bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of inducible nitric oxide is required for monocytic differentiation of u937 cells induced by vitamin e-succinate
Authors: Kim Song-Ja , Bang Ok-Sun , Lee Young-Sup , Kang Shin-Sung
Published: April 2021

Many putative differentiating agents arrest cell growth prior to progression of the cell through differentiation. Vitamin E-succinate is known to be a potent modulator of haematopoietic differentiation as well as an inhibitor of cell growth in vitro and in vivo. In this study, we examined whether vitamin E-succinate could modulate the monocytic differentiation of U937 human monoblasts. Treatment with vitamin E-succinate for 1-4 days inhibited the proliferation of U937 cells. Vitamin E-succinate also induced monocytic differentiation as indicated by the increase in nitro blue tetrazolium reduction activity, and the expression of monocyte specific cell surface antigen, CD11c, and integrins α5 and β1. The monocytic differentiation of U937 cells was also induced when the cells were cultured in fibronectin-coated wells. Monocytic differentiation was enhanced when the cells were treated with both vitamin E-succinate and fibronectin, suggesting that vitamin E-succinate and fibronectin synergistically act on monocytic differentiation of U937 cells. During monocytic differentiation of U937 cells induced by vitamin E-succinate and/or fibronectin, nitric oxide was detected in supernatants. The production of nitric oxide was not detected when monocytic differentiation of U937 cells was induced by phorbol-12 myristate 13-acetate, a well known inducer of macrophage-like cell differentiation. Vitamin E-succinate and/or fibronectin induced monocytic differentiation was blocked by the treatment of nitric oxide synthase inhibitor, N-G-monomethyl-L-arginine. In contrast, treatment of cells with sodium nitroprusside, a chemical nitric oxide donor, stimulated monocytic differentiation of U937 cells at an early time point. Taken together, these results suggest that nitric oxide is an important intermediator at an early stage of vitamin E-succinate- and/or fibronectin-induced monocytic differentiation of U937 cells.
Contiguous phosphorylated and non-phosphorylated domains along axonal neurofilaments
Authors: Brown Anthony
Published: April 2021

I have investigated the phosphorylation state of the medium molecular mass neurofilament protein (NF-M) along axonal neurofilaments. Cultured embryonic sensory neurons were treated with non-ionic detergent to cause the cytoskeletal polymers to splay apart from each other. Neurofilaments were visualized by double-label immunofluorescence microscopy and the proportion of their length that stained with various NF-M antibodies was determined using digital image analysis techniques. Monoclonal antibody RMO255, which binds to NF-M independently of phosphorylation state, stained an average of 98% of the neurofilament length. In contrast, monoclonal antibody RMO55, which binds specifically to a phosphorylated epitope on NF-M, stained some neurofilaments completely, some not at all, and some along part of their length. These partly stained neurofilaments exhibited single or multiple discrete segments of staining along their length separated by segments that were unstained. The average proportion of the neurofilament length that stained with this antibody was lowest proximally (12-22%, n=3) and increased along the axon to reach a maximum distally (58-87%, n=3). A converse pattern (77-87% proximally and 2-9% distally, n=3) was observed for neurons stained with monoclonal antibody FNP7, which binds to specifically to a non-phosphorylated epitope in both NF-M and the high molecular mass neurofilament protein, NF-H. Analysis of the staining of individual neurofilaments revealed a bimodal frequency distribution in which neurofilaments were more likely to be phosphorylated along either all or none of their length than along part of their length. These observations indicate that: (a) phosphorylated and non-phosphorylated neurofilaments can coexist side-by-side in these axons, (b) neurofilaments can be composed of single or multiple contiguous phosphorylated and non-phosphorylated epitope domains along their length, (c) the proportion of the neurofilament length that is phosphorylated at these epitopes increases along the axon in a proximal-to-distal manner, and (d) the pattern of phosphorylation is non-random, generating populations of phosphorylated and non-phosphorylated neurofilaments and discrete phosphorylated and non-phosphorylated domains along individual neurofilaments.
Adsorptive endocytosis mediates the passage of hiv-1 across the blood-brain barrier: Evidence for a post-internalization coreceptor
Authors: Banks William A. , Akerstrom Victoria , Kastin Abba J.
Published: April 2021

HIV-1 induces the AIDS dementia complex and infects brain endothelial and glial cells. Because the endothelial cells comprising the blood-brain barrier (BBB) do not possess CD4 receptors or galactosylceramide binding sites, it is unclear how HIV-1 negotiates the BBB. Previous work has suggested that gp120, the glycoprotein viral coat of HIV-1, is capable of inducing adsorptive endocytosis. Glycoprotein lectins like wheatgerm agglutinin induce adsorptive endocytosis and greatly potentiate the uptake by and passage across mouse endothelial cells in vivo and in vitro. We show here that the wheatgerm agglutinin-induced binding of gp120 is dose-dependent and involves components of the cytoskeleton. The uptake is partially dependent on temperature and energy and is modestly enhanced by potassium depletion. Glycosylation of gp120 is critical for its uptake by adsorptive endocytosis since the non-glycosylated form of gp120 is unaffected by wheatgerm agglutinin. Evidence is presented for the existence of a coreceptor sensitive to protamine sulfate that is primarily involved in membrane fusion after 125I-gp120 has bound to the cell membrane and is probably activated after internalization. This coreceptor probably contains a negatively charged heparin sulfate group and could be a member of the chemokine receptor family.
The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly
Authors: Roghi Christian , Giet Régis , Uzbekov Rustem , Morin Nathalie , Chartrain Isabelle , Le Guellec René , Couturier Anne , Dorée Marcel , Philippe Michel , Prigent Claude
Published: April 2021

