Calcineurin activity is required for the completion of cytokinesis

Successful completion of cytokinesis requires the spatio-temporal regulation of protein phosphorylation and the coordinated activity of protein kinases and phosphatases. Many mitotic protein kinases are well characterized while mitotic phosphatases are largely unknown. Here, we show that the Ca²⁺- and calmodulin-dependent phosphatase, calcineurin (CaN), is required for cytokinesis in mammalian cells, functioning specifically at the abscission stage. CaN inhibitors induce multinucleation in HeLa cells and prolong the time cells spend connected via an extended intracellular bridge. Upon Ca²⁺ influx during cytokinesis, CaN is activated, targeting a set of proteins for dephosphorylation, including dynamin II (dynII). At the intracellular bridge, phospho-dynII and CaN are co-localized to dual flanking midbody rings (FMRs) that reside on either side of the central midbody ring. CaN activity and disassembly of the FMRs coincide with abscission. Thus, CaN activity at the midbody plays a key role in regulating the completion of cytokinesis in mammalian cells.     

TTDN1 is a Plk1-interacting protein involved in maintenance of cell cycle integrity

Polo-like kinase 1 (Plk1) is a highly conserved serine/threonine kinase that plays critical roles in many cell cycle events, especially in mitosis. In the present study, we identified TTDN1 as a potential interacting partner of Plk1 in yeast two-hybrid screens. Sequence analysis indicates that TTDN1 contains a consensus Plk1-binding motif at its C terminus. TTDN1 colocalizes with Plk1 at the centrosome in mitosis and the midbody during cytokinesis. TTDN1 is phosphorylated by Cdk1 in mitosis, and this is required for its interaction with Plk1. Site-directed mutagenesis indicates that TTDN1 is phosphorylated at multiple residues, including Ser93 and Ser104. Mutation of Thr120 of TTDN1 abolishes its interaction with Plk1, suggesting phosphorylation of Thr120 in the consensus Plk1-binding motif is required for its interaction with Plk1. Overexpression of TTDN1 or its knockdown by siRNA causes multi-polar spindles and multiple nuclei, suggesting that TTDN1 plays a role in regulating mitosis and cytokinesis. Received 27 November 2006; received after revision 4 January 2007; accepted 25 January 2007 Y. Zhang, Y. Tian: These authors contribute equally to this work.     

Regulation of caldesmon activity by Cdc2 kinase plays an important role in maintaining membrane cortex integrity during cell division

To study the mitosis-specific phosphorylation of caldesmon (CaD), we generated a mutant of the C-terminal fragment (amino acids 244–538) of human fibroblast CaD (CaD39-6F), as well as a mutant of the full-length CaD (CaD-6F), in which all six potential phosphorylation sites for Cdc2 kinase were abolished. The mitotic CaD39-6F-overexpressing cells required more time to progress from anaphase start to 50% cytokinesis, exhibited larger size, and abnormally formed numerous small blebs. In contrast, overexpression of the wild-type C-terminal fragment of CaD (CaD39) did not result in abnormal bleb formation, but led to larger size and prolonged the time requirement between anaphase start and 50% cytokinesis. Similar abnormal blebs were also observed in the CaD-6F-overexpressing cells. CaD-6F-overexpressing cells did not show larger size but required more time to progress from anaphase start to 50% cytokinesis. These results suggest that mitosis-specific phosphorylation of CaD plays a role in inhibiting bleb formation and that the N-terminal fragment of CaD is required for cell size determination. Received 4 September 2002; received after revision 25 November 2002; accepted 4 December 2002     

Regulation of cytokinesis

At the end of mitosis, daughter cells are separated from each other by cytokinesis. This process involves equal partitioning and segregation of cytoplasm between the two cells. Despite years of study, the mechanism driving cytokinesis in animal cells is not fully understood. Actin and myosin are major components of the contractile ring, the structure at the equator between the dividing cells that provides the force necessary to constrict the cytoplasm. Despite this, there are also tantalizing results suggesting that cytokinesis can occur in the absence of myosin. It is unclear what the roles are of the few other contractile ring components identified to date. While it has been difficult to identify important proteins involved in cytokinesis, it has been even more challenging to pinpoint the regulatory mechanisms that govern this vital process. Cytokinesis must be precisely controlled both spatially and temporally; potential regulators of these parameters are just beginning to be identified. This review discusses the recent progress in our understanding of cytokinesis in animal cells and the mechanisms that may regulate it. Received 24 August 1998; received after revision 9 October 1998; accepted 9 October 1998     

