bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning, sequencing, and nucleolar targeting of the basal-body-binding nucleolar protein BN46/51
Authors: Trimbur Gina M. , Goeckeler Jennifer L. , Brodsky Jeffrey L. , Walsh Charles J.
Published: April 2021

BN46/51 is an acidic protein found in the granular component of the nucleolus of the amebo-flagellate Naegleria gruberi. When Naegleria amebae differentiate into swimming flagellates, BN46/51 is found associated with the basal body complex at the base of the flagella. In order to determine the factors responsible for targeting BN46/51 to a specific subnucleolar region, cDNAs coding for both subunits were isolated and sequenced. Two clones, JG4.1 and JG12.1 representing the 46 kDa and 51 kDa subunits, respectively, were investigated in detail. JG12.1 encoded a polypeptide of 263 amino acids with a predicted size of 30.1 kDa that co-migrated with the 51 kDa subunit of BN46/51 when expressed in yeast. JG4.1 encoded a polypeptide of 249 amino acids with a predicted size of 28.8 kDa that co-migrated with the 46 kDa subunit of BN46/51. JG4.1 was identical to JG12.1 except for the addition of an aspartic acid between positions 94 and 95 of the JG12.1 sequence and the absence of 45 amino acids beginning at position 113. The predicted amino acid sequences were not closely related to any previously reported. However, the sequences did have 26-31% identity to a group of FKPBs (FK506 binding proteins) but lacked the peptidyl-prolyl cis-trans isomerase domain of the FKBPs. Both subunits contained two KKE and three KKX repeats found in other nucleolar proteins and in some microtubule binding proteins. Using ‘Far Western’ blots of nucleolar proteins, BN46/51 bound to polypeptides of 44 kDa and 74 kDa. The 44 kDa component was identified as the Naegleria homologue of fibrillarin. BN46/51 bound specifically to the nucleoli of fixed mammalian cells, cells which lack a BN46/51 related polypeptide. When the JG4.1 and JG12.1 cDNAs were expressed in yeast, each subunit was independently targeted to the yeast nucleolus. We conclude that BN46/51 represents a unique nucleolar protein that can form specific complexes with fibrillarin and other nucleolar proteins. We suggest that the association of BN46/51 with the MTOC of basal bodies may reflect its role in connecting the nucleolus with the MTOC activity for the mitotic spindle. This would provide a mechanism for nucleolar segregation during the closed mitosis of Naegleria amebae.
Polarized targeting of epithelial cell proteins in thyrocytes and MDCK cells
Authors: Prabakaran Daniel , Ahima Rexford S. , Harney John W. , Berry Marla J. , Larsen P. Reed , Arvan Peter
Published: April 2021

Polarized trafficking signals may be interpreted differently in different cell types. In this study, we have compared the polarized trafficking of different proteins expressed endogenously in primary porcine thyroid epithelial cells to similar proteins expressed in MDCK cells. As in MDCK cells, NH4Cl treatment of filter-grown thyrocytes caused mis-sorted soluble proteins to exhibit enhanced secretion to the apical medium. In independent studies, thrombospondin 1 (a thyroid basolaterally secreted protein) was secreted basolaterally from MDCK cells. Likewise, the 5′-deiodinase (a thyroid basolateral membrane protein) encoded by the DIO1 gene was also distributed basolaterally in transfected MDCK cells. Consistent with previous reports, when the secretion of human growth hormone (an unglycosylated regulated secretory protein) was examined from transfected MDCK cells, the release was nonpolarized. However, transfected thyrocytes secreted growth hormone apically in a manner dependent upon zinc addition. Moreover, two additional regulated secretory proteins expressed in thyrocytes, thyroglobulin (the major endogenous glycoprotein) and parathyroid hormone (an unglycosylated protein expressed transiently), were secreted apically even in the absence of zinc. We hypothesize that while cellular mechanisms for interpreting polarity signals are generally similar between thyrocytes and MDCK cells, thyrocytes allow for specialized packaging of regulated secretory proteins for apical delivery, which does not require glycosylation but may involve availability of certain ions as well as appropriate intracellular compartmentation.
Mutational analysis of cysteine-string protein function in insulin exocytosis
Authors: Zhang Hui , Kelley William L. , Chamberlain Luke H. , Burgoyne Robert D. , Lang Jochen
Published: April 2021

Cysteine-string proteins (Csps) are vesicle proteins involved in neurotransmission. They contain at least four domains: an N-terminal J-domain which can interact with the chaperone Hsc70, an adjacent linker region, the defining cysteine rich domain and a variable C terminus. As the relevance of these domains for the function of Csps in exocytosis is unknown, we have performed a mutational analysis of Csp domains using insulin release by large dense core vesicles (LDCVs) as a model of regulated exocytosis. All mutants were apparently palmitoylated and their subcellular distribution was similar to endogenous Csp. Point mutations within the highly conserved HPD motif of the J-domain abolished activation of Hsc70. However, these mutations altered the effect of Csp on exocytosis only after additional truncation of the extreme C terminus as found in the Csp splice variant Csp2. Furthermore, the strikingly conserved linker region adjacent to the J-domain was important for Csp function in exocytosis, but not for the activation of Hsc70 ATPase. The effects of Csp wild-type or mutants were preserved in permeabilized cells excluding an effect on transmembrane ion fluxes. These observations demonstrate a functional difference between the two isoforms and suggest a role for the J-domain co-chaperone function as well as for the newly defined linker region in LDCV exocytosis.
Activation of pp60src is critical for stretch-induced orienting response in fibroblasts
Authors: Sai Xiaorui , Naruse Keiji , Sokabe Masahiro
Published: April 2021

