bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polarization of cell growth in yeast: I. Establishment and maintenance of polarity states
Authors: Pruyne David , Bretscher Anthony 
Published: April 2021

ABSTRACT
The ability to polarize is a fundamental property of cells. The yeast Saccharomyces cerevisiae has proven to be a fertile ground for dissecting the molecular mechanisms that regulate cell polarity during growth. Here we discuss the signaling pathways that regulate polarity. In the second installment of this two-part commentary, which appears in the next issue of Journal of Cell Science, we discuss how the actin cytoskeleton responds to these signals and guides the polarity of essentially all events in the yeast cell cycle.
During the cell cycle, yeast cells assume alternative states of polarized growth, which range from tightly focused apical growth to non-focused isotropic growth. RhoGTPases, and in particular Cdc42p, are essential to guiding this polarity. The distribution of Cdc42p at the cell cortex establishes cell polarity. Cyclin-dependent protein kinase, Ras, and heterotrimeric G proteins all modulate yeast cell polarity in part by altering the distribution of Cdc42p. In turn, Cdc42p generates feedback signals to these molecules in order to establish stable polarity states and coordinate cytoskeletal organization with the cell cycle. Given that many of these signaling pathways are present in both fungi and animals, they are probably ancient and conserved mechanisms for regulating polarity.
The cell biology of osteoclast function
Authors: Väänänen H. Kalervo , Zhao Haibo , Mulari Mika , Halleen Jussi M. 
Published: April 2021

ABSTRACT
Osteoclasts are multinucleated cells responsible for bone resorption. They have developed an efficient machinery for dissolving crystalline hydroxyapatite and degrading organic bone matrix rich in collagen fibers. When initiating bone resorption, osteoclasts become polarized, and three distinct membrane domains appear: a ruffled border, a sealing zone and a functional secretory domain. Simultaneously, the cytoskeleton undergoes extensive re-organisation. During this process, the actin cytoskeleton forms an attachment ring at the sealing zone, the membrane domain that anchors the resorbing cell to bone matrix. The ruffled border appears inside the sealing zone, and has several characteristics of late endosomal membrane. Extensive vesicle transport to the ruffled border delivers hydrochloric acid and proteases to an area between the ruffled border and the bone surface called the resorption lacuna. In this extracellular compartment, crystalline hydroxyapatite is dissolved by acid, and a mixture of proteases degrades the organic matrix. The degradation products of collagen and other matrix components are endocytosed, transported through the cell and exocytosed through a functional secretory domain. This transcytotic route allows osteoclasts to remove large amounts of matrix-degradation products without losing their tight attachment to underlying bone. It also facilitates further processing of the degradation products intracellularly during the passage through the cell.
Inhibition of chondrocyte differentiation in vitro by constitutive and inducible overexpression of the c-fos proto-oncogene
Authors: Thomas David P. , Sunters Andrew , Gentry Aleksandra , Grigoriadis Agamemnon E. 
Published: April 2021

ABSTRACT
We have investigated the role of c-Fos in chondrocyte differentiation in vitro using both constitutive and inducible overexpression approaches in ATDC5 chondrogenic cells, which undergo a well-defined sequence of differentiation from chondroprogenitors to fully differentiated hypertrophic chondrocytes. Initially, we constitutively overexpressed exogenous c-fos in ATDC5 cells. Several stable clones expressing high levels of exogenous c-fos were isolated and those also expressing the cartilage marker type II collagen showed a marked decrease in cartilage nodule formation. To investigate further whether c-Fos directly regulates cartilage differentiation independently of potential clonal variation, we generated additional clones in which exogenous c-fos expression was tightly controlled by a tetracycline-regulatable promoter. Two clones, DT7.1 and DT12.4 were capable of nodule formation in the absence of c-fos. However, upon induction of exogenous c-fos, differentiation was markedly reduced in DT7.1 cells and was virtually abolished in clone DT12.4. Pulse experiments indicated that induction of c-fos only at early stages of proliferation/differentiation inhibited nodule formation, and limiting dilution studies suggested that overexpression of c-fos decreased the frequency of chondroprogenitor cells within the clonal population. Interestingly, rates of proliferation and apoptosis were unaffected by c-fos overexpression under standard conditions, suggesting that these processes do not contribute to the observed inhibition of differentiation. Finally, gene expression analyses demonstrated that the expression of the cartilage markers type II collagen and PTH/PTHrP receptor were down-regulated in the presence of exogenous c-Fos and correlated well with the differentiation status. Moreover, induction of c-fos resulted in the concomitant increase in the expression of fra-1 and c-jun, further highlighting the importance of AP-1 transcription factors in chondrocyte differentiation. These data demonstrate that c-fos overexpression directly inhibits chondrocyte differentiation in vitro, and therefore these cell lines provide very useful tools for identifying novel c-Fos-responsive genes that regulate the differentiation and activity of chondrocytes.
Penaeidins, antimicrobial peptides with chitin-binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge
Authors: Destoumieux Delphine , Muñoz Marcello , Cosseau Céline , Rodriguez Jenny , Bulet Philippe , Comps Michel , Bachère Evelyne 
Published: April 2021

