bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirements for the nuclear export of the small ribosomal subunit
Authors: Moy Terence I. , Silver Pamela A.
Published: April 2021

Eukaryotic ribosome biogenesis requires multiple steps of nuclear transport because ribosomes are assembled in the nucleus while protein synthesis occurs in the cytoplasm. Using an in situ RNA localization assay in the yeast Saccharomyces cerevisiae, we determined that efficient nuclear export of the small ribosomal subunit requires Yrb2, a factor involved in Crm1-mediated export. Furthermore, in cells lacking YRB2, the stability and abundance of the small ribosomal subunit is decreased in comparison with the large ribosomal subunit. To identify additional factors affecting small subunit export, we performed a large-scale screen of temperature-sensitive mutants. We isolated new alleles of several nucleoporins and Ran-GTPase regulators. Together with further analysis of existing mutants,we show that nucleoporins previously shown to be defective in ribosomal assembly are also defective in export of the small ribosomal subunit.
Ihh enhances differentiation of CFK-2 chondrocytic cells and antagonizes PTHrP-mediated activation of PKA
Authors: Deckelbaum Ron A. , Chan George , Miao Dengshun , Goltzman David , Karaplis Andrew C.
Published: April 2021

Indian Hedgehog (Ihh), a member of the hedgehog (HH) family of secreted morphogens, and parathyroid hormone-related peptide (PTHrP) are key regulators of cartilage cell (chondrocyte) differentiation. We have investigated, in vitro, the actions of HH signalling and its possible interplay with PTHrP using rat CFK-2 chondrocytic cells. Markers of chondrocyte differentiation[alkaline phosphatase (ALP) activity, and type II (Col2a1) and type X collagen (Col10a1) expression] were enhanced by overexpression of Ihh or its N-terminal domain (N-Ihh), effects mimicked by exogenous administration of recombinant N-terminal HH peptide. Moreover, a missense mutation mapping to the N-terminal domain of Ihh (W160G) reduces the capacity of N-Ihh to induce differentiation. Prolonged exposure of CFK-2 cells to exogenous N-Shh(5×10-9 M) in the presence of PTHrP (10-8 M) or forskolin (10-7 M) resulted in perturbation of HH-mediated differentiation. In addition, overexpression of a constitutively active form of the PTHrP receptor (PTHR1 H223R) inhibited Ihh-mediated differentiation,implicating activation of protein kinase A (PKA) by PTHR1 as a probable mediator of the antagonistic effects of PTHrP. Conversely, overexpression of Ihh/N-Ihh or exogenous treatment with N-Shh led to dampening of PTHrP-mediated activation of PKA. Taken together, our data suggest that Ihh harbors the capacity to induce rather than inhibit chondrogenic differentiation, that PTHrP antagonizes HH-mediated differentiation through a PKA-dependent mechanism and that HH signalling, in turn, modulates PTHrP action through functional inhibition of signalling by PTHR1 to PKA.
Developmentally regulated trafficking of the lysosomal membrane protein p67 in Trypanosoma brucei
Authors: Alexander David L. , Schwartz Kevin J. , Balber Andrew E. , Bangs James D.
Published: April 2021

p67 is a lysosomal type I membrane glycoprotein of Trypanosoma brucei. In procyclic stage cells p67 trafficks to the lysosome without modification, but in the bloodstream stage Golgi processing adds poly-N-acetyllactosamine to N-glycans. In both stages proteolytic fragmentation occurs in the lysosome, but turnover is approximately nine times faster in bloodstream cells. Trafficking of wildtype p67 and mutants missing the cytoplasmic (p67ΔCD) or cytoplasmic/transmembrane domains (p67ΔTM) was monitored by pulse-chase,surface biotinylation and immunofluorescence. Overexpressed wildtype p67 trafficks normally in procyclics, but some leaks to the cell surface suggesting that the targeting machinery is saturable. p67ΔCD and p67ΔTM are delivered to the cell surface and secreted, respectively. The membrane/cytoplasmic domains function correctly in procyclic cells when fused to GFP indicating that these domains are sufficient for stage-specific lysosomal targeting. In contrast, p67 wildtype and deletion reporters are overwhelmingly targeted to the lysosome and degraded in bloodstream cells. These findings suggest that either redundant developmentally regulated targeting signals/machinery are operative in this stage or that the increased endocytic activity of bloodstream cells prevents export of the deletion reporters.
The DC electrical-field-induced Ca2+ response and growth stimulation of multicellular tumor spheroids are mediated by ATP release and purinergic receptor stimulation
Authors: Sauer Heinrich , Stanelle Ramona , Hescheler Jürgen , Wartenberg Maria
Published: April 2021

