bluuebunny/crossref_metadata_2025 huggingface dataset:
Translocation of cortactin to the cell periphery is mediated by the small GTPase Rac1
Authors: Weed Scott A. , Du Yunrui , Parsons J. Thomas
Published: April 2021

Small GTPases of the Rho family regulate signaling pathways that control actin cytoskeletal structures. In Swiss 3T3 cells, RhoA activation leads to stress fiber and focal adhesion formation, Rac1 to lamellipoda and membrane ruffles, and Cdc42 to microspikes and filopodia. Several downstream molecules mediating these effects have been recently identified. In this report we provide evidence that the intracellular localization of the actin binding protein cortactin, a Src kinase substrate, is regulated by the activation of Rac1. Cortactin redistributes from the cytoplasm into membrane ruffles as a result of growth factor-induced Rac1 activation, and this translocation is blocked by expression of dominant negative Rac1N17. Expression of constitutively active Rac1L61 evoked the translocation of cortactin from cytoplasmic pools into peripheral membrane ruffles. Expression of mutant forms of the serine/threonine kinase PAK1, a downstream effector of Rac1 and Cdc42 recently demonstrated to trigger cortical actin polymerization and membrane ruffling, also led to the translocation of cortactin to the cell cortex, although this was effectively blocked by coexpression of Rac1N17. Collectively these data provide evidence for cortactin as a putative target of Rac1-induced signal transduction events involved in membrane ruffling and lamellipodia formation.
The cdc25B phosphatase is essential for the G 2/M phase transition in human cells
Authors: Lammer1 Christiane , Wagerer Sybille , Saffrich Rainer , Mertens Daniel , Ansorge Wilhelm , Hoffmann Ingrid
Published: April 2021

Cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases. In human cells, cdc25 proteins are encoded by a multigene family, consisting of cdc25A, cdc25B and cdc25C. While cdc25A plays a crucial role at the G 1/S phase transition, cdc25C is involved in the dephosphorylation and activation of the mitotic kinase, cdc2/cyclinB. In addition, cdc25C itself is regulated by cdc2/cyclinB which then creates a positive feedback loop that controls entry into mitosis. In this study we show that the activity of cdc25B appears during late S phase and peaks during G 2 phase. Both in vitro and in vivo cdc25B is activated through phosphorylation during S-phase. Using a cell duplication, microinjection assay we show that ablation of cdc25B function by specific antibodies blocks cell cycle progression in Hs68 cells by inhibition of entry into mitosis. Cdc25B function neither plays a role in later stages of mitosis nor for the inititation of DNA replication. These results indicate that cdc25B is a mitotic regulator that might act as a ‘starter phosphatase’ to initiate the positive feedback loop at the entry into M phase.
Escape of mitochondrial DNA to the nucleus in ymel yeast is mediated by vacuolar-dependent turnover of abnormal mitochondrial compartments
Authors: Campbell Corey L. , Thorsness Peter E.
Published: April 2021

Inactivation of Ymelp, a mitochondrially-localized ATP-dependent metallo-protease in the yeast Saccharomyces cerevisiae, causes a high rate of DNA escape from mitochondria to the nucleus as well as pleiotropic functional and morphological mitochondrial defects. The evidence presented here suggests that the abnormal mitochondria of a ymel strain are degraded by the vacuole. First, electron microscopy of Yme1p-deficient strains revealed mitochondria physically associated with the vacuole via electron dense structures. Second, disruption of vacuolar function affected the frequency of mitochondrial DNA escape from ymel and wild-type strains. Both PEP4 or PRC1 gene disruptions resulted in a lower frequency of mitochondrial DNA escape. Third, an in vivo assay that monitors vacuole-dependent turnover of the mitochondrial compartment demonstrated an increased rate of mitochondrial turnover in ymel yeast when compared to the rate found in wild-type yeast. In this assay, vacuolar alkaline phosphatase, encoded by PHO8, was targeted to mitochondria in a strain bearing disruption to the genomic PHO8 locus. Maturation of the mitochondrially localized alkaline phosphatase pro-enzyme requires proteinase A, which is localized in the vacuole. Therefore, alkaline phosphatase activity reflects vacuole-dependent turnover of mitochondria. This assay reveals that mitochondria of a ymel strain are taken up by the vacuole more frequently than mitochondria of an isogenic wild-type strain when these yeast are cultured in medium necessitating respiratory growth. Degradation of abnormal mitochondria is one pathway by which mitochondrial DNA escapes and migrates to the nucleus.
MAP kinase activitys increases during mitosis in early sea urchin embryos
Authors: Philipova Rada , Whitaker Michael
Published: April 2021