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.
EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCγ signaling pathway
Authors: Xie Heng , Pallero Manuel A. , Gupta Kiran , Chang Philip , Ware Margaret F. , Witke Walter , Kwiatkowski David J. , Lauffenburger Douglas A. , Murphy-Ullrich Joanne E. , Wells Alan
Published: April 2021

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from ~60% to ~30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum.

We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase Cγ (PLCγ) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c′973) which fails to activate PLCγ or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCγ. Because both WT and c′973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility.

In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCγ pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.
Calcium/calmodulin-dependent protein kinase II controls α5β1 integrin-mediated inside-out signaling
Authors: Bouvard Daniel , Molla Annie , Block Marc R.
Published: April 2021

Fibronectin binding on α5β1 integrin is strictly dependent on intracellular calcium. Using an in vitro assay, we previously found that either calcineurin inhibitors or a blocking calcineurin monoclonal antibody added to cell lysates completely abolished the fibronectin/integrin interaction, which suggested that the activity of calcineurin, a calcium/calmodulin-dependent phosphatase, was required to counteract some kinase activity and maintain the high affinity state of α5β1. In this paper, we show that blocking of the calcium/calmodulin kinase II (CaMKII) activity with the specific inhibitor KN-62 or with its pseudosubtrate Autocamtide-2 preserved the high affinity state of the integrin even under experimental conditions that inhibit calcineurin. Conversely, the addition of purified CaMKII to the cell lysate inhibited α5β1 binding to fibronectin in vitro. Consistent with these results, cell adhesion on fibronectin was stimulated by KN-62. Moreover, Scatchard analysis of fibronectin binding on CHO cells revealed that KN-62 decreased the Kd value from 0.3 to 0.05 μM. Finally the expression of exogenous constitutively active CaMKII resulted in a dramatic defect in cell adhesion with no significant modification in α5β1 cell surface expression. In summary our results demonstrate that CaMKII controls the affinity state of the integrin α5β1 in vitro and in living cells.
The replication mechanism of kinetoplast DNA networks in several trypanosomatid species
Authors: Guilbride D. Lys , Englund Paul T.
Published: April 2021

Kinetoplast DNA, a giant network of interlocked DNA circles, replicates by an unusual mechanism. Minicircles are released individually from the network by a topoisomerase II, and then, after replication, their progeny are reattached at antipodal positions on the network periphery. Studies to date have revealed two distinct variations on this model. In Crithidia fasciculata the newly replicated minicircles quickly become uniformly distributed around the network periphery, whereas in Trypanosoma brucei the minicircles accumulate near their two points of attachment. The kinetoplast DNA replication mechanism used by other related trypanosomatid species was until now unknown. Here we used a novel method, involving fluorescence microscopy of isolated networks, to investigate kinetoplast DNA replication in Leishmania tarentolae, Leishmania donovani, Trypanosoma cruzi and Phytomonas serpens. We found that all of these species have a replication mechanism resembling that of C. fasciculata and that the polar replication mechanism observed in T. brucei is so far unique to this species.
Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells
Authors: Ding Da-Qiao , Chikashige Yuji , Haraguchi Tokuko , Hiraoka Yasushi
Published: April 2021

Using a computerized fluorescence microscope system to observe fluorescently stained cellular structures in vivo, we have examined the dynamics of chromosomes and microtubules during the process of meiosis in the fission yeast Schizosaccharomyces pombe. Fission yeast meiotic prophase is characterized by a distinctive type of nuclear movement that is led by telomeres clustered at the spindle-pole body (the centrosome-equivalent structure in fungi): the nucleus oscillates back and forth along the cell axis, moving continuously between the two ends of the cell for some hours prior to the meiotic divisions. To obtain a dynamic view of this oscillatory nuclear movement in meiotic prophase, we visualized microtubules and chromosomes in living cells using jellyfish green fluorescent protein fused with α-tubulin and a DNA-specific fluorescent dye, Hoechst 33342, respectively. Continuous observation of chromosomes and microtubules in these cells demonstrated that the oscillatory nuclear movement is mediated by dynamic reorganization of astral microtubules originating from the spindle-pole body. During each half-oscillatory period, the microtubules extending rearward from the leading edge of the nucleus elongate to drive the nucleus to one end of the cell. When the nucleus reversed direction, its motion during the second half of the oscillation was not driven by the same microtubules that drove its motion during the first half, but rather by newly assembled microtubules. Reversible inhibition of nuclear movement by an inhibitor of microtubule polymerization, thiabendazole, confirmed the involvement of astral microtubules in oscillatory nuclear movement. The speed of the movement fluctuated within a range 0 to 15 μm/minute, with an average of about 5 μm/minute. We propose a model in which the oscillatory nuclear movement is mediated by dynamic instability and selective stabilization of astral microtubules.
Cell adhesion to laminin 1 or 5 induces isoform-specific clustering of integrins and other focal adhesion components
Authors: Dogic Dagmar , Rousselle Patricia , Aumailley Monique
Published: April 2021