It has been demonstrated that adenosine 5′-triphosphate (ATP) is actively secreted by cells, thereby eliciting Ca2+-dependent signal transduction cascades in an autocrine and paracrine manner. In the present study the effects of direct current (DC) electrical fields on ATP release, the intracellular Ca2+ concentration [Ca2+]i and growth of multicellular prostate tumor spheroids were investigated. Treatment of multicellular tumor spheroids by a single DC electrical field pulse with a field strength of 750 Vm-1 for 60 seconds resulted in a transient Ca2+ response, activation of c-Fos and growth stimulation. The initial [Ca2+]i signal was elicited at the anode-facing side of the spheroid and spread with a velocity of approximately 12 μm per second across the spheroid surface. The electrical-field-evoked Ca2+ response as well as c-Fos activation and growth stimulation of tumor spheroids were inhibited by pretreatment with the anion channel blockers NPPB, niflumic acid and tamoxifen. Furthermore, the Ca2+ response elicited by electrical field treatment was abolished following purinergic receptor desensitivation by repetitive treatment of tumor spheroids with ATP and pretreatment with the purinergic receptor antagonist suramin as well as with apyrase. Electrical field treatment of tumor spheroids resulted in release of ATP into the supernatant as evaluated by luciferin/luciferase bioluminescence. ATP release was efficiently inhibited in the presence of anion channel blockers. Our data suggest that electrical field treatment of multicellular tumor spheroids results in ATP release, which concomitantly activates purinergic receptors, elicits a Ca2+ wave spreading through the tumor spheroid tissue and stimulates tumor growth.
Three-dimensional organization of active rRNA genes within the nucleolus
Authors: Cheutin Thierry , O'Donohue Marie-Françoise , Beorchia Adrien , Vandelaer Marc , Kaplan Hervé , Deféver Bruno , Ploton Dominique , Thiry Marc
Published: April 2021

In this work, we have localized transcribing rRNA genes at the ultrastructural level and described their three-dimensional organization within the nucleolus by electron tomography. Isolated nucleoli, which exhibit a reduced transcriptional rate, were used to determine the sites of initial BrUTP incorporation (i.e. rRNA synthesis by the transcriptional machinery). Using pulse-chase experiments with BrUTP and an elongation inhibitor,cordycepin, it was possible to precisely localize the initial sites of BrUTP incorporation. Our data show that BrUTP incorporation initially takes place in the fibrillar centers and that elongating rRNAs rapidly enter the surrounding dense fibrillar component. Furthermore, we investigated the spatial arrangement of RNA polymerase I molecules within the whole volume of the fibrillar centers. Electron tomography was performed on thick sections of cells that had been labeled with anti-RNA polymerase I antibodies prior to embedding. Detailed tomographic analyses revealed that RNA polymerase I molecules are mainly localized within discrete clusters. In each of them, RNA polymerase I molecules were grouped as several coils, 60 nm in diameter. Overall, these findings have allowed us to propose a model for the three-dimensional organization of transcribing rDNA genes within the nucleolus.
Daxx and histone deacetylase II associate with chromatin through an interaction with core histones and the chromatin-associated protein Dek
Authors: Hollenbach Andrew D. , McPherson Craig J. , Mientjes Edwin J. , Iyengar Rekha , Grosveld Gerard
Published: April 2021

Human Daxx is a protein that functions, in part, as a transcriptional co-repressor through its interaction with a growing number of nuclear,DNA-associated proteins. To determine the mechanism by which hDaxx represses transcription, we used conventional chromatography to isolate endogenous hDaxx. We determined that hDaxx has an apparent molecular weight of 360 kDa,which is consistent with the fact that multiple domains of hDaxx are required for transcriptional repression and suggests that hDaxx associates with multiple proteins. Using co-fractionation and co-immunoprecipitation we demonstrate that hDaxx associates with proteins that are critical for transcriptional repression, such as histone deacetylase II, constituents of chromatin such as core histones H2A, H2B, H3 and H4, and Dek, a chromatin-associated protein reported to change the topology of DNA in chromatin in vitro. We also demonstrate a requirement for the SPT domain and the first paired amphipathic helix of hDaxx for its association with histone deacetylase II and acetylated histone H4, respectively. Finally, we provide evidence suggesting that the association of hDaxx with chromatin-related proteins is dependent on the post-translational phosphorylation status of hDaxx. A working model for the repressive action of hDaxx through its association with chromatin related proteins is presented.
Merotelic kinetochore orientation versus chromosome mono-orientation in the origin of lagging chromosomes in human primary cells
Authors: Cimini Daniela , Fioravanti Daniela , Salmon E. D. , Degrassi Francesca
Published: April 2021

Defects in chromosome segregation play a critical role in producing genomic instability and aneuploidy, which are associated with congenital diseases and carcinogenesis. We recently provided evidence from immunofluorescence and electron microscopy studies that merotelic kinetochore orientation is a major mechanism for lagging chromosomes during mitosis in PtK1 cells. Here we investigate whether human primary fibroblasts exhibit similar errors in chromosome segregation and if at least part of lagging chromosomes may arise in cells entering anaphase in the presence of mono-oriented chromosomes. By using in situ hybridization with alphoid probes to chromosome 7 and 11 we showed that loss of a single sister is much more frequent than loss of both sisters from the same chromosome in anatelophases from human primary fibroblasts released from a nocodazole-induced mitotic arrest, as predicted from merotelic orientation of single kinetochores. Furthermore, the lagging of pairs of separated sisters was higher than expected from random chance indicating that merotelic orientation of one sister may promote merotelic orientation of the other. Kinetochores of lagging chromosomes in anaphase human cells were found to be devoid of the mitotic checkpoint phosphoepitopes recognized by the 3F3/2 antibody, suggesting that they attached kinetochore microtubules prior to anaphase onset. Live cell imaging of H2B histone-GFP-transfected cells showed that cells with mono-oriented chromosomes never enter anaphase and that lagging chromosomes appear during anaphase after chromosome alignment occurs during metaphase. Thus, our results demonstrate that the mitotic checkpoint efficiently prevents the possible aneuploid burden due to mono-oriented chromosomes and that merotelic kinetochore orientation is a major limitation for accurate chromosome segregation and a potentially important mechanism of aneuploidy in human cells.
Pax3 induces cell aggregation and regulates phenotypic mesenchymal-epithelial interconversion
Authors: Wiggan O'Neil , Fadel Marc P. , Hamel Paul A.
Published: April 2021