Gene controlled condensation in individual chromosomes

Summary When cells were irradiated with variable doses of gamma rays, 0.33% showed the appearance of single decondensed chromosomes (SDC) at the moment at which all the other chromosomes of the complement exhibited the normal condensed state corresponding to metaphase stages. Several hypotheses are discussed to explain the origin of SDC. It appears that the most reasonable mechanism to explain our observations is to assume that the process of chromosome condensation is independently controlled in each individual chromosome by a gene/s located in each one of the chromosomes of the complement. A radiation-induced deficiency in one of these genes may produce an impairement in the normal process of condensation of the carrier chromosome which would give rise to SDC.This work was supported by grants from CIC and CONICET.Acknowledgments. I wish to thank Dr J.M. Andrieu who kindly performed the irradiation of the specimens.     

Dynamics of ESCRT proteins

Proteins of the ESCRT (endosomal sorting complex required for transport) complex function in membrane fission processes, such as multivesicular body (MVBs) formation, the terminal stages of cytokinesis, and separation of enveloped viruses from the plasma membrane. In mammalian cells, the machinery consists of a network of more than 20?proteins, organized into three complexes (ESCRT-I, -II, and -III), and other associated proteins such as the ATPase vacuolar protein sorting 4 (Vps4). Early biochemical studies of MVBs biogenesis in yeast support a model of sequential recruitment of ESCRT complexes on membranes. Live-cell imaging of ESCRT protein dynamics during viral budding and cytokinesis now reveal that this long-standing model of sequential assembly and disassembly holds true in mammalian cells.     

Nuclear fusion and irregular cytokinesis in binucleate and tetraploid cells ofVicia faba after caffeine treatment

Summary By means of 3 h treatment with 0.2% caffeine solution, binucleate and tetraploid cells were obtained in the lateral root meristem ofVicia faba. During recovery changing rates of fused interphases were noticed. Cell walls were formed in the equatiorial plane of the preceeding division of binucleate and tetraploid cells at interphase and in the course of bimitosis or 4n-mitosis at prophase or metaphase; during bitelophase a constriction of the fused nuclei could be seen. The conclusion is that the basic requirements of cytokinesis are not affected by caffeine.     

Biological effects of lithium: Experimental analysis in plant cytokinesis

Summary The biological effects of lithium ions have been studied, using plant cytokinesis in onion root meristems as the experimental model. Lithium induces binucleate cells by inhibiting cell plate formation. Moreover, lithium and caffeine have additive effects on the induction of binucleate cells. Na⁺, K⁺, Ca⁺⁺ and Mg⁺⁺ antagonize lithium-induced inhibition of cytokinesis.     

Cytokinesis in onion roots: Inhibition by vanadate and caffeine

Summary The effect of vanadate ions on plant cytokinesis has been studied inAllium cepa root meristematic cells. Vanadate induces binucleate cells by inhibiting cell plate formation. Moreover, vanadate and caffeine have additive effects in the induction of binucleate cells.     

Calcium and magnesium in plant cytokinesis and their antagonism with caffeine

Summary The efficiency of caffeine at different concentration on the induction of binucleate cells in onion root-tip was studied. The drug effect is strongly depressed in the Ca⁺⁺ and/or Mg⁺⁺ presence at half-rate of maximum efficiency (0.04%), about 2 mM). We therefore conclude that both cations must play a role in plant cytokinesis.     

Akt-mediated GSK-3β inhibition prevents migration of polyamine-depleted intestinal epithelial cells via Rac1