Laminin 1 (α1β1γ1) and laminin 5 (α3β3γ2) induce cell adhesion with different involvement of integrins: both are ligands for the α6β1 integrin, while α3β1 integrin has affinity for laminin 5 only. These two laminin isoforms therefore provide good models to investigate whether α3β1 and α6β1 integrins play different roles in signal transduction and in focal adhesion formation. Laminin 1 or 5 induced adhesion of normal human skin fibroblasts to a similar extent but promoted different overall cell shapes. On laminin 1 the fibroblasts formed mainly filopodia-like structures, while on laminin 5 they developed lamellipodias. Staining of fibrillar actin with fluorescein-phalloidin revealed a similar organisation of the actin cytoskeleton on both substrates. However, integrin subunits and several cytoskeletal linker proteins, including vinculin, talin, and paxillin, showed an isoform-specific arrangement into focal adhesions. On laminin 1 they were recruited into thick and short aggregates localized at the termini of actin stress fibers, while on laminin 5 they appeared as dots or streaks clustered on a long portion of actin microfilaments.

To test whether the differing affinity of laminin 1 or 5 for α3β1 integrin would explain the formation of morphologically different focal adhesions, cells were seeded on laminin 1 under conditions in which α3β1 integrins were occupied by a function-blocking antibody. This resulted in the formation of focal adhesions similar to that observed on laminin 5, where the integrin is occupied by its natural ligand. These results provide the first evidence for a cross-talk between α3β1 and α6β1 integrins and indicate that occupancy of α3β1 integrins results in a trans-dominant regulation of α6β1 integrin clustering and of focal adhesions. It suggests that recruitment of integrins and cytoskeletal linker proteins are laminin isoform-specific and that tissue specific expression of laminin isoforms might modulate cell behavior by the activation of distinct sets of integrins and by the induction of distinct molecular assemblies within the cell adhesion signaling complexes.
Effects of cytokines on mycobacterial phagosome maturation
Authors: Via L. E. , Fratti R. A. , McFalone M. , Pagán-Ramos E. , Deretic D. , Deretic1 V.
Published: April 2021

One of the major mechanisms permitting intracellular pathogens to parasitize macrophages is their ability to alter maturation of the phagosome or affect its physical integrity. These processes are opposed by the host innate and adaptive immune defenses, and in many instances mononuclear phagocytes can be stimulated with appropriate cytokines to restrict the growth of the microorganisms within the phagosomal compartment. Very little is known about the effects that cytokines have on phagosome maturation. Here we have used green fluorescent protein (GFP)-labeled mycobacteria and a fixable acidotropic probe, LysoTracker Red DND-99, to monitor maturation of the mycobacterial phagosome. The macrophage compartments that stained with the LysoTracker probe were examined first. This dye was found to colocalize preferentially with the late endosomal and lysosomal markers rab7 and Lamp1, and with a fluid phase marker chased into the late endosomal compartments. In contrast, LysoTracker showed only a minor overlap with the early endosomal marker rab5. Pathogenic mycobacteria are believed to reside in nonacidified vacuoles sequestered away from late endosomal compartments as a part of their intracellular survival strategy. We examined the status of mycobacterial phagosomes in macrophages from IL-10 knockout mice, in quiescent cells, and in mononuclear phagocytes stimulated with the macrophage-activating cytokine IFN-γ. When macrophages were derived from the bone marrow of transgenic IL-10 mice lacking this major deactivating cytokine, colocalization of GFP-fluorescing mycobacteria with the LysoTracker staining appeared enhanced, suggestive of increased acidification of the mycobacterial phagosome relative to macrophages from normal mice. When bone marrow-derived macrophages from normal mice or a J774 murine macrophage cell line were stimulated with IFN-γ and LPS, this resulted in increased colocalization of mycobacteria and LysoTracker, but no statistically significant enhancement was observed in IL-10 transgenic animals. These studies are consistent with the interpretation that proinflammatory and anti-inflammatory cytokines affect maturation of mycobacterial phagosomes. Although multiple mechanisms are likely to be at work, we propose the existence of a direct link between cytokine effects on the host cell and phagosome maturation in the macrophage.
Microinjection of activated phosphatidylinositol-3 kinase induces process outgrowth in rat PC12 cells through the Rac-JNK signal transduction pathway
Authors: Kita Yoshihiro , Kimura Koutarou D. , Kobayashi Michimoto , Ihara Sayoko , Kaibuchi Kozo , Kuroda Shinya , Ui Motoyasu , Iba Hideo , Konishi Hiroaki , Kikkawa Ushio , Nagata Satoshi , Fukui Yasuhisa
Published: April 2021

