bluuebunny/crossref_metadata_2025 huggingface dataset:
The dynamics of plasma membrane PtdIns(4,5)P2 at fertilization of mouse eggs
Authors: Halet Guillaume , Tunwell Richard , Balla Tamas , Swann Karl , Carroll John
Published: April 2021

A series of intracellular Ca2+ oscillations are responsible for triggering egg activation and cortical granule exocytosis at fertilization in mammals. These Ca2+ oscillations are generated by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which results from the hydrolysis of phosphatidylinositol 4,5-bisphosphate[PtdIns(4,5)P2]. Using confocal imaging to simultaneously monitor Ca2+ and plasma membrane PtdIns(4,5)P2in single living mouse eggs we have sought to establish the relationship between the kinetics of PtdIns(4,5)P2 metabolism and the Ca2+ oscillations at fertilization. We report that there is no detectable net loss of plasma membrane PtdIns(4,5)P2either during the latent period or during the subsequent Ca2+oscillations. When phosphatidylinositol 4-kinase is inhibited with micromolar wortmannin a limited decrease in plasma membrane PtdIns(4,5)P2 is detected in half the eggs studied. Although we were unable to detect a widespread loss of PtdIns(4,5)P2, we found that fertilization triggers a net increase in plasma membrane PtdIns(4,5)P2 that is localized to the vegetal cortex. The fertilization-induced increase in PtdIns(4,5)P2 follows the increase in Ca2+, is blocked by Ca2+ buffers and can be mimicked, albeit with slower kinetics, by photoreleasing Ins(1,4,5)P3. Inhibition of Ca2+-dependent exocytosis of cortical granules, without interfering with Ca2+ transients, inhibits the PtdIns(4,5)P2 increase. The increase appears to be due to de novo synthesis since it is inhibited by micromolar wortmannin. Finally,there is no increase in PtdIns(4,5)P2 in immature oocytes that are not competent to extrude cortical granules. These studies suggest that fertilization does not deplete plasma membrane PtdIns(4,5)P2 and that one of the pathways for increasing PtdIns(4,5)P2 at fertilization is invoked by exocytosis of cortical granules.
Sequential activation of individual PKC isozymes in integrin-mediated muscle cell spreading: a role for MARCKS in an integrin signaling pathway
Authors: Disatnik Marie-Hélène , Boutet Stéphane C. , Lee Christine H. , Mochly-Rosen Daria , Rando Thomas A.
Published: April 2021

To understand how muscle cell spreading and survival are mediated by integrins, we studied the signaling events initiated by the attachment of muscle cells to fibronectin (FN). We have previously demonstrated that muscle cell spreading on FN is mediated by α5β1 integrin, is associated with rapid phosphorylation of focal adhesion kinase and is dependent on activation of protein kinase C (PKC). Here we investigated the role of individual PKC isozymes in these cellular processes. We show that α,δ and ϵPKC are expressed in muscle cells and are activated upon integrin engagement with different kinetics — ϵPKC was activated early, whereas α and δPKC were activated later. Using isozyme-specific inhibitors, we found that the activation of ϵPKC was necessary for cell attachment to FN. However, using isozyme-specific activators, we found that activation of each of three isozymes was sufficient to promote the spreading of α5-integrin-deficient cells on FN. To investigate further the mechanism by which integrin signaling and PKC activation mediate cell spreading, we studied the effects of these processes on MARCKS, a substrate of PKC and a protein known to regulate actin dynamics. We found that MARCKS was localized to focal adhesion sites soon after cell adhesion and that MARCKS translocated from the membrane to the cytosol during the process of cell spreading. This translocation correlated with different phases of PKC activation and with reorganization of the actin cytoskeleton. Using MARCKS-antisense cDNA, we show that α5-expressing cells in which MARCKS expression is inhibited fail to spread on FN, providing evidence for the crucial role of MARCKS in muscle cell spreading. Together, the data suggest a model in which early activation of ϵPKC is necessary for cell attachment; the later activation of α or δPKC may be necessary for the progression from attachment to spreading. The mechanism of PKC-mediated cell spreading may be via the phosphorylation of signaling proteins, such as MARCKS, that are involved in the reorganization of the actin cytoskeleton.
Resistance of keratinocytes to TGFβ-mediated growth restriction and apoptosis induction accelerates re-epithelialization in skin wounds
Authors: Amendt Christiane , Mann Amrit , Schirmacher Peter , Blessing Manfred
Published: April 2021

The pleiotropic growth factor TGFβ plays an important role in regulating responses to skin injury. TGFβ targets many different cell types and is involved in all aspects of wound healing entailing inflammation,re-epithelialization, matrix formation and remodeling. To elucidate the role of TGFβ signal transduction in keratinocytes during cutaneous wound healing, we have used transgenic mice expressing a dominant negative type II TGFβ receptor exclusively in keratinocytes. We could demonstrate that this loss of TGFβ signaling in keratinocytes led to an accelerated re-epithelialization of full thickness excisional wounds accompanied by an increased proliferation in keratinocytes at the wound edge. Furthermore, we show that impaired TGFβ signaling in keratinocytes reduces apoptosis in re-epithelialized wounds of transgenic animals.

