bluuebunny/crossref_metadata_2025 huggingface dataset:
Reconstitution of microtubule nucleation potential in centrosomes isolated from Spisula solidissimaoocytes
Authors: Schnackenberg Bradley J. , Hull Dawn R. , Balczon Ronald D. , Palazzo Robert E.
Published: April 2021

Treatment of isolated Spisula solidissimacentrosomes with KI removes γ-tubulin, 25 nm rings, and their microtubule nucleation potential, revealing the presence of a filamentous lattice, the ‘centromatrix’. Treatment of this centromatrix with Spisulaoocyte extract results in the binding of γ-tubulin and 25 nm rings, and the recovery of microtubule nucleation potential. Fractionation of this extract resulted in the separation of elements that are required for the recovery of microtubule nucleation potential. We show that some, but not all, of the elements needed cosediment with microtubules. Further, extracts prepared from activated (meiotic) and non-activated (interphase) Spisulaoocytes, CHO cells blocked in S phase, Drosophilaembryos and Xenopusoocytes all support the recovery of microtubule nucleation potential by the Spisulacentromatrix. These results demonstrate that components necessary for centrosome-dependent microtubule nucleation are functionally conserved and abundant in both interphase and meiotic/mitotic cytoplasm.
The Ran decathlon: multiple roles of Ran
Authors: Sazer Shelley , Dasso Mary
Published: April 2021

The Ran GTPase system affects many cellular processes, including the regulation of cell cycle progression, nuclear envelope structure and function, and nucleocytoplasmic transport. The biochemical basis for the involvement of Ran in nuclear import and export has been well documented, but the direct targets of Ran in other cellular processes have not yet been identified. There is, however, mounting evidence that Ran directly affects at least some of these other cellular processes by mechanisms independent of its role in transport. In this Commentary we discuss evidence linking Ran to different aspects of cell function, and how these multiple facets of Ran’s activity may relate to each other.
Progesterone regulates the accumulation and the activation of Eg2 kinase in Xenopus oocytes
Authors: Frank-Vaillant Marie , Haccard Olivier , Thibier Catherine , René Ozon , Arlot-Bonnemains Yannick , Prigent Claude , Jessus Catherine
Published: April 2021

Xenopusprophase oocytes reenter meiotic division in response to progesterone. The signaling pathway leading to Cdc2 activation depends on neosynthesized proteins and a decrease in PKA activity. We demonstrate that Eg2 protein, a Xenopusmember of the Aurora/Ipl1 family of protein kinases, accumulates in response to progesterone and is degraded after parthenogenetic activation. The polyadenylation and cap ribose methylation of Eg2 mRNA are not needed for the protein accumulation. Eg2 protein accumulation is induced by progesterone through a decrease in PKA activity, upstream of Cdc2 activation. Eg2 kinase activity is undetectable in prophase and is raised in parallel with Cdc2 activation. In contrast to Eg2 protein accumulation, Eg2 kinase activation is under Cdc2 control. Furthermore, by using an anti-sense strategy, we show that Eg2 accumulation is not required in the transduction pathway leading to Cdc2 activation. Altogether, our results strongly suggest that Eg2 is not necessary for Cdc2 activation, though it could participate in the organization of the meiotic spindles, in agreement with the well-conserved roles of the members of the Aurora family, from yeast to man.
Microtubules, but not actin filaments, drive daughter cell budding and cell division in Toxoplasma gondii
Authors: Shaw Michael K. , Compton Helen L. , Roos David S. , Tilney Lewis G.
Published: April 2021

We have used drugs to examine the role(s) of the actin and microtubule cytoskeletons in the intracellular growth and replication of the intracellular protozoan parasite, Toxoplasma gondii. By using a 5 minute infection period and adding the drugs shortly after entry we can treat parasites at the start of intracellular development and 6-8 hours prior to the onset of daughter cell budding. Using this approach we found, somewhat surprisingly, that reagents that perturb the actin cytoskeleton in different ways (cytochalasin D, latrunculin A and jasplakinolide) had little effect on parasite replication although they had the expected effects on the host cells. These actin inhibitors did, however, disrupt the orderly turnover of the mother cell organelles leading to the formation of a large residual body at the posterior end of each pair of budding parasites. Treating established parasite cultures with the actin inhibitors blocked ionophore-induced egression of tachyzoites from the host cells, demonstrating that intracellular parasites were susceptible to the effects of these inhibitors. In contrast, the anti-microtubule drugs oryzalin and taxol, and to a much lesser extent nocodazole, which affect microtubule dynamics in different ways, blocked parasite replication by disrupting the normal assembly of the apical conoid and the microtubule inner membrane complex (IMC) in the budding daughter parasites. Centrosome replication and assembly of intranuclear spindles, however, occurred normally. Thus, daughter cell budding per se is dependent primarily on the parasite microtubule system and does not require a dynamic actin cytoskeleton, although disruption of actin dynamics causes problems in the turnover of parasite organelles.
Ezrin links syndecan-2 to the cytoskeleton
Authors: Granés Francesc , Ureña Jesús Mariano , Rocamora Nativitat , Vilaró Senén
Published: April 2021