We have previously shown that sustained phosphatidylinositol (PI)-3 kinase activity is necessary for neurite outgrowth of PC12 cells induced by nerve growth factor (NGF). Microinjection of a constitutively active mutant of PI-3 kinase induced process formation suggesting that PI-3 kinase is indeed involved in the neurite outgrowth. However, the processes appeared to be incomplete neurites as they had very poor organization of F-actin and GAP43 antigen. The microtubule network was enhanced in the process-bearing cells and process formation was inhibited by colchicine suggesting that microtubules play an important role in process formation downstream of PI-3 kinase. These cell responses were inhibited by dominant-negative mutants of Ras and Sek1/SAPK but not by a dominant-negative mutant Ras and PD98059, a MAP kinase kinase (MEK) inhibitor, suggesting that not the Ras-MAP kinase pathway but the Ras-Jun N-terminal kinase (JNK) pathway is involved in process formation.
Activation of p53-mediated cell cycle checkpoint in response to micronuclei formation
Authors: Sablina Anna A. , Ilyinskaya Galina V. , Rubtsova Svetlana N. , Agapova Larissa S. , Chumakov Peter M. , Kopnin; Boris P.
Published: April 2021

Inactivation of p53 tumor-suppressor leads to genetic instability and, in particular, to accumulation of cells with abnormal numbers of chromosomes. In order to better define the role of p53 function in maintaining genome integrity we investigated the involvement of p53 in the control of proliferation of micronucleated cells resulting from abnormal chromosome segregation. Using cell lines expressing temperature-sensitive (ts) p53 or containing p53 genetic suppressor element (p53-GSE) we showed that inhibition of p53 function increases the frequency of cells with micronuclei. Immunofluorescence study revealed that in REF52 cell cultures with both spontaneous and colcemid-induced micronuclei the proportion of p53-positive cells is considerably higher among micronucleated variants as compared with their mononuclear counterparts. Analysis of 12(1)ConA cells expressing the β-galactosidase reporter gene under the control of a p53-responsive promoter showed activation of p53-regulated transcription in the cells with micronuclei. Importantly, the percentage of cells manifesting specific p53 activity in colcemid-treated cultures increased with an augmentation of the number of micronuclei in the cell. Activation of p53 in micronucleated cells was accompanied by a decrease in their ability to enter S-phase as was determined by comparative analysis of 5-bromodeoxyuridine (5-BrdU) incorporation by the cells with micronuclei and their mononuclear counterparts. Inhibition of p53 function in the cells with tetracycline-regulated p53 gene expression, as well as in the cells expressing ts-p53 or p53-GSE, abolished cell cycle arrest in micronucleated cells. These results along with the data showing no increase in the frequency of chromosome breaks in REF52 cells after colcemid treatment suggest the existence of p53-mediated cell cycle checkpoint(s) preventing proliferation of micronucleated cells derived as a result of abnormal chromosome segregation during mitosis.
Modulation of hepatocyte growth factor-induced scattering of HT29 colon carcinoma cells: Involvement of the MAPK pathway
Authors: Herrera Roman
Published: April 2021

Hepatocyte growth factor (HGF)/scatter factor modulates the motility of HT29 colon carcinoma cells in vitro by inducing morphological changes that depend on the type of extra-cellular matrix (ECM) ligand; HGF-induced scattering of HT29 cells is observed if cells are grown on plastic coated with serum proteins but not laminin. The absence of scattering correlates with a lack of cell spreading on laminin and it is not due to impaired HGF induced tyrosine phosphorylation of the E-cadherin/desmosome component, γ-catenin, or lack of activation of mitogen activated protein kinase (MAPK). Treatment of HT29 cells with phorbol 12-myristate, 13-acetate (PMA), but not arachidonic acid, restored the ability of the cells to spread on laminin in an integrin-dependent manner. Moreover, the addition of both PMA and HGF restored the ability of these cells to scatter on laminin in a synergistic manner. This event correlated with increased tyrosine phosphorylation of paxillin and activation of MAPK. Moreover, when the MEK (MAPK kinase)/MAPK pathway was blocked by the MEK inhibitor PD098059, HGF-induced scattering of HT29 cells was blocked. Thus, HGF modulation of HT29 cell motility is regulated by both integrin and growth factor-dependent signaling and implicates MAPK in the modulation of intercellular adhesion and epithelial cell motility.
A novel Rab GTPase, Rab33B, is ubiquitously expressed and localized to the medial Golgi cisternae
Authors: Zheng Jun Ying , Koda Toshiaki , Fujiwara Toshiyuki , Kishi Masahiko , Ikehara Yukio , Kakinuma Mitsuaki
Published: April 2021

