bluuebunny/crossref_metadata_2025 huggingface dataset:
Immortalised mouse submandibular epithelial cell lines retain polarised structural and functional properties
Authors: Laoide Bríd M. , Courty Yves , Gastinne Isabelle , Thibaut Cécile , Kellermann Odile , Rougeon François
Published: April 2021

The mouse submandibular gland (SMG) is an excellent model for the study of many important biological phenomena such as hormonal regulation of differentiation, neurotransmitter control of secretion, epithelial transport, exocytosis and endocytosis as well as the regulation of mouse SMG specific gene expression, in particular, NGF, EGF and renin. The postnatal development and sexual dimorphism of the mouse gland permits the isolation of male SMGs of different ages, corresponding to different stages of differentiation, particularily with respect to the cytodifferentiation of ductal cell types. We have immortalized SMG epithelial cell lines using mice transgenic for the large T antigen of SV40 or polyoma viruses. Epithelial clusters from the dissected glands were placed in culture and cell lines were established from the immortalized population. Two cell lines, SIMS and SIMP, which retain structural and functional characteristics, are described here. The cell lines are immortalised but not transformed, as judged by the absence of anchorage independent growth potential and the lack of tumour formation in athymic nude mice. Confocal and electron microscopy examination demonstrate that SIMP and SIMS cells express E-cadherin and ZO-1 and have features of polarised epithelial cells. In addition, they form spherical cysts with a wide lumen when grown in type I collagen gels. When grown on a filter support SIMS cells form a tight monolayer, exhibit vectorial transport function and show exclusive Na+,K+-ATPase localisation to the basolateral domain. We determined the cell type restricted expression of cytokeratin markers in the mouse SMG in vivo and we demonstrate that SIMS and SIMP cell lines express duct-specific cytokeratins. Finally, the expression of a set of differentiation markers, including EGF, NGF and renin, was detected by RT-PCR and by indirect immunofluorescence staining in these lines. Thus, these polarised ductal cell lines, as well as having important intrinsic properties, represent well characterised mouse epithelial models which, until now, have not been readily available for cellular studies.
Integrin α6Bβ1 is involved in kidney tubulogenesis in vitro
Authors: Falk Mats , Salmivirta Katriina , Durbeej Madeleine , Larsson Erik , Ekblom Marja , Vestweber Dietmar , Ekblom Peter
Published: April 2021

Laminin-1 has previously been shown to be of major importance for the development of kidney tubules. Antibodies against fragments E8 and E3 of laminin-1 perturb kidney development in vitro. We here studied expression of integrins α6β1 and α6β4, two known laminin receptors, during kidney development. Integrin β1 subunit could be detected by immunofluorescence on all cell types of embryonic mouse kidney, but we could not detect integrin β4 subunit in embryonic kidney by immunofluorescence or by in situ hybridization. The presence of integrin α6 subunit in all epithelia of embryonic kidney was demonstrated by immunofluorescence and by in situ hybridization. RT-PCR showed that α6B is the major splice variant in embryonic kidney. During in vitro conversion of nephrogenic mesenchyme to epithelial tubules, a strong increase in the expression of the 6 kb mRNA for α6 integrin subunit was seen by northern blotting at the onset of epithelial morphogenesis, on day two of culture. Immunoprecipitation of extracts from embryonic kidney with antibodies against α6 subunit yielded bands corresponding to the expected size of β1 integrin subunit but not of β4 subunit. Monoclonal antibodies against either α6 or β1 subunit but not against E-cadherin blocked kidney tubulogenesis in vitro. This suggests that integrin α6Bβ1 is involved in kidney tubulogenesis in vitro. Another possibility is that the antibodies against integrin α6 and β1 subunit cause abnormal signalling by the integrin.
p230 is associated with vesicles budding from the trans-Golgi network
Authors: Gleeson Paul A. , Anderson Timothy J. , Stow Jennifer L. , Griffiths Gareth , Toh Ban Hock , Matheson Fiona
Published: April 2021

Transport vesicle formation requires the association of cytosolic proteins with the membrane. We have previously described a brefeldin-A sensitive, hydrophilic protein (p230), containing a very high frequency of heptad repeats, found in the cytosol and associated with Golgi membranes. We show here that p230 is localised on the trans-Golgi network, by immunogold labeling of HeLa cell cryosections using α2,6 sialyltransferase as a compartment-specific marker. The role of G protein activators on the binding of p230 to Golgi membranes and in vesicle biogenesis has been investigated. Treatment of streptolysin-O permeabilised HeLa cells with either GTPγS or AlF4− resulted in accumulation of p230 on Golgi membranes. Furthermore, immunolabeling of isolated Golgi membranes treated with AlF4−, to induce the accumulation of vesicles, showed that p230 is predominantly localised to the cytoplasmic surface of trans-Golgi network-derived budding structures and small coated vesicles. p230-labeled vesicles have a thin (∼10 nm) electron dense cytoplasmic coat and could be readily distinguished from clathrin-coated vesicles. Dual immunogold labeling of perforated cells, or of cryosections of treated Golgi membranes, revealed that p230 and the trans-Golgi network-associated p200, which we show here to be distinct molecules, appear to be localised on separate populations of vesicles budding from the trans-Golgi network. These results strongly suggest the presence of distinct populations of non-clathrin coated vesicles derived from the trans-Golgi network. As p230 recycles between the cytosol and buds/vesicles of TGN membranes, a process regulated by G proteins, we propose that p230 is involved in the biogenesis of a specific population of non-clathrin coated vesicles.
Expression of fibulin-2 by fibroblasts and deposition with fibronectin into a fibrillar matrix
Authors: Sasaki Takako , Wiedemann Hanna , Matzner Monika , Chu Mon-Li , Timpl Rupert
Published: April 2021