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120-130 kDa) increased and peaked at 30 minutes. Immuno-precipitation experiments revealed that paxillin, pp125FAK, and pp130CAS were included in the 70 kDa, and 120-130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60src, the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60src increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an anti-sense S-oligodeoxynucleotide (S-ODN) against pp60src, but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60src, we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1 than in 3Y1 fibroblasts. These results strongly suggest that cyclic stretch induces the activation of pp60src and that pp60src is indispensable for the tyrosine phosphorylation of pp130CAS, pp125FAK and paxillin followed by the orienting response in 3Y1 fibroblasts.
RA regulation of keratin expression and myogenesis suggests different ways of regenerating muscle in adult amphibian limbs
Authors: Corcoran Jonathan P. , Ferretti Patrizia
Published: April 2021

Formation of a regeneration blastema following limb amputation is believed to occur through a process of dedifferentiation. It has been suggested, however, that the cells contributed to the blastema by the stump muscle are satellite-like cells, rather than cells originated by dedifferentiation. We have previously shown that simple epithelial keratins 8 and 18 are expressed in the mesenchymal progenitor cells of the regenerating amphibian limb and in cultured cells with myogenic potential, and that their expression appears to be causally related to changes in proliferation and differentiation. We show here that retinoic acid (RA) affects the expression of these keratins differently in myogenic cells originated from normal limb and limb blastema. Furthermore, we find that the effects of RA on proliferation, myogenic differentiation and adhesion of these cells also differ. In fact, whereas RA does not affect keratin expression, proliferation or myogenic differentiation in blastemal cells, it does decrease keratin levels and thymidine incorporation and increase myogenesis in cells from normal limb. Conversely, RA increases cell adhesion only in blastemal cells. Significantly, these effects of RA on cultured cells are consistent with those observed in vivo. Overall the results presented here suggest that in the urodele limb there are two distinct cell populations with myogenic potential, one originating from dedifferentiation and one equivalent to the satellite cells of the mammalian muscle, which are likely to be primarily involved in blastema formation and muscle repair, respectively.
Influence of transforming growth factor β1 (TGF-β1) on the behaviour of porcine thyroid epithelial cells in primary culture through thrombospondin-1 synthesis
Authors: Claisse D. , Martiny I. , Chaqour B. , Wegrowski Y. , Petitfrere E. , Schneider C. , Haye B. , Bellon G.
Published: April 2021

Transforming growth factor β1 (TGF-β1) is a secreted polypeptide that is thought to play a major role in the regulation of folliculogenesis and differentiation of thyroid cells. On porcine thyroid follicular cells cultured on plastic substratum, TGF-β1, in a concentration-dependent way, promoted the disruption of follicles, cell spreading, migration and confluency by a mechanism that did not involve cell proliferation. TGF-β1 strongly activated the production of thrombospondin-1 and αvβ3 integrin in a concentration-dependent manner whereas the expression of thyroglobulin was unaffected. Anisomycin, an inhibitor of protein synthesis, inhibited the effect of TGF-β1 on cell organization. Thrombospondin-1 reproduced the effect of TGF-β1. In the presence of thrombospondin-1 cells did not organize in follicle-like structures but, in contrast, spreaded and reached confluency independently of cell proliferation. This effect is suppressed by an RGD-containing peptide. The adhesive properties of thrombospondin-1 for thyroid cells were shown to be mediated by both the aminoterminal heparin-binding domain and the RGD domain of thrombospondin-1. Adhesion was shown to involve αvβ3 integrin. The results show that TGF-β1 exerted an influence upon function and behaviour of follicle cells partly mediated by the synthesis of thrombospondin-1 and of its receptor αvβ3 integrin.
Extracellular signal-regulated kinase functions in the urokinase receptor-dependent pathway by which neutralization of low density lipoprotein receptor-related protein promotes fibrosarcoma cell migration and Matrigel invasion
Authors: Webb Donna J. , Nguyen Diem H. D. , Gonias Steven L.
Published: April 2021