A cDNA array identified the transcription factor early growth response factor 1 (Egr1) as a target gene for TGFβ in late phases of the wound healing process. As a member of the immediate-early gene family, Egr1 is upregulated shortly after injury and induces the expression of growth factor genes. We could demonstrate that Egr1 expression is also upregulated in skin wounds which have already undergone re-epithelialization. In conclusion, we attribute the enhanced re-epithelialization in our transgenics to the resistance of keratinocytes to TGFβ-mediated growth restriction and apoptosis induction. We also propose a new role for TGFβ induced Egr1 in late phase wound repair.
Agonist-induced phasic and tonic responses in smooth muscle are mediated by InsP3
Authors: McCarron John G. , Craig John W. , Bradley Karen N. , Muir Thomas C.
Published: April 2021

Many cellular functions are regulated by agonist-induced InsP3-evoked Ca2+ release from the internal store. In non-excitable cells, predominantly, the initial Ca2+release from the store by InsP3 is followed by a more sustained elevation in [Ca2+]i via store-operated Ca2+ channels as a consequence of depletion of the store. Here, in smooth muscle, we report that the initial transient increase in Ca2+, from the internal store, is followed by a sustained response also as a consequence of depletion of the store (by InsP3), but, influx occurs via voltage-dependent Ca2+ channels. Contractions were measured in pieces of whole distal colon and membrane currents and [Ca2+]i in single colonic myocytes. Carbachol evoked phasic and tonic contractions; only the latter were abolished in Ca2+-free solution. The tonic component was blocked by the voltage-dependent Ca2+ channel blocker nimodipine but not by the store-operated channel blocker SKF 96365. InsP3 receptor inhibition, with 2-APB, attenuated both the phasic and tonic components. InsP3 may regulate tonic contractions via sarcolemma Ca2+ entry. In single cells,depolarisation (to ∼-20 mV) elevated [Ca2+]i and activated spontaneous transient outward currents (STOCs). CCh suppressed STOCs, as did caffeine and InsP3. InsP3 receptor blockade by 2-APB or heparin prevented CCh suppression of STOCs; protein kinase inhibition by H-7 or PKC19-36did not. InsP3 suppressed STOCs by depleting a Ca2+ store accessed separately by the ryanodine receptor (RyR). Thus depletion of the store by RyR activators abolished the InsP3-evoked Ca2+ transient. RyR inhibition (by tetracaine) reduced only STOCs but not the InsP3transient. InsP3 contributes to both phasic and tonic contractions. In the former, muscarinic receptor-evoked InsP3 releases Ca2+ from an internal store accessed by both InsP3 and RyR. Depletion of this store by InsP3 alone suppresses STOCs, depolarises the sarcolemma and permits entry of Ca2+ to generate the tonic component. Therefore, by lowering the internal store Ca2+ content,InsP3 may generate a sustained smooth muscle contraction. These results provide a mechanism to account for phasic and tonic smooth muscle contraction following receptor activation.
Distinct effects of thrombopoietin depending on a threshold level of activated Mpl in BaF-3 cells
Authors: Millot Gaël A. , Vainchenker William , Duménil Dominique , Svinarchuk Fédor
Published: April 2021

Thrombopoietin (TPO) plays a critical role in megakaryopoiesis through binding to its receptor Mpl. This involves activation of various intracellular signaling pathways, including phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) pathways. Their precise role in TPO-mediated proliferation, survival and differentiation is not fully understood. In the present study, we show that TPO induces different biological responses in Mpl-transduced BaF-3 cells, depending on the cell surface density of Mpl and the resulting activation level of signaling pathways. TPO mediates cell proliferation in cells expressing high levels of Mpl but only mediates survival without proliferation in cells expressing low levels of the receptor. By using the kinase inhibitors PD98059 and LY294002,we further showed that the activation level of the PI3K and MAPK p42/44 pathways is a determining factor for the proliferative effect. In cells expressing low levels of Mpl, the survival effect was strongly dependent on the activation level of the PI3K/AKT, but not the MAPK p42/44 pathway. Moreover, this effect was correlated with the phosphorylation level of BAD but not with the expression level of Bcl-XL. However, PI3K pathway inhibition did not increase apoptosis when BaF-3 cells proliferated in response to TPO, indicating a compensating mechanism from other Mpl signaling pathways in this case.
Trimeric assembly of the C-terminal region of Thrombospondin-1 or Thrombospondin-2 is necessary for cell spreading and fascin spike organisation
Authors: Anilkumar Narayanapanicker , Annis Douglas S. , Mosher Deane F. , Adams Josephine C.
Published: April 2021