The extracellular matrix protein fibulin-2 was shown to be a typical product of cultured human and mouse fibroblasts by several immunological assays. It is secreted and deposited in cells and tissues as a disulfide-bonded oligomer identical in size to the previously described recombinant fibulin-2. Most of the fibroblast fibulin-2 is deposited into a dense fibrillar meshwork which requires treatment with EDTA and/or 6 M urea for solubilization. Fibulin-2 and fibronectin are synthesized at equivalent levels and both colocalize in the fibrils as shown by immunofluorescence. Metabolic labelling and pulse-chase studies demonstrated fibulin-2 oligomers in detergent extracts of cells and their rapid translocation to extracellular EDTA-sensitive assembly forms. Unlike for fibronectin and fibulin-1 only a little fibulin-2 was found in the cell culture medium. Immunogold staining of confluent human fibroblasts showed localization of fibulin-2 to a fine meshwork or bundles of amorphous microfibrils in the matrix. This also demonstrated a distinct colocalization of fibulin-2 and fibronectin at the electron microscope level, indicating that the interaction between these two protein shown in in vitro assays may also exist in situ. No distinct colocalization of both proteins could, however, be observed with crossstriated fibrils of collagen I and collagen VI microfibrils.
Subcellular localization of Trypanosoma cruzi glycoprotein Gp72
Authors: Haynes Paul A. , Russell David G. , Cross George A. M.
Published: April 2021

We have investigated the subcellular location of the Trypanosoma cruzi surface glycoprotein, Gp72, by introducing epitope-tagged copies of gp72 into gp72 null-mutant cells. A tagged Gp72, containing three tandemly repeated copies of a human influenza hemagglutinin nonapeptide (HA) adjacent to the mature Gp72 amino terminus, was able to complement the null mutant phenotype, as well as being recognized in western blots by both anti-HA antibody and the carbohydrate-specific monoclonal antibody WIC29.26. Integration of this epitope-tagged gp72 into the chromosomal gp72 locus produced a clonal cell line, 72HAN3.1G7, which was used for studies of the subcellular location of the epitope-tagged Gp72. Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 epimastigotes showed that Gp72 was evenly distributed over the cell body and somewhat concentrated in the proximal region of the flagellum. No fluorescence could be detected in the distal tip of the flagellum. Immunoelectron microscopy of fixed 72HAN3.1G7 epimastigotes revealed that Gp72 was predominantly membrane-associated and located on the cell surface. Indirect immunofluorescence microscopy of live 72HAN3.1G7 epimastigote cells showed a similar pattern of fluorescence on the flagellum, but no fluorescence was detected on the cell body, which was attributed to masking by other cell-surface components. Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 amastigotes revealed that Gp72, which has long been considered to be expressed only in epimastigotes and metacyclic trypomastigotes, can be expressed in amastigotes, but it no longer contains the WIC29.26 carbohydrate epitope.
A membrane-associated protein complex with selective binding to the clathrin coat adaptor AP1
Authors: Mallet William G. , Brodsky Frances M.
Published: April 2021

Adaptors are the membrane-binding components of clathrin-coated vesicles. The interaction of the trans-Golgi coat adaptor AP1 with membrane-associated proteins was analyzed by affinity chromatography. Proteins of 83 and 52 kDa bound specifically to the core domain of AP1 and showed no interaction with AP2 or other clathrin-coated vesicle proteins. The AP1-binding proteins were tightly membrane-associated, though behaved as peripheral membrane proteins. They were detected in membranes depleted of clathrin-coated vesicles and not in coated vesicles, suggesting that the interaction of these proteins with AP1 may precede coated vesicle budding. Co-fractionation of the AP1-binding proteins with trans-Golgi network membrane was also observed. Upon gel filtration, both AP1-binding proteins eluted in a high molecular mass complex which was labile at high concentrations of Tris. The 83 kDa protein bound to AP1 affinity resin in the absence of the 52 kDa protein. In contrast, the separated 52 kDa protein did not bind AP1, suggesting that the 83 kDa protein is the AP1-binding component of the complex. Characterization of this protein complex defines a novel membrane-associated component that specifically interacts with AP1 and may contribute to its function in forming clathrin-coated vesicles.
Most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles
Authors: Paoletti Anne , Moudjou Mohammed , Paintrand Michel , Salisbury Jeffrey L. , Bornens Michel
Published: April 2021