The low density lipoprotein receptor-related protein (LRP) has been reported to regulate cellular migration. In this study, an antisense RNA expression strategy was used to reduce LRP to undetectable levels in HT 1080 fibrosarcoma cells. The LRP-deficient cells demonstrated increased levels of cell-surface uPAR, higher levels of uPA in conditioned medium, increased migration on vitronectin-coated surfaces, and increased invasion of Matrigel. LRP-deficient cells also demonstrated increased levels of phosphorylated extracellular signal-regulated kinase (ERK) in the absence of exogenous stimulants. Antibodies which block binding of endogenously produced uPA to uPAR reduced ERK phosphorylation and migration of LRP-deficient cells to the levels observed with control cells. Inhibitors of ERK activation, including PD098059 and dominant-negative MEK1, also decreased the migration of LRP-deficient but not control cells. By contrast, constitutively active MEK1 stimulated the migration of control but not LRP-deficient cells. Although Matrigel invasion by LRP-deficient cells was inhibited by the proteinase inhibitor, aprotinin, PD098059 in combination with aprotinin was necessary for an optimal effect. Expression of the VLDL receptor in LRP-deficient cells reversed the changes in cellular migration and invasion. These studies demonstrate that binding of endogenously produced uPA to uPAR may serve as a major determinant of basal levels of activated ERK and, by this mechanism, regulate cellular migration and invasion. By regulating the uPA/uPAR system, LRP may also regulate ERK activation, cellular migration, and invasion.
COPI vesicles accumulating in the presence of a GTP restricted Arf1 mutant are depleted of anterograde and retrograde cargo
Authors: Pepperkok Rainer , Whitney J. Andrew , Gomez Marie , Kreis Thomas E.
Published: April 2021

Microinjection of the slowly hydrolyzable GTP analogue GTPγS or the ectopic expression of a GTP restricted mutant of the small GTPase arf1 (arf1[Q71L]) leads to the rapid accumulation of COPI coated vesicles and buds in living cells. This effect is blocked at 15°C and by microinjection of antibodies against β-COP. Anterograde and retrograde membrane protein transport markers, which have been previously shown to be incorporated into COPI vesicles between the endoplasmic reticulum and Golgi complex, are depleted from the GTPγS or arf1[Q71L] induced COPI coated vesicles and buds. In contrast, in control cells 30 to 60% of the COPI carriers co-localize with these markers. These in vivo data corroborate recent in vitro work, suggesting that GTPγS and arf1[Q71L] interfere with the sorting of membrane proteins into Golgi derived COPI vesicles, and provide the first in vivo evidence for a role of GTP hydrolysis by arf1 in the sorting of cargo into COPI coated vesicles and buds.
Skh1, the MEK component of the Mkh1 signaling pathway in Schizosaccharomyces pombe
Authors: Loewith Robbie , Hubberstey Andrew , Young Dallan
Published: April 2021

We previously reported the identification of Mkh1, a MEK kinase in Schizosaccharomyces pombe that is required for cell wall integrity, and we presented genetic evidence that Pmk1/Spm1, a MAP kinase, functions downstream from Mkh1 in the same pathway. Here, we report the identification of Skh1, a MEK (MAP kinase kinase) in S. pombe. The sequence of Skh1 is nearly identical to that of the recently reported Pek1 sequence. We present biochemical and genetic evidence that Skh1 is the MEK component of the Mkh1-Spm1 MAP kinase cascade. Our yeast two-hybrid results indicate that Mkh1, Skh1, and Spm1 physically interact to form a ternary complex. Deletion of mkh1, skh1 or spm1 results in identical phenotypes, including sensitivity to β-glucanase treatment, growth inhibition on media containing KCl, and filamentous growth on medium containing caffeine. Double mutant strains exhibit phenotypes that are identical to the single mutant strains. Furthermore, expression of an activated HA-Skh1DD protein suppressed these defects in mkh1Δ cells, and overexpression of Spm1 suppressed these defects in skh1Δ cells. We also show that HA-Spm1 is hyper-phosphorylated on tyrosine residues in cells coexpressing the activated HA-Skh1DD protein. Furthermore, we found the phosphorylated/activated form of GFP-HA-Spm1 at detectable levels in wild-type cells, but not at appreciable levels in mkh1Δ or skh1Δ cells expressing this fusion protein. Together, our results indicate that Mkh1, Skh1 and Spm1 constitute a MAPK cascade in fission yeast.
Clathrin plays a novel role in the regulation of cell polarity, pseudopod formation, uropod stability and motility in Dictyostelium
Authors: Wessels Deborah , Reynolds Josh , Johnson Olof , Voss Edward , Burns Royce , Daniels Karla , Garrard Elizabeth , O’Halloran Teresa J. , Soll David R.
Published: April 2021

Although the traditional role of clathrin has been in vesicle trafficking and the internalization of receptors, a novel role in cytokinesis was recently revealed in an analysis of a clathrin-minus Dictyostelium mutant (chc−). chc− cells grown in suspension were demonstrated to be defective in assembling myosin II into a normal contractile ring. To test whether this defect reflected a more general one of cytoskeletal dysfunction, chc− cells were analyzed for cell polarity, pseudopod formation, uropod stability, cell locomotion, chemotaxis, cytoskeletal organization and vesicle movement. chc− cells crawled, chemotaxed, localized F-actin in pseudopods, organized their microtubule cytoskeleton in a relatively normal fashion and exhibited normal vesicle dynamics. Although chc− cells extended pseudopods from the anterior half of the cell with the same frequency as normal chc+ cells, they extended pseudopods at twice the normal frequency from the posterior half of the cell. The uropods of chc− cells also exhibited spatial instability. These defects resulted in an increase in roundness, a reduction in polarity, a reduction in velocity, a dramatic increase in turning, a high frequency of 180° direction reversals and a decrease in the efficiency of chemotaxis. All defects were reversed in a rescued strain. These results are the first to suggest a novel role for clathrin in cell polarity, pseudopod formation, uropod stability and locomotion. It is hypothesized that clathrin functions to suppress pseudopod formation and to stabilize the uropod in the posterior half of a crawling cell, two behavioral characteristics that are essential for the maintenance of cellular polarity, efficient locomotion and efficient chemotaxis.
Src tyrosine kinase activity down-regulates Rho-dependent responses during Shigella entry into epithelial cells and stress fibre formation
Authors: Duménil Guillaume , Sansonetti Philippe , Nhieu Guy Tran Van
Published: April 2021