Thrombospondin-1 (TSP-1) and the highly related protein thrombospondin-2(TSP-2) are trimeric extracellular molecules that have complex roles in wound healing, angiogenesis and matrix organisation. At the cellular level, TSP-1 supports cell adhesion and migration by the organisation of fascin spike cytoskeletal structures. To define the molecular requirements for assembly of fascin spikes by thrombospondins, we developed a panel of recombinant protein units of TSP-1 and TSP-2; these were designed according to the domain boundaries and included matched monomeric and trimeric units. These proteins were tested for their effects on cell attachment and fascin spike organisation using C2C12 skeletal myoblasts and vascular smooth muscle cells. In monomeric units, cell attachment activity was localised to the type 1 repeats or type 3 repeats/C-terminal globule, and both regions need to be present in the same molecule for maximal activity. On a molar basis, cell-attachment activities with monomeric units were low compared with intact TSP-1, and no monomeric unit induced cell spreading. Trimeric versions of the type 1 repeats were more adhesive but did not induce cell spreading. Strikingly, trimers that contained the type 3 repeats/C-terminal globule of either TSP-1 or TSP-2 supported cell spreading and fascin spike organisation, producing a similar activity to intact TSP-1. We conclude that trimeric assembly of the highly conserved TSP C-terminal region is necessary for organisation of the fascin-based cytoskeletal structures that are needed for thrombospondin-induced cell motility.
Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells
Authors: Scalettar Bethe A. , Rosa Patrizia , Taverna Elena , Francolini Maura , Tsuboi Takashi , Terakawa Susumu , Koizumi Schuichi , Roder John , Jeromin Andreas
Published: April 2021

Neuronal calcium sensor-1 (NCS-1) and its non-mammalian homologue,frequenin, have been implicated in a spectrum of cellular processes, including regulation of stimulated exocytosis of synaptic vesicles and secretory granules (SGs) in neurons and neuroendocrine cells and regulation of phosphatidylinositol 4-kinase beta activity in yeast. However, apart from these intriguing putative functions, NCS-1 and frequenin are relatively poorly understood. Here, the distribution, dynamics and function of NCS-1 were studied using PC12 cells that stably express NCS-1-EYFP (NCS-1 fused to enhanced yellow fluorescent protein) or that stably overexpress NCS-1. Fluorescence and electron microscopies show that NCS-1-EYFP is absent from SGs but is present on small clear organelles, some of which are just below the plasma membrane. Total internal reflection fluorescence microscopy shows that NCS-1-EYFP is associated with synaptic-like microvesicles (SLMVs) in growth cones. Overexpression studies show that NCS-1 enhances exocytosis of synaptotagmin-labeled regulated secretory organelles (RSOs) under basal conditions and during stimulation by UTP. Significantly, these studies implicate NCS-1 in the enhancement of both basal and stimulated phosphoinositide-dependent exocytosis of RSOs in PC12 cells, and they show that NCS-1 is distributed strategically to interact with putative targets on the plasma membrane and on SLMVs. These studies also reveal that SLMVs undergo both fast directed motion and highly hindered diffusive motion in growth cones, suggesting that cytoskeletal constituents can both facilitate and hinder SLMV motion. These results also reveal interesting similarities and differences between transport organelles in differentiated neuroendocrine cells and neurons.
Activation of GLUT1 by metabolic and osmotic stress: potential involvement of AMP-activated protein kinase (AMPK)
Authors: Barnes Kay , Ingram Jean C. , Porras Omar H. , Barros L. Felipe , Hudson Emma R. , Fryer Lee G. D. , Foufelle Fabienne , Carling David , Hardie D. Grahame , Baldwin Stephen A.
Published: April 2021

In the rat liver epithelial cell line Clone 9, the Vmax for glucose uptake is actuely increased by inhibition of oxidative phosphorylation and by osmotic stress. By using a membrane-impermeant photoaffinity labelling reagent together with an isoform-specific antibody, we have, for the first time, provided direct evidence for the involvement of the GLUT1 glucose transporter isoform in this response. Transport stimulation was found to be associated with enhanced accessibility of GLUT1 to its substrate and with photolabelling of formerly `cryptic' exofacial substrate binding sites in GLUT1 molecules. The total amount of cell surface GLUT1 remained constant. The precise mechanism for this binding site `unmasking' is unclear but appears to involve AMP-activated protein kinase: in the current study, osmotic and metabolic stresses were found to result in activation of the α1 isoform of AMP-activated protein kinase, and transport stimulation could be mimicked both by 5-aminoimidazole-4-carboxamide ribonucleoside and by infection of cells with a recombinant adenovirus encoding constitutively active AMP-activated protein kinase. The effect of 5-aminoimidazole-4-carboxamide ribonucleoside, as for metabolic stress, was on the Vmax rather than on the Km for transport and did not affect the cell-surface concentration of GLUT1. The relevant downstream target(s) of AMP-activated protein kinase have not yet been identified, but stimulation of transport by inhibition of oxidative phosphorylation or by 5-aminoimidazole-4-carboxamide ribonucleoside was not prevented by either inhibitors of conventional and novel protein kinase C isoforms or inhibitors of nitric oxide synthase. These enzymes, which have been implicated in stress-regulated pathways in other cell types, are therefore unlikely to play a role in transport regulation by stress in Clone 9 cells.
Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells
Authors: Vouret-Craviari Valérie , Bourcier Christine , Boulter Etienne , Van Obberghen-Schilling Ellen
Published: April 2021