A MBP kinase activity increases at mitosis during the first two embryonic cell cycles of the sea urchin embryo. The activity profile of the MBP kinase is the same both in whole cell extracts and after immunoprecipitation with an anti-MAP kinase antibody (2199). An in-gel assay of MBP activity also shows the same activity profile. The activity is associated with the 44 kDa protein that cross-reacts with anti-MAP kinase antibodies. The 44 kDa protein shows cross-reactivity to anti-phosphotyrosine and MAP kinase-directed anti-phosphotyrosine/phosphothreonine antibodies at the times that MBP kinase activity is high. The 2199 antibody co-precipitates some histone H1 kinase activity, but the MBP kinase activity cannot be accounted for by histone H1 kinase-dependent phosphorylation of MBP. The MAP kinase 2199 antibody was used to purify the MBP kinase activity. Peptide sequencing after partial digestion shows the protein to be homologous to MAP kinases from other species. These data demonstrate that MAP kinase activation during nuclear division is not confined to meiosis, but also occurs during mitotic cell cycles. MAP kinase activity in immunoprecipitates also increases immediately after fertilization, which in the sea urchin egg occurs at interphase of the cell cycle. Treating unfertilized eggs with the calcium ionophore A23187 stimulates the increase in MAP kinase activity, demonstrating that a calcium signal can activate MAP kinase and suggesting that the activation of MAP kinase at fertilization is due to the fertilization-induced increase in cytoplasmic free calcium concentration. This signalling pathway must differ from the pathway responsible for calcium-induced inactivation of MAP kinase activity that is found in eggs that are fertilized in meiotic metaphase.
Evidence for MAP kinase activation during mitotic division
Authors: Céline De Nadai Sandrine Chiri , Ciapa Brigitte
Published: April 2021

MAP kinases have been implicated in the control of a broad spectrum of cellular events in many types of cells. In somatic cells, MAP kinase activation seems to be triggered after exit from a quiescent state (in G0 or G2) only and then inactivated by entry into a proliferative state. In oocytes of various species, a one-time activation of MAP kinase that is apparently not repeated during the succeeding mitotic cycles occurs after meiotic activation. However, several reports suggest that a myelin basic protein (MBP) kinase activity, unrelated to that of maturation promoting factor, can sometimes be detected during mitotic divisions in various types of cells and oocytes. We have reinvestigated this problem in order to determine the origin and the role of MBP kinase that is stimulated at time of mitosis in the fertilized eggs of the sea urchin Paracentrotus lividus. We used anti-ERK1 antibodies or substrates specific for different MAP kinases, and performed in-gel phosphorylation experiments. Our results suggest that an ERK1-like protein was responsible for part of the MBP kinase activity that is stimulated during the first mitotic divisions. Furthermore, we observed that wortmannin, an inhibitor of PI 3-kinase that arrests the fertilized sea urchin eggs at the prometaphase stage, inhibited the inactivation of MAP kinase normally observed when the eggs divide, suggesting a role for PI 3-kinase in the deactivation process of MAP kinase. We also discuss how the activities of MPF and MAP kinase may be interconnected to regulate the first mitotic divisions of the early sea urchin embryo.
Characterisation of α-dystrobrevin in muscle
Authors: Nawrotzki Ralph , Loh Nellie Y. , Ruegg Markus A. , Davies Kay E. , Blake Derek J.
Published: April 2021

Dystrophin-related and associated proteins are important for the formation and maintenance of the mammalian neuromuscular junction. Initial studies in the electric organ of Torpedo californica showed that the dystrophin-related protein dystrobrevin (87K) co-purifies with the acetylcholine receptors and other postsynaptic proteins. Dystrobrevin is also a major phosphotyrosine-containing protein in the postsynaptic membrane. Since inhibitors of tyrosine protein phosphorylation block acetylcholine receptor clustering in cultured muscle cells, we examined the role of α-dystrobrevin during synapse formation and in response to agrin. Using specific antibodies, we show that C2 myoblasts and early myotubes only produce α-dystrobrevin-1, the mammalian orthologue of Torpedo dystrobrevin, whereas mature skeletal muscle expresses three distinct α-dystrobrevin isoforms. In myotubes, α-dystrobrevin-1 is found on the cell surface and also in acetylcholine receptor-rich domains. Following agrin stimulation, α-dystrobrevin-1 becomes re-localised beneath the cell surface into macroclusters that contain acetylcholine receptors and another dystrophin-related protein, utrophin. This redistribution is not associated with tyrosine phosphorylation of α-dystrobrevin-1 by agrin. Furthermore, we show that α-dystrobrevin-1 is associated with both utrophin in C2 cells and dystrophin in mature skeletal muscle. Thus α-dystrobrevin-1 is a component of two protein complexes in muscle, one with utrophin at the neuromuscular junction and the other with dystrophin at the sarcolemma. These results indicate that α-dystrobrevin-1 is not involved in the phosphorylation-dependent, early stages of receptor clustering, but rather in the stabilisation and maturation of clusters, possibly via an interaction with utrophin.
A cytoplasmic dynein required for mitotic aster formation in vivo
Authors: Inoue Satoshi , Yoder O. C. , Turgeon B. Gillian , Aist James R.
Published: April 2021