Centrin is a member of the calcium-binding EF-hand protein superfamily present in centrosomes of widely divergent species. Investigating the cellular distribution of human centrin by both immunofluorescence and cell fractionation, we report that centrin is biochemically complex in human cells, displaying as much as ten isoforms in 2-D electrophoresis. This suggests that centrin may be subject to multiple regulations. Strikingly, more than 90% of centrin is not associated with the centrosome fraction. The centrosome-associated centrin, however, displays a specific pattern in 2-D electrophoresis and is concentrated within the distal lumen of the centrioles, where a complex structure has been previously described. This precise localization allows the resolution of centrioles at the optical level throughout the cell cycle and provides a valuable tool for monitoring centriole duplication. To get insights on centrin function, we performed injection experiments of recombinant heterologous centrin in two-cell stage frog embryos in an attempt to produce dominant negative effects. We report that green algae and human centrin delay cleavage and promote the formation of abnormal blastomeres in which the distribution of microtubule asters and of nuclei is dramatically impaired. This suggests that centrin could be involved in the centrosome reproduction cycle, in the coordination of cytoplasmic and nuclear division or in cytokinesis.
Bone-resorbing osteoclasts reveal a dynamic division of basal plasma membrane into two different domains
Authors: Salo Jari , Metsikkö Kalervo , Palokangas Harri , Lehenkari Petri , Väänänen H. Kalervo
Published: April 2021

Bone-resorbing multinucleate osteoclasts exhibit a ruffled border membrane apposing the bone and a basal membrane contacting the circulation. A junctional complex called the sealing zone separates these two membrane domains, but the defined nature of these membrane domains has remained obscure. We now show, using enveloped viral glycoproteins and lectins as tools, that osteoclasts exhibit a novel membrane domain in the basal surface when they are polarized for resorption. Influenza haemagglutinin, which is apically targeted in epithelial cells, is targeted to a restricted area at the top of the basal surface, while vesicular stomatitis virus G-protein which is basolaterally targeted in epithelia, occupies the rest of the basal surface. Neither of these viral glycoproteins is gathered to the ruffled border nor sealing zone area, but they share in a specific way the basal surface. To show that the division of basal membrane into two different domains also occurs in non-infected cells, we have analyzed the distribution of receptors for these viruses and binding sites of some lectins. Both of these methods show that also some endogenous proteins are located in different domains in the basal surface in active osteoclasts. We also show that these two different membrane domains can be distinquished in scanning electron microscopy level due to the villus appearance of the central basal domain.
Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication
Authors: Krude Torsten , Musahl Christine , Laskey Ronald A. , Knippers Rolf
Published: April 2021

Members of the Mcm-protein family have recently been shown to be involved in restricting DNA replication to a single cycle in Xenopus laevis egg extracts. In this study, we extended these observations to human somatic cells and analysed the localisation of the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 in replicating HeLa cell nuclei. These Mcm-proteins are entirely nuclear in interphase cells and apparently exist in two populations: a nucleosolic population, and a population bound to a nuclear structure, most likely chromatin. The bound population is detected throughout the nucleus in late G1 and early S, and at discrete subnuclear sites following further progression of S-phase. We use high resolution confocal microscopy to determine the subnuclear sites of chromatin-bound Mcm proteins in comparison to the sites of replicating DNA. Importantly, hCdc21, hCdc46 and P1Mcm3 do not colocalise with replication foci, instead these proteins appear to coincide with subnuclear sites of unreplicated chromatin. During progression of S-phase hCdc21, hCdc46 and P1Mcm3 are displaced from their site on chromatin at the time when this site is replicated. Consequently, early replicating sites do not contain bound hCdc21, hCdc46 or P1Mcm3 during later stages of S-phase. Furthermore, G2 nuclei and condensed chromatin in mitotic cells do not contain bound hCdc21, hCdc46 or P1Mcm3.

Thus, the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 are not concentrated at sites of DNA replication. Instead, they appear to be present only on unreplicated chromatin and are displaced from replicating chromatin, consistent with a role in monitoring unreplicated chromatin and ensuring only a single round of DNA replication per cell cycle.
Hyaluronan contributes to the enlargement of hypertrophic lacunae in the growth plate
Authors: Pavasant Prasit , Shizari Tony , Underhill Charles B.
Published: April 2021

Histochemical staining of the epiphysial growth plate revealed that free hyaluronan (i.e. available to the staining probe) was restricted to the zone of hypertrophy, where it was located in the pericellular space between the chondrocytes and the edge of the lacunae. Furthermore, the amount of hyaluronan staining was directly proportional to the size of the lacunae. Autoradiographic analysis of growth plates cultured with isotopically labeled glucosamine indicated that at least a portion of this hyaluronan was newly synthesized by the hypertrophic chondrocytes. Since hyaluronan can adsorb large amounts of water, it is possible that it exerted a hydrostatic pressure on the surrounding territorial matrix and thereby caused the expansion of hypertrophic lacunae. To assess this possibility, segments of the growth plate were placed in organ culture under different conditions. Under normal culture conditions, a band of hyaluronan staining migrated across the segments coinciding with the enlargement of lacunae in these regions, and the segments, as a whole, increased in size. In contrast, when the segments were cultured in the presence of hyaluronidase, which degraded the pericellular hyaluronan, the lacunae did not undergo enlargement and the overall size of the segments did not increase. These results suggest that the production of hyaluronan contributes to the enlargement of hypertrophic lacunae which is important for determining both the body’s stature and proportions.
Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs
Authors: Dutartre Hélène , Davoust Jean , Gorvel Jean-Pierre , Chavrier Philippe
Published: April 2021

In mammalian cells, Rho GTPases control the reorganisation of the actin cytoskeleton in response to growth factors. In the cytoplasm, the polymerisation of actin filaments and their organisation into complex architectures is orchestrated by numerous proteins which act either directly, by interacting with actin, or by producing secondary messengers which serve as mediators between signal transduction pathways and the microfilament organisation. We sought to determine whether the intracellular distribution of some of these regulatory components may be controlled by the Rho GTPase CDC42Hs.