The syndecan family of heparan sulfate proteoglycans is known to associate with the actin cytoskeleton, possibly transducing signals from the extracellular matrix. In the search for proteins that could mediate the association of syndecan-2 with the actin cytoskeleton we found that ezrin, a protein which links membrane receptors to the cytoskeleton, coimmunoprecipitated with syndecan-2 in COS-1 cells. In vitro assays indicated a direct association between the amino-terminal domain of ezrin and the cytoplasmic domain of syndecan-2. Confocal microscopy showed colocalization of ezrin and syndecan-2 in actin-rich microspikes in COS-1 cells. The syndecan-2/ezrin protein complex was resistant to 0.2% Triton X-100 extraction but the syndecan-2/amino-terminal domain of ezrin complex was not, which indicated that carboxi-terminal domain of ezrin is involved in the cytoskeleton anchorage of this protein complex. Additionally we observed that the activation of rhoA GTPase increased syndecan-2 insolubility in 0.2% Triton X-100 and syndecan-2/ezrin association. Taken together, these results indicate that ezrin connects syndecan-2 to the actin cytoskeleton.
Mucin-like molecules form a negatively charged coat that protects Trypanosoma cruzitrypomastigotes from killing by human anti-α-galactosyl antibodies
Authors: Pereira-Chioccola Vera Lucia , Acosta-Serrano Alvaro , de Almeida Igor Correia , Ferguson Michael A. J. , Souto-Padron Thais , Rodrigues Maurício M. , Travassos Luiz R. , Schenkman Sergio
Published: April 2021

In the presence of sialic acid donors Trypanosoma cruziacquires up to 107sialic acid residues on its surface, in a reaction catalyzed by its unique trans-sialidase. Most of these sialic acid residues are incorporated into mucin-like glycoproteins. To further understand the biological role of parasite sialylation, we have measured the amount of mucin in this parasite. We found that both epimastigote and trypomastigote forms have the same number of mucin molecules per surface area, although trypomastigotes have less than 10% of the amount of glycoinositol phospholipids, the other major surface glycoconjugate of T. cruzi. Based on the estimated surface area of each mucin, we calculated that these molecules form a coat covering the entire trypomastigote cell. The presence of the surface coat is shown by transmission electron microscopy of Ruthenium Red-stained parasites. The coat was revealed by binding of antibodies isolated from Chagasic patients that react with high affinity to α-galactosyl epitopes present in the mucin molecule. When added to the trypomastigote, these antibodies cause an extensive structural perturbation of the parasite coat with formation of large blebs, ultimately leading to parasite lysis. Interestingly, lysis is decreased if the mucin coat is heavily sialylated. Furthermore, addition of MgCl2reverses the protective effect of sialylation, suggesting that the sialic acid negative charges stabilize the surface coat. Inhibition of sialylation by anti-trans-sialidase antibodies, found in immunized animals, or human Chagasic sera, also increase killing by anti-α-galactosyl antibodies. Therefore, the large amounts of sialylated mucins, forming a surface coat on infective trypomastigote forms, have an important structural and protective role.
Molecular motors − a paradigm for mutant analysis
Authors: Endow Sharyn A.
Published: April 2021

Molecular motors perform essential functions in the cell and have the potential to provide insights into the basis of many important processes. A unique property of molecular motors is their ability to convert energy from ATP hydrolysis into work, enabling the motors to bind to and move along cytoskeletal filaments. The mechanism of energy conversion by molecular motors is not yet understood and may lead to the discovery of new biophysical principles. Mutant analysis could provide valuable information, but it is not obvious how to obtain mutants that are informative for study. The analysis presented here points out several strategies for obtaining mutants by selection from molecular or genetic screens, or by rational design. Mutants that are expected to provide important information about the motor mechanism include ATPase mutants, which interfere with the nucleotide hydrolysis cycle, and uncoupling mutants, which unlink basic motor activities and reveal their interdependence. Natural variants can also be exploited to provide unexpected information about motor function. This general approach to uncovering protein function by analysis of informative mutants is applicable not only to molecular motors, but to other proteins of interest.
A functional knock-out of titin results in defective myofibril assembly
Authors: van der Ven Peter F. M. , Bartsch Jörg W. , Gautel Mathias , Jockusch Harald , Fürst Dieter O.
Published: April 2021

