bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of histone H3 is correlated with changes in the maintenance of sister chromatid cohesion during meiosis in maize, rather than the condensation of the chromatin
Authors: Kaszás Étienne , Cande W. Zacheus
Published: April 2021

Meiotic chromosome condensation is a unique process, characterized by dramatic changes in chromosome morphology that are required for the correct progression of pairing, synapsis, recombination and segregation of sister chromatids. We used an antibody that recognizes a ser 10 phosphoepitope on histone H3 to monitor H3 phosphorylation during meiosis in maize meiocytes. H3 phosphorylation has been reported to be an excellent marker for chromosome condensation during mitotic prophase in animal cells. In this study, we find that on maize mitotic chromosomes only pericentromeric regions are stained; there is little staining on the arms. During meiosis, chromosome condensation from leptotene through diplotene occurs in the absence of H3 phosphorylation. Instead, the changes in H3 phosphorylation at different stages of meiosis correlate with the differences in requirements for sister chromatid cohesion at different stages. Just before nuclear envelope breakdown, histone H3 phosphorylation is seen first in the pericentromeric regions and then extends through the arms at metaphase I; at metaphase II only the pericentromeric regions are stained. In afd1 (absence of first division), a mutant that is defective in many aspects of meiosis including sister chromatid cohesion and has equational separation at metaphase I, staining is restricted to the pericentromeric regions during metaphase I and anaphase I; there is no staining at metaphase II or anaphase II. We conclude that changes in the level of phosphorylation of ser10 in H3 correspond to changes in the cohesion of sister chromatids rather than the extent of chromosome condensation at different stages of meiosis.
Fc receptor mediated endocytosis of small soluble immunoglobulin G immune complexes in Kupffer and endothelial cells from rat liver
Authors: Løvdal T. , Andersen E. , Brech A. , Berg T.
Published: April 2021

Soluble circulating immunoglobulin G immune complexes are mainly eliminated by the liver, predominantly by uptake in the Kupffer cells, but also the liver endothelial cells seem to be of importance. In the present study we have followed the intracellular turnover of immune complexes after Fcγ receptor mediated endocytosis in cultured rat liver endothelial cells and Kupffer cells by means of isopycnic centrifugation, DAB cross-linking and morphological techniques. For the biochemical experiments the antigen, dinitrophenylated bovine serum albumin (BSA), was labeled with radioiodinated tyramine cellobiose that cannot cross biological membranes and therefore traps labeled degradation products at the site of formation. The endocytic pathway followed by immune complexes was compared with that followed by scavenger receptor ligands, such as formaldehyde treated BSA and dinitrophenylated BSA, and the mannose receptor ligand ovalbumin.

Both Kupffer cells and liver endothelial cells took up and degraded the immune complexes, but there was a clear delay in the degradation of immune complexes as compared to degradation of ligands taken up via scavenger receptors. The kinetics of the endocytosis of scavenger receptor ligand was unaffected by simultaneous uptake of immune complexes. Experiments using both biochemical and morphological techniques indicated that the delayed degradation was due to a late arrival of the immune complexes at the lysosomes, which partly was explained by retroendocytosis of immune complexes. Electron microscopy studies revealed that the immune complexes were retained in the early endosomes that remained accessible to other endocytic markers such as ovalbumin. In addition, the immune complexes were seen in multivesicular compartments apparently devoid of other endocytic markers. Finally, the immune complexes were degraded in the same lysosomes as the ligands of scavenger receptors. Thus, immune complexes seem to follow an endocytic pathway that is kinetically or maybe morphologically different from that followed by scavenger and mannose receptor ligands.
Failure of pronuclear migration and repeated divisions of polar body nuclei associated with MTOC defects in Polo eggs of Drosophila
Authors: Riparbelli M. G. , Callaini G. , Glover D. M.
Published: April 2021