Soluble mediators such as thrombin and sphingosine-1-phosphate regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas sphingosine-1-phosphate induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac1 activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac1 is inhibited. In contrast, sphingosine-1-phosphate addition leads to comparatively weak and delayed activation of RhoA and it activates Rac1. In addition, we show here that sphingosine-1-phosphate treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in sphingosine-1-phosphate-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple sphingosine-1-phosphate receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.
Role of the β1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia
Authors: Gustavsson Anna , Armulik Annika , Brakebusch Cord , Fässler Reinhard , Johansson Staffan , Fällman Maria
Published: April 2021

Invasin of Yersinia pseudotuberculosis binds to β1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the β1-integrin-mediated internalization of Yersinia, a β1-integrin-deficient cell line, GD25, transfected with wild-type β1A, β1B or different mutants of the β1A subunit was used. Both β1A and β1B bound to invasin-expressing bacteria, but only β1A was able to mediate internalization of the bacteria. The cytoplasmic region of β1A, differing from β1B, contains two NPXY motifs surrounding a double threonine site. Exchanging the tyrosines of the two NPXYs to phenylalanines did not inhibit the uptake, whereas a marked reduction was seen when the first tyrosine (Y783) was exchanged to alanine. A similar reduction was seen when the two nearby threonines (TT788-9) were exchanged with alanines. It was also noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex structures was unable to mediate uptake of invasin-expressing bacteria.
Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes
Authors: Malhi Harmeet , Irani Adil N. , Gagandeep Singh , Gupta Sanjeev
Published: April 2021

The liver can regenerate itself through the progenitor cells it harbors. Here we demonstrate isolation of epithelial progenitor/stem cells from the fetal human liver, which contains a large number of hepatoblasts. Progenitor liver cells displayed clonogenic capacity, expressed genes observed in hepatocytes, bile duct cells and oval cells, and incorporated genes transferred by adenoviral or lentiviral vectors. Under culture conditions,progenitor cells proliferated for several months, with each cell undergoing more than forty divisions, but they retained normal karyotypes. Progenitor cells differentiated into mature hepatocytes in mice with severe combined immunodeficiency, both when in an ectopic location and when in the liver itself. Cells integrated in the liver parenchyma and proliferated following liver injury. An abundance of progenitor cells in the fetal human liver is consistent with models indicating depletion of progenitor/stem cells during aging and maturation of organs. The studies indicate that isolation of progenitor cells from fetal organs will be appropriate for establishing novel systems to investigate basic mechanisms and for cell and gene therapy.
Tristetraprolin and LPS-inducible CXC chemokine are rapidly induced in presumptive satellite cells in response to skeletal muscle injury
Authors: Sachidanandan Chetana , Sambasivan Ramkumar , Dhawan Jyotsna
Published: April 2021

Myogenic precursor cells known as satellite cells persist in adult skeletal muscle and are responsible for its ability to regenerate after injury. Quiescent satellite cells are activated by signals emanating from damaged muscle. Here we describe the rapid activation of two genes in response to muscle injury; these transcripts encode LPS-inducible CXC chemokine (LIX), a neutrophil chemoattractant, and Tristetraprolin (TTP), an RNA-binding protein implicated in the regulation of cytokine expression. Using a synchronized cell culture model we show that C2C12 myoblasts arrested in G0 exhibit some molecular attributes of satellite cells in vivo: suppression of MyoD and Myf5 expression during G0 and their reactivation in G1. Synchronization also revealed cell cycle dependent expression of CD34, M-cadherin, HGF and PEA3, genes implicated in satellite cell biology. To identify other genes induced in synchronized C2C12 myoblasts we used differential display PCR and isolated LIX and TTP cDNAs. Both LIX and TTP mRNAs are short-lived, encode molecules implicated in inflammation and are transiently induced during growth activation in vitro. Further, LIX and TTP are rapidly induced in response to muscle damage in vivo. TTP expression precedes that of MyoD and is detected 30 minutes after injury. The spatial distribution of LIX and TTP transcripts in injured muscle suggests expression by satellite cells. Our studies suggest that in addition to generating new cells for repair, activated satellite cells may be a source of signaling molecules involved in tissue remodeling during regeneration.
NBD-labeled phosphatidylcholine enters the yeast vacuole via the pre-vacuolar compartment
Authors: Hanson Pamela K. , Grant Althea M. , Nichols J. Wylie
Published: April 2021