Invasion of epithelial cells by Shigella, the causative agent of bacillary dysentery, is dependent upon the formation of characteristic membrane ruffles that engulf the bacteria in a macropinocytic-like process. We show here that Cdc42 and Rac GTPases, but not Rho, are critical for actin polymerisation, whereas Rho is necessary for the recruitment of ezrin and Src at the site of entry. Remarkably, cells expressing constitutively active Src did not show ezrin recruitment at Shigella entry foci. In these cells, formation of stress fibres induced by LPA stimulation, or microinjection of activated Rho (V14Rho), was inhibited. Src-mediated tyrosyl-phosphorylation of p190RhoGAP correlated with changes in the ability of p190RhoGAP to interact with Rho, suggesting that Src regulates Rho function via p190RhoGAP. We propose that Rho activation is required for proper organisation of Shigella entry foci and for Src recruitment, and that Src tyrosine kinase activity, in turn, down-regulates the function of Rho at the site of Shigella entry. The significance of this negative regulatory loop on Rhodependent responses is discussed.
Who is mentoring the mentors?
Authors: A Caveman
Published: April 2021

Can you imagine a battalion of troops commanded by a milkman, a bank headed by a car mechanic, or an airplane flown by a used-car salesmanâ€¦?
The nuclear pore complex: mediator of translocation between nucleus and cytoplasm
Authors: Allen T. D. , Cronshaw J. M. , Bagley S. , Kiseleva E. , Goldberg M. W.
Published: April 2021

The enclosure of nuclear contents in eukaryotes means that cells require sites in the boundary that mediate exchange of material between nucleus and cytoplasm. These sites, termed nuclear pore complexes (NPCs), number 100-200 in yeast, a few thousand in mammalian cells and ∼50 million in the giant nuclei of amphibian oocytes. NPCs are large (125 MDa) macromolecular complexes that comprise 50-100 different proteins in vertebrates. In spite of their size and complex structure, NPCs undergo complete breakdown and reformation at cell division. Transport through NPCs can be rapid (estimated at several hundred molecules/pore/second) and accommodates both passive diffusion of relatively small molecules, and active transport of complexes up to several megadaltons in molecular mass. Each pore can facilitate both import and export. The two processes apparently involve multiple pathways for different cargoes, and their transport signals, transport receptors and adapters, and the molecules (and their regulators) that underpin the transport mechanisms. Over the past few years there has been an increasing interest in the pore complex: structural studies have been followed by elucidation of the biochemical aspects of nuclear import, and subsequent investigations into nuclear export. The current challenge is to understand the interactions between the structural elements of the pore complex and the mechanisms that drive the physical processes of translocation through it.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs0712.html & Video 2
The amino- and carboxyl-terminal tails of β-catenin reduce its affinity for desmoglein 2
Authors: Wahl James K. , Nieset Jill E. , Sacco-Bubulya Paula A. , Sadler Tammy M. , Johnson Keith R. , Wheelock Margaret J.
Published: April 2021

Beta-catenin and plakoglobin are members of the armadillo family of proteins and were first identified as components of intercellular adhering junctions. In the adherens junction β-catenin and plakoglobin serve to link classical cadherins to the actin-based cytoskeleton. In the desmosome plakoglobin links the desmosomal cadherins, the desmogleins and the desmocollins, to the intermediate filament cytoskeleton. β-catenin is not a component of the desmosome. Previously we have shown that the central armadillo repeat region of plakoglobin is the site for desmosomal cadherin binding. We hypothesized that the unique amino- and/or carboxyl-terminal ends of β-catenin may regulate its exclusion from the desmosomal plaque. To test this hypothesis we used chimeras between β-catenin and plakoglobin to identify domain(s) that modulate association with desmoglein 2. Chimeric constructs, each capable of associating with classical cadherins, were assayed for association with the desmosomal cadherin desmoglein 2. Addition of either the N- or C-terminal tail of β-catenin to the armadillo repeats of plakoglobin did not interfere with desmoglein 2 association. However, when both β-catenin amino terminus and carboxyl terminus were added to the plakoglobin armadillo repeats, association with desmoglein 2 was diminished. Removal of the first 26 amino acids from this construct restored association. We show evidence for direct protein-protein interactions between the amino- and carboxyl-terminal tails of β-catenin and propose that a sequence in the first 26 amino acids of β-catenin along with its carboxyl-terminal tail decrease its affinity for desmoglein and prevent its inclusion in the desmosome.
Tight junctions are membrane microdomains
Authors: Nusrat A. , Parkos C. A. , Verkade P. , Foley C. S. , Liang T. W. , Innis-Whitehouse W. , Eastburn K. K. , Madara J. L.
Published: April 2021