Small GTP-binding proteins of the Rab family play important roles at defined steps of vesicular transport in protein secretion and the endocytosis pathway. In mammals, more than 30 proteins belonging to the Rab family have been reported to date. We report here the molecular cloning and characterization of a novel Rab protein, Rab33B. The amino acid sequence of Rab33B shows 55.3% identity to the Rab33A protein (previously called S10), and these two proteins share unique amino acid sequences at the effector domain. The genomic organization of rab33B was the same as rab33A: it consists of two exons. Thus, these two proteins make a subclass within the Rab family. Northern blot analysis showed that rab33B is expressed ubiquitously in mouse tissues, in contrast to rab33A whose expression is restricted to the brain and the immune system. A 26 kDa protein was detected by western blotting using a Rab33B-specific monoclonal antibody. Using immunofluorescence studies, Rab33B was shown to co-localize with α-mannosidase II, a Golgi-specific marker. Immunoelectron microscopy analysis further defined the localization of Rab33B to the medial Golgi cisternae. These results suggest Rab33B plays a role in intra-Golgi transport.
E-cadherin mediated cell adhesion recruits SAP97 into the cortical cytoskeleton
Authors: Reuver Susanne M. , Garner Craig C.
Published: April 2021

Members of the SAP family of synapse-associated proteins have recently emerged as central players in the molecular organization of synapses. In this study, we have examined the mechanism that localizes one member, SAP97, to sites of cell-cell contact. Utilizing epithelial CACO-2 cells and fibroblast L-cells as model systems, we demonstrate that SAP97 is associated with the submembranous cortical cytoskeleton at cell-cell adhesion sites. Furthermore, we show that its localization into this structure is triggered by E-cadherin. Although SAP97 can be found in an E-cadherin/catenin adhesion complex, this interaction seems to be mediated by the attachment of SAP97 to the cortical cytoskeleton. Our results are consistent with a model in which SAP97 is recruited to sites of cell-cell contact via an E-cadherin induced assembly of the cortical cytoskeleton.
Reversal of myogenic terminal differentiation by SV40 large T antigen results in mitosis and apoptosis
Authors: Endo Takeshi , Nadal-Ginard Bernardo
Published: April 2021

Terminally differentiated skeletal muscle myotubes are arrested in the Go phase of the cell cycle, and this arrest is not reversed by stimulation with serum or growth factors. The myotubes have been shown to be refractory to apoptosis even under low serum conditions. When the SV40 large T antigen is induced in the C2SVTts11 myotubes, which stably harbor the T antigen gene linked to an inducible promoter, the terminally differentiated cells reenter the cell cycle to resume nuclear DNA replication representing S phase. We show here that the large Texpressing myotubes further proceeded to M phase represented by the appearance of mitotic figures with centrosomes, condensed chromosomes, and mitotic spindles. The myotubes eventually cleaved and midbodies were formed at the cleavage sites of the cytoplasm. In some cases actin filaments, reminiscent of the contractile rings, accumulated at the cleavage furrows. Thus, terminally differentiated myotubes remain able to resume at least one round of the cell cycle and consequently are considered to be capable of dedifferentiation. A subset of myotubes expressing large T did not undergo mitosis. Some of them were degenerative and contained deformed giant nuclei and pulverized nuclei. The others suffered apoptotic cell death, which was identified by morphological changes of the nuclei and the labeling with dUTP at the ends of chromatin DNA fragments. The induction of apoptosis was unlikely to be confined to a particular phase of the cell cycle. These results imply that terminally differentiated myotubes also retain a complete set of machinery for apoptosis.
Up-regulation of p27Kip1, p21WAF1/Cip1 and p16Ink4a is associated with, but not sufficient for, induction of squamous differentiation
Authors: Harvat Beth L. , Wang Amy , Seth Prem , Jetten A. M.
Published: April 2021

Irreversible growth arrest is an early and integral part of squamous cell differentiation in normal human epidermal keratinocytes (NHEKs) and is assumed to be linked to the control of expression of differentiation-specific genes. In this study, we examine the link between the molecular events associated with growth arrest and the expression of differentiation genes. NHEKs that have been induced to undergo growth arrest and differentiation by suspension culture contain populations in both G1 and G2/M of the cell cycle. The irreversible growth arrest state in NHEKs is characterized by an accumulation of the hypophosphorylated forms of Rb and p130, with subsequent down-regulation of levels of Rb, up-regulation of p130 and associated down-regulation of E2F-regulated genes such as cyclin A. These events correlate with an inhibition of G1 cdk activity, mediated in part by an increase in the cdk inhibitors p21WAF1/Cip1, p27Kip1 and p16Ink4a. Flow cytometric and immunoblot analysis demonstrated that the timing of the up-regulation of p27, p16 and p130 corresponds closely with the induction of the squamous-specific genes cornifin α (SPRR-1) and transglutaminase type I, suggesting a close link between control of growth arrest and differentiation. However, growth arrest induced by over-expression of p27, p21 or p16 by recombinant adenovirus is not sufficient to induce expression of the differentiation genes, or to invoke the pattern of cell cycle regulatory protein expression characteristic of the differentiation-specific irreversible growth arrest. We conclude that growth arrest mediated by activation of the Rb pathway is not sufficient to trigger terminal squamous differentiation and additional signals which can be generated during suspension culture are required to promote the complete differentiation program.
Identification of laminin-10/11 as a strong cell adhesive complex for a normal and a malignant human epithelial cell line
Authors: Ferletta Maria , Ekblom Peter
Published: April 2021