At low temperature, the short-chain fluorescent-labeled phospholipids,1-myristoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl]-phosphatidylcholine (M-C6-NBD-PC) and its phosphatidylethanolamine analog, M-C6-NBD-PE, are internalized by flip across the plasma membrane of S. cerevisiae and show similar enrichment in intracellular membranes including the mitochondria and nuclear envelope/ER. At higher temperatures (24-37°C), or if low temperature internalization is followed by warming, M-C6-NBD-PC, but not M-C6-NBD-PE, is trafficked to the lumen of the vacuole. Sorting of M-C6-NBD-PC to the vacuole is blocked by energy-depletion and by null mutations in the VPS4 and VPS28 genes required for vesicular traffic from the pre-vacuolar compartment (PVC) to the vacuole. This sorting is not blocked by a temperature-sensitive mutation in SEC12,which inhibits ER to Golgi transport, a null mutation in VPS8, which inhibits Golgi to PVC transport, or temperature-sensitive and null mutations in END4, which inhibit endocytosis from the plasma membrane. Monomethylation or dimethylation of the primary amine head-group of M-C6-NBD-PE is sufficient for sorting to the yeast vacuole in both wild-type yeast and in strains defective in the phosphatidylethanolamine methylation pathway. These data indicate that methylation of M-C6-NBD-PE produces the crucial structural component required to sort these phospholipid analogues to the vacuole via the PVC.
Ephrin-B1 transduces signals to activate integrin-mediated migration,attachment and angiogenesis
Authors: Huynh-Do Uyen , Vindis Cécile , Liu Hua , Cerretti Douglas Pat , McGrew Jeffrey T. , Enriquez Miriam , Chen Jin , Daniel Thomas O.
Published: April 2021

Ephrin-B/EphB family proteins are implicated in bidirectional signaling and were initially defined through the function of their ectodomain sequences in activating EphB receptor tyrosine kinases. Ephrin-B1-3 are transmembrane proteins sharing highly conserved C-terminal cytoplasmic sequences. Here we use a soluble EphB1 ectodomain fusion protein (EphB1/Fc) to demonstrate that ephrin-B1 transduces signals that regulate cell attachment and migration. EphB1/Fc induced endothelial ephrin-B1 tyrosine phosphorylation, migration and integrin-mediated (αvβ3 andα 5β1) attachment and promoted neovascularization, in vivo, in a mouse corneal micropocket assay. Activation of ephrin-B1 by EphB1/Fc induced phosphorylation of p46 JNK but not ERK-1/2 or p38 MAPkinases. By contrast, mutant ephrin-B1s bearing either a cytoplasmic deletion (ephrin-B1ΔCy) or a deletion of four C-terminal amino acids(ephrin-B1ΔPDZbd) fail to activate p46 JNK. Transient expression of intact ephin-B1 conferred EphB1/Fc migration responses on CHO cells, whereas the ephrin-B1ΔCy and ephrin-B1ΔPDZbd mutants were inactive. Thus ephrin-B1 transduces `outside-in' signals through C-terminal protein interactions that affect integrin-mediated attachment and migration.
The PtdIns3Pphosphatase myotubularin is a cytoplasmic protein that also localizes to Rac1-inducible plasma membrane ruffles
Authors: Laporte Jocelyn , Blondeau Francois , Gansmuller Anne , Lutz Yves , Vonesch Jean-Luc , Mandel Jean-Louis
Published: April 2021

Myotubularin, the phosphatase mutated in X-linked myotubular myopathy, was shown to dephosphorylate phosphatidylinositol 3-monophosphate(PtdIns3P) and was also reported to interact with nuclear transcriptional regulators from the trithorax family. We have characterized a panel of specific antibodies and investigated the subcellular localization of myotubularin. Myotubularin is not detected in the nucleus, and localizes mostly as a dense cytoplasmic network. Overexpression of myotubularin does not detectably affect vesicle trafficking in the mammalian cells investigated, in contrast to previous observations in yeast models. Both mutation of a key aspartate residue of myotubularin and dominant activation of Rac1 GTPase lead to the recruitment of myotubularin to specific plasma membrane domains. Localization to Rac1-induced ruffles is dependent on the presence of a domain highly conserved in the myotubularin family (that we named RID). We thus propose that myotubularin may dephosphorylate a subpool of PtdIns3P(or another related substrate) at the plasma membrane.
Distance-dependent cellular palmitoylation of de-novo-designed sequences and their translocation to plasma membrane subdomains
Authors: Navarro-Lérida Inmaculada , Álvarez-Barrientos Alberto , Gavilanes Francisco , Rodriguez-Crespo Ignacio
Published: April 2021