With this aim, we have established HeLa-derived human cell lines in which expression of a constitutively activated mutant of CDC42Hs is inducible. Morphological analysis by immunofluorescence labelling and confocal laser scanning microscopy revealed a massive reorganisation of F-actin in cortical microspikes as well as podosome-like structures located at the ventral face of the cells. Concomitantly, the cells became giant and multinucleate indicating that cytokinesis was impaired. The actin bundling protein T-plastin, the vasodilatator-stimulated phosphoprotein (VASP), a profilin ligand, as well as the 85 kDa regulatory subunit of the phosphoinosite 3-kinase redistributed with F-actin into the CDC42Hs-induced structures.
Overexpression of occludin, a tight junction-associated integral membrane protein, induces the formation of intracellular multilamellar bodies bearing tight junction-like structures
Authors: Furuse Mikio , Fujimoto Kazushi , Sato Naruki , Hirase Tetsuaki , Tsukita Sachiko , Tsukita Shoichiro
Published: April 2021

Occludin is an integral membrane protein localizing at tight junctions with four transmembrane domains. When chicken occludin was overexpressed in insect cells by recombinant baculovirus infection, peculiar multilamellar structures accumulated in the cytoplasm. Partial isolation of these structures indicated that the introduced chicken occludin was highly enriched in these structures. Thin section electron microscopy revealed that each lamella was transformed from intracellular membranous cisternae whose luminal space was completely collapsed, and that in each lamella, outer leaflets of opposing membranes appeared to be fused with no gaps, like tight junctions. Furthermore, in the freeze-fracture replicas of these multilamellar structures, short tight junction-like intramembranous particle strands were occasionally observed, which were specifically labeled by anti-occludin mAb. These observations favor the idea that occludin plays a key role in the formation of tight junctions.
Time-resolved, in vivo studies of mitotic spindle formation and nuclear lamina breakdown in Drosophila early embryos
Authors: Paddy Michael R. , Saumweber Harald , Agard David A. , Sedat John W.
Published: April 2021

Time-resolved, two-component, three-dimensional fluorescence light microscopy imaging in living Drosophila early embryos is used to demonstrate that a large fraction of the nuclear envelope lamins remain localized to a rim in the nuclear periphery until well into metaphase. The process of lamin delocalization and dispersal, typical of ‘open’ forms of mitosis, does not begin until about the time the final, metaphase geometry of the mitotic spindle is attained. Lamin dispersal is completed about the time that the chromosomal movements of anaphase begin. This pattern of nuclear lamina breakdown appears to be intermediate between traditional designations of ‘open’ and ‘closed’ mitoses. These results thus clarify earlier observations of lamins in mitosis in fixed Drosophila early embryos, clearly showing that the observed lamin localization does not result from a structurally defined ‘spindle envelope’ that persists throughout mitosis.

During this extended time interval of lamin localization in the nuclear periphery, the lamina undergoes an extensive series of structural rearrangements that are closely coupled to, and likely driven by, the movements of the centrosomes and microtubules that produce the mitotic spindle. Furthermore, throughout this time the nuclear envelope structure is permeable to large macromolecules, which are excluded in interphase. While the functional significance of these structural dynamics is not yet clear, it is consistent with a functional role for the lamina in mitotic spindle formation.
Enhanced E-cadherin expression and increased calcium-dependent cell-cell adhesion in Human T-cell leukemia virus type I Tax-expressing PC12 cells
Authors: Kitajima I. , Kawahara K. , Hanyu N. , Shin H. , Tokioka T. , Soejima Y. , Tsutsui J. , Ozawa M. , Shimayama T. , Maruyama I.
Published: April 2021

Human T-cell leukemia virus type I (HTLV-I) Tax protein induces the expression of host cellular genes, some of which are crucial in cell proliferation and differentiation. We examined the mechanisms by which HTLV-I Tax protein induces phenotypic changes in PC12 cells. We demonstrated that the HTLV-I Tax gene induces epithelioid changes and increases cell-cell contact in PC12 cells. No change in the expression of the neural cell adhesion molecule was observed between HTLV-I Tax-expressing PC12 cells and PC12 cells transfected with a control plasmid. However, HTLV-I Tax-expressing PC12 cells demonstrated a marked change in the abundance and distribution of E-cadherin, which was concentrated at regions of cellular contact and accompanied by changes in calcium-dependent cell adhesion. Although E-cadherin is expressed at low levels in PC12 and PC12 transfected with a control plasmid cells, the steady state level of E-cadherin in tax-expressing PC12 cells increases significantly, apparently as a result of regulation at the transcriptional level. Diminished expression of Tax protein in Tax-expressing PC12 cells exposed to antisense oligonucleotides for the Tax gene suppresses E-cadherin expression and decreases cell-cell adhesion. These findings imply that HTLV-I Tax protein enhanced E-cadherin expression modulates calciumdependent cell-cell adhesion mechanisms.
NuMA assembles into an extensive filamentous structure when expressed in the cell cytoplasm
Authors: Saredi Alejandro , Howard Louisa , Compton Duane A.
Published: April 2021