Laminins are heterotrimeric proteins of basement membranes. More than 50 different trimers may exist. Laminin-10 (α5β1γ1) rather than laminin-1 (α1β1γ1) could be the most abundant isoform in the adult stage, and laminin-10 is made by several developing epithelial sheets. We show here that a much used commercial human preparation contains laminin-10 (α5β1γ1), some laminin-11 (α5β2γ1), but no laminin-1. Moreover, the laminin-10/11 mixture was found to be a strong adhesive for two human cell lines derived from epithelia. Antibodies against integrin β1, α6 or α3 (at 50 µg/ml) or dystroglycan did not inhibit cell attachment to laminin-10/11, although lower concentrations of anti-dystroglycan and integrin α6 antibodies inhibited cell binding to laminin-1.
Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells
Authors: Toomre Derek , Keller Patrick , Olivo Jamie White, Jean-Christophe , Simons Kai
Published: April 2021

The mechanisms and carriers responsible for exocytic protein trafficking between the trans-Golgi network (TGN) and the plasma membrane remain unclear. To investigate the dynamics of TGN-to-plasma membrane traffic and role of the cytoskeleton in these processes we transfected cells with a GFP-fusion protein, vesicular stomatitis virus G protein tagged with GFP (VSVG3-GFP). After using temperature shifts to block VSVG3-GFP in the endoplasmic reticulum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma membrane transport were visualized in real time by confocal and video microscopy. Both small vesicles (<250 nm) and larger vesicular-tubular structures (>1.5 µm long) are used as transport containers (TCs). These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of ∼0.7 µm/second. Automatic computer tracking objectively determined the dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To directly determine the role of microtubules in post-Golgi traffic, rhodamine-tubulin was microinjected and both labeled cargo and microtubules were simultaneously visualized in living cells. These studies demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.

Key words: VSVG, GFP, Golgi, TGN, Cytoskeleton, Microtubule, Exocytosis
The tyrosinase tail mediates sorting to the lysosomal compartment in MDCK cells via a di-leucine and a tyrosine-based signal
Authors: Simmen Thomas , Schmidt Andrea , Hunziker Walter , Beermann Friedrich
Published: April 2021

Tyrosinase is a type I membrane protein found in melanosomes, which are lysosomal-like organelles and specific for pigment cells. A mutation of mouse tyrosinase, platinum (cp), leads to truncation of tyrosinase’s cytosolic tail, and results in misrouting to the cell periphery. In this study, we expressed chimeras of wild-type and mutant cytosolic tails of mouse tyrosinase fused to rat lysosome-associated membrane protein-1 luminal and transmembrane domain to study sorting of tyrosinase in Madin-Darby canine kidney cells. The study shows that the mouse tyrosinase cytosolic tail is necessary and sufficient to mediate sorting of a heterologous type I membrane protein to compartments of the lysosomal lineage. Whereas deletions of 7 or 10 C-terminal amino acids of the tail still result in sorting to lysosomes, a deletion mutant corresponding to platinum (cp) tail fails to sort correctly and corroborates the in situ findings in cp homozygous mutant mice. Correct sorting of tyrosinase-lysosome-associated membrane protein-1 chimeras is mediated by the interplay of a di-leucine signal and a tyrosine motif of the Y-X-X-Ø type.
The ubiquitin-proteasome system and endocytosis
Authors: Strous Ger J. , Govers Roland
Published: April 2021

Internalization of membrane proteins has been studied for more than three decades without solving all the underlying mechanisms. Our knowledge of clathrin-mediated endocytosis is certainly sufficient to understand the basic principles. However, more detailed insight is required to recognize why different proteins enter clathrin-coated pits with different rates and affinities. In addition to clathrin coat components, at least two adaptor systems and even more accessory proteins have been described to preselect membrane proteins before they can enter cells. Recent experimental data have identified the ubiquitin-proteasome Nonetheless, mean peeling velocity (µm/second) generally increased with detachmentsystem as a regulatory system for endocytosis. This system is well-known for its basic regulatory function in protein degradation, and controls a magnitude of key events. The ubiquitin-proteasome system is now identified as a regulator of the endocytosis of selected membrane proteins. In this review, we will discuss the complexity and implications of this mechanism for receptor-mediated endocytosis.
Muscle cell peeling from micropatterned collagen: direct probing of focal and molecular properties of matrix adhesion
Authors: Ra Hyun J. , Picart Catherine , Feng Huisheng , Sweeney H. Lee , Discher Dennis E.
Published: April 2021

