. Here, we report on the expression of the cysteine protease cathepsin K in thyroid epithelial cells. The cDNA for porcine thyroid cathepsin K showed homologies ranging from 71% to 94% to the cDNA of cathepsin K from various species and cell types. The deduced amino acid sequence of porcine thyroid cathepsin K predicted a 37 kDa preproenzyme, with the active site residues Cys-140, His-277 and Asn-297, and one potential N-glycosylation site. The localization of cathepsin K was not restricted to lysosomes. Rather, secreted cathepsin K was predominantly found within the follicular lumen and in association with the apical plasma membrane of thyroid epithelial cells. Enzyme cytochemistry showed that cell-surface associated cathepsin K was proteolytically active at neutral pH. In vitro, recombinant cathepsin K liberated thyroxine from thyroglobulin by limited proteolysis at neutral pH. We postulate that its localization enables cathepsin K to contribute to the extracellular proteolysis of thyroglobulin, i.e. thyroid hormone liberation, at the apical surface of thyroid epithelial cells in situ.
All three PACSIN isoforms bind to endocytic proteins and inhibit endocytosis
Authors: Modregger Jan , Ritter Brigitte , Witter Brigitte , Paulsson Mats , Plomann Markus
Published: April 2021

The PACSINs are a family of cytoplasmic phosphoproteins that play a role in vesicle formation and transport. We report the cloning and cDNA sequencing of PACSIN 3 and the analysis of all three PACSIN isoforms with regard to tissue distribution, ligand binding properties and influence on endocytosis. PACSIN 3 differs from the other family members in having a short proline-rich region and lacking asparagine-proline-phenylalanine motifs. In contrast to the neurospecific PACSIN 1 and the ubiquitously expressed PACSIN 2, PACSIN 3 is mainly detected in lung and muscle tissues. All isoforms potentially oligomerize and bind to dynamin, synaptojanin 1 and N-WASP via their Src homology 3 domains. The PACSIN proteins colocalize with dynamin, but not with clathrin, implying a specific role with a distinct subpopulation of dynamin at defined cellular sites. Transferrin endocytosis is blocked in a dose-dependent manner in cells overexpressing the PACSIN variants, but the inhibitory effect can be abolished by mutating specific amino acid residues in the Src homology 3 domains. These characteristics of the PACSIN protein family suggest a general function in recruitment of the interacting proteins to sites of endocytosis.
Identification of Rgp1p, a novel Golgi recycling factor, as a protein required for efficient localization of yeast casein kinase 1 to the plasma membrane
Authors: Panek Heather R. , Conibear Elizabeth , Bryan Joshua D. , Colvin Richard T. , Goshorn Chan D. , Robinson Lucy C.
Published: April 2021

The Yck1p and Yck2p casein kinase 1 isoforms in yeast are essential peripheral plasma membrane-associated protein kinases with roles in endocytosis, cellular morphogenesis and cytokinesis. The membrane targeting of these cytoplasmically oriented protein kinases requires normal secretory pathway function, but specific targeting factors have not been identified. To learn more about Yckp targeting, we characterized mutations that cause synthetic lethality with impairment of Yck function. We report here that these include mutations in two gene products that function in protein trafficking. One of these is the previously described t-SNARE Tlg2p, which participates in recycling of proteins to the Golgi. The other is a previously uncharacterized protein, Rgp1p, which appears to have a similar function. Loss of either Tlg2p or Rgp1p causes inefficient localization of Yck2p, suggesting that its transport may be directed, in part, by a targeting factor that must be recycled back to the Golgi.
Association of mammalian SMC1 and SMC3 proteins with meiotic chromosomes and synaptonemal complexes
Authors: Eijpe M. , Heyting C. , Gross B. , Jessberger R.
Published: April 2021

In somatic cells, the heterodimeric Structural Maintenance of Chromosomes (SMC) proteins are involved in chromosome condensation and gene dosage compensation (SMC2 and 4), and sister chromatid cohesion and DNA recombination (SMC1 and 3). We report here evidence for an involvement of mammalian SMC1 and SMC3 proteins in meiosis. Immunofluorescence analysis of testis sections showed intense chromatin association in meiotic prophase cells, weaker staining in round spermatids and absence of the SMC proteins in elongated spermatids. In spermatocyte nuclei spreads, the SMC1 and SMC3 proteins localize in a beaded structure along the axial elements of synaptonemal complexes of pachytene and diplotene chromosomes. Both SMC proteins are present in rat spermatocytes and enriched in preparations of synaptonemal complexes. Several independent experimental approaches revealed interactions of the SMC proteins with synaptonemal complex-specific proteins SCP2 and SCP3. These results suggest a model for the arrangement of SMC proteins in mammalian meiotic chromatin.
Evidence for deterministic chaos in aperiodic oscillations of proliferative activity in long-term cultured Fao hepatoma cells
Authors: Wolfrom C. , Chau N. P. , Maigné J. , Lambert J. C. , Ducot B. , Guerroui S. , Deschatrette J.
Published: April 2021

The proliferative activity of long-term cultured mammalian cells exhibits traits of a complex dynamic system, with a succession of spontaneous rises and falls in proliferation rate. We analyzed three successive series of proliferation data for the Fao hepatoma cell line in long-term cultures. In the three series the proliferation rate displayed apparently disordered oscillations, which each lasted about 3-5 passages, with variable amplitude and were therefore unpredictable. Such non-linear kinetics raises the major issue of whether these fluctuations are random, or determined and coordinated. We used a graphical method of analysis of the data, which demonstrated that all troughs of proliferation were mathematically related to a common value in each series. This common value was itself related to the maximum level of proliferation of the cell line. Non-linear analysis thus confirmed that the fluctuations in proliferation rate of tumoral Fao cells are, at least in part, determined. This pattern evokes chaotic dynamics and is evidence for the flexible coordination of the complex system linking positive and negative growth regulators in long-term cultured cells.
Ras-independent oncogenic transformation by an EGF-receptor mutant
Authors: Boerner Julie L. , McManus Michael J. , Martin G. Steven , Maihle Nita J.
Published: April 2021

Mutations in the ligand-binding domain of the epidermal growth factor receptor have been identified in several types of human cancers, including malignant gliomas. These mutations render signaling by this receptor to be constitutively ligand-independent. In fibroblasts transformed with ligand-independent epidermal growth factor receptor mutants, there is a correlation between the formation of a unique phosphotyrosine protein complex and oncogenic transformation. This phosphoprotein complex includes Grb2, Shc, Sos, tyrosine-phosphorylated form of caldesmon, and two, as yet, unidentified proteins. The presence of Grb2, Shc, and Sos in this complex implicates Ras in ligand-independent signaling by these oncogenic epidermal growth factor receptor mutants. We, therefore, have used retroviral co-infections of cultured primary fibroblasts to determine if Ras activation is required for phosphoprotein complex formation, stress fiber loss, or transformation. As predicted, expression of a dominant-negative Ras mutant (N17Ras) completely abrogates ligand-stimulated soft agar colony growth of primary fibroblasts. In contrast, N17Ras expression has no effect on v-ErbB mediated stress fiber disassembly, soft agar colony growth, or phosphoprotein complex assembly. In addition, our data suggest that ligand-dependent Ras activation may be suppressed by oncogenic v-ErbB expression. Together these observations suggest that oncogenic signaling by v-ErbB does not require Ras activation, and implicate an alternative signal transduction pathway in ligand-independent epidermal growth factor receptor oncogenic signaling.