Paired box-containing transcription factors play fundamental roles in pattern formation during embryonic development of diverse organisms ranging from Drosophila to mammals. Although mutations to Pax3 and other Pax-family genes in both mice and humans result in numerous tissue-specific morphological defects, little is known about the cellular processes that Pax genes regulate. We show that extopic Pax3 expression in two distinct phenotypically mesenchymal mammalian cell lines induces the formation of multi-layered condensed cell aggregates with epithelial characteristics. For one of these lines, we showed further that Pax3-induced cell aggregation is accompanied by specific morphological changes, including a significant reduction in cell size, altered cell shape and dramatic alterations to both membrane and cytoskeleton architecture. In addition to mediating a phenotypic mesenchymal-to-epithelial transition, Pax3 also establishes the conditions in these cells for a subsequent hepatocyte growth factor/scatter factor(HGF/SF)-induced phenotypic epithelial-to-mesenchymal transition. Thus, our data show a novel morphogenetic activity for Pax3 which, when absent in vivo,is predicted to give rise to the observed structural defects in somites and the neural tube during embryonic development.
Induction of chondrocyte growth arrest by FGF: transcriptional and cytoskeletal alterations
Authors: Rozenblatt-Rosen Orit , Mosonego-Ornan Efrat , Sadot Einat , Madar-Shapiro Liora , Sheinin Yuri , Ginsberg Doron , Yayon Avner
Published: April 2021

The effect of fibroblast growth factor (FGF) on mature chondrocytes, the cells responsible for axial skeletal development, is growth attenuation rather than stimulation. This singular response has been linked to signaling via FGF receptor 3 (FGFR3), partly because mutations causing chronic FGFR3 activation lead to various human disorders of bone growth. In order to study how FGF inhibits growth, we analyzed its effect on a rat chondrocyte-derived cell line. We show that the FGF-induced growth arrest occurs at the G1 phase,accompanied by profound changes in gene expression and cytoskeletal organization. Within minutes of binding, FGF induces tyrosine kinase activity in the focal substrate adhesions where it colocalizes with vinculin. Upon FGF stimulation, FGFR3 is selectively removed from the focal adhesions, which is followed by their disassembly and disruption of the organized cytoskeleton. Multiple genes are induced following FGF stimulation in chondrocytes, which has been shown by DNA array screening and confirmed for some by immunoblotting. These genes include regulators of cell differentiation and proliferation such as c-jun, JunD, cyclin-D1, NFκB1 and of plasma-membrane microdomain morphology, such as ezrin. The transcription factor Id1 is downregulated, consistent with the cells' exit from the mitotic cycle. Moreover, following FGF stimulation, levels of FGFR3 mRNA and protein decline, as does downstream signaling through the MAPK pathway. The importance of this FGFR3-mediated on-off control is illustrated in transgenic mice expressing mutant, hyperactive FGFR3, where abnormally high levels of NFκB are expressed throughout their bone growth-plates. A working model is presented of the signaling network involved in regulating FGF-induced chondrocyte differentiation and receptor downregulation.
A family of transmembrane microneme proteins ofToxoplasma gondiicontain EGF-like domains and function as escorters
Authors: Meissner Markus , Reiss Matthias , Viebig Nicola , Carruthers Vern B. , Toursel Catherine , Tomavo Stanislas , Ajioka James W. , Soldati Dominique
Published: April 2021

TgMIC6, TgMIC7, TgMIC8 and TgMIC9 are members of a novel family of transmembrane proteins localized in the micronemes of the protozoan parasite Toxoplasma gondii. These proteins contain multiple epidermal growth factor-like domains, a putative transmembrane spanning domain and a short cytoplasmic tail. Sorting signals to the micronemes are encoded in this short tail. We established previously that TgMIC6 serves as an escorter for two soluble adhesins, TgMIC1 and TgMIC4. Here, we present the characterization of TgMIC6 and three additional members of this family, TgMIC7, -8 and -9. Consistent with having sorting signals localized in its C-terminal tail,TgMIC6 exhibits a classical type I membrane topology during its transport along the secretory pathway and during storage in the micronemes. TgMIC6 is processed at the N-terminus, probably in the trans-Golgi network, and the cleavage site has been precisely mapped. Additionally, like other members of the thrombospondin-related anonymous protein family, TgMIC2, TgMIC6 and TgMIC8 are proteolytically cleaved near their C-terminal domain upon discharge by micronemes. We also provide evidence that TgMIC8 escorts another recently described soluble adhesin, TgMIC3. This suggests that the existence of microneme protein complexes is not an exception but rather the rule. TgMIC6 and TgMIC8 are expressed in the rapidly dividing tachyzoites, while TgMIC7 and TgMIC9 genes are predominantly expressed in bradyzoites, where they presumably also serve as escorters.
Fission yeast Pds5 is required for accurate chromosome segregation and for survival after DNA damage or metaphase arrest
Authors: Wang Shao-Win , Read Rebecca L. , Norbury Chris J.
Published: April 2021