ABSTRACT
Penaeidins are members of a new family of antimicrobial peptides isolated from a crustacean, which present both Gram-positive antibacterial and antifungal activities. We have studied the localization of synthesis and storage of penaeidins in the shrimp Penaeus vannamei. The distribution of penaeidin transcripts and peptides in various tissues reveals that penaeidins are constitutively synthesized and stored in the shrimp haemocytes. It was shown by immunocytochemistry, at both optical and ultrastructural levels, that the peptides are localized in granulocyte cytoplasmic granules. The expression and localization of penaeidins were further analysed in shrimp subjected to microbial challenge. We found that (1) penaeidin mRNA levels decrease in circulating haemocytes in the first 3 hours following stimulation and (2) an increase in plasma penaeidin concentration occurs after microbial challenge, together with (3) a penaeidin immunoreactivity in cuticular tissue, which can be related to the chitin-binding activity we demonstrate here for penaeidins.
Type 1 protein phosphatase is required for maintenance of cell wall integrity, morphogenesis and cell cycle progression in Saccharomyces cerevisiae
Authors: Andrews Paul D. , Stark Michael J. R. 
Published: April 2021

ABSTRACT
GLC7 encodes the catalytic subunit of type 1 protein serine/threonine phosphatase (PP1) in the yeast Saccharomyces cerevisiae. Here we have characterized the temperature-sensitive glc7–10 allele, which displays aberrant bud morphology and an abnormal actin cytoskeleton at the restrictive temperature. At 37°C glc7-10 strains accumulated a high proportion of budded cells with an unmigrated nucleus, duplicated spindle pole bodies, a short spindle, delocalized cortical actin and 2C DNA content, indicating a cell cycle block prior to the metaphase to anaphase transition. glc7–10 was suppressed by growth on high osmolarity medium and exhibited temperature-sensitive cell lysis upon hypo-osmotic stress. Pkc1p, the yeast protein kinase C homolog which is thought to regulate the Mpk1p MAP kinase pathway involved in cell wall remodelling and polarized cell growth, was found to act as a dosage suppressor of glc7–10. Although neither activation of BCK1 (MEKK) by the dominant BCK1-20 mutation nor increased dosage of MKK1 (MEK) or MPK1 (MAP kinase) mimicked PKC1 as a glc7–10 dosage suppressor, extra copies of genes encoding upstream components of the Pkc1p pathway such as ROM2, RHO2, HCS77/WSC1/SLG1 and MID2 also suppressed glc7–10 effectively. Conversely, mpk1Δ glc7–10 and bck1Δ glc7–10 double mutants displayed a synthetic cell lysis defect compared with each single mutant and glc7–10 was hypersensitive to reduced PKC1 function, displaying highly aberrant morphologies and inviability even at the normally permissive temperature of 26°C. Dephosphorylation by PP1 therefore functions positively to promote cell integrity, bud morphology and polarization of the actin cytoskeleton and glc7–10 cells require higher levels of Pkc1p activity to sustain these functions.
Polarization of cell growth in yeast II. The role of the cortical actin cytoskeleton
Authors: Pruyne David , Bretscher Anthony 
Published: April 2021