An astral pulling force helps to elongate the mitotic spindle in the filamentous ascomycete, Nectria haematococca. Evidence is mounting that dynein is required for the formation of mitotic spindles and asters. Obviously, this would be an important mitotic function of dynein, since it would be a prerequisite for astral force to be applied to a spindle pole. Missing from the evidence for such a role of dynein in aster formation, however, has been a dynein mutant lacking mitotic asters. To determine whether or not cytoplasmic dynein is involved in mitotic aster formation in N. haematococca, a dynein-deficient mutant was made. Immunocytochemistry visualized few or no mitotic astral microtubules in the mutant cells, and studies of living cells confirmed the veracity of this result by revealing the absence of mitotic aster functions in vivo: intra-astral motility of membranous organelles was not apparent; the rate and extent of spindle elongation during anaphase B were reduced; and spindle pole body separation almost stopped when the anaphase B spindle in the mutant was cut by a laser microbeam, demonstrating unequivocally that no astral pulling force was present. These unique results not only provide a demonstration that cytoplasmic dynein is required for the formation of mitotic asters in N. haematococca; they also represent the first report of mitotic phenotypes in a dynein mutant of any filamentous fungus and the first cytoplasmic dynein mutant of any organism whose mitotic phenotypes demonstrate the requirement of cytoplasmic dynein for aster formation in vivo.
α-SNAP but not γ-SNAP is required for ER-Golgi transport after vesicle budding and the Rab1-requiring step but before the EGTA-sensitive step
Authors: Peter Frank , Wong Siew Heng , Subramaniam V. Nathan , Tang Bor Luen , Hong Wanjin
Published: April 2021

N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) have been implicated in diverse vesicular transport events; yet their exact role and site of action remain to be established. Using an established in vitro system, we show that antibodies against α-SNAP inhibit vesicle transport from the ER to the cis-Golgi and that recombinant α-SNAP enhances/stimulates the process. Cytosol immunodepleted of α-SNAP does not support normal transport unless supplemented with recombinant α-SNAP but not γ-SNAP. In marked contrast, cytosol immunodepleted of γ-SNAP supports ER-Golgi transport to the normal level. Neither antibodies against γ-SNAP nor recombinant γ-SNAP have any effect on ER-Golgi transport. These results clearly establish an essential role for α-SNAP but not γ-SNAP in ER-Golgi transport. When the transport assay is performed with cytosol immunodepleted of α-SNAP, followed by incubation with cytosol immunodepleted of a COPII subunit, normal transport is achieved. In marked contrast, no transport is detected when the assay is first performed with cytosol depleted of the COPII subunit followed by α-SNAP-depleted cytosol, suggesting that α-SNAP is required after a step that requires COPII (the budding step). In combination with cytosol immunodepleted of Rab1, it is seen that α-SNAP is required after a Rab1-requiring step. It has been shown previously that EGTA blocks ER-Golgi transport at a step after vesicle docking but before fusion and we show here that α-SNAP acts before the step that is blocked by EGTA. Our results suggest that α-SNAP may be involved in the pre-docking or docking but not the fusion process.
Suppression of tumorigenicity in an aggressive cervical carcinoma induced by protein zero, a nervous system IgCAM
Authors: Spiryda Lisa B. , Colman David R.
Published: April 2021

In mammals, protein zero (P0), a neural IgCAM, is expressed solely in the peripheral nervous system where it mediates self-adhesion of Schwann cell membranes as compact myelin is generated. We show that when P0 is expressed in HeLa, a cervical carcinoma cell line, cells regain adhesion-mediated growth control, including the acquisition of contact inhibition and loss of anchorage-independent growth. Additionally, P0-expressing HeLa cells lose the ability to invade an artificial matrix, which correlates with decreased secretion of matrix-degrading enzymes. Lastly, and of great interest, unlike the aggressively metastatic cell line from which they were derived, P0-HeLa cells are neither tumorigenic nor metastatic when injected into athymic nude mice. By all these criteria, P0 expression appears to efficiently suppress in the long term, the transformed state of this carcinoma cell line. N-cadherin and its intracellular partners plakoglobin, α- and β-catenin were significantly upregulated in the P0-HeLa cells. It appears therefore that P0 induces epithelialization and suppression of tumorigenicity in HeLa through the activation of the cadherin/catenin signaling systems. We conclude that the forced expression of bona fide adhesion molecules, such as P0, may serve as ‘upstream’ inducers of an essentially dormant but undamaged adhesion program in carcinoma cells that ultimately triggers the re-acquisition of normal epithelial characteristics, thereby suppressing tumorigenicity. Therapeutically, it may be that intercellular adhesion, no matter how it is induced, may serve as a single master event that is able to induce reversion of the carcinomatous state.
DNA replication-dependent intranuclear relocation of double minute chromatin
Authors: Itoh Nobuo , Shimizu Noriaki
Published: April 2021

Double minutes (DMs) seen in a substantial fraction of human tumors are the cytogenetic manifestation of gene amplification which renders the tumor cells advantageous for growth and survival. DMs are acentric and atelomeric chromatin composed of circular DNA. In this study, we found they showed a remarkable relocation inside the nucleus which was spatially and temporally coupled to DNA replication. Using the human COLO 320DM tumor line, we detected DMs by fluorescence in situ hybridization followed by confocal examination. The location of multi-copy DMs was evaluated statistically by an easy method developed in this study. By examination of a synchronized culture, we found that DMs preferentially located at the nuclear periphery during G1-phase of the cell cycle, which is consistent with the location at M-phase. The peripheral DMs were in contact with the nuclear lamina which was shown by the simultaneous detection of DMs and lamin protein. The peripheral location persisted until the cells reached the G1/S-boundary, then the DMs relocated promptly to inward once the DNA replication started. The relocation was obvious using two different probes that detect DMs, or using two different methods for the cell fixation. Furthermore, the simultaneous detection of DMs and the site of DNA replication suggested that the inward relocation of peripheral DMs initiated just prior to the onset of DNA replication at the periphery. On the other hand, if the same amplified sequences were placed in a chromosome as an homogeneously staining region, they did not show any significant relocation during S-phase. From these and reported results, there may exist a generalized inward motion of some kind of chromatin that precedes the replication of their DNA. DMs might magnify the motion by their acentric, atelomeric or small circular nature.
Synaptojanin family members are implicated in endocytic membrane traffic in yeast
Authors: Singer-Krüger Birgit , Nemoto Yasuo , Daniell Laurie , Ferro-Novick Susan , Camilli Pietro De
Published: April 2021