Using recursive PCR, we created an artificial protein sequence that consists of a consensus myristoylation motif (MGCTLS) followed by the triplet AGS repeated nine times and fused to the GFP reporter. This linker-GFP sequence was utilized as a base to produce multiple mutants that were used to transfect COS-7 cells. Constructs where a `palmitoylable' cysteine residue was progressively moved apart from the myristoylation site to positions 3, 9, 15 and 21 of the protein sequence were made, and these mutants were used to investigate the effect of protein myristoylation on subsequent palmitoylation,subcellular localization, membrane association and caveolin-1 colocalization. In all cases, dual acylation of the GFP chimeras correlated with translocation to Triton X-100-insoluble cholesterol/sphingomyelin-enriched subdomains. Whereas a strong Golgi labeling was observed in all the myristoylated chimeras, association with the plasma membrane was only observed in the dually acylated constructs. Taking into account the conflicting data regarding the existence and specificity of cellular palmitoyl-transferases, our results provide evidence that de-novo-designed sequences can be efficiently S-acylated with palmitic acid in vivo, strongly supporting the hypothesis that non-enzymatic protein palmitoylation can occur within mammalian cells. Additionally, this palmitoylation results in the translocation of the recombinant construct to low-fluidity domains in a myristate-palmitate distance-dependent manner.
Central and peripheral circadian oscillator mechanisms in flies and mammals
Authors: Glossop Nicholas R. J. , Hardin Paul E.
Published: April 2021

Circadian oscillators are cell-autonomous time-keeping mechanisms that reside in diverse tissues in many organisms. In flies and mice, the core molecular components that sustain these oscillators are highly conserved, but the functions of some of these components appear to have diverged significantly. One possible reason for these differences is that previous comparisons have focused primarily on the central oscillator of the mouse and peripheral oscillators in flies. Recent research on mouse and Drosophila peripheral oscillators shows that the function of the core components between these organisms may be more highly conserved than was first believed, indicating the following: (1) that central and peripheral oscillators in flies do not necessarily have the same molecular mechanisms;(2) that mammalian central oscillators are regulated differently from peripheral oscillators; and (3) that different peripheral oscillators within and across species show striking similarities. The core feedback loop in peripheral oscillators might therefore be functionally well conserved, and central oscillators could be specialized versions of a basic oscillator design.
A novel centrosome-associated protein with affinity for microtubules
Authors: Stein Pascal A. , Toret Christopher P. , Salic Adrian N. , Rolls Melissa M. , Rapoport Tom A.
Published: April 2021

We have identified a novel mammalian protein, MIR1, with microtubule-binding activity. MIR1 is a relative of MID1/midin, the protein implicated in Opitz G/BBB syndrome. In tissue culture cells, MIR1 is enriched at the centrosome. MIR1 dissociates from centrosomes at the G2/M transition and is recruited back to spindle poles during anaphase. When overexpressed during interphase, MIR1 binds along microtubule filaments, which become stabilized, bundled and detached from the centrosome. In mitosis,overexpressed MIR1 dissociates from microtubules but still affects the normally focused localization of γ-tubulin in spindle poles. Tight binding to microtubules in interphase appears to require an oligomeric state of MIR1, and phosphorylation in mitosis at predicted cyclin-dependent kinase(cdk) sites weakens the interaction.
Cell differentiation induces TIF1β association with centromeric heterochromatin via an HP1 interaction
Authors: Cammas Florence , Oulad-Abdelghani Mustapha , Vonesch Jean-Luc , Huss-Garcia Yolande , Chambon Pierre , Losson Régine
Published: April 2021

The transcriptional intermediary factor 1 (TIF1) family protein TIF1βis a corepressor for Krüppel-associated box (KRAB)-domain-containing zinc finger proteins and plays a critical role in early embryogenesis. Here, we examined TIF1β distribution in the nucleus of mouse embryonic carcinoma F9 cells during retinoic-acid-induced primitive endodermal differentiation. Using confocal immunofluorescence microscopy, we show that, although TIF1β is diffusely distributed throughout the nucleoplasm of undifferentiated cells, it relocates and concentrates into distinct foci of centromeric heterochromatin in differentiated cells characterized by a low proliferation rate and a well developed cytokeratin network. This relocation was not observed in isoleucine-deprived cells, which are growth arrested, or in compound RXRα-/-/RARγ-/- null mutant cells, which are resistant to RA-induced differentiation. Amino-acid substitutions in the PxVxL motif of TIF1β, which abolish interaction with members of the heterochromatin protein 1 (HP1) family, prevent its centromeric localization in differentiated cells. Collectively, these data provide compelling evidence for a dynamic nuclear compartmentalization of TIF1βthat is regulated during cell differentiation through a mechanism that requires HP1 interaction.
Secretion of human latent TGF-β-binding protein-3 (LTBP-3) is dependent on co-expression of TGF-β
Authors: Penttinen Carita , Saharinen Juha , Weikkolainen Krista , Hyytiäinen Marko , Keski-Oja Jorma
Published: April 2021