The meiotic spindle of Drosophila oocytes is acentriolar but develops an unusual central microtubule organising centre (MTOC) at the end of meiosis I. In polo oocytes, this common central pole for the two tandem spindles of meiosis II was poorly organised and in contrast to wild-type failed to maintain its associated Pav-KLP motor protein. Furthermore, the polar body nuclei failed to arrest at metaphase, and the four products of female meiosis all underwent repeated haploid division cycles on anastral spindles. This was linked to a failure to form the astral array of microtubules with which the polar body chromosomes are normally associated. The MTOC associated with the male pronucleus was also defective in polo eggs, and the sperm aster did not grow. Migration of the female pronucleus did not take place and so a gonomeric spindle could not form. We discuss these findings in relation to the known roles of polo like kinases in regulating the behaviour of MTOCs.
A concise yet informative stroll through matrix metalloproteinases and TIMPs
Authors: Shapiro S.D.
Published: April 2021

Matrix Metalloproteinases and TIMPs by J. Frederick Woessner and Hideaki Nagase Oxford University Press (2000) pp. 223. ISBN 0–19-850268-0 35.00 Ever since Gross discovered that collagenase was responsible for resorption of the tadpole tail, there has been a small group of outstanding scientists that have dedicated their careers to the study of matrix metalloproteinases (MMPs). The family of MMPs has now grown to over 20, and they have been implicated in multiple biological processes drawing the attention of scientists of many disciplines. Two leaders in the field who have ushered in the modern era of MMP biology are Fred Woessner and Hideake Nagase and they share their expertise in Matrix Metalloproteinases and TIMPs. In a concise, yet thorough manner, these authors provide the basic biochemical and biological basis for the study of MMPs. This information, laced with a strong sense of historical perspective, is conveyed in the same interesting manner in which they educated this reviewer and many others over late night scotch at MMP Gordon Conferences. For the interested novice, one will come away no longer needing a score card to keep track of MMP-1 through MMP-22 (perhaps more by now). One will understand which cells produce which MMPs and TIMPs in response to which stimuli. The reader will understand the multiple levels of regulation of MMP activity through gene transcription, proenzyme activation, and inhibition by TIMPs. The book is filled with readable tables depicting important concepts in classification, evolution, and substrate specificity. The authors provide extensive key references for further reading as only they can. The only area not extensively covered is the rapidly emerging in vivo function of MMPs that comes from transgenic and gene targeted mice and animal models. Perhaps this will be the sequel to this primer. As the biological role of these enzymes expands and it becomes more difficult for scientists to ignore MMPs, this book provides a meaningful and painless way to become fluent in the field. Upon completion of the text, readers will feel comfortable incorporating MMPs into their research endeavors. Hopefully this work will spark investigators to ask how these enzymes relate to one's own research interests thus broadening our general biological knowledge.
Inducible expression of claudin-1-myc but not occludin-VSV-G results in aberrant tight junction strand formation in MDCK cells
Authors: McCarthy K. M. , Francis S. A. , McCormack J. M. , Lai J. , Rogers R. A. , Skare I. B. , Lynch R. D. , Schneeberger E. E.
Published: April 2021