The synaptojanins represent a subfamily of inositol 5’-phosphatases that contain an NH2-terminal Sac1p homology domain. A nerve terminal-enriched synaptojanin, synaptojanin 1, was previously proposed to participate in the endocytosis of synaptic vesicles and actin function. The genome of Saccharomyces cerevisiae contains three synaptojanin-like genes (SJL1, SJL2 and SJL3), none of which is essential for growth. We report here that a yeast mutant lacking SJL1 and SJL2 (Δsjl1 Δsjl2) exhibits a severe defect in receptor-mediated and fluid-phase endocytosis. A less severe endocytic defect is present in a Δsjl2 Δsjl3 mutant, while endocytosis is normal in a Δsjl1 Δsjl3 mutant. None of the mutants are impaired in invertase secretion. The severity of the endocytic impairment of the sjl double mutants correlates with the severity of actin and polarity defects. Furthermore, the deletion of SJL1 suppresses the temperature-sensitive growth defect of sac6, a mutant in yeast fimbrin, supporting a role for synaptojanin family members in actin function. These findings provide a first direct evidence for a role of synaptojanin family members in endocytosis and provide further evidence for a close link between endocytosis and actin function.
The recycling pathway of protein ERGIC-53 and dynamics of the ER-Golgi intermediate compartment
Authors: Klumperman Judith , Schweizer Anja , Clausen Henrik , Tang Bor Luen , Hong Wanjin , Oorschot Viola , Hauri Hans-Peter
Published: April 2021

To establish recycling routes in the early secretory pathway we have studied the recycling of the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53 in HepG2 cells. Immunofluorescence microscopy showed progressive concentration of ERGIC-53 in the Golgi area at 15°C. Upon rewarming to 37°C ERGIC-53 redistributed into the cell periphery often via tubular processes that largely excluded anterograde transported albumin. Immunogold labeling of cells cultured at 37°C revealed ERGIC-53 predominantly in characteristic β-COP-positive tubulo-vesicular clusters both near the Golgi apparatus and in the cell periphery. Concentration of ERGIC-53 at 15°C resulted from both accumulation of ERGIC-53 in the ERGIC and movement of ERGIC membranes closer to the Golgi apparatus. Upon rewarming to 37°C the labeling of ERGIC-53 in the ERGIC rapidly returned to normal levels whereas ERGIC-53’s labeling in the cis-Golgi was unchanged. Temperature manipulations had no effect on the average number of ERGIC-53 clusters. Density gradient centrifugation indicated that the surplus ERGIC-53 accumulating in the ERGIC at 15°C was rapidly transported to the ER upon rewarming. These results suggest that the ERGIC is a dynamic membrane system composed of a constant average number of clusters and that the major recycling pathway of ERGIC-53 bypasses the Golgi apparatus.
The role of sequences unique to nuclear intermediate filaments in the targeting and assembly of human lamin B: evidence for lack of interaction of lamin B with its putative receptor
Authors: Mical Timothy I. , Monteiro Mervyn J.
Published: April 2021

The mechanism by which human nuclear lamin B is targeted and assembled has been studied by transfecting into mammalian cells lamin mutants deleted of three sequences unique to lamins. Nuclear lamins contain an extra 42 amino acids (aa) in their rod domains and NLS and CAAX motifs in their tail domains, which distinguishes them from cytoplasmic IF proteins. These three sequences act in concert to ensure correct temporal and spatial assembly of lamin B. Deletion of any one of these three sequences from lamin B did not significantly disrupt nuclear lamina targeting, but when two or more of these sequences were deleted, targeting was severely compromised. The CAAX motif is necessary for the efficient integration of lamin B into an already formed nuclear lamina, since lamin B CAAX–mutants had reduced targeting to the lamina when arrested in S phase of the cell cycle. CAAX-deficient mutant lamin B proteins were soluble and not associated with membranes at mitosis, proving that the CAAX motif is responsible for association of human lamin B with membranes. In addition, CAAX–mutant lamin B proteins fractionated independently of the lamin B-receptor (LBR), indicating that these two proteins do not bind directly to each other.
Maize meiotic spindles assemble around chromatin and do not require paired chromosomes
Authors: Chan Annette , Cande W. Zacheus
Published: April 2021