To quantitatively elucidate attributes of myocyte-matrix adhesion, muscle cells were controllably peeled from narrow strips of collagen-coated glass. Initial growth of primary quail myoblasts on collagen strips was followed by cell alignment, elongation and end-on fusion between neighbors. This geometric influence on differentiation minimized lateral cell contact and cell branching, enabling detailed study of myocyte-matrix adhesion. A micropipette was used to pull back one end of a quasi-cylindrical cell while observing in detail the non-equilibrium detachment process. Peeling velocities fluctuated as focal roughness, µm in scale, was encountered along the detachment front. Nonetheless, mean peeling velocity ( µm/second) generally increased with detachment force (nN), consistent with forced disruption of adhesion bonds. Immunofluorescence of β1-integrins correlated with the focal roughness and appeared to be clustered in axially extended focal contacts. In addition, the peeling forces and rates were found to be moderately well described by a dynamical peeling model for receptor-based adhesion (Dembo, M., Torney, D. C., Saxman, K. and Hammer, D. (1988). Proc. R. Soc. Lond. B 234, 55-83). Estimates were thereby obtained for the spontaneous, molecular off-rate and the receptor complex stiffness (approx.10 -5 -10 -6N/m) of adherent myocytes. Interestingly, the local stiffness is within the range of flexible proteins of the spectrin superfamily. The overall approach lends itself to elucidating the developing function of other structural and adhesive components of cells, particularly skeletal muscle cells with specialized components, such as the spectrinhomolog dystrophin and its membrane-linked receptor dystroglycan.
A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock
Authors: Weighardt Florian , Cobianchi Fabio , Cartegni Luca , Chiodi Ilaria , Villa Antonello , Riva Silvano , Biamonti Giuseppe
Published: April 2021

A two-hybrid screening in yeast for proteins interacting with the human hnRNP A1, yielded a nuclear protein of 917 amino acids that we termed hnRNP A1 associated protein (HAP). HAP contains an RNA binding domain (RBD) flanked by a negatively charged domain and by an S/K-R/E-rich region. In in vitro pull-down assays, HAP interacts with hnRNP A1, through its S/K-R/E-rich region, and with several other hnRNPs. HAP was found to be identical to the previously described Scaffold Attachment Factor B (SAF-B) and to HET, a transcriptional regulator of the Heat Shock Protein 27 gene. We show that HAP is a bona fide hnRNP protein, since anti-HAP antibodies immunoprecipitate from HeLa cell nucleoplasm the complete set of hnRNP proteins. Unlike most hnRNP proteins, the subnuclear distribution of HAP is profoundly modified in heat-shocked HeLa cells.

Heat-shock treatment at 42°C causes a transcription-dependent recruitment of HAP to a few large nuclear granules that exactly coincide with sites of accumulation of Heat Shock Factor 1 (HSF1). The recruitment of HAP to the granules is temporally delayed with respect to HSF1 and persists for a longer time during recovery at 37°C. The hnRNP complexes immunoprecipitated from nucleoplasm of heat-shocked cells with anti-HAP antibodies have an altered protein composition with respect to canonical complexes. Altogether our results suggest an involvement of HAP in the cellular response to heat shock, possibly at the RNA metabolism level.
Chlamydia infection of epithelial cells expressing dynamin and Eps15 mutants: clathrin-independent entry into cells and dynamin-dependent productive growth
Authors: Boleti Haralabia , Benmerah Alexandre , Ojcius David M. , Cerf-Bensussan Nadine , Dautry-Varsat Alice
Published: April 2021

Chlamydiae enter epithelial cells via a mechanism that still remains to be fully elucidated. In this study we investigated the pathway of entry of C. psittaci GPIC and C. trachomatis LGV/L2 into HeLa cells and demonstrated that it does not depend on clathrin coated vesicle formation. We used mutant cell lines defective in clathrin-mediated endocytosis due to overexpression of dominant negative mutants of either dynamin I or Eps15 proteins. When clathrindependent endocytosis was inhibited by overexpression of the dynK44A mutant of dynamin I (defective in GTPase activity), Chlamydia entry was not affected. However, in these cells there was a dramatic inhibition in the proliferation of Chlamydia and the growth of the chlamydia vacuole (inclusion). When clathrin-dependent endocytosis was inhibited by overexpression of an Eps15 dominant negative mutant, the entry and growth of Chlamydia was unaltered. These results indicate that the effect on the growth of Chlamydia in the dynK44A cells was not simply due to a deprivation of nutrients taken up by endocytosis. Instead, the dominant-negative mutant of dynamin most likely affects the vesicular traffic between the Chlamydia inclusion and intracellular membrane compartments. In addition, cytochalasin D inhibited Chlamydia entry by more than 90%, indicating that chlamydiae enter epithelial cells by an actin-dependent mechanism resembling phagocytosis. Finally, dynamin is apparently not involved in the formation of phagocytic vesicles containing Chlamydia.
Myofibrillogenesis in the developing chicken heart: assembly of Z-disk, M-line and the thick filaments
Authors: Ehler Elisabeth , Rothen Barbara M. , Hämmerle Sibylle P. , Komiyama Masatoshi , Perriard Jean-Claude
Published: April 2021