Occludin and 18 distinct members of the claudin family are tetra-span transmembrane proteins that are localized in cell-specific tight junctions (TJs). A previous study showed that expression of chick occludin in Madin-Darby canine kidney (MDCK) cells raised transepithelial electrical resistance (TER) and, paradoxically, increased mannitol flux. In the present study, we employed epitope tagged canine occludin expression, under the control of the tetracycline repressible transactivator, to determine the extent to which the unexpected parallel increase in TER and mannitol flux was related to a structural mismatch between avian and canine occludins, which are only 50% identical. To determine whether the paradoxical changes in permeability was specific to occludin, we assessed the effect of over-expressing epitope tagged murine claudin-1. Our data revealed that over-expression of either of the epitope tagged mammalian tight junction proteins increased TER, mannitol and FITC-dextran flux. We observed a 2- and up to 5.6-fold over-expression of occludin-VSV-G and claudin-1-myc, respectively, with no change in ZO-1, endogenous occludin or claudin-1 expression. Confocal microscopy revealed that occludin-VSV-G, claudin-1-myc and ZO-1 co-localized at the TJ. In addition, claudin-1-myc formed aberrant strands along the lateral cell surface without an underlying ZO-1 scaffold. In fracture labeled replicas these strands consisted of claudin-1-myc with little accompanying occludin. These observations suggest that in epithelial cells claudin-1 can assemble into TJ strands without the participation of either ZO-1 or occludin. The proximity of the myc tag to the COOH-terminal YV sequence of claudin-1 appeared to interfere with its interaction with ZO-1, since over-expression of non-tagged claudin-1 increased TER but had a minimal effect on solute flux and no aberrant strands formed. From our data we conclude that differences in structure between avian and mammalian occludin do not account for the observed paradoxical increase in mannitol flux. Levels of ZO-1 remained unchanged despite substantial increases in induced TJ integral protein expression, suggesting that an imbalance between levels of ZO-1 and occludin or claudin-1 leads to altered regulation of pores through which non-charged solute flux occurs. We suggest that ion and solute flux are differentially regulated at the TJ.
Cell cycle-dependent repetitive Ca2+ waves induced by a cytosolic sperm extract in mature ascidian eggs mimic those observed at fertilization
Authors: McDougall Alex , Levasseur Mark , O’Sullivan Antony J. , Jones Keith T.
Published: April 2021

Sperm-triggered Ca2+ oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca2+ oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca2+ oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca2+ oscillations that are all waves in ascidians. The first of these Ca2+ waves begins at the point of sperm-egg fusion while a second phase of Ca2+ waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca2+ waves during meiosis and thereby ensuring meiotic exit.

We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca2+-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca2+ waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca2+ waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca2+ oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca2+ oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca2+ release even though they responded to microinjection of the Ca2+-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca2+-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca2+-releasing activity.

These data support a model of egg activation whereby the sperm introduces a Ca2+-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca2+ waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.
Rab5 regulates the kiss and run fusion between phagosomes and endosomes and the acquisition of phagosome leishmanicidal properties in RAW 264.7 macrophages
Authors: Duclos Sophie , Diez Roberto , Garin Jérome , Papadopoulou Barbara , Descoteaux Albert , Stenmark Harald , Desjardins Michel
Published: April 2021

Phagolysosome biogenesis is essential for the killing and degradation of intracellular pathogens. It involves the fusion of phagosomes with various endocytic organelles, a process known to be regulated in part by Rab proteins. We generated RAW 264.7 macrophages expressing an active mutant of Rab5 (Rab5(Q79L)) to determine the role of Rab5 in phagocytosis and phagolysosome biogenesis. Our results indicate that Rab5 stimulates phagocytosis of latex beads but not Fc or C3 receptor-mediated phagocytosis. Rab5 also acts to restrict the complete fusion of phagosomes with endosomes, a phenomenon allowing exchange of solutes from the two compartments without complete intermixing of their membrane (kiss and run). In Rab5(Q79L)-expressing macrophages, uncontrolled fusion events occurred, leading to the appearance of giant phagosomes. These phagosomes could initiate their maturation and acquire LAMP1, but failed to generate the microbicidal conditions needed to kill intracellular parasites. These results identify Rab5 as a key molecule regulating phagosome-endosome fusion and as an essential component in the innate ability of macrophages to restrict the growth of intracellular parasites.
Synthesis and sorting of proteoglycans
Authors: Prydz Kristian , Dalen Knut Tomas
Published: April 2021

Proteoglycans are widely expressed in animal cells. Interactions between negatively charged glycosaminoglycan chains and molecules such as growth factors are essential for differentiation of cells during development and maintenance of tissue organisation. We propose that glycosaminoglycan chains play a role in targeting of proteoglycans to their proper cellular or extracellular location. The variability seen in glycosaminoglycan chain structure from cell type to cell type, which is acquired by use of particular Ser-Gly sites in the protein core, might therefore be important for post-synthesis sorting. This links regulation of glycosaminoglycan synthesis to the post-Golgi fate of proteoglycans.
The PI 3-kinase isoforms p110α and p110β have differential roles in PDGF- and insulin-mediated signaling
Authors: Hooshmand-Rad Roya , Hájková Lucie , Klint Peter , Karlsson Roger , Vanhaesebroeck Bart , Claesson-Welsh Lena , Heldin Carl-Henrik
Published: April 2021