To understand how the meiotic spindle is formed and maintained in higher plants, we studied the organization of microtubule arrays in wild-type maize meiocytes and three maize meiotic mutants, desynaptic1 (dsy1), desynaptic2 (dsy2), and absence of first division (afd). All three meiotic mutations have abnormal chromosome pairing and produce univalents by diakinesis. Using these three mutants, we investigated how the absence of paired homologous chromosomes affects the assembly and maintenance of the meiotic spindle. Before nuclear envelope breakdown, in wild-type meiocytes, there were no bipolar microtubule arrays. Instead, these structures formed after nuclear envelope breakdown and were associated with the chromosomes. The presence of univalent chromosomes in dsy1, dsy2, and afd meiocytes and of unpaired sister chromatids in the afd meiocytes did not affect the formation of bipolar spindles. However, alignment of chromosomes on the metaphase plate and subsequent anaphase chromosome segregation were perturbed. We propose a model for spindle formation in maize meiocytes in which microtubules initially appear around the chromosomes during prometaphase and then the microtubules self-organize. However, this process does not require paired kinetochores to establish spindle bipolarity.
Deficient epithelial-fibroblast heterocellular gap junction communication can be overcome by co-culture with an intermediate cell type but not by E-cadherin transgene expression
Authors: Woodward T. L. , Sia M. A. , Blaschuk O. W. , Turner J. D. , Laird D. W.
Published: April 2021

Epithelial, fibroblast and intermediate cell lines were employed to examine the mechanism(s) essential for heterocellular gap junction intercellular communication in vitro. These cell lines were characterized extensively for cell type based on morphology, intermediate cytoskeletal proteins, cell adhesion molecules and their associated proteins, tight junction proteins as well as functional differentiation. All cell types expressed connexin43 and were dye-coupled in homocellular culture. Epithelial and intermediate cells or fibroblasts and intermediate cells readily assembled heterocellular connexin43-positive gap junction plaques when co-cultured, while gap junction plaques in mixed cultures of epithelial cells and fibroblasts were rare. Dye microinjection studies were used to show that there was little gap junction intercellular communication between epithelial cells and fibroblasts. However, intermediate cells were able to communicate with epithelial cells and, to a lesser extent, fibroblasts and could transfer dye to both epithelial cells and fibroblasts when all three cell types were cultured together. Fibroblasts that were stably transfected with a cDNA encoding E-cadherin had a greater tendency to aggregate and exhibited a more epithelial-like phenotype but heterocellular gap junction intercellular communication with epithelial cells, which endogenously express E-cadherin, was not enhanced. These results suggest that mutual expression of E-cadherin is insufficient to stimulate gap junction formation between epithelial cells and fibroblasts. Moreover, our results also demonstrate that communication gaps between epithelial cells and fibroblasts can be bridged by intermediate cells, a process that may be important in mammary gland development, growth, differentiation and cancer.
Expression and potential role of Fsrg1, a murine bromodomain-containing homologue of the Drosophila gene female sterile homeotic
Authors: Rhee Kunsoo , Brunori Michele , Besset Valérie , Trousdale Rhonda , Wolgemuth Debra J.
Published: April 2021

We have isolated a cDNA which is a murine homologue of the Drosophila gene female sterile homeotic (fsh). This homologue, which we have designated Fsrg1*, contains two bromodomains and an ET motif characteristic of the Fsh sub-class of bromodomain-containing proteins. Northern blot hybridization analysis of adult tissues revealed that Fsrg1 was expressed at low levels rather ubiquitously, but most abundantly in the testis and ovary. Polyclonal antibodies raised against an Fsrg1 fusion protein were used to characterize the Fsrg1 gene product in tissues. Constructs were also generated in which the Fsrg1 cDNA was tagged with epitopes for hemaglutinin and used in transfection experiments. Immunoblot analysis revealed that the Fsrg1 protein migrates with a relative molecular mass of ˜110 kDa, although the cDNA sequence would predict a protein of ˜88 kDa. The migration at ˜110 kDa was observed for both in vivo protein and protein produced in cultured cells. The Fsrg1 protein was localized to the nucleus when expressed in cultured cells, consistent with the presence of a nuclear localization signal motif in the Fsrg1 sequence. No kinase activity was detected for this nuclear protein as assessed in either autokinase or specific substrate assays. In situ hybridization analysis revealed strikingly high expression of Fsrg1 in granulosa cells of growing follicles in the adult ovary and suggested its possible involvement in folliculogenesis. Additional clues to its potential function were provided by the demonstration of its high level of expression in epithelia of tissues which undergo hormonally-modulated remodeling.
Localization and regulation of the cdk-activating kinase (Cak1p) from budding yeast
Authors: Kaldis Philipp , Pitluk Zachary W. , Bany I. Amy , Enke Deborah A. , Wagner Marisa , Winter Edward , Solomon Mark J.
Published: April 2021

Eukaryotic cell cycles are controlled by the activities of cyclin-dependent kinases (cdks). The major cdk in budding yeast, Saccharomyces cerevisiae, is Cdc28p. Activation of Cdc28p requires phosphorylation on threonine 169 and binding to a cyclin. Thr-169 is phosphorylated by the cdkactivating kinase (CAK), Cak1p, which was recently identified as the physiological CAK in budding yeast. Here we present our further characterization of yeast Cak1p. We have found that Cak1p is dispersed throughout the cell as shown by immunofluorescence; biochemical subcellular fractionation confirmed that most of the Cak1p is found in the cytoplasm. Cak1p is a monomeric enzyme in crude yeast lysates. Mutagenesis of potential sites of activating phosphorylation had little effect on the activity of Cak1p in vitro or in vivo. Furthermore, Cak1p contains no posttranslational modifications detectable by two-dimensional isoelectric focusing. We found that Cak1p is a stable protein during exponential growth but that its expression decreases considerably when cells enter stationary phase. In contrast, Cak1p levels oscillate dramatically during meiosis, reflecting regulation at both the transcriptional and post-translational level. The localization and regulation of Cak1p are in contrast to those of the known vertebrate CAK, p40MO15.
Nucleoskeleton and initiation of DNA replication in metazoan cells
Authors: Ortega J. Miguel , DePamphilis Melvin L.
Published: April 2021