Latent TGF-β-binding proteins (LTBPs) were initially identified through their binding to the growth factor. Three of the four known LTBPs are able to associate covalently with the small latent forms of TGF-β and mediate their efficient secretion. LTBPs have subsequently been found to associate with the extracellular matrix. We report here the cDNA cloning and characterization of the human LTBP-3 protein, which is the smallest LTBP. The hLTBP-3 gene consists of 28 exons, including one alternatively spliced exon. The splice variant contains an additional epidermal-growth-factor-like repeat in the C-terminus. The gene is transcribed to produce a ∼4.6 kb mRNA, which is expressed at high levels in human heart, skeletal muscle, prostate and ovaries and in certain osteosarcoma and fibroblastic cell lines. Antibodies were generated against recombinant fragment of hLTBP-3 and used to detect the protein and its secretion from cultured COS-7 and osteosarcoma cells. Immunoblotting analysis indicated that efficient secretion of overexpressed hLTBP-3 from COS-7 cells required co-expression of TGF-β1, which resulted in the secretion of high molecular weight complexes of ∼240 kDa. hLTBP-3 protein was secreted from cultured osteosarcoma cells as high molecular weight complexes rather than in the free form. Similar complexes were recognized with antibodies specific toβ1•LAP. These findings indicate that human LTBP-3 has an essential role in the secretion and targeting of TGF-β1.
Paclitaxel-dependent mutants have severely reduced microtubule assembly and reduced tubulin synthesis
Authors: Barlow Steven B. , Gonzalez-Garay Manuel L. , Cabral Fernando
Published: April 2021

A subset of mutant cell lines selected for resistance to the antitumor drug paclitaxel are unable to progress normally through mitosis unless the drug is present in the growth medium. Without paclitaxel the cells form defective spindles, undergo aberrant mitoses, fail to complete cell division and eventually die. Analysis of these drug-dependent cells revealed a low amount of microtubule polymer and less tubulin production than wild-type cells. Ribonuclease protection experiments indicated that the decreased tubulin protein was due to decreased tubulin mRNA. Enhancing microtubule assembly by treating the cells with paclitaxel, restored tubulin to levels comparable with those of paclitaxel-treated wild-type cells, which demonstrated that the drug-dependent cells do not have a permanent impairment in their capacity to synthesize tubulin. Paclitaxel-resistant (but not dependent) cells have a smaller reduction in microtubule polymer with little or no decrease in tubulin production, whereas colcemidresistant cells have increased microtubule assembly but also exhibit little or no change in tubulin production. Finally,a mutant cell line producing an unstable β-tubulin protein has normal growth as well as normal synthesis and polymerization of tubulin, despite an approximately 30% decrease in steady state tubulin content. These studies establish a lower limit of tubulin assembly needed for cell survival and indicate that tubulin assembly must fall below this point to trigger a significant decrease in tubulin synthesis.
The PDZ-interacting domain of TRPC4 controls its localization and surface expression in HEK293 cells
Authors: Mery Laurence , Strauß Bettina , Dufour Jean F. , Krause Karl H. , Hoth Markus
Published: April 2021

Mammalian homologs of the Drosophila TRP protein have been shown to form cation-permeable channels in the plasma membrane but very little is known about the mechanisms that control their cell surface localization. Recently it has been demonstrated that the last three C-terminal amino acids(TRL) of TRPC4 comprise a PDZ-interacting domain that binds to the scaffold protein EBP50 [ezrin/moesin/radixin-binding phosphoprotein 50]. In this report, we have examined the influence of the TRL motif on the subcellular distribution of TRPC4 in human embryonic kidney (HEK) 293 cells. We have also analyzed the consequences of the interaction between EBP50 and the membrane-cytoskeletal adaptors of the ezrin/radixin/moesin (ERM) family for the cell surface expression of TRPC4. Using immunofluorescence microscopy, we found that the mutant lacking the TRL motif accumulated into cell outgrowths and exhibited a punctate distribution pattern whereas the wild-type channel was evenly distributed on the cell surface. Deletion of the PDZ-interacting domain also decreased the expression of TRPC4 in the plasma membrane by 2.4-fold, as assessed by cell surface biotinylation experiments. Finally, in a large percentage of cells co-expressing TRPC4 and an EBP50 mutant lacking the ERM-binding site, TRPC4 was not present in the plasma membrane but co-localized with the truncated scaffold in a perinuclear compartment (most probably representing the Golgi apparatus) and in vesicles associated with actin filaments. Our data demonstrate that the PDZ-interacting domain of TRPC4 controls its localization and surface expression in transfected HEK293 cells. They also point to a yet unexplored role of the EBP50-ERM complex in the regulation of protein insertion into the plasma membrane.
Leukotriene D4 induces stress-fibre formation in intestinal epithelial cells via activation of RhoA and PKCδ
Authors: Massoumi Ramin , Larsson Christer , Sjölander Anita
Published: April 2021