Phosphoinositide 3´-kinases constitute a family of lipid kinases implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. Phosphoinositide 3´-kinases that bind to the platelet-derived growth factor receptor are composed of two subunits: the p85 subunit acts as an adapter and couples the catalytic p110 subunit to the activated receptor. There are different isoforms of p85 as well as of p110, the individual roles of which have been elusive. Using microinjection of inhibitory antibodies specific for either p110α or p110β we have investigated the involvement of the two p110 isoforms in platelet-derived growth factor- and insulin-induced actin reorganization in porcine aortic endothelial cells. We have found that antibodies against p110α, but not antibodies against p110β, inhibit platelet-derived growth factor-stimulated actin reorganization, whereas the reverse is true for inhibition of insulin-induced actin reorganization. These data indicate that the two phosphoinositide 3´-kinase isoforms have distinct roles in signal transduction pathways induced by platelet-derived growth factor and insulin.
c-Cbl localizes to actin lamellae and regulates lamellipodia formation and cell morphology
Authors: Scaife Robin M. , Langdon Wallace Y.
Published: April 2021

Adhesive and locomotive properties of cells have key roles in normal physiology and disease. Cell motility and adhesion require the assembly and organization of actin microfilaments into stress fibers, lamellipodia and filopodia, and the formation of these structures is mediated by signalling through Rho GTPases. Here we identify c-Cbl (a multi-adaptor proto-oncogene product involved in protein tyrosine kinase signalling) as an important regulator of the actin cytoskeleton. By immunofluorescence microscopy we have determined that c-Cbl co-localizes with the adaptor protein Crk to submembranous actin lamellae in NIH 3T3 fibroblasts and that c-Cbl’s actin localization requires specific SH3-binding sequences. Further, we have found that truncation of this SH3-binding domain in c-Cbl profoundly alters the morphology of NIH 3T3 fibroblasts by inhibiting the formation of actin lamellae, lamellipodia and membrane ruffles. The induction of lamellipodia and membrane ruffles are also inhibited during cell spreading and migration, conditions when these structures are normally most prominent. The inhibitory effect of truncated c-Cbl expression on lamellipodia formation can be reversed by mutational inactivation of its divergent SH2 domain, by the co-expression of constitutively active Rac or by the overexpression of c-Cbl. This study therefore identifies a cytoskeletal role for c-Cbl which may involve the regulation of Crk and Rac, and which is dependent on targeting of c-Cbl to actin lamellae and the ability to recruit signalling protein(s) associated with its divergent SH2 domain.
Increased recycling of α5β1 integrins by lung endothelial cells in response to tumor necrosis factor
Authors: Gao Baochong , Curtis Theresa M. , Blumenstock Frank A. , Minnear Fred L. , Saba Thomas M.
Published: April 2021

Tumor necrosis factor α (TNF-α) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-α may influence cell-matrix interactions by altering the clustering as well as internalization of the α5β1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-α caused an increase in the intracellular staining of α5β1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery. Flow cytometric analysis of endothelial cells incubated at 37°C after antibody-labeling of their surface α5β1 integrins at 4°C confirmed an increase in the rate of α5β1 integrin internalization which was at least 3 times greater after TNF-α exposure, based on the half-life for antibody-labeled surface integrins to reach equilibrium with non-labeled integrins within the intracellular pool. Interestingly, the total cell surface expression of α5β1 integrins was relatively constant after TNF-α exposure despite the enhanced rate of internalization, suggesting an accelerated recycling of the internalized α5β1 integrins back to the cell surface. This response was confirmed by the measurement of labeled integrin recycling, which showed a significant (P<0.01) increase in the rate of recycling of the internalized integrins in TNF-treated endothelial cells. Enhanced internalization and subsequent recycling of α5β1 integrins by endothelial monolayers exposed to TNF-α may facilitate the redistribution of cell-surface integrins in response to this inflammatory cytokine and may also modify cell-matrix interactions leading to reduced integrity and increased protein permeability of the lung endothelial monolayers.
Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes
Authors: Khayat Zayna A. , Tong Peter , Yaworsky Karen , Bloch Robert J. , Klip Amira
Published: April 2021