To determine whether or not initiation sites for DNA replication in mammalian cells are defined by association with nuclear structure, attachments between the nucleoskeleton and the hamster DHFR gene initiation zone were examined. Nucleoskeletons were prepared by encapsulating cells in agarose and then extracting them with a nonionic detergent in a physiological buffer. The fraction of DNA that remained following endonuclease digestion was resistant to salt, sensitive to Sarkosyl, and essentially unchanged by glutaraldehyde crosslinking. Although newly replicated DNA was preferentially attached to the nucleoskeleton, no specific sequence was preferentially attached within a 65 kb locus containing the DHFR gene, two origins of bi-directional replication and at least one nuclear matrix attachment region. Instead, the entire region went from preferentially unattached to preferentially attached as cells progressed from G1 to late S-phase. Thus, initiation sites in mammalian chromosomes are not defined by attachments to the nucleoskeleton. To further assess the relationship between the nucleoskeleton and DNA replication, plasmid DNA containing the DHFR initiation region was replicated in a Xenopus egg extract. All of the DNA associated with the nucleoskeleton prior to S-phase without preference for a particular sequence and was released upon mitosis. However, about half of this DNA was trapped rather than bound to the nucleoskeleton. Thus, attachments to the nucleoskeleton can form in the absence of either DNA replication or transcription, but if they are required for replication, they are not maintained once replication is completed.
Chromatin binding and polymerization of the endogenous Xenopus egg lamins: The opposing effects of glycogen and ATP
Authors: Lourim David , Krohne Georg
Published: April 2021

We have previously identified and quantitated three B-type lamin isoforms present in the nuclei of mature Xenopus laevis oocytes, and in cell-free egg extracts. As Xenopus egg extracts are frequently used to analyze nuclear envelope assembly and lamina functions, we felt it was imperative that the polymerization and chromatin-binding properties of the endogenous B-type egg lamins be investigated. While we have demonstrated that soluble B-type lamins bind to chromatin, we have also observed that the polymerization of egg lamins does not require membranes or chromatin. Lamin assembly is enhanced by the addition of glycogen/glucose, or by the depletion of ATP from the extract. Moreover, the polymerization of egg cytosol lamins and their binding to demembranated sperm or chromatin assembled from naked λ-DNA is inhibited by an ATP regeneration system. These ATP-dependent inhibitory activities can be overcome by the coaddition of glycogen to egg cytosol. We have observed that glycogen does not alter ATP levels during cytosol incubation, but rather, as glycogen-enhanced lamin polymerization is inhibited by okadaic acid, we conclude that glycogen activates protein phosphatases. Because protein phosphatase 1 (PP1) is the only phosphatase known to be specifically regulated by glycogen our data indicate that PP1 is involved in lamin polymerization. Our results show that ATP and glycogen effect lamin polymerization and chromatin binding by separate and opposing mechanisms.
A role for microtubule dynamics in phagosome movement
Authors: Blocker Ariel , Griffiths Gareth , Olivo Jean-Christophe , Hyman Anthony A. , Severin Fedor F.
Published: April 2021

We have shown previously that intracellular phagosome movement requires microtubules. Here we provide evidence that within cells phagosomes display two different kinds of microtubule-based movements in approximately equal proportions. The first type occurs predominantly in the cell periphery, often shortly after the phagosome is formed, and at speeds below 0.1 µm/second. The second is faster (0.2-1.5 µm/second) and occurs mainly after phagosomes have reached the cell interior. Treating cells with nanomolar concentrations of taxol or nocodazole alters microtubule dynamics without affecting either total polymer mass or microtubule organisation. Such treatments slow the accumulation of phagosomes in the perinuclear region and reduce the number of slow movements by up to 50% without affecting the frequency of fast movements. This suggests that a proportion of slow movements are mediated by microtubule dynamics while fast movements are powered by microtubule motors. In macrophages, interphase microtubules radiate from the microtubule organising centre with their plus-end towards the cell periphery. To understand the behaviour of ‘early’ phagosomes at the cell periphery we investigated their ability to bind microtubule plus-ends in vitro. We show that early phagosomes have a strong preference for microtubule plus-ends, whereas ‘late’ phagosomes do not, and that plus-end affinity requires the presence of microtubule-associated proteins within cytosol. We suggest that phagosomes can bind to the plus-ends of dynamic microtubules and move by following their shrinkage or growth.
Long-range and selective autoregulation of cell-cell or cell-matrix adhesions by cadherin or integrin ligands
Authors: Levenberg Shulamit , Katz Ben-Zion , Yamada Kenneth M. , Geiger Benjamin
Published: April 2021