NuMA is a 236 kDa protein that participates in the organization of the mitotic spindle despite its strict localization in the nucleus during interphase. To test how cells progress through mitosis when NuMA is localized in the cytoplasm instead of the nucleus, we have deleted the nuclear localization sequence of NuMA using site-directed mutagenesis and transiently expressed this mutant protein (NuMA-ΔNLS) in BHK-21 cells. During interphase, NuMA-ΔNLS accumulates in the cytoplasm as a large mass approximately the same size as the cell nucleus. When cells enter mitosis, NuMA-ΔNLS associates normally with the mitotic spindle without causing any apparent deleterious effects on the progression of mitosis. Examination of the cytoplasmic mass formed by NuMA-ΔNLS using transmission electron microscopy (TEM) revealed an extensive network of ∼5 nm filaments that are further organized by the presence of dynamic microtubules into a dense web of solid, ∼23 nm cables. Using flow cytometry, we have isolated the intact filamentous mass formed by NuMA-ΔNLS from lysates of transiently transfected cells. These isolated structures are constructed of networks of interconnected 5 nm filaments and are composed exclusively of NuMA. These data demonstrate that NuMA is capable of assembling into an extensive filamentous structure supporting the possibility that NuMA serves a structural function either in the nucleus during interphase or at the polar ends of the mitotic spindle.
Examination of the endosomal and lysosomal pathways in Dictyostelium discoideum myosin I mutants
Authors: Temesvari Lesly A. , Bush John M. , Peterson Michelle D. , Novak Kristine D. , Titus Margaret A. , Cardelli James A.
Published: April 2021

The role of myosin Is in endosomal trafficking and the lysosomal system was investigated in a Dictyostelium discoideum myosin I double mutant myoB-/C-, that has been previously shown to exhibit defects in fluid-phase endocytosis during growth in suspension culture (Novak et al., 1995). Various properties of the endosomal pathway in the myoB-/C- double mutant as well as in the myoB- and myoC- single mutants, including intravesicular pH, and intracellular retention time and exocytosis of a fluid phase marker, were found to be indistinguishable from wild-type parental cells. The intimate connection between the contractile vacuole complex and the endocytic pathway in Dictyostelium, and the localization of a myosin I to the contractile vacuole in Acanthamoeba, led us to also examine the structure and function of this organelle in the three myosin I mutants. No alteration in contractile vacuole structure or function was observed in the myoB-, myoC- or myoB-/C- cell lines. The transport, processing, and localization of a lysosomal enzyme, α-mannosidase, were also unaltered in all three mutants. However, the myoB- and myoB-/C- cell lines, but not the myoC- cell line, were found to oversecrete the lysosomal enzymes α-mannosidase and acid phosphatase, during growth and starvation. None of the mutants oversecreted proteins following the constitutive secretory pathway. Two additional myosin I mutants, myoA- and myoA-/B-, were also found to oversecrete the lysosomally localized enzymes α-mannosidase and acid phosphatase. Taken together, these results suggest that these myosins do not play a role in the intracellular movement of vesicles, but that they may participate in controlling events that occur at the actin-rich cortical region of the cell. While no direct evidence has been found for the association of myosin Is with lysosomes, we predict that the integrity of the lysosomal system is tied to the fidelity of the actin cortex, and changes in cortical organization could influence lysosomal-related membrane events such as internalization or transit of vesicles to the cell surface.
Primate homologues of rat TGN38: primary structure, expression and functional implications
Authors: Ponnambalam Sreenivasan , Girotti Milena , Yaspo Marie-Laure , Owen Charles E. , Perry Anthony C. F. , Suganuma Tatsuo , Nilsson Tommy , Fried Mike , Banting George , Warren Graham
Published: April 2021

cDNAs encoding the human and macaque homologues of rat TGN38 have been cloned and sequenced. The proteins have a highly conserved N terminus (comprising the signal peptide) and C terminus (comprising part of the lumenal domain, the membrane spanning region and cytoplasmic tail) but vary in the other part of the lumenal domain, which contains the repeat region. Whereas rat TGN38 contains 6 tandem repeats of an 8mer, both primate proteins possess 14 tandem repeats of a 14mer sequence. The human protein, like rat TGN38, is localised primarily to the TGN but is present on the cell surface and returns via endosomes. This behaviour is consistent with conservation of the membrane spanning region and the cytoplasmic tail, which contain the retention and retrieval signals, respectively, for localisation in the TGN. The unexpected differences in the lumenal domain can best be rationalised by the fact that both types of repeat domains have most of the properties of mucins. We suggest that TGN38 homologues are mucin-like molecules that regulate membrane traffic to and from the TGN.
The GPI-anchored adhesion molecule F3 induces tyrosine phosphorylation: involvement of the FNIII repeats
Authors: Cervello Melchiorre , Matranga Valeria , Durbec Pascal , Rougon Geneviève , Gomez Sophie
Published: April 2021

The glycosyl-phosphatidylinositol (GPI)-anchored F3 molecule, a member of the Ig superfamily made up of Ig and FNIII-like domains, is involved in cell-cell adhesion, neuronal pathfinding and fasciculation. Little is known about the mechanism(s) that governs the F3-mediated cellcell recognition. In particular, it is not known whether F3 transduces signals across the membrane.