ABSTRACT
The actin cytoskeleton provides the structural basis for cell polarity in Saccharomyces cerevisiaeas well as most other eukaryotes. In Part I of this two-part commentary, presented in the previous issue of Journal of Cell Science, we discussed the basis by which yeast establishes and maintains different states of polarity through Rho GTPases and cyclin-dependent protein kinase signaling. Here we discuss how, in response to those signals, the actin cytoskeleton guides growth of the yeast cell. A polarized array of actin cables at the cell cortex is the primary structural determinant of polarity. Motors such as class V myosins use this array to transport secretory vesicles, mRNA and organelles towards growth sites, where they are anchored by a cap of cytoskeletal and regulatory proteins. Cortical actin patches enhance and maintain this polarity, probably through endocytic recycling, which allows reuse of materials and prevents continued growth at old sites. The dynamic arrangement of targeting and recycling provides flexibility for the precise control of morphogenesis.
ERGIC-53 and traffic in the secretory pathway
Authors: Hauri Hans-Peter , Kappeler Felix , Andersson Helena , Appenzeller Christian 
Published: April 2021

ABSTRACT
The ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53 is a mannose-specific membrane lectin operating as a cargo receptor for the transport of glycoproteins from the ER to the ERGIC. Lack of functional ERGIC-53 leads to a selective defect in secretion of glycoproteins in cultured cells and to hemophilia in humans. Beyond its interest as a transport receptor, ERGIC-53 is an attractive probe for studying numerous aspects of protein trafficking in the secretory pathway, including traffic routes, mechanisms of anterograde and retrograde traffic, retention of proteins in the ER, and the function of the ERGIC. Understanding these fundamental processes of cell biology will be crucial for the elucidation and treatment of many inherited and acquired diseases, such as cystic fibrosis, Alzheimer’s disease and viral infections.
Migration of human vascular smooth muscle cells involves serum-dependent repeated cytosolic calcium transients
Authors: Scherberich Arnaud , Campos-Toimil Manuel , Rondé Philippe , Takeda Kenneth , Beretz Alain 
Published: April 2021

ABSTRACT
Migration of vascular smooth muscle cells (VSMC) is a key event in the formation of neointima during atherosclerosis. Fura-2 loaded VSMCs were used to investigate calcium homeostasis during cell migration. Multiple spontaneous transient increases in cytosolic free calcium [Ca2+]iwere observed in single human VSMCs migrating on type I collagen. Such [Ca2+]itransients were dependent on the presence of serum or PDGF-BB. Removal of serum, or loading cells with BAPTA, abolished the transients and decreased cell migration speed. The transients were not affected by disruption of cell polarization by dihydrocytochalasin B. Adhesion was used to investigate the specific role of cell-substrate interactions in the generation of transients. Transients are seen in VSMCs adhering either on collagen or on poly-L-lysine, suggesting that generation of transients is not strictly dependent on integrins. Buffering [Ca2+]iwith BAPTA led to accumulation of β1 integrins at the cellular tail, and to increased release of integrin on the extracellular matrix. These results demonstrate a role for [Ca2+]itransients in the rapid, serum-dependent migration of VSMCs. These [Ca2+]itransients are present in migrating VSMCs only when two simultaneous events occur: (1) substrate independent spreading and (2) stimulation of cells by serum components such as PDGF-BB.
The replication capacity of intact mammalian nuclei in Xenopus egg extracts declines with quiescence, but the residual DNA synthesis is independent of Xenopus MCM proteins
Authors: Sun Wei-hsin , Hola Marek , Pedley Kevin , Tada Shusuke , Blow J. Julian , Todorov Ivan T. , Kearsey Stephen E. , Brooks Robert F. 
Published: April 2021