In this study we demonstrate that local stimulation of cell surface cadherins or integrins induces a selective enhancement of adherens junction or focal contact assembly, respectively, throughout the cell. N-cadherin transfected CHO cells (CHO-Ncad) were incubated with different ligands including N-cadherin extracellular domain (NEC), anti-N-cadherin antibodies, fibronectin and concanavalin A (ConA), conjugated to synthetic beads. Electron microscopic examination indicated that both cadherin- and integrin-reactive beads bound tightly to the cell surface and were apparently endocytosed after several hours of incubation. The ConA-beads remained largely at the cell surface. Immunofluorescence labeling of the cells with antibodies to different adhesion-associated molecules indicated that both NEC- and anti-N-cadherin-conjugated beads induced a major increase in the level of junction-associated cadherin and β-catenin labeling and a modest increase in junctional vinculin labeling, compared to untreated cells or cells bound to ConA-beads. FN-conjugated beads, on the other hand, significantly enhanced vinculin labeling at focal contacts and suppressed cadherin and β-catenin staining in cell-cell junctions. The cadherin-reactive beads specifically stimulated tyrosine phosphorylation at cell-cell junctions, while the FN-beads increased the levels of focal contact-associated phosphotyrosine, as shown by immunofluorescence labeling of the cells for phosphotyrosine. Inhibition of this phosphorylation by genistein resulted in a complete suppression of the effects of both types of beads. These findings indicate that specific cadherin- and integrin-mediated surface interactions can trigger positively cooperative long-range signaling events which lead to the selective assembly of cell-cell or cell-matrix adhesions, and that these signals involve tyrosine phosphorylation.
The spa2-related protein, sph1p, is important for polarized growth in yeast
Authors: Roemer Terry , Vallier Laura , Sheu Yi-Jun , Snyder Michael
Published: April 2021

The Saccharomyces cerevisiae protein Sph1p is both structurally and functionally related to the polarity protein, Spa2p. Sph1p and Spa2p are predicted to share three 100-amino acid domains each exceeding 30% sequence identity, and the amino-terminal domain of each protein contains a direct repeat common to Homo sapiens and Caenorhabditis elegans protein sequences. sph1- and spa2-deleted cells possess defects in mating projection morphology and pseudohyphal growth. sph1Δ spa2Δ double mutants also exhibit a strong haploid invasive growth defect and an exacerbated mating projection defect relative to either sph1Δ or spa2Δ single mutants. Consistent with a role in polarized growth, Sph1p localizes to growth sites in a cell cycle-dependent manner: Sph1p concentrates as a cortical patch at the presumptive bud site in unbudded cells, at the tip of small, medium and large buds, and at the bud neck prior to cytokinesis. In pheromone-treated cells, Sph1p localizes to the tip of the mating projection. Proper localization of Sph1p to sites of active growth during budding and mating requires Spa2p. Sph1p interacts in the two-hybrid system with three mitogen-activated protein (MAP) kinase kinases (MAPKKs): Mkk1p and Mkk2p, which function in the cell wall integrity/cell polarization MAP kinase pathway, and Ste7p, which operates in the pheromone and pseudohyphal signaling response pathways. Sph1p also interacts weakly with STE11, the MAPKKK known to activate STE7. Moreover, two-hybrid interactions between SPH1 and STE7 and STE11 occur independently of STE5, a proposed scaffolding protein which interacts with several members of this MAP kinase module. We speculate that Spa2p and Sph1p may function during pseudohyphal and haploid invasive growth to help tether this MAP kinase module to sites of polarized growth. Our results indicate that Spa2p and Sph1p comprise two related proteins important for the control of cell morphogenesis in yeast.
Coexpression of both types of desmosomal cadherin and plakoglobin confers strong intercellular adhesion
Authors: Marcozzi Cristiana , Burdett Ian D. J. , Buxton Roger S. , Magee Anthony I.
Published: April 2021

Desmosomes are unique intercellular junctions in that they invariably contain two types of transmembrane cadherin molecule, desmocollins and desmogleins. In addition they possess a distinct cytoplasmic plaque structure containing a few major proteins including desmoplakins and the armadillo family member plakoglobin. Desmosomal cadherins are putative cell-cell adhesion molecules and we have tested their adhesive capacity using a transfection approach in mouse L cells. We find that L cells expressing either one or both of the desmosomal cadherins desmocollin 2a or desmoglein 1 display weak cell-cell adhesion activity that is Ca2+-dependent. Both homophilic and heterophilic adhesion could be detected. However, co-expression of plakoglobin with both desmosomal cadherins, but not with desmoglein 1 alone, resulted in a dramatic potentiation of cell-cell aggregation and the accumulation of detergent-insoluble desmosomal proteins at points of cell-cell contact. The effect of plakoglobin seems to be due directly to its interaction with the desmosomal cadherins rather than to its signalling function. The data suggest that the desmosome may obligatorily contain two cadherins and is consistent with a model in which desmocollins and desmogleins may form side by side heterodimers in contrast to the classical cadherins that are homodimeric. Plakoglobin may function by potentiating dimer formation, accretion of dimers to cell-cell contact sites or desmosomal cadherin stability.
Tight junctions
Authors: Balda Maria S. , Matter Karl
Published: April 2021

Tight junctions are the most apical intercellular junctions of epithelial and endothelial cells and create a regulatable semipermeable diffusion barrier between individual cells. On a cellular level, they form an intramembrane diffusion fence that restricts the intermixing of apical and basolateral membrane components. In addition to these well defined functions, more recent evidence suggests that tight junctions are also involved in basic cellular processes like the regulation of cell growth and differentiation.
Activation of a PP2A-like phosphatase and dephosphorylation of τ protein characterize onset of the execution phase of apoptosis
Authors: Mills Jason C. , Lee Virginia M.-Y. , Pittman Randall N.
Published: April 2021