Here we show that in F3-transfected CHO cells (1A cells) an increase in tyrosine phosphorylation occurs during F3-mediated aggregation. Moreover, under aggregation conditions F3 immunoprecipitated from 32P-metabolically labeled 1A cells associated with three major phosphorylated proteins. Interestingly, genistein inhibited the F3-mediated aggregation. Increased tyrosine phosphorylation was also observed using antibody-mediated F3-crosslinking. Furtheremore, F3 expressed both in 1A cells and in post-natal mouse cerebellum forms non-covalent soluble complexes with protein tyrosine kinase(s). In cerebellum the F3-associated kinase was identified as fyn. By contrast, a truncated F3 protein, expressed in CHO cells, from which all the FN type III repeats have been deleted, does not associate with a kinase. Cross-linking of the F3-truncated form does not induce modulation of tyrosine phosphorylation. Taken together these data demonstrate that F3 is a molecule that transduces signals through both association with protein tyrosine kinase and modulation of protein tyrosine phosphorylation. The presence of FN type III domains is essential for the activation of the intracellular signaling pathway.
Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility
Authors: Bré Marie-Hélène , Redeker Virginie , Quibell Martin , Darmanaden-Delorme Julienne , Bressac Christophe , Cosson Jacky , Huitorel Philippe , Schmitter Jean-Marie , Rossier Jean , Johnson Tony , Adoutte André , Levilliers Nicolette
Published: April 2021

Two monoclonal antibodies, AXO 49 and TAP 952, probed with carboxy-terminal peptides from Paramecium axonemal tubulin and with polyglycylated synthetic peptides, are found to recognize differently tubulin polyglycylation, the most recently identified posttranslational modification discovered in Paramecium axonemal tubulin. With these antibodies, we show that tubulin polyglycylation is widely distributed in organisms ranging from ciliated protozoa to mammals; it arose early in the course of evolution, but seems to be absent in primitive protozoa such as the Euglenozoa. Tubulin polyglycylation is the last posttranslational modification which takes place in the course of Drosophila spermatogenesis and its occurrence corresponds to the end of spermatozoan maturation. An involvement of polyglycylated tubulin in axoneme motility is suggested since AXO 49 and TAP 952 specifically inhibit the reactivated motility of sea urchin spermatozoa.
Cycloheximide-induced activation of mouse eggs: effects on cdc2/cyclin B and MAP kinase activities
Authors: Moos Jiri , Kopf Gregory S. , Schultz Richard M.
Published: April 2021

Fertilization of metaphase II-arrested mouse eggs results in resumption of meiosis and a decrease in both cdc2/cyclin B kinase and MAP kinase activities; the decrease in cdc2/cyclin B kinase activity precedes the decrease in MAP kinase activity. Cycloheximide treatment of metaphase II-arrested mouse eggs also results in resumption of meiosis but bypasses the fertilization-induced Ca2+ transient. However, it is not known if cycloheximide treatment results in the same temporal changes in cdc2/cyclin B kinase and MAP kinase activities that are intimately associated with resumption of meiosis. We report that cycloheximidetreated mouse eggs manifest similar temporal changes in the decrease in both cdc2/cyclin B kinase and MAP kinase activities that occur following fertilization, although cortical granule exocytosis is not stimulated. The decrease in cdc2/cyclin B kinase activity, however, does not seem to be required for the decrease in MAP kinase activity, since the decrease in MAP kinase activity still occurs in cycloheximide-treated eggs that are also incubated in the presence of nocodazole, which inhibits cyclin B degradation and hence the decrease in cdc2/cyclin B kinase. Following removal of these drugs, cdc2/cyclin B kinase activity remains high, MAP kinase activity increases to levels similar to that in the metaphase II-arrested eggs, and a spindle(s) forms with the chromosomes aligned on a metaphase plate. Results of these experiments suggest that some other protein with a relatively short half-life, e.g. cmos, a known upstream activator of MAP kinase, may be responsible for events leading to the decrease in MAP kinase activity.
Plant clathrin heavy chain: sequence analysis and restricted localisation in growing pollen tubes
Authors: Blackbourn Hugh D. , Jackson Antony P.
Published: April 2021

Clathrin-coated vesicles were isolated from soybean (Glycine max L.) cells in suspension culture and their purity was assessed using SDS-PAGE, peptide sequencing and electron microscopy. Antibodies raised to these coated vesicles were used to immunoscreen a soybean cDNA library in λgt11 and isolate a partial clone of the clathrin heavy chain (HC) gene. Full-length cDNA for soybean clathrin HC was deduced by 5‘ and 3‘ cDNA amplification. The cDNA encodes an amino acid sequence of 1,700 residues, which is slightly larger than rat clathrin HC and may account for the reduced mobility of plant clathrin on SDS-PAGE. Insertion of these extra residues is largely confined to the amino and carboxy termini. Other domains within the heavy chain arms, including those implicated in light chain binding and trimerisation, are rela tively well conserved between eukaryotes. A computer algorithm to determine α-helical coiled-coil structures reveals that only one domain, aligning to residues 1,460-1,489 in rat clathrin HC, has a high probability for coiled-coil structure in all five eukaryotic clathrin HC sequences. This provides further evidence that the interaction between clathrin heavy and light chains is mediated by three bundles of coiled-coils near to the carboxy terminus. In analysing the role of plant clathrin in endocytotic trafficking, as against trafficking from the Golgi apparatus to the vacuole, our attention was focused on membrane recyling in tip-growing pollen tubes. These rapidly growing cells are highly secretory and require a high level of plasma membrane recycling to maintain the tube tip architecture. Monoclonal antibodies to plant clathrin HC confirmed that coated vesicles are rela tively abundant in tip-growing pollen tubes of Lilium longiflorum. This analysis also demonstrated that a high propor tion of the clathrin present is in an assembled state, suggesting a highly dynamic trafficking pathway. Immuno fluorescence analysis of pollen tubes revealed that clathrin localises to the plasma membrane at the apex of the pollen tube tip, which is consistent with high levels of clathrinmediated membrane recycling. The use of these reagents in conjunction with tip-growing pollen tubes has created a unique opportunity to examine the basis for constitutive endocytosis, so that the more complex question of receptor-mediated pathways in plants can also be assessed.
γ-Tubulin in mammalian cells: the centrosomal and the cytosolic forms
Authors: Moudjou Mohammed , Bordes Nicole , Paintrand Michel , Bornens Michel
Published: April 2021