Sister chromatid cohesion, which is established during the S phase of the eukaryotic cell cycle and persists until the onset of anaphase, is essential for the maintenance of genomic integrity. Cohesion requires the multi-protein complex cohesin, as well as a number of accessory proteins including Pds5/BIMD/Spo76. In the budding yeast Saccharomyces cerevisiae Pds5 is an essential protein that localises to chromosomes in a cohesin-dependent manner. Here we describe the characterisation in the fission yeast Schizosaccharomyces pombe of pds5+, a novel,non-essential orthologue of S. cerevisiae PDS5. The S. pombePds5 protein was localised to punctate nuclear foci in a manner that was dependent on the Rad21 cohesin component. This, together with additional genetic evidence, points towards an involvement of S. pombe Pds5 in sister chromatid cohesion. S. pombe pds5 mutants were hypersensitive to DNA damage and to mitotic metaphase delay, but this sensitivity was apparently not due to precocious loss of sister chromatid cohesion. These cells also suffered increased spontaneous chromosome loss and meiotic defects and their viability was dependent on the spindle checkpoint protein Bub1. Thus, while S. pombe Pds5 has an important cohesin-related role, this differs significantly from that of the equivalent budding yeast protein.
pH-dependent regulation of lysosomal calcium in macrophages
Authors: Christensen Kenneth A. , Myers Jesse T. , Swanson Joel A.
Published: April 2021

Calcium measurements in acidic vacuolar compartments of living cells are few, primarily because calibration of fluorescent probes for calcium requires knowledge of pH and the pH-dependence of the probe calcium-binding affinities. Here we report pH-corrected measurements of free calcium concentrations in lysosomes of mouse macrophages, using both ratiometric and time-resolved fluorescence microscopy of probes for pH and calcium. Average free calcium concentration in macrophage lysosomes was 4-6×10-4 M, less than half of the extracellular calcium concentration, but much higher than cytosolic calcium levels. Incubating cells in varying extracellular calcium concentrations did not alter lysosomal pH, and had only a modest effect on lysosomal calcium concentrations, indicating that endocytosis of extracellular fluid provided a small but measurable contribution to lysosomal calcium concentrations. By contrast, increases in lysosomal pH, mediated by either bafilomycin A1 or ammonium chloride, decreased lysosomal calcium concentrations by several orders of magnitude. Re-acidification of the lysosomes allowed rapid recovery of lysosomal calcium concentrations to higher concentrations. pH-dependent reductions of lysosomal calcium concentrations appeared to result from calcium movement out of lysosomes into cytoplasm,since increases in cytosolic calcium levels could be detected upon lysosome alkalinization. These studies indicate that lysosomal calcium concentration is high and is maintained in part by the proton gradient across lysosomal membranes. Moreover, lysosomes could provide an intracellular source for physiological increases in cytosolic calcium levels.
All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction
Authors: Chung Jee Y. , Park Young Chul , Ye Hong , Wu Hao
Published: April 2021

The tumor necrosis factor (TNF) receptor associated factors (TRAFs) have emerged as the major signal transducers for the TNF receptor superfamily and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAFs collectively play important functions in both adaptive and innate immunity. Recent functional and structural studies have revealed the individuality of each of the mammalian TRAFs and advanced our understanding of the underlying molecular mechanisms. Here, we examine this functional divergence among TRAFs from a perspective of both upstream and downstream TRAF signal transduction pathways and of signaling-dependent regulation of TRAF trafficking. We raise additional questions and propose hypotheses regarding the molecular basis of TRAF signaling specificity.
Domains of the Pavarotti kinesin-like protein that direct its subcellular distribution: effects of mislocalisation on the tubulin and actin cytoskeleton duringDrosophilaoogenesis
Authors: Minestrini Gianluca , Máthé Endre , Glover David M.
Published: April 2021

The kinesin-like protein encoded by pavarotti (Pav-KLP) is essential for cytokinesis and associates with the central part of the late mitotic spindle and interphase nuclei in somatic cells (Adams et al., 1988). Here we define regions of the molecule that regulate its subcellular localisation and study the consequences of overexpressing mutant forms of the protein during oogenesis in Drosophila. Pav-KLP normally associates with the oocyte nucleus, but when over-expressed at moderate levels, its GFP tagged form also accumulates in nurse cell nuclei. At high expression levels this leads to loss of the microfilaments that tether these nuclei, so that they block the ring canals and prevent the `dumping' of nurse cell cytoplasm into the oocyte, which results in sterility. Localisation to these nuclei is prevented by mutations in either the conserved ATP-binding site of the motor domain or the nuclear localisation sequences in the C-terminal domain. Both such mutations lead to the formation of stable arrays of cytoplasmic microtubules and the progressive disruption of the actin cytoskeleton. The latter is evident by a breakdown of the cortical actin causing disruption of cell membranes; this breakdown ultimately results in the accumulation of cytoplasmic aggregates containing tubulin, actin and at least some of their binding proteins. Pav-KLP is also found associated with the ring canals,actin-rich structures built from remnants of the cytokinesis ring. The stalk domain alone is sufficient for the exclusive association of Pav-KLP to these structures, and this has no consequences for fertility. We discuss whether disruption of actin structures by full-length cytoplasmic forms of Pav-KLP is a consequence of the resulting stabilised cytoplasmic microtubules per se or accumulation of the motor protein at ectopic cortical sites to sequester molecules that regulate actin behaviour.
Poly-γ-glutamate synthesis during formation of nematocyst capsules inHydra
Authors: Szczepanek Susanne , Cikala Mihai , David Charles N.
Published: April 2021