Tight junctions (TJ) of polarized epithelial cells regulate barrier function at mucosal surfaces. Structural proteins of TJs include hyperphosphorylated occludin (HO) and the peripheral membrane protein, ZO-1. Since TJs are dynamically regulated, and lipid-modified signal transduction proteins localize to TJs, we considered the possibility that the TJ itself is composed of microdomains with unique structure. Differential detergent extraction and isopycnic sucrose density gradients were utilized to isolate TJ-enriched membranes from a polarized intestinal epithelial cell line, T84. Here we report that major pools of hyperphosphorylated occludin (HO) and ZO-1 are found in raft-like membrane microdomains with characteristics of the previously described detergent-insoluble glycolipid rafts (DIGs). Properties of such gradient fractions included Triton X-100 (TX-100) insolubility, light scattering at 600 nm, buoyant density of approximately 1.08 g/cm3 and increased cholesterol content compared to high density fractions. Similar results were obtained using natural epithelium. Unlike the TJ proteins HO and ZO-1, other basolateral transmembrane proteins including E-cadherin, c-met and beta 1 integrin were not increased in DIG-like fractions. Immunoprecipitation studies revealed coprecipitation of a pool of occludin with caveolin-1, a scaffolding protein abundant in DIGs. Coprecipitation results were supported by immunofluorescence and immunogold labeling studies demonstrating caveolin-1 localization in the apical membrane and focal colocalization with occludin in TJs. TJ disassembly by calcium chelation resulted in displacement of TJ proteins from the ‘raft-like’ compartment. Our findings suggest that raft-like compartments play an important role in the spatial organization of TJs and probably in regulation of paracellular permeability in epithelial cells.
Disassembly of membrane-associated NSF 20S complexes is slow relative to vesicle fusion and is Ca2+-independent
Authors: Swanton Eileithyia , Bishop Naomi , Sheehan John , High Stephen , Woodman Philip
Published: April 2021

N-ethylmaleimide-sensitive fusion protein (NSF) and its co-factor soluble NSF attachment protein (α-SNAP) are essential components of the synaptic vesicle fusion machinery and form part of a structurally-conserved 20S protein complex. However, their precise function, relative to fusion itself, is not clear. Using a UV-activated cross-linking approach, we have measured the rate at which a single round of NSF-driven ATP hydrolysis leads to 20S complex disassembly within synaptic membranes. Although this rate is substantially faster than previous

estimates of NSF-dependent ATP hydrolysis, it remains much lower than published rates for fusion of synaptic vesicles. Furthermore, the stability of 20S complexes is unaffected by Ca2+ at concentrations that elicit rapid membrane fusion. We conclude that the ATPase activity of NSF does not contribute directly to vesicle fusion, but more likely plays an earlier role in the synaptic vesicle cycle.
Expression of Eph receptors and ephrins is differentially regulated by E-cadherin
Authors: Orsulic Sandra , Kemler Rolf
Published: April 2021

E-cadherin is the main cell adhesion molecule of early embryonic and adult epithelial cells. Downregulation of E-cadherin is associated with epithelial-mesenchymal transition during embryonic mesoderm formation and tumor progression. To identify genes whose expression is affected by the loss of E-cadherin, we compared mRNA expression patterns between wild-type and E-cadherin null mutant embryonic stem (ES) cells. We found that expression of several Eph receptors and ephrins is dependent on E-cadherin. Rescue of E-cadherin null ES cells with E-cadherin cDNA restores the wild-type expression pattern of Eph family members. Rescue of E-cadherin null ES cells with N-cadherin cDNA does not restore the wild-type expression pattern, indicating that the

regulation of differential expression of Eph family members is specific to E-cadherin. Constitutive ectopic expression of E-cadherin in non-epithelial NIH3T3 cells results in the production of the EphA2 receptor. In epithelial cells, E-cadherin is required for EphA2 receptor localization at cell-cell contacts; in the absence of functional E-cadherin, EphA2 localizes to the perinuclear region. Our results indicate that E-cadherin may be directly or indirectly required for the membrane localization of Eph receptors and their membrane-bound ligands.
Requirement for C-terminal end of fibroblast growth factor receptor 4 in translocation of acidic fibroblast growth factor to cytosol and nucleus
Authors: Klingenberg Olav , Więdłocha Antoni , Rapak Andrzej , Khnykin Denis , Citores Lucia , Olsnes Sjur
Published: April 2021

The ability of COS cells to bind and internalise acidic fibroblast growth factor (aFGF) was studied after transient transfection of the cells with wild-type and mutated fibroblast growth factor receptor 4. In one case the tyrosine kinase of the receptor was inactivated by a point mutation in the active site, whereas in other cases parts of the receptor were deleted to remove various parts of the cytoplasmic domain. In all cases the receptors were expressed at the cell surface at a high level and the cells bound labelled growth factor efficiently and internalised it by endocytosis. Translocation of externally added aFGF across cellular membranes to reach the cytosol and nucleus was measured as transport of labelled growth factor to the nuclear fraction obtained by centrifugation, by farnesylation of growth factor modified to carry a CAAX motif, and by phosphorylation of the growth factor at a site specific for protein kinase C. Whereas both full-length receptors (with and without an active kinase domain) facilitated translocation of the growth factor to the cytosol and nucleus, as assessed by these methods, the mutants of the receptor where the C terminus was deleted, were unable to do so. In contrast, a receptor containing only the 57 most C-terminal amino acids of the cytoplasmic domain in addition to the juxtamembrane, transmembrane and extracellular domains, was in fact able to mediate translocation of aFGF to the cytosol. These data indicate that information contained in the C terminus of the receptor is required for translocation.
Galectin-8 binding to integrins inhibits cell adhesion and induces apoptosis
Authors: Hadari Yaron R. , Goren Rinat Arbel- , Levy Yifat , Amsterdam Avraham , Alon Ronen , Zakut Rina , Zick Yehiel
Published: April 2021