The centrosome is one of the cellular organelles for which the mechanism by which it operates still remains to be unravelled. The finding of the association with the centrosome of γ-tubulin, a protein which belongs to the tubulin superfamily, has provided a long sought after biochemical tool with which to address centrosome function. We have generated a specific anti-γ-tubulin polyclonal antibody to study the biochemical properties and the cellular distribution of the human lymphoblastic γ-tubulin. Using cell fractionation and mass isolation of centrosomes, we observed that in contrast to the figures suggested by immunofluorescence, a minimum figure of 80% of total γ-tubulin exists as a cytosolic form. The centrosomal form, for which at least half is not strongly associated with the centrosome, behaves in two-dimensional gel electrophoresis identically to the soluble form (as at least two spots of a pI of around 6). Post-embedding immunolocalization reveals that γ-tubulin is distributed in the pericentriolar matrix but is also closely associated with centrioles. Using a combination of gel filtration, ion exchange chromatography, equilibrium sucrose gradient centrifugation and immunoprecipitation, we show that the major part of cytosolic γ-tubulin might be involved in complexes heavier than the Tcp1 particle. We further demonstrate, by co-immunoprecipitation of γ-tubulin and Tcp1 with either anti-Tcp1 or anti-γ-tubulin antibodies, that a small part of γ-tubulin participates in Tcp1-γ-tubulin particles. Interestingly, the soluble form of γ-tubulin co-purifies with taxol-stabilized microtubules and its association with microtubules resisted salt, ATP and GTP treatments. The existence of a centrosomal form and a large pool of cytosolic γ-tubulin-containing complexes in somatic cells suggests that the overall γ-tubulin cellular distribution does not seem to be as straightforward as it was drawn earlier.
Transcriptional regulation of αB-crystallin in astrocytes: Analysis of HSF and AP1 activation by different types of physiological stress
Authors: Head Mark W. , Hurwitz Lina , Goldman James E.
Published: April 2021

The coordinated cellular responses to physiological stress are known to be effected in part by the activation of heat shock factor 1, a transcriptional activator protein capable of binding to, and inducing transcription from genes containing heat shock elements. Other stress responsive signal transduction pathways also exist including the stress activated protein kinase cascade that regulates the activity of the transcription factor AP1. We have examined the expression of the low molecular stress proteins, heat shock protein 27 and αB-crystallin in astrocytes in response to physiological stress of different types and asked what component of this induction is effected at the transcriptional level and whether activation of heat shock factor 1 and AP1 might account for these events. We have found that stress regulated induction of αB-crystallin has a strong transcriptional component and that it may be effected by at least two different transcriptional mechanisms. In one set of phenomena, represented here by cadmium exposure, αB-crystallin and heat shock protein 27 are coordinately regulated and this occurs in the presence of activated heat shock factor 1. In the second series of phenomena, represented here by hypertonic stress, αB-crystallin is induced in the absence of heat shock factor activation and in the absence of any corresponding change in heat shock protein 27 expression. Although hypertonic stress does activate an AP1-like binding activity, the AP1 consensus binding site in the αB-crystallin promoter does not appear to be a target for this hypertonic stress inducible activity. These data suggest that the hypertonic stress response is effected through a heat shock factor independent mechanism and that hypertonic stress regulated induction of αB-crystallin does not directly depend on the SAPK pathway and AP1 activity.
Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells
Authors: Gabrielli Brian G. , Souza Colin P. C. De , Tonks Ian D. , Clark Joanne M. , Hayward Nicholas K. , Ellem Kay A. O.
Published: April 2021

The formation of the mitotic spindle is an essential prerequisite for successful mitosis. The dramatic changes in the level of microtubule (Mt) nucleation at the centrosomes and Mt dynamics that occur in prophase are presumed to be initiated through the activity of cdc2/cyclin B. Here we present data that the cdc25B isoform functions to activate the cytoplasmic pool of cdc2/cyclin B responsible for these events. In contrast to cdc25C, cdc25B is present at low levels in HeLa cells during interphase, but sharply increases in prophase, when cdc25B accumulation in the cytoplasm correlates with prophase spindle formation. Overexpression of wild type and dominant negative mutants of cdc25B and cdc25C shows that prophase Mt nucleation is a consequence of cytoplasmic cdc25B activity, and that cdc25C regulates nuclear G2/M events. Our data also suggest that the functional status of the centrosome can regulate nuclear mitotic events.
MAP2d promotes bundling and stabilization of both microtubules and microfilaments
Authors: Ferhat Lotfi , Represa Alfonso , Bernard Anne , Ben-Ari Yezekiel , Khrestchatisky Michel
Published: April 2021