Nematocysts are explosive organelles found in all Cnidaria. Explosion of nematocyst capsules is driven by the high pressure within the capsule formed by the high concentration of poly-γ-glutamate in the capsule matrix. Poly-γ-glutamate is a polyanion that binds cations tightly, including the fluorescent cationic dyes acridine orange and DAPI(4′,6-diamidino-2-phenylindole). We have used acridine orange and DAPI staining to localize poly-γ-glutamate within capsules and to follow the biosynthesis of poly-γ-glutamate during capsule formation. The results indicate that poly-γ-glutamate biosynthesis occurs late in capsule formation after invagination of the tubule and that it is accompanied by swelling of the capsule due to increasing osmotic pressure. The matrix in all four capsule types is homogeneously filled with poly-γ-glutamate. In vivo this poly-γ-glutamate is complexed with monovalent cations. In addition, poly-γ-glutamate is formed within the tubule lumen of stenoteles. We argue that this poly-γ-glutamate is required to drive the two-step explosion process in stenotele nematocysts.
P35-sensitive caspases, MAP kinases and Rho modulate β-adrenergic induction of apoptosis in mollusc immune cells
Authors: Lacoste Arnaud , Cueff Anne , Poulet Serge A.
Published: April 2021

Apoptosis is an important mechanism for the preservation of a healthy and balanced immune system in vertebrates. Little is known, however, about how apoptotic processes regulate invertebrate immune defenses. In the present study, we show that noradrenaline, a catecholamine produced by the neuroendocrine system and by immune cells in molluscs, is able to induce apoptosis of oyster Crassostrea gigas hemocytes. The apoptosis-inducing effect of noradrenaline was mimicked by isoproterenol and blocked by propranolol, which indicates that noradrenaline triggers apoptosis via a β-adrenergic signaling pathway. Exposure to the pan-caspase inhibitor Z-VAD-FMK or expression of the caspase inhibitor P35 under the transcriptional control of a mollusc hsp70 gene promoter reduced the number of apoptotic cells among noradrenaline-treated hemocytes. These results suggest that P35-sensitive caspases are involved in the apoptotic process triggered by β-adrenergic signaling. Complementary experiments suggest that mitogen-activated protein kinases and Rho, a member of the Ras GTPase family, may be involved in antiapoptotic mechanisms that modulate the apoptotic effect of noradrenaline. Taken together, these results provide a first insight into apoptotic processes in mollusc immune cells.
Mechanisms of CFTR regulation by syntaxin 1A and PKA
Authors: Chang Steven Y. , Di Anke , Naren Anjaparavanda P. , Palfrey H. Clive , Kirk Kevin L. , Nelson Deborah J.
Published: April 2021

Activation of the chloride selective anion channel CFTR is stimulated by cAMP-dependent phosphorylation and is regulated by the target membrane t-SNARE syntaxin 1A. The mechanism by which SNARE proteins modulate CFTR in secretory epithelia is controversial. In addition, controversy exists as to whether PKA activates CFTR-mediated Cl- currents (ICFTR) by increasing the number of channels in the plasma membrane and/or by stimulating membrane-resident channels. SNARE proteins play a well known role in exocytosis and have recently been implicated in the regulation of ion channels; therefore this investigation sought to resolve two related issues:(a) is PKA activation or SNARE protein modulation of CFTR linked to changes in membrane turnover and (b) does syntaxin 1A modulate CFTR via direct effects on the gating of channels residing in the plasma membrane versus alterations in membrane traffic. Our data demonstrate that syntaxin 1A inhibits CFTR as a result of direct protein-protein interactions that decrease channel open probability (Po) and serves as a model for other SNARE protein-ion channel interactions. We also show that PKA activation can enhance membrane trafficking in some epithelial cell types, and this is independent from CFTR activation or syntaxin 1A association.
Differential expression of receptors for Shiga and Cholera toxin is regulated by the cell cycle
Authors: Majoul Irina , Schmidt Tobias , Pomasanova Maria , Boutkevich Evgenia , Kozlov Yuri , Söling Hans-Dieter
Published: April 2021

Cholera and Shiga toxin bind to the cell surface via glycolipid receptors GM1 and Gb3, respectively. Surprisingly, the majority of Vero cells from a non-synchronized population bind either Cholera or Shiga toxin but not both toxins. The hypothesis that the differential expression of toxin receptors is regulated by the cell cycle was tested. We find that Cholera toxin binds preferentially in G0/G1, with little binding through S-phase to telophase,whereas Shiga toxin binds maximally through G2 to telophase but does not bind during G0/G1 and S-phase. The changes result from the corresponding changes in Gb3 and GM1 synthesis, not from variations of receptor transport to the cell surface. The changes do not reflect competition of Gb3 and GM1 synthesis for lactosylceramide. Cells as diverse as Vero cells, PC12 cells and astrocytes show the same cell-cycle-dependent regulation of glycosphingolipid receptors,suggesting that this novel phenomenon is based on a conserved regulatory mechanism.
Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target
Authors: Beningo Karen A. , Wang Yu-li
Published: April 2021

Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 μm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.
The editosome for cytidine to uridine mRNA editing has a native complexity of 27S: identification of intracellular domains containing active and inactive editing factors
Authors: Sowden Mark P. , Ballatori Nazzareno , Jensen Karen L. de Mesy , Reed Lakesha Hamilton , Smith Harold C.
Published: April 2021

Apolipoprotein B mRNA cytidine to uridine editing requires the assembly of a multiprotein editosome comprised minimally of the catalytic subunit,apolipoprotein B mRNA editing catalytic subunit 1 (APOBEC-1), and an RNA-binding protein, APOBEC-1 complementation factor (ACF). A rat homolog has been cloned with 93.5% identity to human ACF (huACF). Peptide-specific antibodies prepared against huACF immunoprecipitated a rat protein of similar mass as huACF bound to apolipoprotein B (apoB) RNA in UV cross-linking reactions, thereby providing evidence that the p66, mooring sequence-selective, RNA-binding protein identified previously in rat liver by UV cross-linking and implicated in editosome assembly is a functional homolog of huACF. The rat protein (p66/ACF) was distributed in both the nucleus and cytoplasm of rat primary hepatocytes. Within a thin section, a significant amount of total cellular p66/ACF was cytoplasmic, with a concentration at the outer surface of the endoplasmic reticulum. Native APOBEC-1 co-fractionated with p66/ACF in the cytoplasm as 60S complexes. In the nucleus, the biological site of apoB mRNA editing, native p66/ACF, was localized to heterochromatin and fractionated with APOBEC-1 as 27S editosomes. When apoB mRNA editing was stimulated in rat primary hepatocytes with ethanol or insulin, the abundance of p66/ACF in the nucleus markedly increased. It is proposed that the heterogeneity in size of complexes containing editing factors is functionally significant and reflects functionally engaged editosomes in the nucleus and an inactive cytoplasmic pool of factors.
Substrate proteolysis is inhibited by dominant-negative Nedd4 and Rsp5 mutants harboring alterations in WW domain 1
Authors: Shcherbik Natalia , Kumar Sharad , Haines Dale S.
Published: April 2021

Mammalian Nedd4 and its budding yeast orthologue Rsp5 are members of a large family of HECT-domain-containing ubiquitin ligases. Besides possessing a Ca2+/lipid-binding domain, both ligases have multiple protein-interacting modules termed WW domains. The C-terminal WW domains mediate interactions with substrates, but the function of the first WW domain remains unclear. We found that expression of a WW domain 1 Nedd4 mutant inhibits the growth of budding yeast by affecting the rsp5-ole1pathway. The WW domain 1 mutant-induced phenotype is suppressed by ole1 cDNA overexpression or oleic acid supplementation of growth media and ole1 RNA levels are reduced in cells expressing this Nedd4 mutant. Also, the WW domain 1 Nedd4 mutant associates via WW domains 2 and 3 with Spt23, a Rsp5 target and ole1 transactivator. The dominant-negative activity of this mutant is associated with promoting accumulation of unprocessed Spt23 and inhibiting generation of processed and presumably active protein. Also, Spt23 processing is inhibited by a Nedd4 mutant that lacks ubiquitin ligase activity and Spt23-binding-competent Rsp5 mutants harboring WW domain 1 or ligase domain mutations. Interestingly, in mammalian cells, wild-type Nedd4 promotes proteasome-mediated degradation of the precursor form of Spt23. WW domain 1 and ligase domain Nedd4 mutants block its degradation. These results indicate that WW domain 1 of these ligases interacts with cofactors that are required for ubiquitin/proteasome-dependent proteolysis of bound substrates.
Cell-type-specific and selectively induced expression of members of the p24 family of putative cargo receptors
Authors: Rötter Jutta , Kuiper Roland P. , Bouw Gerrit , Martens Gerard J. M.
Published: April 2021

Members of the p24 family of type I transmembrane proteins are highly abundant in transport vesicles and are thought to be involved in selective protein transport between the endoplasmic reticulum and the Golgi complex. The p24 proteins have been grouped into four subfamilies (α, β,γ, and δ) and appear to assemble into tetrameric complexes that contain only one representative from each subfamily. Here we molecularly dissected the p24 family in a single cell type, namely in the intermediate pituitary melanotrope cells of the amphibian Xenopus laevis. The biosynthetic activity of these cells for production of their major cargo protein proopiomelanocortin (POMC) can be physiologically manipulated via the process of background adaptation (∼30-fold induction, with highly active cells in black toads and virtually inactive cells in white animals). Extensive cDNA library screening revealed the identity of six p24 proteins expressed in the Xenopus melanotrope cells, namely one member of the p24α(α3), one of the p24β (β1), two of the p24γ (γ2, γ3) and two of the p24δ (δ1, δ2) subfamily. Two other Xenopus p24 proteins, Xp24α2 and-γ1, were not expressed in the melanotrope cells, pointing to cell-type specific p24 expression. Of the six melanotrope p24 proteins, the expression of four (Xp24α3, -β1,-γ3 and -δ2) was 20- to 30-fold induced in active versus inactive melanotropes, whereas that of the other two members(Xp24γ2 and -δ1) had not or only slightly increased. The four proteins were induced only in the intermediate melanotrope cells and not in the anterior pituitary cells, and displayed similar overall tissue distributions that differed from those of Xp24γ1,-γ2 and -δ1. Together, our results reveal that p24 expression can be cell-type specific and selectively induced, and suggest that in Xenopus melanotrope cells anα 3/β1/γ3/δ2p24 complex is involved in POMC transport through the early stages of the secretory pathway.
Twinfilin, a molecular mailman for actin monomers
Authors: Palmgren Sandra , Vartiainen Maria , Lappalainen Pekka
Published: April 2021