ABSTRACT
In eukaryotes, the initiation of DNA synthesis requires the assembly of a pre-replicative complex (pre-RC) at origins of replication. This involves the sequential binding of ORC (origin-recognition-complex), Cdc6 and MCM proteins, a process referred to as licensing. After origin firing, the Cdc6 and MCM proteins dissociate from the chromatin, and do not rebind until after the completion of mitosis, thereby restricting replication to a single round in each cell cycle. Although nuclei normally become licensed for replication as they enter G1, the extent to which the license is retained when cells enter the quiescent state (G0) is controversial. Here we show that the replication capacity of nuclei from Swiss 3T3 cells, in Xenopusegg extracts, is not lost abruptly with the onset of quiescence, but instead declines gradually. The decline in replication capacity, which affects both the number of nuclei induced to replicate and their subsequent rate of DNA synthesis, is accompanied by a fall in the level of chromatin-bound MCM2. When quiescent nuclei are incubated in egg extracts, they do not bind further MCMs unless the nuclei are first permeabilized. The residual replication capacity of intact nuclei must therefore be dependent on the remaining endogenous MCMs. Although high levels of Cdk activity are known to block MCM binding, we show that the failure of intact nuclei in egg extracts to increase their bound MCMs is not due to their uptake and accumulation of Cdk complexes. Instead, the failure of binding must be due to exclusion of some other binding factor from the nucleus, or to the presence within nuclei of an inhibitor of binding other than Cdk activity. In contrast to the situation in Xenopusegg extracts, following serum stimulation of intact quiescent cells, the level of bound MCMs does increase before the cells reach S phase, without any disruption of the nuclear envelope.
Induced detachment of acentric chromatin from mitotic chromosomes leads to their cytoplasmic localization at G1and the micronucleation by lamin reorganization at S phase
Authors: Tanaka Tsubasa , Shimizu Noriaki 
Published: April 2021

ABSTRACT
Acentric and atelomeric double minute chromatin found in human cancer cells are eliminated from cells by selective incorporation into the micronuclei. We showed previously that most of the micronuclei were formed at S phase and mediated by the nuclear bud-shaped structures that selectively entrap double minutes. In this paper, we have examined the behavior of double minutes in relation to the nuclear lamin protein in cell cycle-synchronized human COLO 320DM tumor cells. At the G1phase, we observed that a portion of double minutes was localized at the cytoplasm and showed no association with lamin. The frequency of this localization was increased by hydroxyurea, an inducer of micronuclei, if treated at the preceding S phase. The acentric double minutes were normally segregated to daughter cells by attaching to the mitotic chromosomes, and the hydroxyurea-treatment induced their detachment, possibly through the introduction of the double strand break. When the cells entered S phase, our data suggested that the lamin protein accumulated around the cytoplasmic double minutes at the proximity of the nucleus leading to the formation of the nuclear bud-shaped structure and the initiation of DNA replication. This association of cytoplasmic double minutes with lamin coincided with the large-scale rearrangement of the intranuclear lamin protein. The implication of these findings as well as their application to a broad spectrum of other acentric, atelomeric and autonomously replicating molecules are discussed.
The actin cytoskeleton of Dictyostelium: a story told by mutants
Authors: Noegel Angelika A. , Schleicher Michael 
Published: April 2021

ABSTRACT
Actin-binding proteins are effectors of cell signalling and coordinators of cellular behaviour. Research on the Dictyostelium actin cytoskeleton has focused both on the elucidation of the function of bona fide actin-binding proteins as well as on proteins involved in signalling to the cytoskeleton. A major part of this work is concerned with the analysis of Dictyostelium mutants. The results derived from these investigations have added to our understanding of the role of the actin cytoskeleton in growth and development. Furthermore, the studies have identified several cellular and developmental stages that are particularly sensitive to an unbalanced cytoskeleton. In addition, use of GFP fusion proteins is revealing the spatial and temporal dynamics of interactions between actin-associated proteins and the cytoskeleton.
Epithelial stem cell-mediated development of the human respiratory mucosa in SCID mice
Authors: Delplanque Aurélie , Coraux Christelle , Tirouvanziam Rabindra , Khazaal Ibrahim , Puchelle Edith , Ambros Peter , Gaillard Dominique , Péault Bruno 
Published: April 2021