The rapid migration of intestinal epithelial cells (IEC) is important for the healing of mucosal wounds. We have previously shown that polyamine depletion inhibits migration of IEC-6 cells. Akt activation and its downstream target GSK-3β have been implicated in the regulation of migration. Here we investigated the significance of elevated phosphatidylinositol 3-kinase (PI3K)/Akt signaling on migration of polyamine-depleted cells. Polyamine-depleted cells had high Akt (Ser473) and GSK-3β (Ser9) phosphorylation. Pretreatment with 20 μM LY294002 (PI3K inhibitor) for 30 min inhibited phosphorylation of Akt, increased migration by activating Rac1 in polyamine-depleted IEC-6 cells, and restored the actin structure similar to that in cells grown in control medium. Treatment of cells with a GSK-3β inhibitor (AR-A014418) altered the actin cytoskeleton and inhibited migration, mimicking the effects of polyamine depletion. Thus, our results indicate that sustained activation of Akt in response to polyamine depletion inhibits migration through GSK-3β and Rac1. Received 25 August 2006; received after revision 3 October 2006; accepted 16 October 2006     

Inhibition of cytokinesis in plant cells by 6-dimethylaminopurine and puromycin

Summary Binucleate cells are produced in garlic root tip cells with puromycin and with 6-dimethylaminopurine (6-DMAP), the purine component of puromycin. The possibility that the effect of puromycin on cytokinesis is due to 6-DMAP-which appears to be without short term effects on protein synthesis-is discussed.This work was supported by AI 031871 from the Centre National de la Recherche Scientifique (M-C.B.) and the University René Descartes (M-C.B. and F.L.), Paris.The authors would like to thank Miss G. Daouse and Miss M. Delage for excellent technical assistance.     

Importance of G2 for the cytokinesis of plant cells: Specific blocking by deoxyguanosine

Summary The modalities of the deoxyguanosine blocking effect on meristematic root cells of Allium sativum L. reveals that, during G₂ phase, fundamental processes leading to cytokinesis take place.     

Basic mechanisms of pigment bleaching and loss of structural resistance in spruce (Picea abies) needles: advances in phytomedical diagnostics

Summary Preliminary phytochemical diagnostic data in addition to scanning electron microscopy in spruce (Picea abies) needles from 18 different locations in Upper Bavaria support the following hypothesis: both physiological changes in the photosynthesizing cells and structural changes on the needle surfaces (loss of integrity of the wax layer over the stomatal cavities) are the result of the interaction of biomolecules with photooxidants. Damaged needles lose their resistance towards fungal invaders which finally cause necrotisation and abscission of the needles.     

Cytokinesis in development and disease: variations on a common theme

Cytokinesis is a crucial step in cell proliferation, and remarkably, it is also an important mechanism for developmental regulation in the generation of diverse cell types in eukaryotic organisms. Successful cytokinesis relies on the assembly and activation of an actomyosin-based contractile ring and membrane deposition/fusion in a spatially and temporally precise manner. As such, the molecular pathways governing cytokinesis are highly complex, involving a large number of components forming intricate interactive networks. The complexity of this system, however, may have also provided a rich platform for evolutionary ‘tinkering’ to achieve specific morphogenetic and developmental outcomes. Furthermore, failed or altered cytokinesis appears to contribute to the development of cancer in unexpected ways. Received 25 June 2007; received after revision 20 July 2007; accepted 16 August 2007     

Soluble polysialylated NCAM: a novel player of the innate immune system in the lung

Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is well studied in the nervous system and described as a dynamic modulator of plastic processes like precursor cell migration, axon fasciculation, and synaptic plasticity. Here, we describe a novel function of polysialylated NCAM (polySia-NCAM) in innate immunity of the lung. In mature lung tissue of healthy donors, polySia was exclusively attached to the transmembrane isoform NCAM-140 and located to intracellular compartments of epithelial cells. In patients with chronic obstructive pulmonary disease, however, increased polySia levels and processing of the NCAM carrier were observed. Processing of polysialylated NCAM was reproduced in a mouse model by bleomycin administration leading to an activation of the inflammasome and secretion of interleukin (IL)-1β. As shown in a cell culture model, polySia-NCAM-140 was kept in the late trans-Golgi apparatus of lung epithelial cells and stimulation by IL-1β or lipopolysaccharide induced metalloprotease-mediated ectodomain shedding, resulting in the secretion of soluble polySia-NCAM. Interestingly, polySia chains of secreted NCAM neutralized the cytotoxic activity of extracellular histones as well as DNA/histone-network-containing “neutrophil extracellular traps”, which are formed during invasion of microorganisms. Thus, shedding of polySia-NCAM by lung epithelial cells may provide a host-protective mechanism to reduce tissue damage during inflammatory processes.     