We examined the temporal reorganization of actin microfilaments by insulin and its participation in the localization of signaling molecules and glucose transporters in L6 myotubes expressing myc-tagged glucose transporter 4 (GLUT4myc). Scanning electron microscopy revealed a dynamic distortion of the dorsal cell surface (membrane ruffles) upon insulin treatment. In unstimulated cells, phalloidin-labeled actin filaments ran parallel to the longitudinal axis of the cell. Immunostaining of the p85 regulatory subunit of phosphatidylinositol 3-kinase was diffusely punctate, and GLUT4myc was perinuclear. After 3 minutes of insulin treatment, actin reorganized to form structures; these structures protruded from the dorsal surface of the myotubes by 10 minutes and condensed in the myoplasm into less prominent foci at 30 minutes. The p85 polypeptide colocalized with these structures at all time points. Actin remodeling and p85 relocalization to actin structures were prevented by cytochalasin D or latrunculin B. GLUT4myc recruitment into the actin-rich projections was also observed, but only after 10 minutes of insulin treatment. Irrespective of insulin stimulation, the majority of p85 and a portion (45%) of GLUT4 were recovered in the Triton X-100-insoluble material that was also enriched with actin. In contrast, vp165, a transmembrane aminopeptidase that morphologically colocalized with GLUT4 vesicles, was fully soluble in Triton X-100 extracts of both insulin-treated and control myotubes. Transient transfection of dominant inhibitory Rac1 (N17) into L6 myotubes prevented formation of dorsal actin structures and blocked insulin-induced GLUT4myc translocation to the cell surface. We propose that insulin-dependent formation of actin structures facilitates the association of PI3-K (p85) with GLUT4 vesicles and, potentially, the arrival of GLUT4 at the cell surface.
The path of RNA through nuclear pores: apparent entry from the sides into specialized pores
Authors: Iborra Francisco J. , Jackson Dean A. , Cook Peter R.
Published: April 2021

The path that RNA takes through nuclear pores was mapped using two high-resolution techniques. Unexpectedly, no RNA in HL60 cells was detected by immunogold labelling in the central axis of the pore complex on its way to the transporter at the nuclear membrane; instead, it was distributed around the sides, apparently entering just before the membrane. In rat liver nuclei, poly(A)+ RNA, hnRNPs A1 and C, mrnp 41, ASF, and a phosphorylated subset of SR proteins were also distributed like mRNA, as were various transport factors and their cargoes (NTF2, Ran, RCC1, karyopherin β, Rch1, transportin α, m2,2,7-trimethylG). Many pores were associated with particular transport factors/cargoes to the exclusion of others; some were associated with poly(A)+ RNA or phosphorylated SR proteins (but not NTF2), others with NTF2 (but not poly(A)+ RNA or the SR proteins). Electron spectroscopic imaging confirmed these results. Some pores contained phosphorus-rich RNA apparently entering from the sides; others lacked any phosphorus, and were surrounded by a ribosome-free zone in the cytoplasm. The results also suggest that pores have different functional zones where SR proteins are dephosphorylated, and where hnRNP C is removed from messages.
Clathrin-coated lattices and buds on MHC class II compartments do not selectively recruit mature MHC-II
Authors: Ramm Georg , Pond Leslie , Watts Colin , Stoorvogel Willem
Published: April 2021