Twinfilin is a ubiquitous actin-monomer-binding protein that is composed of two ADF-homology domains. It forms a 1:1 complex with ADP-actin-monomers,inhibits nucleotide exchange on actin monomers and prevents assembly of the monomer into filaments. The two ADF-H domains in twinfilin probably have 3D structures similar to those of the ADF/cofilin proteins and overlapping actin-binding sites. Twinfilin also interacts with PtdIns(4,5)P2, which inhibits its actin-monomer-sequestering activity in vitro. Mutations in the twinfilin gene result in defects in the bipolar budding pattern in S. cerevisiae and in a rough eye phenotype and aberrant bristle morphology in Drosophila melanogaster. These phenotypes are caused by the uncontrolled polymerization of actin filaments in the absence of twinfilin. Studies on budding yeast suggest that twinfilin contributes to actin filament turnover by localizing actin monomers, in their `inactive'ADP-form, to the sites of rapid filament assembly. This is mediated through direct interactions between twinfilin and capping protein. Therefore,twinfilin might serve as a link between rapid actin filament depolymerization and assembly in cells.
The small GTPase Rab22 interacts with EEA1 and controls endosomal membrane trafficking
Authors: Kauppi Maria , Simonsen Anne , Bremnes Bjørn , Vieira Amandio , Callaghan Judy , Stenmark Harald , Olkkonen Vesa M.
Published: April 2021

Rab22a is a small GTPase that is expressed ubiquitously in mammalian tissues and displays the highest sequence homology to Rab5. In BHK-21 cells,overexpression of the wild-type Rab22a caused formation of abnormally large vacuole-like structures containing the early-endosomal antigen EEA1 but not Rab11, a marker of recycling endosomes or the late-endosomal/lysosomal markers LAMP-1 and lyso-bis-phosphatidic acid. In HeLa cells, overexpressed Rab22a was found on smaller EEA1-positive endosomes, but a portion of the protein was also found in the Golgi complex. Using the yeast two-hybrid system and a biochemical pull-down assay, the GTP-bound form of Rab22a was found to interact with the N-terminus of EEA1. In HeLa cells overexpressing Rab22a or its mutants affected in the GTPase cycle, no significant changes were observed in the uptake of Alexa-transferrin. However, the GTPase-deficient Rab22a Q64L mutant caused a redistribution of transferrin-positive endosomes to the leading edges of cells and a fragmentation of the Golgi complex. In BHK cells,the Q64L mutant caused the accumulation of a fluid phase marker,TRITC-dextran, and a lysosomal hydrolase, aspartylglucosaminidase, in abnormal vacuole-like structures that contained both early and late endosome markers. Both the wild-type Rab22a and the Q64L mutant were found to interfere with the degradation of EGF. These results suggest that Rab22a may regulate the dynamic interactions of endosomal compartments and it may be involved in the communication between the biosynthetic and early endocytic pathways.
A requirement for the Abnormal Spindle protein to organise microtubules of the central spindle for cytokinesis inDrosophila
Authors: Riparbelli Maria Giovanna , Callaini Giuliano , Glover David M. , Avides Maria do Carmo
Published: April 2021

Drosophila abnormal spindle (asp) mutants exhibit a mitotic metaphase checkpoint arrest with abnormal spindle poles, which reflects a requirement for Asp for the integrity of microtubule organising centres (MTOCs). In male meiosis, the absence of a strong spindle integrity checkpoint enables asp mutant cells to proceed through anaphase and telophase. However, the central spindle region is not correctly organised and cells frequently fail to complete cytokinesis. This contrasts with meiosis in wild-type males where at late anaphase a dense array of microtubules forms in the central spindle region that has Asp localised at its border. We speculate that Asp is associated with the minus ends of microtubules that have been released from the spindle poles to form the central spindle. A parallel situation arises in female meiosis where Asp not only associates with the minus ends of microtubules at the acentriolar poles but also with the central spindle pole body that forms between the two tandem spindles of meiosis II. Upon fertilisation, Asp is also recruited to the MTOC that nucleates the sperm aster. Asp is required for growth of the microtubules of the sperm aster,which in asp mutants remains diminutive and so prevents migration of the pronuclei.
The expression, lamin-dependent localization and RNAi depletion phenotype for emerin inC. elegans
Authors: Gruenbaum Yosef , Lee Kenneth K. , Liu Jun , Cohen Merav , Wilson Katherine L.
Published: April 2021