Targeting of the Akt/PKB kinase to the actin skeleton

Serine/threonine kinase Akt/PKB intracellular distribution undergoes rapid changes in response to agonists such as Platelet-derived growth factor (PDGF) or Insulin-like growth factor (IGF). The concept has recently emerged that Akt subcellular movements are facilitated by interaction with nonsubstrate ligands. Here we show that Akt is bound to the actin skeleton in in situ cytoskeletal matrix preparations from PDGF-treated Saos2 cells, suggesting an interaction between the two proteins. Indeed, by immunoprecipitation and subcellular fractioning, we demonstrate that endogenous Akt and actin physically interact. Using recombinant proteins in in vitro binding and overlay assays, we further demonstrate that Akt interacts with actin directly. Expression of Akt mutants strongly indicates that the N-terminal PH domain of Akt mediates this interaction. More important, we show that the partition between actin bound and unbound Akt is not constant, but is modulated by growth factor stimulation. In fact, PDGF treatment of serum-starved cells triggers an increase in the amount of Akt associated with the actin skeleton, concomitant with an increase in Akt phosphorylation. Conversely, expression of an Akt mutant in which both Ser473 and Thr308 have been mutated to alanine completely abrogates PDGF-induced binding. The small GTPases Rac1 and Cdc42 seem to facilitate actin binding, possibly increasing Akt phosphorylation.Received 10 September 2003; accepted 25 September 2003     

Shedding by rod photoreceptors after sunrise in fish

Diurnal shedding by retinal rods was studied in wild cutthroat trout,Oncorhyncus clarki, hatchery rainbow trout,Oncorhyncus mykiss, and the plains killifish,Fundulus zebrinus, by counting the shed tips of rod outer segments ingested as phagosomes by pigment epithelial cells. After sunrise, phagosomes increased in all species, but fewer occurred in trout, and these were elevated from 3 to 9 hours after sunrise. Shedding occurred earlier in the light period and was more robust in killifish, with phagosomes elevated from 1.5 to 6 hours after sunset. The data suggest that both production of phagosomes by shedding and their subsequent disposal are slower at the lower temperatures experienced by trout. Otherwise, rod shedding produced under natural lighting is not appreciably different than that provoked by sudden onset of artificial light.     

OSBP-related protein-2 (ORP2): a novel Akt effector that controls cellular energy metabolism

ORP2 is a ubiquitously expressed OSBP-related protein previously implicated in endoplasmic reticulum (ER)—lipid droplet (LD) contacts, triacylglycerol (TG) metabolism, cholesterol transport, adrenocortical steroidogenesis, and actin-dependent cell dynamics. Here, we characterize the role of ORP2 in carbohydrate and lipid metabolism by employing ORP2-knockout (KO) hepatoma cells (HuH7) generated by CRISPR-Cas9 gene editing. The ORP2-KO and control HuH7 cells were subjected to RNA sequencing, analyses of Akt signaling, carbohydrate and TG metabolism, the extracellular acidification rate, and the lipidome, as well as to transmission electron microscopy. The loss of ORP2 resulted in a marked reduction of active phosphorylated Akt(Ser473) and its target Glycogen synthase kinase 3β(Ser9), consistent with defective Akt signaling. ORP2 was found to form a physical complex with the key controllers of Akt activity, Cdc37, and Hsp90, and to co-localize with Cdc37 and active Akt(Ser473) at lamellipodial plasma membrane regions, in addition to the previously reported ER–LD localization. ORP2-KO reduced glucose uptake, glycogen synthesis, glycolysis, mRNA-encoding glycolytic enzymes, and SREBP-1 target gene expression, and led to defective TG synthesis and storage. ORP2-KO did not reduce but rather increased ER–LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al. in FASEB J 32:1281–1295, 2018), this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation.     

Differentiation of human megakaryocytes in culture starting from the primordial circulating cells in the newborn]

Human neonatal blood mononuclear cells were seeded in plasma clot containing high dose of a crude erythropietin. Pure megakaryocyte colonies were observed rarely and most of the colonies were mixed, megakaryocytes being located between subcolonies of erythrocytic bursts. The megakaryocytic nature of large cells could be clearly confirmed by the presence of platelet peroxidase, demarcation membranes, and alpha granules detected by electron microscopy; in addition mature micromegakaryocytes were recognized, shedding platelets.