Two low molecular weight MAP2 variants have been described, MAP2c and MAP2d. These variants are produced from a single gene by alternative splicing, and in their C-terminal regions contain, respectively, 3 and 4 tandem repeats, some of which are known to be involved in binding to microtubules. Substantial differences in the developmental expression pattern of MAP2c and MAP2d suggest they have different functions in neural cells. In order to investigate the respective roles of these MAP2 variants, we have analyzed the effects of MAP2c and MAP2d expression on microtubule and microfilament organization in transiently transfected cells. Our results show that both variants stabilize microtubules, but only MAP2d stabilizes microfilaments.
Immunofluorescence studies of human fibroblasts demonstrate the presence of the complex of elongation factor-1βγd in the endoplasmic reticulum
Authors: Sanders Jo , Brandsma Margreet , Janssen George M. C. , Dijk Jan , Möller Wim
Published: April 2021

The eukaryotic elongation factor-1 (EF-1) consists of four subunits, EF-1α, EF-1β, EF-1γ and EF-1d which induce efficient transfer of aminoacyltRNA to the ribosome. In this process EF-1α·GTP acts as the carrier of the aminoacyltRNA on its way to the ribosome. After release of aminoacyltRNA to the ribosome under concomitant hydrolysis of GTP, the inactive EF-1α·GDP form is recycled to EF-1α·GTP by EF-1βγd. In eukaryotic cells the concentration of EF-1α exceeds that of the complex βγd by a factor of 5-10. In order to delineate the intracellular localization of the different subunits of EF-1, antibodies against the EF-1 subunits have been elicited and indirect immunofluorescence microscopy experiments were performed.

In human fibroblasts, the guanine nucleotide exchange part of EF-1, EF-1βγd, was found to colocalize with the endoplasmic reticulum (ER), displaying a distinct finestructure in its staining pattern. The guanine nucleotidebinding subunit of EF-1, EF-1α, shows a more diffuse distribution throughout the cytoplasm and is, in addition, associated with the nucleus.
Trafficking of cell-surface amyloid β-protein precursor: I. Secretion, endocytosis and recycling as detected by labeled monoclonal antibody
Authors: Koo Edward H. , Squazzo Sharon L. , Selkoe Dennis J. , Koo Catherine H.
Published: April 2021

Amyloid βprotein, the principal constituent of amyloid fibrils found in senile plaques and blood vessels in Alzheimer’s disease, is constitutively produced and released into medium of cultured cells. Amyloid βprotein is derived by proteolysis of the βamyloid precursor protein by unclear mechanisms. βamyloid precursor protein is a transmembrane protein which can be processed to release a large secretory product or processed in the endosomal/lysosomal pathway without secretion. Previous studies have shown that from the cell surface, βamyloid precursor protein may be released after cleavage or internalized without cleavage, the latter in a pathway that both produces amyloid βprotein and also targets some molecules to the lysosomal compartment. Analysis of βamyloid precursor protein trafficking is confounded by the concomitant secretion and internalization of molecules from the cell surface. To address this issue, we developed an assay, based on the binding of a radioiodinated monoclonal antibody, to measure the release and internalization of cell surface βamyloid precursor protein in transfected cells. With this approach, we showed that surface βamyloid precursor protein is either rapidly released or internalized, such that the duration at the cell surface is very short. Approximately 30% of cell surface βamyloid precursor protein molecules are released. Following internalization, a fraction of molecules are recycled while the majority of molecules are rapidly sorted to the lysosomal compartment for degradation. When the C terminus of βamyloid precursor protein is deleted, secretion is increased by approximately 2.5-fold as compared to wildtype molecules. There is a concomitant decrease in internalization in these mutant molecules as well as prolongation of the resident time on the cell surface. This observation is consistent with recent evidence that signals within the cytoplasmic domain mediate βamyloid precursor protein internalization.
Trafficking of cell-surface amyloid β-protein precursor: II. Endocytosis, recycling, and lysosomal targeting detected by immunolocalization
Authors: Yamazaki Tsuneo , Koo Edward H. , Selkoe Dennis J.
Published: April 2021

Amyloid βprotein (Aβ) is a proteolytic fragment of the amyloid βprotein precursor (βPP). Progressive cerebral deposition of Aβ is an early and invariant feature of Alzheimer’s disease. The cellular trafficking of βPP is of particular interest because understanding the production of Aβ requires a comprehensive elucidation of the metabolic pathways of this protein. In addition, βPP is a type I integral membrane glycoprotein that belongs to a class of molecules with both full length and secreted products. Recent evidence suggests that βPP can be processed in an endosomal/lysosomal pathway. In the latter organelles, a number of βPP carboxyterminal derivatives are found, but the precise pathway and kinetics of βPP trafficking from the cell surface remain unclear. To address these questions, we visualized directly the βPP internalization pathway by following the localization and distribution of βPP monoclonal antibodies added to intact βPP-transfected Chinese hamster ovary cells. Using immunofluorescence and immunoelectron microscopy, βPP was shown to be rapidly internalized via coated pits and vesicles, after which the molecules were transported to endosomes, prelysosomes, and lysosomes. Using a modified immunodetection protocol, we demonstrated the rapid recycling of endocytosed βPP to the cell surface and its ultimate targeting to lysosomes. Because we recently found that endocytosis of cell surface βPP is one route for the constitutive production of Aβ, the recycling pathway for cell surface βPP demonstrated here is a probable route for production of the critical Aβ fragment.