The execution phase is an evolutionarily conserved stage of apoptosis that occurs with remarkable temporal and morphological uniformity in most if not all cell types regardless of the condition used to induce death. Characteristic features of apoptosis such as membrane blebbing, DNA fragmentation, chromatin condensation, and cell shrinkage occur during the execution phase; therefore, there is considerable interest in defining biochemical changes and signaling events early in the execution phase. Since onset of the execution phase is asynchronous across a population with only a small fraction of cells in this stage at any given time, characterizing underlying biochemical changes is difficult. An additional complication is recent evidence suggesting that the execution phase occurs after cells commit to die; thus, agents that modulate events in the execution phase may alter the morphological progression of apoptosis but will not affect the time-course of death. In the present study, we use a single cell approach to study and temporally order biochemical and cytoskeletal events that occur specifically in the execution phase. Microtubules de-acetylate and disassemble as terminally differentiated PC12 cells enter the execution phase following removal of nerve growth factor. Using phosphorylation sensitive antibodies to τ, we show that this microtubule-stabilizing protein becomes dephosphorylated near the onset of the execution phase. Low concentrations of okadaic acid inhibit dephosphorylation suggesting a PP2A-like phosphatase is responsible. Transfecting τ into CHO cells to act as a ‘reporter’ protein shows a similar dephosphorylation of τ by a PP2A-like phosphatase during the execution phase following induction of apoptosis with UV irradiation. Therefore, activation of PP2A phosphatase occurs at the onset of the execution phase in two very different cell types following different initiators of apoptosis which is consistent with activation of PP2A phosphatase being a common feature of the execution phase of apoptosis. Experiments using either taxol to inhibit microtubule disassembly or okadaic acid to inhibit τ dephosphorylation suggest that microtubule disassembly is necessary for τ dephosphorylation to occur. Thus, we propose that an early step in the execution phase (soon after a cell commits to die) is microtubule disassembly which frees or activates PP2A to dephosphorylate τ as well as other substrates.
Retrieved constituents of large dense-cored vesicles and synaptic vesicles intermix in stimulation-induced early endosomes of noradrenergic neurons
Authors: Partoens P. , Slembrouck D. , Quatacker J. , Baudhuin P. , Courtoy P. J. , Potter W. P. De
Published: April 2021

Two storage compartments in cultured noradrenergic neurons derived from the superior cervical ganglion from fetal pig have been defined using sucrose density gradient centrifugation and electron microscopy: (1) large dense-cored vesicles (LDV) contain noradrenaline and dopamine-β-hydroxylase (DβH); (2) small electron-lucent vesicles contain acetylcholine and p38 and represent the noradrenergic small synaptic vesicles (SSV); no small dense-cored vesicles (SDV) could be detected. Our results demonstrate that internalized LDV membrane constituents are retrieved into early endosomes, as shown by the colocalization of retrieved DβH with the endosomal markers Rab5 and HRP in sucrose density gradients and on confocal microscopical images. Recycling of the SSV membranes via an endosomal intermediate is also confirmed in noradrenergic neurons. Finally, colocalization of retrieved DβH and retrieved p38 in stimulated neurons indicates that the two sets of constituents intermix. These data provide the first experimental evidence for a common early endosome in which SSV and LDV membrane constituents are internalized after exocytosis and imply that endosomal sorting is an important process for the generation of different secretory vesicles in the noradrenergic nerve terminal.
Dissection of the translocation and chaperoning functions of yeast BiP/Kar2p in vivo
Authors: Holkeri Heidi , Paunola Eija , Jämsä Eija , Makarow Marja
Published: April 2021

We used the rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli β-lactamase to dissect the functions of Saccharomyces cerevisiae BiP/Kar2p in vivo. Both were fused to the Hsp150Δ-polypeptide, which promotes proper folding of heterologous proteins which otherwise are misfolded in the yeast ER. Hsp150Δ-NGFRe and Hsp150Δ-β-lactamase acquired disulfides and were properly folded and secreted to the culture medium. When disulfide formation was prevented by incubating cells with dithiothreitol (DTT), Hsp150Δ-NGFRe remained in the endoplasmic reticulum (ER). The occupancy of an otherwise partially used N-glycosylation site of reduced NGFRe was complete suggesting that, normally, folding and disulfide formation occurred as rapidly as N-glycosylation. Removal of DTT resulted in remarkably rapid disulfide formation and secretion, suggesting only mild conformational distortion of reduced NGFRe. In contrast, reduced Hsp150Δ-β-lactamase was severely misfolded and attained a secretion competent conformation more slowly after reoxidation. When kar2-159 cells were incubated at permissive temperature 24°C with DTT, the reporter proteins were retained in the ER. After shift of the cells to 34°C to inactivate BiP/Kar2p irreversibly, and subsequent removal of DTT, most pre-accumulated Hsp150Δ-NGFRe was rapidly secreted, whereas Hsp150Δ-β-lactamase was secretion incompetent. Thus, Hsp150Δ-NGFRe did not require BiP/Kar2p for conformational maturation, though translocation was dependent on BiP/Kar2p. Apparently proteins differ in their post-translocational requirements for BiP/Kar2p, indicating that translocation and chaperoning are distinct functions.