Newly synthesized major histocompatibility complex class II molecules (MHC-II) are transported to MHC-II-containing endosomal and lysosomal compartments (MIICs) for the degradation of associated invariant chain and peptide loading. Subsequently MHC-II is transported to the plasma membrane, in part through direct fusion of MIICs with the plasma membrane. In search of potential alternative pathway(s) we studied the 3-dimensional structure of MIICs and the subcellular distribution of MHC-II by immuno electronmicroscopy on whole-mount preparations and cryosections of Mel JuSo cells. Intracellular MHC-II and invariant chain mainly localized to lamp-1 positive compartments suggesting that the majority of MHC-II exits the endocytic tract at lysosomes. Clathrin-coated lattices and buds were found to be associated with these organelles, but MHC-II was not found to be enriched in the clathrin-coated domains. Moreover, leupeptin, a drug that interferes with Ii-processing and delays delivery of newly synthesized MHC-II to the plasma membrane, was not found to decrease the relative amount of MHC-II in clathrin-coated areas. Together these data indicate clathrin-mediated exit site(s) from lysosomes but suggest that they do not selectively recruit mature MHC-II, consistent with the notion that transport to the plasma membrane occurs independently of the cytoplasmic domains of the MHC-II α and β chains.
Visualizing myosin's power stroke in muscle contraction
Authors: Reedy M.C.
Published: April 2021

The long-standing swinging crossbridge or lever arm hypothesis for the motor action of myosin heads finds support in recent results from 3-D tomograms of insect flight muscle (IFM) fast frozen during active contraction and from both fluorescence polarization and X-ray diffraction during rapid stretches or releases of isometrically contracting fibers. The latter provide direct evidence for lever arm movements synchronous with force changes. Rebuilding the atomic model of nucleotide-free subfragment 1 (S1) to fit fast-frozen, active IFM crossbridges suggests a two-stage power stroke in which the catalytic domain rolls on actin from weak to strong binding; this is followed by a 5-nm lever arm swing of the light chain domain, which gives a total interaction distance of approx. 12 nm. Comparison of S1 crystal structures with in situ myosin heads suggests that actin binding may be necessary in order to view the full repertoire of myosin motor action. The differing positions of the catalytic domains of actin-attached myosin heads in contracting IFM suggest that both the actin-myosin binding energy and the hydrolysis of ATP may be used to cock the crossbridge and drive the power stroke.
Integrin cytoplasmic domain-binding proteins
Authors: Liu Shouchun , Calderwood David A. , Ginsberg Mark H.
Published: April 2021

Integrins are a large family of cell surface receptors that mediate cell adhesion and influence migration, signal transduction, and gene expression. The cytoplasmic domains of integrins play a pivotal role in these integrin-mediated cellular functions. Through interaction with the cytoskeleton, signaling molecules, and other cellular proteins, integrin cytoplasmic domains transduce signals from both the outside and inside of the cell and regulate integrin-mediated biological functions. Identification and functional analyses of integrin cytoplasmic domain-binding proteins have been pursued intensively. In recent years, more cellular proteins have been reported to directly interact with integrin cytoplasmic domains and some of these interactions may play important roles in integrin-mediated biological responses. Integrin β chains, for example, interact with actin-binding proteins (e.g. talin and filamin), which form mechanical links to the cytoskeleton. These and other proteins (e.g. FAK, ILK and novel proteins such as TAP20) might also link integrins to signaling mechanisms and, in some cases (e.g. JAB1) mediate integrin-dependent gene regulation.
The apoptosis mediator mDAP-3 is a novel member of a conserved family of mitochondrial proteins
Authors: Berger T. , Brigl M. , Herrmann J.M. , Vielhauer V. , Luckow B. , Schlondorff D. , Kretzler M.
Published: April 2021