The interaction of cells with the extracellular matrix regulates cell adhesion, motility, growth, survival and differentiation through integrinmediated signal transduction. Here we demonstrate that galectin-8, a secreted mammalian β-galactoside binding protein, inhibits adhesion of human carcinoma (1299) cells to plates coated with integrin ligands, and induces cell apoptosis. Pretreatment of the cells with Mn2+, which increases the affinity of integrins for their ligands, abolished the inhibitory effects of galectin-8. The inhibitory effects of galectin-8 were specific and were not mimicked by plant lectins or other galectins (galectin-1 and galectin-3). In accordance with its antiadhesive effects, transfection of galectin-8 cDNA into 1299 cells significantly reduced (by 75%) colony formation, when compared to the number of colonies formed by cells transfected with an empty vector. Affinity chromatography over immobilized galectin-8 indicated that few membrane proteins interacted with galectin-8 in a sugardependent manner. Microsequencing and western immunoblotting revealed that α3β1 integrin derived from 1299 as well as other cells (e.g. HeLa and human endothelial cells) is a major galectin-8 bindingprotein. Furthermore, immunoprecipitation and immunohistochemical studies suggested that endogenous galectin-8, secreted from 1299 cells, forms complexes with α3β1 integrins expressed on the surface of 1299 cells. Galectin-8 also interacts with other members of the integrin family, like α6β1 integrins. In contrast, galectin-8 only minimally interacts with α4 or β3 integrins. We propose that galectin-8 is an integrin bindingprotein that interacts to a different extent with several, but not all members of the integrin family. Binding of galectin-8 modulates integrin interactions with the extracellular matrix and thus regulates cell adhesion and cell survival.
Toxoplasma gondii catalase: are there peroxisomes in Toxoplasma?
Authors: Ding M. , Clayton C. , Soldati D.
Published: April 2021

The intracellular protozoan parasite Toxoplasma gondii, like all members of the phylum Apicomplexa, is known to possess many organelles: in addition to mitochondria and the compartments of the secretory pathway, there is a reduced chloroplast (the apicoplast) and the phylumspecific components of the apical complex: dense granules, micronemes and rhoptries. Conspicuously missing so far are microbodies, organelles that can be found in nearly all eukaryotic organisms. Microbodies show a large variation with regard to their size, number and contents, depending on the organism and cell type. One marker enzyme of this single membranebound organelle is catalase, which is responsible for the degradation of hydrogen peroxide to water and oxygen. The EST project in T. gondii revealed the existence of two overlapping clones which showed similarity with catalase, and these were used to clone the corresponding gene. The predicted sequence of T. gondii catalase has -AKM at the C terminus, which falls within the consensus of the PTS1 peroxisomal targeting signal. Southern blot analysis confirmed the presence of a single copy gene. Northern and western blot analyses showed that the catalase gene is transcribed and translated. Immunofluorescence assays using an antibody raised against a catalase peptide identified a distinct structure towards the apical end, but other catalasespecific antibodies failed to confirm this localisation. Cell fractionations indicated that the majority of the enzyme was in the cytosol. The fusion of the C-terminal twelve amino acids, including AKM, or the canonical peroxisomal targeting signal, -SKL, to GFP resulted in predominantly cytosolic localization in T. gondii. There was therefore no evidence for membranebound peroxisomes in Toxoplasma.
When it is time to have an old dogma put down
Authors: A Caveman
Published: April 2021

Consider the life cycle of a scientific idea - from birth, through development, to deathâ€¦
Intranuclear trafficking of transcription factors: implications for biological control
Authors: Stein Gary S. , André J. van Wijnen , Stein Janet L. , Lian Jane B. , Montecino Martin , Choi Je-Yong , Zaidi Kaleem , Javed Amjad
Published: April 2021

The subnuclear organization of nucleic acids and cognate regulatory factors suggests that there are functional interrelationships between nuclear structure and gene expression. Nuclear proteins that are localized in discrete domains within the nucleus include the leukemiaassociated acute myelogenous leukemia (AML) and promyelocytic leukemia (PML) factors, the SC-35 RNA-processing factors, nucleolar proteins and components of both transcriptional and DNA replication complexes. Mechanisms that control the spatial distribution of transcription factors within the three-dimensional context of the nucleus may involve the sorting of regulatory information, as well as contribute to the assembly and activity of sites that support gene expression. Molecular, cellular, genetic and biochemical approaches have identified distinct protein segments, termed intranucleartargeting signals, that are responsible for directing regulatory factors to specific subnuclear sites. Gene rearrangements that remove or alter intranucleartargeting signals are prevalent in leukemias and have been linked to altered localization of regulatory factors within the nucleus. These modifications in the intranuclear targeting of transcription factors might abrogate fidelity of gene expression in tumor cells by influencing the spatial organization and/or assembly of machineries involved in the synthesis and processing of gene transcripts.
The role of retinoic acid receptors in neurite outgrowth from different populations of embryonic mouse dorsal root ganglia
Authors: Corcoran Jonathan , Shroot Braham , Pizzey John , Maden Malcolm
Published: April 2021