Titin, also called connectin, is a giant muscle protein that spans the distance from the sarcomeric Z-disc to the M-band. Titin is thought to direct the assembly of sarcomeres and to maintain sarcomeric integrity by interacting with numerous sarcomeric proteins and providing a mechanical linkage. Since severe defects of such an important molecule are likely to result in embryonic lethality, a cell culture model should offer the best practicable tool to probe the cellular functions of titin. The myofibroblast cell line BHK-21/C13 was described to assemble myofibrils in culture. We have now characterized the sub-line BHK-21-Bi, which bears a small deletion within the titin gene. RNA analysis revealed that in this mutant cell line only a small internal portion of the titin mRNA is deleted. However, western blots, immunofluorescence microscopy and immunoprecipitation experiments showed that only the N-terminal, approx. 100 kDa central Z-disc portion of the 3 MDa titin protein is expressed, due to the homozygous deletion in the gene. Most importantly, in BHK-21-Bi cells the formation of thick myosin filaments and the assembly of myofibrils are impaired, although sarcomeric proteins are expressed. Lack of thick filament formation and of ordered actin-myosin arrays was confirmed by electron microscopy. Myogenisation induced by transfection with MyoD yielded myofibrils only in myotubes formed from wild type and not from mutant cells, ruling out that a principal failure in myogenic commitment of the BHK-21-Bi cells might cause the observed effects. These experiments provide the first direct evidence for the crucial role of titin in both thick filament formation as a molecular ruler and in the coordination of myofibrillogenesis.
A fission yeast general translation factor reveals links between protein synthesis and cell cycle controls
Authors: Grallert Beáta , Kearsey Stephen E. , Lenhard Michael , Carlson Cathrine Rein , Nurse Paul , Boye Erik , Labib Karim
Published: April 2021

In two independent screens we isolated fission yeast mutations with phenotypes suggesting defects in B-cyclin function or expression. These mutations define a single gene which we call ded1. We show that ded1 encodes a general translation factor that is related in sequence and function to RNA helicases required for translation in other species. Levels of the B-cyclins Cig2 and Cdc13 are dramatically reduced upon inactivation of Ded1, and this reduction is independent of degradation by the anaphase promoting complex. When a ded1 mutant is grown under semi-restrictive conditions, the translation of Cig2 (and to a lesser extent Cdc13), is impaired relative to other proteins. We show that B-cyclin translation is specifically inhibited upon nitrogen starvation of wild-type cells, when B-cyclin/Cdc2 inactivation is a prerequisite for G1 arrest and subsequent mating. Our data suggest that translational inhibition of B-cyclin expression represents a third mechanism, in addition to cyclin degradation and Rum1 inhibition, that contributes to Cdc2 inactivation as cells exit from the mitotic cell cycle and prepare for meiosis.
A novel RGD-independent fibronectin assembly pathway initiated by α4β1 integrin binding to the alternatively spliced V region
Authors: Sechler Jan L. , Cumiskey Anne Marie , Gazzola Deana M. , Schwarzbauer Jean E.
Published: April 2021

Fibronectin (FN) matrix assembly is a multi-step process that involves binding to integrin receptors, FN-FN interactions and connections to the actin cytoskeleton. Ultimately, FN is converted into stable matrix fibrils that are detergent-insoluble. RGD-binding integrins such as α5β1 play a major role in the assembly of fibrillar FN. Here we show that α4β1 binding to the alternatively spliced V (IIICS) region of FN initiates an alternative assembly pathway. Activation of α4β1 with exogenous agents such as Mn2+ or a β1-stimulatory antibody TS2/16 was sufficient to induce initiation of FN fibrillogenesis by Ramos B lymphoma cells and by CHO(B2)α4 cells. Using recombinant FNs lacking specific sequences, we show that assembly is independent of the RGD sequence but requires the V25/CS-1 segment. Previously, we have characterized an activated recombinant FN (FNΔIII1-7) that rapidly forms detergent-insoluble multimers upon binding to α5β1 integrin. α4β1 also formed FNΔIII1-7 multimers without the aid of exogenous stimulants, suggesting that an activated form of FN can override the need for activation of the integrin. In contrast to assembly by α5β1, actin filaments remained largely cortical and no change in cell growth rate was observed with α4β1-mediated assembly. These results show that binding sites on FN other than the RGD sequence/synergy site and distant from the cell binding domain can promote FN assembly. Thus, there appear to be multiple, integrin-specific mechanisms for assembly of FN matrix.
Determination of cell polarity in germinated spores and hyphal tips of the filamentous ascomycete Ashbya gossypii requires a rhoGAP homolog
Authors: Wendland J. , Philippsen P.
Published: April 2021

In the filamentous ascomycete Ashbya gossypii, like in other filamentous fungi onset of growth in dormant spores occurs as an isotropic growth phase generating spherical germ cells. Thereafter, a switch to polarized growth results in the formation of the first hyphal tip. The initial steps of hyphal tip formation in filamentous fungi, therefore, resemble processes taking place prior to and during bud emergence of unicellular yeast-like fungi. We investigated whether phenotypic similarities between these distinct events extended to the molecular level. To this end we isolated and characterized the A. gossypii homolog of the Saccharomyces cerevisiae BEM2 gene which is part of a network of rho-GTPases and their regulators required for bud emergence and bud growth in yeast. Here we show that the AgBem2 protein contains a GAP-(GTPase activating protein) domain for rho-like GTPases at its carboxy terminus, and that this part of AgBem2p is required for complementation of an Agbem2 null strain. Germination of spores resulted in enlarged Agbem2 germ cells that were unable to generate the bipolar branching pattern found in wild-type germ cells. In addition, mutant hyphae were swollen due to defects in polarized cell growth indicated by the delocalized distribution of chitin and cortical actin patches. Surprisingly, the complete loss of cell polarity which lead to spherical hyphal tips was overcome by the establishment of new cell polarities and the formation of multiple new hyphal tips. In conclusion these results and other findings demonstrate that establishment of cell polarity, maintenance of cell polarity, and polarized hyphal growth in filamentous fungi require members of rho-GTPase modules.
During both interphase and mitosis, DNA topoisomerase II interacts with DNA as well as RNA through the protein’s C-terminal domain
Authors: Rzepecki Ryszard , Fisher Paul A.
Published: April 2021