ABSTRACT
We have developed an in vivo assay for progenitor cells of the human tracheobronchial epithelium relying on the transplantation of human prenatal respiratory tissues into severe combined immunodeficiency mice. Engrafted embryonic or fetal open tracheobronchial rudiments are rapidly closed at each end by a neoformed membrane that we named the operculum. After 2-4 weeks, differentiated human respiratory epithelium covers both the native airway matrix and the new operculum. Human epithelial cells dissociated from either emerging embryonic lung primordia or mature xenografts were seeded in host human airway grafts, of which native epithelium had been eliminated by several cycles of freezing and thawing. All grafts seeded with donor epithelial cells and implanted back into SCID mice recovered a surface mucociliary epithelium expressing expected markers and secreting mucus. Spontaneous epithelium regrowth was never observed in control unseeded, denuded grafts. In some experiments, donor epithelial cells and host denuded airway were sex-mismatched and the donor origin of newly formed epithelial structures was confirmed by sex chromosome detection. After two rounds of seeding and reimplantation, a normal epithelium was observed to line the 3rd generation operculum. These observations substantiate a functional assay for human candidate airway epithelium stem cells.
Live fluorescence imaging reveals early recruitment of emerin, LBR, RanBP2, and Nup153 to reforming functional nuclear envelopes
Authors: Haraguchi Tokuko , Koujin Takako , Hayakawa Tomohiro , Kaneda Toru , Tsutsumi Chihiro , Imamoto Naoko , Akazawa Chihiro , Sukegawa Jun , Yoneda Yoshihiro , Hiraoka Yasushi 
Published: April 2021

ABSTRACT
We determined the times when the nuclear membrane, nuclear pore complex (NPC) components, and nuclear import function were recovered during telophase in living HeLa cells. Simultaneous observation of fluorescently-labeled NLS-bearing proteins, lamin B receptor (LBR)-GFP, and Hoechst33342-stained chromosomes revealed that nuclear membranes reassembled around chromosomes by 5 minutes after the onset of anaphase (early telophase) whereas nuclear import function was recovered later, at 8 minutes. GFP-tagged emerin also accumulated on chromosomes 5 minutes after the onset of anaphase. Interestingly, emerin and LBR initially accumulated at distinct, separate locations, but then became uniform 8 minutes after the onset of anaphase, concurrent with the recovery of nuclear import function. We further determined the timing of NPC assembly by immunofluorescence staining of cells fixed at precise times after the onset of anaphase. Taken together, these results showed that emerin, LBR, and several NPC components (RanBP2, Nup153, p62), but not Tpr, reconstitute around chromosomes very early in telophase prior to the recovery of nuclear import activity.
The large intracytoplasmic loop of the glucose transporter GLUT2 is involved in glucose signaling in hepatic cells
Authors: Guillemain Ghislaine , Loizeau Martine , Pinçon-Raymond Martine , Girard Jean , Leturque Armelle 
Published: April 2021

ABSTRACT
The hypothesis that the glucose transporter GLUT2 can function as a protein mediating transcriptional glucose signaling was addressed. To divert the putative interacting proteins from a glucose signaling pathway, two intracytoplasmic domains of GLUT2, the C terminus and the large loop located between transmembrane domains 6 and 7, were transfected into mhAT3F hepatoma cells. Glucose-induced accumulation of two hepatic gene mRNAs (GLUT2 and L-pyruvate kinase) was specifically inhibited in cells transfected with the GLUT2 loop and not with the GLUT2 C terminus. The dual effects of glucose were dissociated in cells expressing the GLUT2 loop; in fact a normal glucose metabolism into glycogen occurred concomitantly with the inhibition of the glucose-induced transcription. This inhibition by the GLUT2 loop could be due to competitive binding of a protein that normally interacts with endogenous GLUT2. In addition, the GLUT2 loop, tagged with green fluorescent protein (GFP), was located within the nucleus, whereas the GFP and GFP-GLUT2 C-terminal proteins remained in the cytoplasm. In living cells, a fraction (50%) of the expressed GFP-GLUT2 loop translocated rapidly from the cytoplasm to the nucleus in response to high glucose concentration and conversely in the absence of glucose. We conclude that, via protein interactions with its large loop, GLUT2 may transduce a glucose signal from the plasma membrane to the nucleus.