Emerin belongs to the LEM-domain family of nuclear membrane proteins, which are conserved in metazoans from C. elegans to humans. Loss of emerin in humans causes the X-linked form of Emery-Dreifuss muscular dystrophy(EDMD), but the disease mechanism is not understood. We have begun to address the function of emerin in C. elegans, a genetically tractable nematode. The emerin gene (emr-1) is conserved in C. elegans. We detect Ce-emerin protein in the nuclear envelopes of all cell types except sperm, and find that Ce-emerin co-immunoprecipitates with Ce-lamin from embryo lysates. We show for the first time in any organism that nuclear lamins are essential for the nuclear envelope localization of emerin during early development. We further show that four other types of nuclear envelope proteins, including fellow LEM-domain protein Ce-MAN1, as well as Ce-lamin, UNC-84 and nucleoporins do not depend on Ce-emerin for their localization. This result suggests that emerin is not essential to organize or localize the only lamin (B-type) expressed in C. elegans. We also analyzed the RNAi phenotype resulting from the loss of emerin function in C. elegans under laboratory growth conditions, and found no detectable phenotype throughout development. We propose that C. elegans is an appropriate system in which to study the molecular mechanisms of emerin function in vivo.
Kinesinsklp5+ andklp6+ are required for normal chromosome movement in mitosis
Authors: West Robert R. , Malmstrom Terra , McIntosh J. Richard
Published: April 2021

Proper mitotic chromosome segregation requires dynamic interactions between spindle microtubules and kinetochores. Here we demonstrate that two related fission yeast kinesins, klp5+ and klp6+, are required for normal chromosome segregation in mitosis. Null mutants frequently lack a normal metaphase chromosome alignment. Chromosome pairs move back and forth along the spindle for an extended period prior to sister chromatid separation, a phenotype reminiscent of the loss of CENP-E in metazoans. Ultimately, sister chromatids segregate, regardless of chromosome position along the spindle, and viable daughter cells are usually produced. The initiation of anaphase B is sometimes delayed, but the rate of spindle elongation is similar to wildtype. Despite a delay, anaphase B often begins before anaphase A is completed. The klp5Δ and klp6Δ null mutants are synthetically lethal with a deletion of the spindle assembly checkpoint gene, bub1+, several mutants in components of the anaphase promoting complex, and a cold sensitive allele of the kinetochore and microtubule-binding protein, Dis1p. Klp5p-GFP and Klp6p-GFP localize to kinetochores from prophase to the onset of anaphase A, but relocalize to the spindle midzone during anaphase B. These data indicate that Klp5p and Klp6p are kinetochore kinesins required for normal chromosome movement in prometaphase.
Generation of diacylglycerol molecular species through the cell cycle: a role for 1-stearoyl, 2-arachidonyl glycerol in the activation of nuclear protein kinase C-βII at G2/M
Authors: Deacon Elizabeth M. , Pettitt Trevor R. , Webb Paul , Cross Timothy , Chahal Hema , Wakelam Michael J. O. , Lord Janet M.
Published: April 2021

Protein kinase C (PKC) is a family of 11 isoenzymes that are differentially involved in the regulation of cell proliferation. PKC-βII, a mitotic lamin kinase, has been shown previously to translocate to the nucleus at G2/M and this was coupled to the generation of nuclear diacylglycerol. However, it is not clear how isoenzyme selective translocation and nuclear targeting is achieved during cell cycle. To investigate further the role of nuclear diacylglycerol we measured PKC isoenzyme translocation and analysed diacylglycerol species at different stages of the cell cycle in U937 cells synchronized by centrifugal elutriation. Translocation of PKC-βII to the membrane fraction, an indicator of activation, occurred at S and G2/M, although PKC-βII was targeted to the nucleus only at G2/M. Levels of nuclear diacylglycerol, specifically tetraunsaturated species, increased during G2/M. By contrast, there were no obvious changes in nuclear phosphatidic acid species or mass. 1-stearoyl, 2-arachidonyl glycerol (SAG), the major polyunsaturated nuclear diacylglycerol, was able to activate classical PKC isoenzymes (PKC-α andβ), but was less effective for activation of novel isoenzymes(PKC-δ), in an in vitro PKC assay. We propose that PKC-βII nuclear translocation during G2/M phase transition is mediated in part by generation of SAG at the nucleus.
F-actin serves as a template for cytokeratin organization in cell free extracts
Authors: Weber Kari L. , Bement William M.
Published: April 2021

The microtubule, F-actin, and intermediate filament systems are often studied as isolated systems, yet the three display mutual interdependence in living cells. To overcome limitations inherent in analysis of polymer-polymer interactions in intact cells, associations between these systems were assessed in Xenopus egg extracts. In both fixed and unfixed extract preparations, cytokeratin associated with F-actin cables that spontaneously assembled in the extracts. Time-course experiments revealed that at early time points cytokeratin cables were invariably associated with F-actin cables,while at later time points they could be found without associated F-actin. In extract samples where F-actin assembly was prevented, cytokeratin formed unorganized aggregates rather than cables. Dynamic imaging revealed transport of cytokeratin by moving F-actin as well as examples of cytokeratin release from F-actin. Experimental alteration of F-actin network organization by addition of α-actinin resulted in a corresponding change in the organization of the cytokeratin network. Finally, pharmacological disruption of the F-actin network in intact, activated eggs disrupted the normal pattern of cytokeratin assembly. These results provide direct evidence for an association between F-actin and cytokeratin in vitro and in vivo, and indicate that this interaction is necessary for proper cytokeratin assembly after transition into the first mitotic interphase of Xenopus.