DNA topoisomerase II (topo II) is thought to be a nuclear enzyme; during interphase most was insoluble and could be recovered in the pellet after centrifugation of cell homogenates at 10,000 g (P-10). Upon entry into mitosis, the majority of topo II did not associate with condensed chromosomes but was apparently solubilized and redistributed throughout the cell. Although two non-chromosomal subfractions of mitotic topo II were defined by centrifugation at 130,000 g, the vast majority (>90%) was recovered in the pellet (P-130). In vivo nucleic acid interactions with topo II were monitored by a recently developed approach of UV-photo-crosslinking, immunoprecipitation and 32P-labeling. P-10 (interphase) topo II was largely associated with DNA. P-130 (mitotic non-chromosomal) topo II was primarily associated with RNA. These nucleic acid interactions with both interphase and mitotic topo II occurred through the catalytically inert and as yet, poorly understood C-terminal domain of the protein. P-10 topo II was highly active enzymatically. Activity, measured by the ability of topo II to decatenate kDNA minicircles, was reduced by treatment with phosphatase. In contrast, P-130 topo II was relatively inactive but activity could be increased by phosphatase treatment. In vivo, P-130 topo II was more heavily phosphorylated than P-10 topo II; in both, only the C-terminal domain of topo II was detectably modified. Our observations suggest that cell cycle-dependent changes in the distribution, nucleic acid interactions and enzymatic activity of topo II are regulated, at least in part, by phosphorylation/dephosphorylation.
Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1
Authors: Noë Veerle , Fingleton Barbara , Jacobs Kathleen , Crawford Howard C. , Vermeulen Stefan , Steelant Wim , Bruyneel Erik , Matrisian Lynn M. , Mareel Marc
Published: April 2021

The function of many transmembrane molecules can be altered by cleavage and subsequent release of their ectodomains. We have investigated ectodomain cleavage of the cell-cell adhesion and signal-transducing molecule E- cadherin. The E-cadherin ectodomain is constitutively shed from the surface of MCF-7 and MDCKts.srcC12 cells in culture. Release of the 80 kDa soluble E-cadherin fragment is stimulated by phorbol-12-myristate-13-acetate and is inhibited by overexpression of the tissue inhibitor of metalloproteinases-2. The metalloproteinases matrilysin and stromelysin-1 both cleave E-cadherin at the cell surface and release sE-CAD into the medium. The soluble E- cadherin fragment thus released inhibits E-cadherin functions in a paracrine way, as indicated by induction of invasion into collagen type I and inhibition of E-cadherin- dependent cell aggregation. Our results, therefore, suggest a novel mechanism by which metalloproteinases can influence invasion.
Subversion of integrins by enteropathogenic Yersinia
Authors: Isberg Ralph R. , Barnes Penelope
Published: April 2021

Enteropathogenic Yersinia are gram-negative bacterial species that translocate from the lumen of the intestine and are able to grow within deep tissue sites. During the earliest stages of disease, the organism is able to bind integrin receptors that are presented on the apical surface of M cells in the intestine, which allows its internalization and subsequent translocation into regional lymph nodes. The primary integrin substrate is the outer-membrane protein invasin, which binds with extraordinarily high affinity to at least five different integrins that have the β1 chain. Bacterial uptake into host cells is modulated by the affinity of receptor-substrate interaction, receptor concentration and the ability of the substrate to aggregate target receptors.
Overexpression of Akt inhibits NGF-induced growth arrest and neuronal differentiation of PC12 cells
Authors: Bang Ok-Sun , Park Eui Kyun , Yang Sung-Il , Lee Sun-Ryung , Franke Thomas F. , Kang Shin-Sung
Published: April 2021

To investigate the role of Akt in nerve growth factor (NGF)-induced neuronal differentiation, PC12 cells ectopically expressing wild-type or dominant-inhibitory forms of Akt were analyzed. NGF-induced neurite outgrowth was greatly accelerated in cells expressing dominant-inhibitory Akt, compared to parental PC12 cells, but was almost completely blocked in cells expressing wild-type Akt. Since neuronal differentiation requires an arrest of cell growth, several aspects of cell growth of the different cell lines were compared. Cells expressing wild-type Akt were not susceptible to the growth-arresting effect of NGF, whereas parental PC12 cells and notably cells expressing mutant Akt were so affected. Accompanying this, the expressions of CDKs and p21WAF1 were down- and up-regulated, respectively, in both parental PC12 cells and cells expressing mutant Akt. When treated with some growth arrest-inducing agents such as sodium nitroprusside, forskolin and butyrolactone I, cells expressing wild-type Akt regained their responsiveness to the effects of NGF on differentiation. In summary, our results indicate that Akt overrides the growth-arresting effect of NGF and thereby, negatively regulates neuronal differentiation.
Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits
Authors: Bertinato Jesse , Schild-Poulter Caroline , Haché Robert J. G.
Published: April 2021