Programmed cell death is essential for organ development and regeneration. To identify molecules relevant for this process, full length cDNA cloning of a short, developmentally regulated murine cDNA fragment, MERM-3, was performed and showed a 1.7 kb mRNA encoding a 45 kDa protein with an ATP/GTP binding motive (P-loop). Sequence analysis revealed an 82% amino acid identity to the human death associated protein 3 (hDAP-3), a positive mediator of apoptosis. The full length sequence being the murine orthologue of hDAP-3 is therefore referred to as mDAP-3. In situ hybridization and northern blot analysis showed an abundant mRNA expression with a pronounced expression in highly proliferative epithelial compartments. For mDAP-3, cytochrome c release and induction of cell death could be demonstrated by overexpression of a mDAP-3/EGFP fusion protein. DAP-3 mediated apoptosis was shown to depend on a functional P-loop. Intracellular localization studies using the mDAP-3/EGFP fusion protein, cell fractionation and protease protection experiments localized mDAP-3 to the mitochondrial matrix. DAP-3, in contrast to cytochrome c, retained its mitochondrial localization during apoptosis induction. A mutant of a putative yeast orthologue of mDAP-3, YGL129c, here referred to as yDAP-3, has been shown to exhibit disrupted mitochondrial function. yDAP-3 deficient mutants could be shown to progressively loose mitochondrial DNA. Loss of mitochondrial DNA in yDAP-3 was partially prevented by transfection of the yDAP-3 deficient mutant with mDAP-3, indicating functional complementation by murine DAP-3 in the yeast system. These data identify mDAP-3 as one of the first proapoptotic factors in the mitochondrial matrix and provide evidence for a critical, evolutionary conserved role of members of the DAP-3 protein family for mitochondrial biogenesis.
Regulation of the formation of tumor cell pseudopodia by the Na+/H+ exchanger NHE1
Authors: Lagana A. , Vadnais J. , Le P. U. , Nguyen T. N. , Laprade R. , Nabi I. R. , Noël J.
Published: April 2021

The Na+/H+ exchanger NHE1 is involved in intracellular pH homeostasis and cell volume regulation and accumulates with actin in the lamellipodia of fibroblasts. In order to determine the role of NHE1 following epithelial transformation and the acquisition of motile and invasive properties, we studied NHE1 expression in polarized MDCK cells, Moloney Sarcoma virus (MSV) transformed MDCK cells and an invasive MSV-MDCK cell variant (MSV-MDCK-INV). Expression of NHE1 was significantly increased in MSV-MDCK-INV cells relative to MSV-MDCK and MDCK cells. NHE1 was localized with β-actin to the tips of MSV-MDCK-INV cell pseudopodia by immunofluorescence. Sensitivity of NHE1-mediated 22Na uptake to ethylisopropylamiloride, a specific inhibitor of NHE1, was increased in MSV-MDCK cells relative to MDCK cells. Changes in intracellular pH induced upon EIPA treatment were also of higher magnitude in MSV-MDCK and MSV-MDCK-INV cells compared to wild-type MDCK cells, especially in Hepes-buffered DMEM medium. Inhibition of NHE1 by 50 μM ethylisopropylamiloride induced the disassembly of actin stress fibers and redistribution of the actin cytoskeleton in all cell types. However, in MSV-MDCK-INV cells, the effect of ethylisopropylamiloride treatment was more pronounced and associated with the increased reversible detachment of the cells from the substrate. Videomicroscopy of MSV-MDCK-INV cells revealed that within 20 minutes of addition, ethylisopropylamiloride induced pseudopodial retraction and inhibited cell motility. The ability of ethylisopropylamiloride to prevent nocodazole-induced formation of actin stress fibers in MSV-MDCK cells was more pronounced in Hepes medium relative to NaHCO3 medium, showing that NHE1 can regulate actin stress fiber assembly in transformed MSV-MDCK cells via its intracellular pH regulatory effect. These results implicate NHE1 in the regulation of the actin cytoskeleton dynamics necessary for the adhesion and pseudopodial protrusion of motile, invasive tumor cells.
Real time imaging reveals a peroxisomal reticulum in living cells
Authors: Schrader Michael , King Stephen J. , Stroh Tina A. , Schroer Trina A.
Published: April 2021