Dorsal root ganglion (DRG) neurons can be categorised into at least three types, based upon their neurotrophin requirement for survival. We have analysed the expression of the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) in NGF, NT-3 and BDNF dependent neurons isolated from embryonic day (E)13.5 mouse DRG. We show that each population of neurons expressed each of the three RXRs, α, β and γ; however, whilst the NGF and NT-3 dependent neurons expressed each of the RARs α, β and γ, the BDNF dependent neurons only expressed RAR α and β. When retinoic acid was added to each of the neuronal classes only the NGF and NT-3 dependent neurons responded by extending neurites, and this response involved the upregulation of RAR β2. This specificity was confirmed by the use of receptor-selective agonists as only a RAR β-selective compound stimulated neurite outgrowth. These results suggest a role for RA acting via RAR β2 in the outgrowth of neurites.
CD44 expression and regulation during mammary gland development and function
Authors: Hebbard Lionel , Steffen Anja , Zawadzki Volker , Fieber Christina , Howells Norma , Moll Jürgen , Ponta Helmut , Hofmann Martin , Sleeman Jonathan
Published: April 2021

The CD44v6 epitope has been widely reported to be expressed in human mammary carcinomas, yet its prognostic significance is controversial and its function in mammary tumors and mammary glands is unknown. To begin to resolve these issues, we analysed in detail the normal postnatal expression patterns and regulation of the CD44v6 epitope in murine mammary glands. We demonstrate that significant CD44v6 epitope expression is first seen during puberty, and that after puberty CD44v6 epitope expression follows the estrous cycle. CD44v6 epitope expression is observed in the myoepithelium and also less widely in luminal epithelial cells. During lactation, CD44v6 epitope expression is turned off and reappears during involution. The CD44 variant isoform bearing the v6 epitope is CD44v1-v10. Using HC11, a mammary epithelial cell line with stem cell characteristics, and facilitated by the cloning of the murine CD44 promoter, we show that growth factors and hormones which regulate ductal growth and differentiation modulate CD44 transcription. Together our data suggest that the CD44v6 epitope is expressed in mammary epithelial stems cells and in lineages derived from these cells, and that CD44v6 expression is regulated in part by hormones and growth factors such as IGF-1 and EGF which regulate the growth and differentiation of the mammary epithelium. The function of these same growth factors and hormones is often perturbed in mammary carcinomas, and we suggest that CD44v6 expression in tumors reflects this perturbation. We conclude that the expression of the CD44v6 epitope observed in some mammary tumors reflects the stem cell origin of breast tumors, and that whether or not the CD44v6 epitope is expressed in a mammary tumor is determined by the differentiation status of the tumor cells.
When you get to the top, avoid turning to the dark side
Authors: A Caveman
Published: April 2021

Picture the seasoned campaigner sitting at a desk, thinking about their rise through the ranks - the mustering of equipment and personnel, the mad rush to be first in position, the occasional setbacks, images of out-flanking maneuvers by competitors, the problems around a difficult experiment, the sacrifices, the momentary defeats long forgottenâ€¦
Membrane gamma-glutamyl transpeptidase activity of melanoma cells: effects on cellular H2O2 production, cell surface protein thiol oxidation and NF-κB activation status
Authors: Maellaro Emilia , Dominici Silvia , Bello Barbara Del , Valentini Marta A. , Pieri Lisa , Perego Paola , Supino Rosanna , Zunino Franco , Lorenzini Evelina , Paolicchi Aldo , Comporti Mario , Pompella Alfonso
Published: April 2021

The metabolism of glutathione by membrane-bound gamma-glutamyl transpeptidase (GGT) has been recently recognized as a basal source of hydrogen peroxide in the extracellular space. Significant levels of GGT activity are expressed by malignant tumours, and in melanoma cell lines they were found to correlate with the malignant behaviour. As hydrogen peroxide and other oxidants can affect signal transduction pathways at several levels, the present study was aimed to verify: (i) the occurrence of GGT-dependent production of hydrogen peroxide in melanoma cells; (ii) the effects of GGT-dependent prooxidant reactions on known redox-sensitive cellular targets, i.e. protein thiols, the nuclear transcription factor NF-κB and p53. Two melanoma Me665/2 cell clones, exhibiting traces of (clone 2/21) or high (clone 2/60) GGT activity, were studied. The occurrence of GGT-dependent production of hydrogen peroxide was apparent in 2/60 cells, in which it was accompanied by lower levels of cell surface protein thiols. In 2/60 cells, GGT expression was also associated with higher levels of NF-κB activation, as compared to GGT-poor 2/21 cell clone. Indeed, stimulation or inhibition of GGT activity in 2/60 cells resulted in progressive activation or inactivation of NF-κB, respectively. An analysis of the p53 gene product indicated lack of protein expression in 2/60 cells, whereas a mutant protein was highly expressed in 2/21 cells. Taken together, these results indicate that the expression of GGT activity can provide melanoma cells with an additional source of hydrogen peroxide, and that such prooxidant reactions are capable to modify protein thiols at the cell surface level. In addition, GGT expression results in an up-regulation of the transcription factor NF-κB, which could explain the higher metastatic behaviour reported for GGT-rich melanoma cells as compared to their GGT-poor counterparts.
Dynamic, Rho1p-dependent localization of Pkc1p to sites of polarized growth
Authors: Andrews Paul D. , Stark Michael J. R.
Published: April 2021