The Ku antigen is a heteromeric (Ku70/Ku80), mostly nuclear protein. Ku participates in multiple nuclear processes from DNA repair to V(D)J recombination to telomere maintenance to transcriptional regulation and serves as a DNA binding subunit and allosteric regulator of DNA-dependent protein kinase. While some evidence suggests that subcellular localization of Ku may be subject to regulation, how Ku gains access to the nucleus is poorly understood. In this work, using a combination of indirect immunofluorescence and direct fluorescence, we have demonstrated that transfer of the Ku heterodimer to the nucleus is determined by basic nuclear localization signals in each of the Ku subunits that function independently. A bipartite basic nuclear localization signal between amino acids 539-556 of Ku70 was observed to be required for nuclear import of full-length Ku70 monomer, while a short Ku80 motif of four amino acids from 565-568 containing three lysines was required for the nuclear import of full- length Ku80. Ku heterodimers containing only one nuclear localization signal accumulated in the nucleus as efficiently as wild-type Ku, while site directed mutagenesis inactivating the basic motifs in each subunit, resulted in a Ku heterodimer that was completely localized to the cytoplasm. Lastly, our results indicate that mutations in Ku previously proposed to abrogate Ku70/Ku80 heterodimerization, markedly reduced the accumulation of Ku70 without affecting heterodimer formation in mammalian cells.
Lamins in disease: why do ubiquitously expressed nuclear envelope proteins give rise to tissue-specific disease phenotypes?
Authors: Hutchison Christopher J. , Alvarez-Reyes Mauricio , Vaughan Owen A.
Published: April 2021

The nuclear lamina is a filamentous structure composed of lamins that supports the inner nuclear membrane. Several integral membrane proteins including emerin, LBR, LAP1 and LAP2 bind to nuclear lamins in vitro and can influence lamin function and dynamics in vivo. Results from various studies suggest that lamins function in DNA replication and nuclear envelope assembly and determine the size and shape of the nuclear envelope. In addition, lamins also bind chromatin and certain DNA sequences, and might influence chromosome position. Recent evidence has revealed that mutations in A-type lamins give rise to a range of rare, but dominant, genetic disorders, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction-system disease and Dunnigan-type familial partial lipodystrophy. An examination of how lamins A/C, emerin and other integral membrane proteins interact at the INM provides the basis for a novel model for how mutations that promote disease phenotypes are likely to influence these interactions and therefore cause cellular pathology through a combination of weakness of the lamina or altered gene expression.
Can’t clone, won’t clone: Essential Molecular Biology, Vol. 1, 2nd edn edited by T. A. Brown
Authors: Evans Sara K. , Lundblad Victoria
Published: April 2021

I have heard it said (though not to my face) that practical molecular biology is somewhat akin to cookery, and I have to admit (though not to my fellow Molecular Biologists) that there is an element of truth in this. Of course, our ovens are cooler, and our pie dishes smaller, but often it is a case of mixing ingredients in the right proportions and baking at 37°C for an hour.
Cyclin specificity: how many wheels do you need on a unicycle?
Authors: Miller Mary E. , Cross Frederick R.
Published: April 2021

Cyclin-dependent kinase (CDK) activity is essential for eukaryotic cell cycle events. Multiple cyclins activate CDKs in all eukaryotes, but it is unclear whether multiple cyclins are really required for cell cycle progression. It has been argued that cyclins may predominantly act as simple enzymatic activators of CDKs; in opposition to this idea, it has been argued that cyclins might target the activated CDK to particular substrates or inhibitors. Such targeting might occur through a combination of factors, including temporal expression, protein associations, and subcellular localization.
HEMCAM/CD146 downregulates cell surface expression of β1 integrins
Authors: Alais Sandrine , Allioli Nathalie , Pujades Cristina , Duband Jean-Loup , Vainio Olli , Imhof Beat A. , Dunon Dominique
Published: April 2021

HEMCAM/gicerin, an immunoglobulin superfamily protein, is involved in homophilic and heterophilic adhesion. It interacts with NOF (neurite outgrowth factor), a molecule of the laminin family. Alternative splicing leads to mRNAs coding for HEMCAM with a short (HEMCAM-s) or a long cytoplasmic tail (HEMCAM-l). To investigate the cellular function of these two variants, we stably transfected murine fibroblasts with either form of HEMCAM. Expression of each isoform of this protein in L cells delayed proliferation and modified their adhesion properties to purified extracellular matrix proteins. Expression of either HEMCAM-s or HEMCAM-l inhibited integrin-dependent adhesion and spreading of fibroblasts to laminin 1, showing that this phenomenon did not depend on the cytoplasmic region. By contrast, L-cell adhesion and spreading to fibronectin depended on the HEMCAM isoform expressed. Flow cytometry and immunoprecipitation studies revealed that the expression of HEMCAM downregulated expression of the laminin-binding integrins α3β1, α6β1 and α7β1, and fibronectin receptor α5β1 from the cell surface. Semi-quantitative PCR and northern blot experiments showed that the expression of α6β1 integrin modified by HEMCAM occurred at a translation or maturation level. Thus, our data demonstrate that HEMCAM regulates fibroblast adhesion by controlling β1 integrin expression.