Myofibrillogenesis in situ was investigated by confocal microscopy of immunofluorescently labelled whole mount preparations of early embryonic chicken heart rudiments. The time-course of incorporation of several components into myofibrils was compared in triple-stained specimens, taken around the time when beating starts. All sarcomeric proteins investigated so far were already expressed before the first contractions and myofibril assembly happened within a few hours. No typical stress fibre-like structures or premyofibrils, structures observed in cultured cardiomyocytes, could be detected during myofibrillogenesis in the heart. Sarcomeric proteins like α-actinin, titin and actin were found in a defined localisation pattern even in cardiomyocytes that did not yet contain myofibrils, making up dense body-like structures. As soon as the heart started to beat, all myofibrillar proteins were already located at their exact position in the sarcomere. The maturation of the sarcomeres was characterised by a short delay in the establishment of the pattern for M-line epitopes of titin with respect to Z-disk epitopes and the incorporation of the M-line component myomesin, which preceded that of myosin binding protein-C. Thus dense body-like structures, made up of titin, α-actinin and actin filaments serve as the first organised complexes also during myofibrillogenesis in situ and titin functions as a ruler for sarcomere assembly as soon as its C termini have become localised. We suggest that assembly of thin and thick filament occurs independently during myofibrillogenesis in situ and that myomesin might be important for integrating thick filaments with the M-line end of titin.
The putative nuclear receptor mediator TIF1α is tightly associated with euchromatin
Authors: Remboutsika Eumorphia , Lutz Yves , Gansmuller Anne , Vonesch Jean-Luc , Losson Régine , Chambon Pierre
Published: April 2021

Ligand-dependent transcriptional regulation by nuclear receptors is believed to be mediated by intermediary factors (TIFs) acting on remodelling of the chromatin structure and/or the activity of the transcriptional machinery. The putative transcriptional mediator TIF1α is a nuclear protein kinase that has been identified via its interaction with liganded nuclear receptors, including retinoic acid (RAR), retinoid X (RXR) and estrogen (ER) receptors. Here, we demonstrate that TIF1α is a non-histone chromosomal protein tightly associated with highly accessible euchromatic regions of the genome. Immunofluorescence confocal microscopy reveals that TIF1α exhibits a finely granular distribution in euchromatin of interphase nuclei, while it is mostly excluded from condensed chromatin and metaphase chromosomes. Immunoelectron microscopy shows that, in contrast to the heterochromatin protein HP1α, most of TIF1α is associated with euchromatin, where it is preferentially localised on regions known to be sites for RNA polymerase II (perichromatin fibrils and borders between euchromatin and heterochromatin). Early mouse embryos as well as embryonal carcinoma (EC) and embryonic stem (ES) cells express high levels of TIF1α. These levels dramatically decrease during organogenesis and upon differentiation of P19 EC cells, indicating that TIF1α is preferentially expressed in undifferentiated pluripotent cells in the course of development. Therefore, TIF1α could belong to a novel class of chromatin-associated TIFs that facilitate the access of transregulators (e.g. liganded nuclear receptors) to their cognate sites in target genes, thereby participitating in the epigenetic control of transcription during embryonic development and cell differentiation.
Polarised expression pattern of focal contact proteins in highly motile antigen presenting dendritic cells
Authors: Madruga Jaime , Koritschoner Nicolás P. , Diebold Sandra S. , Kurz Steffen M. , Zenke Martin
Published: April 2021

Dendritic cells are professional antigen presenting cells that capture antigens and migrate to lymphoid tissues to elicit specific T cell responses. Here we used an in vitro differentiation system for generating highly motile dendritic cells from chicken bone marrow progenitors by employing the conditional v-Rel estrogen receptor (ER) fusion protein v-RelER. Molecular mechanisms of dendritic cell motility were investigated. Differentiation of v-relER progenitors into dendritic cells is associated with a reduction in cell-cell and cell-extracellular matrix interactions as cells acquire motility. We demonstrate that v-relER progenitors and dendritic cells express several adhesion receptors and components of adhesion complexes. Differentiation of v-relER cells was accompanied by downregulation of focal adhesion kinase (FAK), a key molecule of adhesion complexes, but ectopic FAK expression did not affect cell adhesion and motility. Interestingly, v-relER dendritic cells exhibit a polarised expression pattern of actin and vimentin, with actin being highly concentrated at the leading edge of the cells where lamellipodia are formed. FAK, paxillin and tyrosine phosphorylated proteins are found at both poles of the cell and colocalise with actin at the leading edge, while surface β1 integrin is confined to the uropod at the rear. CD34+ stem cell-derived human dendritic cells also exhibited an elongated bipolar morphology, mode of migration and a polarised pattern of actin-vimentin expression similar to v-relER dendritic cells.