In eukaryotes, the Rho GTPases and their effectors are key regulators of the actin cytoskeleton, membrane trafficking and secretion, cell growth, cell cycle progression and cytokinesis. Budding yeast Pkc1p, a protein kinase C-like enzyme involved in cell wall biosynthesis and cytoskeletal polarity, is structurally and functionally related to the Rho- associated kinases (PRK/ROCK) of mammalian cells. In this study, localization of Pkc1p was monitored in live cells using a GFP fusion (Pkc1p-GFP). Pkc1p-GFP showed dynamic spatial and temporal localization at sites of polarized growth. Early in the cell cycle, Pkc1p-GFP was found at the pre-bud site and bud tips, becoming delocalized as the cell progressed further and finally relocalizing around the mother-daughter bud neck in an incomplete ring, which persisted until cell separation. Bud localization was actin-dependent but stability of Pkc1p- GFP at the neck was actin-independent, although localization at both sites required functional Rho1p. In addition, Pkc1p-GFP showed rapid relocalization after cell wall damage. These results suggest that the roles of Pkc1p in both polarized growth and the response to cell wall stress are mediated by dynamic changes in its localization, and suggest an additional potential role in cytokinesis.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs1621.html, Movie 2, Movie 3, Movie 4, Movie 5, Movie 6, Movie 7, Movie 8, Movie 9
Slow muscle induction by Hedgehog signalling in vitro
Authors: Norris Wendie , Neyt Christine , Ingham Phillip W. , Currie Peter D.
Published: April 2021

Muscles are composed of several fibre types, the precise combination of which determines muscle function. Whereas neonatal and adult fibre type is influenced by a number of extrinsic factors, such as neural input and muscle load, there is little knowledge of how muscle cells are initially determined in the early embryo. In the zebrafish, fibres of the slow twitch class arise from precociously specified myoblasts that lie close to the midline whereas the remainder of the myotome differentiates as fast myosin expressing muscle. In vivo evidence has suggested the Sonic Hedgehog glycoprotein, secreted from the notochord, controls the formation of slow twitch and fast twitch muscle fates. Here we describe an in vitro culture system that we have developed to test directly the ability of zebrafish myoblasts to respond to exogenous Sonic Hedgehog peptide. We find that Sonic Hedgehog peptide can control the binary cell fate choice of embryonic zebrafish myoblasts in vitro. We have also used this culture system to assay the relative activities of different Hedgehog-family proteins and to investigate the possible involvement of heterotrimeric G-proteins in Hedgehog signal transduction.
The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus
Authors: Opdam Frank J. M. , Echard Arnaud , Croes Huib J. E. , van den Hurk José A. J. M. , van de Vorstenbosch Rinske A. , Ginsel Leo A. , Goud Bruno , Fransen Jack A. M.
Published: April 2021

Members of the Rab subfamily of small GTPases play an important role in the regulation of intracellular transport routes. Rab6A has been shown to be a regulator of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER). Here, we report on the identification of a Rab6 isoform, termed Rab6B. The corresponding full-length cDNA was isolated from a Caco-2 cell library. The deduced amino acid sequence showed 91% identity with the Rab6A protein and revealed that sequence divergence is dispersed over a large region of the COOH-terminal domain. Rab6B is encoded by an independent gene which is located on chromosome 3 region q21-q23. In contrast to Rab6A whose expression is ubiquitous, northern blot analysis, immunohistochemistry, and immunofluorescence demonstrated that Rab6B is expressed in a tissue and cell-type specific manner. Rab6B is predominantly expressed in brain and the neuroblastoma cell line SK-N-SH. In brain, Rab6B was found to be specifically expressed in microglia, pericytes and Purkinje cells. Endogenous Rab6B localises to the Golgi apparatus and to ERGIC-53-positive vesicles. Comparable studies between Rab6A and Rab6B revealed distinct biochemical and cellular properties. Rab6B displayed lower GTP-binding activities and in overexpression studies, the protein is distributed over Golgi and ER membranes, whereas Rab6A is more restricted to the Golgi apparatus. Since the GTP-bound form of Rab6B (Rab6B Q72L) does interact with all known Rab6A effectors, including Rabkinesin-6, the results suggest a cell-type specific role for Rab6B in retrograde membrane traffic at the level of the Golgi complex.
A confectioner's approach to success and progress, and why Carlos Santana and i like the same cake
Authors: A Caveman
Published: April 2021

In science, there are two cakes from which we all strive to eat a littleâ€¦
Cellular Samurai: katanin and the severing of microtubules
Authors: Quarmby Lynne
Published: April 2021

Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI- 1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.