Movies available on-line: http://www.biologists.com/JCS/movies/jcs1886.html, movie 1A, movie 1B, movie 2A, movie 2B
Prolactin signalling to milk protein secretion but not to gene expression depends on the integrity of the Golgi region
Authors: Lkhider Mustapha , Pétridou Barbara , Aubourg Alain , Ollivier-Bousquet Michèle
Published: April 2021

Prolactin added to the incubation medium of lactating mammary epithelial cells is transported from the basal to the apical region of cells through the Golgi region and concomitantly stimulates arachidonic acid release and protein milk secretion. We report that when PRL is added after disorganisation of the Golgi apparatus by brefeldin A treatment, prolactin signalling to expression of genes for milk proteins and prolactin endocytosis are not affected. However, prolactin transport to the apical region of cells (transcytosis), as well as prolactin-induced arachidonic acid release and subsequent stimulation of the secretion of caseins, which are located in a post-Golgi compartment, are inhibited. This inhibition was not a consequence of damage to the secretory machinery, as under the same conditions, protein secretion could be stimulated by the addition of arachidonic acid to the incubation medium. Thus, it is possible to discriminate between prolactin-induced actions that are dependent (signalling to milk protein secretion) or independent (signalling to milk gene expression) on the integrity of the Golgi apparatus. These results suggest that these two biological actions may be transduced via distinct intracellular pathways, and support the hypothesis that prolactin signals may be emitted at various cellular sites.
Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane
Authors: Lusa Sari , Blom Titta S. , Eskelinen Eeva-Liisa , Kuismanen Esa , Månsson Jan-Eric , Simons Kai , Ikonen Elina
Published: April 2021

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [3H]cholesterol released from [3H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.
Normal membrane localization and actin association of the NF2 tumor suppressor protein are dependent on folding of its N-terminal domain
Authors: Brault Estelle , Gautreau Alexis , Lamarine Marc , Callebaut Isabelle , Thomas Gilles , Goutebroze Laurence
Published: April 2021

The neurofibromatosis type 2 (NF2) tumor suppressor protein, known as schwannomin or merlin, is involved in linking membrane proteins to the cytoskeleton. Like the related ERM proteins, schwannomin has long been suspected of exhibiting a complex 3D organization caused by the association of different regions within the protein. Intramolecular interactions characterized to date are linking N-terminal sequences of the protein to C-terminal sequences. Here, we demonstrate, by a biochemical approach, the existence of a structured domain entirely contained within the N-terminal half of schwannomin. This structure, which is resistant to chymotryptic digestion, encompasses the FERM domain (residues 19-314), but excludes the 18 extreme N-terminal residues specific to schwannomin. The structure is disrupted by some, but not all, naturally occurring NF2 mutations. We investigated the significance of this structured domain in schwannomin cellular functions and found that normal schwannomin localization beneath the plasma membrane is directly dependent on proper folding of the N-terminal domain. In addition, folding of the N-terminal domain influences schwannomin interaction with actin through two novel actin-binding sites located in this region. These results suggest that loss of activity of several naturally occurring schwannomin mutants is due to disruption of the fold of the N-terminal domain, leading to loss of both membrane localization and actin association.
The hSkn-1a POU transcription factor enhances epidermal stratification by promoting keratinocyte proliferation
Authors: Hildesheim Jeffrey , Kühn Ulrich , Yee Carole L. , Foster Ruth A. , Yancey Kim B. , Vogel Jonathan C.
Published: April 2021

Skn-1a is a POU transcription factor that is primarily expressed in the epidermis and is known to modulate the expression of several genes associated with keratinocyte differentiation. However, the formation of a stratified epidermis requires a carefully controlled balance between keratinocyte proliferation and differentiation, and a role for Skn-1a in this process has not been previously demonstrated. Here, our results show, surprisingly, that human Skn-1a contributes to epidermal stratification by primarily promoting keratinocyte proliferation and secondarily by enhancing the subsequent keratinocyte differentiation. In organotypic raft cultures of both primary human keratinocytes and immortalized HaCaT keratinocytes, human Skn-1a expression is associated with increased keratinocyte proliferation and re-epithelialization of the dermal substrates, resulting in increased numbers of keratinocytes available for the differentiation process. In these same raft cultures, human Skn-1a expression enhances the phenotypic changes of keratinocyte differentiation and the upregulated expression of keratinocyte differentiation genes. Conversely, expression of a dominant negative human Skn-1a transcription factor lacking the C-terminal transactivation domain blocks keratinocytes from proliferating and stratifying. Keratinocyte stratification is dependent on a precise balance between keratinocyte proliferation and differentiation, and our results suggest that human Skn-1a has an important role in maintaining epidermal homeostasis by promoting keratinocyte proliferation.
End13p/Vps4p is required for efficient transport from early to late endosomes in Saccharomyces cerevisiae
Authors: Zahn Regina , Stevenson Brian J. , Schröder-Köhne Stephan , Zanolari Bettina , Riezman Howard , Munn Alan L.
Published: April 2021