We have directly imaged the dynamic behavior of a variety of morphologically different peroxisomal structures in HepG2 and COS-7 cells transfected with a construct encoding GFP bearing the C-terminal peroxisomal targeting signal 1. Real time imaging revealed that moving peroxisomes interacted with each other and were engaged in transient contacts, and at higher magnification, tubular peroxisomes appeared to form a peroxisomal reticulum. Local remodeling of these structures could be observed involving the formation and detachment of tubular processes that interconnected adjacent organelles. Inhibition of cytoplasmic dynein based motility by overexpression of the dynactin subunit, dynamitin (p50), inhibited the movement of peroxisomes in vivo and interfered with the reestablishment of a uniform distribution of peroxisomes after recovery from nocodazole treatment. Isolated peroxisomes moved in vitro along microtubules in the presence of a microtubule motor fraction. Our data reveal that peroxisomal behavior in vivo is significantly more dynamic and interactive than previously thought and suggest a role for the dynein/dynactin motor in peroxisome motility.
Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover
Authors: Ren Xiang-Dong , Kiosses William B. , Sieg David J. , Otey Carol A. , Schlaepfer David D. , Schwartz Martin Alexander
Published: April 2021

Focal adhesion kinase (FAK) is activated and localized at focal adhesions upon cell adhesion to extracellular matrices. Cells lacking FAK show increased focal adhesion number and decreased cell migration, functions that are regulated by the small GTPase Rho. We now report that fibroblasts from FAK−/− mice failed to transiently inhibit Rho activity when plated on fibronectin. Re-expression of FAK restored normal Rho regulation. Turnover of focal adhesions correlated inversely with Rho activity. The presence or absence of FAK was mimicked by inhibiting or activating Rho, respectively. These data suggest that loss of FAK resulting in constitutive activation of Rho and inhibition of focal adhesion turnover can account for deficiencies in cell migration and embryonic lethality of the FAK knockout.
IRE1 and efferent signaling from the endoplasmic reticulum
Authors: Urano Fumihiko , Bertolotti Anne , Ron David
Published: April 2021

Genetic analysis of the cellular adaptation to malfolded proteins in the endoplasmic reticulum (the unfolded protein response – UPR) has revealed a novel signaling pathway initiated by activation of IRE1, an ER-resident protein kinase and endonuclease. In yeast, Ire1p activates gene expression by promoting a non-conventional splicing event that converts the mRNA encoding the Hac1p transcription factor from an inefficiently translated inactive mRNA to an actively translated one. Hac1p binds to the promoters of genes encoding chaperones and other targets of the UPR and activates them. Recently, mammalian IRE1 homologues have been identified and their response to ER stress is regulated by binding to the ER chaperone BiP. The mechanisms by which mammalian IRE1 activates gene expression have not been completely characterized and mammalian HAC1 homologues have not been identified. Surprisingly, mammalian IRE1s are able to activate both JUN N-terminal kinases and an alternative ER-stress signaling pathway mediated by the transcription factor ATF6. This indicates that the mammalian UPR is more complex than that found in yeast.
The localized assembly of extracellular matrix integrin ligands requires cell-cell contact
Authors: Martin-Bermudo Maria D. , Brown Nicholas H.
Published: April 2021

The assembly of an organism requires the interaction between different layers of cells, in many cases via an extracellular matrix. In the developing Drosophila larva, muscles attach in an integrin-dependent manner to the epidermis, via a specialized extracellular matrix called tendon matrix. Tiggrin, a tendon matrix integrin ligand, is primarily synthesized by cells distant to the muscle attachment sites, yet it accumulates specifically at these sites. Previous work has shown that the PS integrins are not required for tiggrin localization, suggesting that there is redundancy among tiggrin receptors. We have examined this by testing whether the PS2 integrin can recruit tiggrin to ectopic locations within the Drosophila embryo. We found that neither the wild type nor modified forms of the PS2 integrin, which have higher affinity for tiggrin, can recruit tiggrin to new cellular contexts. Next, we genetically manipulated the fate of the muscles and the epidermal muscle attachment cells, which demonstrated that muscles have the primary role in recruiting tiggrin to the tendon matrix and that cell-cell contact is necessary for this recruitment. Thus we propose that the inherent polarity of the muscle cells leads to a molecular specialization of their ends, and interactions between the ends produces an integrin-independent tiggrin receptor. Thus, interaction between cells generates an extracellular environment capable of nucleating extracellular matrix assembly.
Polarization of cell growth in yeast: I. Establishment and maintenance of polarity states
Authors: Pruyne David , Bretscher Anthony
Published: April 2021

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

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

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

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

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

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.