end13-1 was isolated in a screen for endocytosis mutants and has been shown to have a post-internalisation defect in endocytic transport as well as a defect in vacuolar protein sorting (Vps− phenotype), leading to secretion of newly synthesised vacuolar proteins. Here we demonstrate that END13 is identical to VPS4, encoding an AAA (ATPase associated with a variety of cellular activities)-family ATPase. We also report that the end13-1 mutation is a serine 335 to phenylalanine substitution in the AAA-ATPase domain of End13p/Vps4p. It has been reported that mutant cells lacking End13p/Vps4p (end13(vps4)Δ) accumulate endocytosed marker dyes, plasma membrane receptors and newly synthesised vacuolar hydrolase precursors in an endosomal compartment adjacent to the vacuole (prevacuolar compartment, or PVC). We find, however, that the end13 mutants have defects in transport of endocytosed fluorescent dyes, plasma membrane receptors and ligands from small peripherally located early endosomes to larger late endosomes, which are often located adjacent to the vacuole. Our results indicate that End13p/Vps4p may play an important role in multiple steps of membrane traffic through the endocytic pathway.
Relationships between EEA1 binding partners and their role in endosome fusion
Authors: Mills Ian G. , Urbé Sylvie , Clague Michael J.
Published: April 2021

Homotypic fusion between early endosomes requires the phosphatidylinositol 3-phosphate (PI3P)-binding protein, Early Endosomal Autoantigen 1 (EEA1). We have investigated the role of other proteins that interact with EEA1 in the fusion of early endosomes derived from Baby Hamster Kidney (BHK) cells. We confirm a requirement for syntaxin 13, but additionally show that the assay is equally sensitive to reagents specifically targeted against syntaxin 6. Binding of EEA1 to immobilised GST-syntaxin 6 and 13 was directly compared; only syntaxin 6 formed a stable complex with EEA1. Early endosome fusion requires the release of intravesicular calcium, and calmodulin plays a vital role in the fusion pathway, as judged by sensitivity to antagonists. We demonstrate that both EEA1 and syntaxin 13 interact with calmodulin. In the case of EEA1, binding to calmodulin requires an IQ domain, which is adjacent to a C-terminal FYVE domain that specifically binds to PI3P. We have assessed the influence of protein binding partners on EEA1 interaction with PI3P and find that both calmodulin and rab5-GTP are antagonistic to PI3P binding, whilst syntaxins 6 and 13 have no effect. These studies reveal a complex network of interactions between the proteins required for endosome fusion.
Life without a cell membrane: regeneration of protoplasts from disintegrated cells of the marine green alga Bryopsis plumosa
Authors: Kim Gwang Hoon , Klotchkova Tatiana A. , Kang Yoon-Mi
Published: April 2021

When the multi-nucleate giant cells of the green alga Bryopsis plumosa (Huds.) Ag. are injured, the protoplasm is extruded from the cells and can generate spontaneously numerous new cells. The cell organelles aggregate rapidly in seawater and become covered with a gelatinous envelope within 15 minutes. A lipid cell membrane is formed inside the envelope within 9 to 12 hours and about 15% of the original cell membrane is recycled to make the membrane of new protoplasts. Cytochemical studies using Nile Red and various enzymes revealed that the primary envelope is initially composed of polysaccharides, and then transformed into a polysaccharide-lipid complex. Fluorescein diacetate staining showed that the primary envelope has some characteristics of a cell membrane including semi-permeability and selective transport of materials. The aggregation of cell organelles appears to be mediated by two kinds of materials, one present in vacuolar sap and the other on the surface of the cell organelles. About a thousand new cells were generated from a single disintegrated branch and 40% of them eventually developed into mature plants.
The non-catalytic domain of the Xenopus laevis auroraA kinase localises the protein to the centrosome
Authors: Giet Régis , Prigent Claude
Published: April 2021

Aurora kinases are involved in mitotic events that control chromosome segregation. All members of this kinase subfamily possess two distinct domains, a highly conserved catalytic domain and an N-terminal non-catalytic extension that varies in size and sequence. To investigate the role of this variable non-catalytic region we overexpressed and purified Xenopus laevis auroraA (pEg2) histidine-tagged N-terminal peptide from bacterial cells. The peptide has no effect on the in vitro auroraA kinase activity, but it inhibits both bipolar spindle assembly and stability in Xenopus egg extracts. Unlike the full-length protein, the N-terminal domain shows only low affinity for paclitaxel-stabilised microtubules in vitro, but localises to the centrosomes in a microtubule-dependent manner. When expressed in Xenopus XL2 cells, it is able to target the green fluorescent protein to centrosomes. Surprisingly, this is also true of the pEg2 catalytic domain, although to a lesser extent. The centrosome localisation of the N-terminal peptide was disrupted by nocodazole whereas localisation of the catalytic domain was not, suggesting that in order to efficiently localise to the centrosome, pEg2 kinase required the non-catalytic N-terminal domain and the presence of microtubules.
MT1-MMP in rat testicular development and the control of Sertoli cell proMMP-2 activation
Authors: Longin Juliette , Guillaumot Patricia , Chauvin Marie-Agnès , Morera Anne-Marie , Le Magueresse-Battistoni Brigitte
Published: April 2021

Metalloproteases (MMPs) are likely to be involved in the restructuring events occurring in the testis throughout development. We here demonstrate that membrane-type 1 (MT1)-MMP, a physiological activator of proMMP-2 under TIMP-2 control, is present within the testis together with MMP-2 and TIMP-2. In the prepubertal testis MT1-MMP immunoreactivity was uniformly distributed, whereas in the adult it was confined to the apical compartment of the tubules, where meiosis and spermiogenesis occur. We further showed that the two cell lineages (somatic and germinal) expressed MT1-MMP and TIMP-2, whereas MMP-2 was of somatic origin. To get a better picture into proMMP-2 activation, use was made of a model of cultured Sertoli cells treated with FSH or co-cultured with germ cells to mimic an immature or a mature developmental period, respectively. We found that follicle-stimulating hormone enhanced the expression of MMP-2 and TIMP-2 but not of MT1-MMP, and promoted the activation of proMMP-2. In co-cultures, a tremendous elevation and activation of MMP-2 was observed, which might relate to the processed MT1-MMP form solely detected in germ cells. That MMP-2 synthesis and activation are under local (germ cells) and hormonal (follicle-stimulating hormone) regulation emphasizes the importance of MMPs in testicular physiology.
c-Cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route
Authors: de Melker Annemieke A. , van der Horst Gerda , Calafat Jero , Jansen Hans , Borst Jannie
Published: April 2021

Cbl family members have an evolutionarily conserved role in attenuating receptor tyrosine kinase function. Their negative regulatory capacity depends on a Ring finger domain that interacts with ubiquitin conjugating enzymes. Cbl molecules constitute a novel type of E3 or ubiquitin ligase family that is recruited to phosphotyrosine motifs. Ubiquitination of the receptor system is coupled to its downregulation, but it is unclear at which point in the endocytic pathway Cbl molecules come into play. Using low temperature and a dynamin mutant, we find that c-Cbl associates with and ubiquitinates the activated epidermal growth factor (EGF) receptor at the plasma membrane in the absence of endocytosis. With the aid of confocal microscopy and immunogold electron microscopy, we could demonstrate that c-Cbl associates with the EGF receptor at the plasma membrane prior to receptor recruitment into clathrin-coated pits and remains associated throughout the clathrin-mediated endocytic pathway. c-Cbl and the EGF receptor also colocalize in internal vesicles of multivesicular endosomes. Our data are consistent with a role for c-Cbl in clathrin-mediated endocytosis of tyrosine kinase receptors, as well as their intracellular sorting.
Store-operated influx of Ca2+ in pancreatic β-cells exhibits graded dependence on the filling of the endoplasmic reticulum
Authors: Dyachok Oleg , Gylfe Erik
Published: April 2021

The store-operated pathway for Ca2+ entry was studied in individual mouse pancreatic β-cells by measuring the cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mn2+ ([Mn2+]i) with the fluorescent indicator fura-2. Influx through the store-operated pathway was initially shut off by pre-exposure to 20 mM glucose, which maximally stimulates intracellular Ca2+ sequestration. To avoid interference with voltage-dependent Ca2+ entry the cells were hyperpolarized with diazoxide and the channel blocker methoxyverapamil was present. Activation of the store-operated pathway in response to Ca2+ depletion of the endoplasmic reticulum was estimated from the sustained elevation of [Ca2+]i or from the rate of increase in [Mn2+]i due to influx of these extracellular ions. Increasing concentrations of the inositol 1,4,5-trisphosphate-generating agonist carbachol or the sarco(endo)plasmatic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) cause gradual activation of the store-operated pathway. In addition, the carbachol- and CPA-induced influx of Mn2+ depended on store filling in a graded manner. The store-operated influx of Ca2+/Mn2+ was inhibited by Gd3+ and 2-aminoethoxydiphenyl borate but neither of these agents discriminated between store-operated and voltage-dependent entry. The finely tuned regulation of the store-operated mechanisms in the β-cell has direct implications for the control of membrane potential and insulin secretion.
The organisation and functions of local Ca2+ signals
Authors: Bootman Martin D. , Lipp Peter , Berridge Michael J.
Published: April 2021

Calcium (Ca2+) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca2+ to play a pivotal role in cell biology results from the facility that cells have to shape Ca2+ signals in space, time and amplitude. To generate and interpret the variety of observed Ca2+ signals, different cell types employ components selected from a Ca2+ signalling ‘toolkit’, which comprises an array of homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca2+ signals that suit their physiology. Recent studies have demonstrated the importance of local Ca2+ signals in defining the specificity of the interaction of Ca2+ with its targets. Furthermore, local Ca2+ signals are the triggers and building blocks for larger global signals that propagate throughout cells.