The intestinal epithelial barrier, which is regulated by the actin cytoskeleton, exhibits permeability changes during inflammation. Here we show that activation of the CysLT1 receptor by the inflammatory mediator leukotriene D4 (LTD4) causes a rapid increase in stress-fibre formation in intestinal epithelial cells. This effect was mimicked by cytotoxic necrotising factor-1 (CNF-1)-induced activation of RhoA,overexpression of constitutively active RhoA (L63-RhoA) and phorbol-ester-induced activation of protein kinase C (PKC). In accordance,inhibition of RhoA, by C3 exoenzyme or by dominant-negative RhoA (N19-RhoA),as well as GF109203X-induced inhibition of PKC, suppressed the LTD4-induced stress-fibre formation. Introduction of the dominant-negative regulatory domain of PKCδ, but not the corresponding structures from PKCα, βII or ϵ, blocked the LTD4-induced stress-fibre formation. Evaluating the relationship between PKCδ and RhoA in LTD4-induced stress-fibre formation,we found that C3 exoenzyme inhibited the rapid LTD4-elicited translocation of PKCδ to the plasma membrane. Furthermore, CNF-1-induced stress-fibre formation was blocked by GF109203X and by overexpression of the regulatory domain of PKC-δ, whereas PKC-induced stress-fibre production was not affected by N19-RhoA. We conclude that PKC-δ is located downstream of RhoA and that active RhoA and PKCδ are both necessary for LTD4-induced stress-fibre formation.
The C-terminal IgI domains of myosin-binding proteins C and H (MyBP-C and MyBP-H) are both necessary and sufficient for the intracellular crosslinking of sarcomeric myosin in transfected non-muscle cells
Authors: Welikson Robert E. , Fischman Donald A.
Published: April 2021

Using the COS cell transfection assay developed previously, we examined which domains of myosin-binding proteins C and H (MyBP-C and MyBP-H) are involved in intracellular interactions with sarcomeric myosin heavy chain(MyHC). Earlier studies demonstrated that overexpression of sarcomeric MyHC in COS cells results in the cytoplasmic assembly of anisotropic, spindle-like aggregates of myosin-containing filaments in the absence of other myofibrillar proteins. When the same sarcomeric MyHC was co-expressed with either MyBP-C or MyBP-H, prominent cable-like co-polymers of MyHC and the MyBPs formed in the cytoplasm instead of the spindle-like aggregates formed by MyHC alone. In vitro binding assays have shown that the C-terminal IgI domain of both MyBP-C(domain C10) and MyBP-H (domain H4) contains the light meromyosin(LMM)-binding sites of each molecule, but this domain cannot explain all of the intracellular properties of the molecules. For example, domains C7-C10 of MyBP-C and domains H1-H4 of MyBP-H are required for the faithful targeting of these proteins to the A-bands of myofibrils in skeletal muscle. Using truncation mutants of both MyBPs tagged with either green fluorescent protein(GFP) or c-myc, we now demonstrate that the last four domains of both MyBP-C and MyBP-H colocalize with the full-length proteins in the MyHC/MyBP cable polymers when co-transfected with MyHC in COS cells. Deletion of the C-terminal IgI domain in either MyBP-C or MyBP-H abrogated cable formation,but the expressed proteins could still colocalize with MyHC-containing filament aggregates. Co-expression of only the C-terminal IgI domain of MyBP-C with sarcomeric MyHC was sufficient for cable formation and colocalization with myosin. We conclude that the C-terminal IgI domains of both MyBP-H and MyBP-C are both necessary and sufficient for inducing MyHC/MyBP cable formation in this COS cell system. However, there must be other myosin-binding sites in MyBP-C and MyBP-H that explain the co-distribution of these proteins with myosin filaments in the absence of cable formation. These latter sites are neither sufficient nor required for cable formation.
Protein phosphatase 1 – targeted in many directions
Authors: Cohen Patricia T. W.
Published: April 2021

Protein phosphatase 1 (PP1) is a major eukaryotic protein serine/threonine phosphatase that regulates an enormous variety of cellular functions through the interaction of its catalytic subunit (PP1c) with over fifty different established or putative regulatory subunits. Most of these target PP1c to specific subcellular locations and interact with a small hydrophobic groove on the surface of PP1c through a short conserved binding motif – the RVxF motif – which is often preceded by further basic residues. Weaker interactions may subsequently enhance binding and modulate PP1 activity/specificity in a variety of ways. Several putative targeting subunits do not possess an RVxF motif but nevertheless interact with the same region of PP1c. In addition, several ‘modulator’ proteins bind to PP1c but do not possess a domain targeting them to a specific location. Most are potent inhibitors of PP1c and possess at least two sites for interaction with PP1c, one of which is identical or similar to the RVxF motif.Regulation of PP1c in response to extracellular and intracellular signals occurs mostly through changes in the levels, conformation or phosphorylation status of targeting subunits. Understanding of the mode of action of PP1c complexes may facilitate development of drugs that target particular PP1c complexes and thereby modulate the phosphorylation state of a very limited subset of proteins.
NCAM regulates cell motility
Authors: Prag Søren , Lepekhin Eugene A. , Kolkova Kateryna , Hartmann-Petersen Rasmus , Kawa Anna , Walmod Peter S. , Belman Vadym , Gallagher Helen C. , Berezin Vladimir , Bock Elisabeth , Pedersen Nina
Published: April 2021

Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells independently of homophilic NCAM interactions.

Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59fyn with which NCAM-140 interacts.

Furthermore, we showed that the extracellular part of NCAM acted as a paracrine inhibitor of NCAM-negative cell locomotion through a heterophilic interaction with a cell-surface receptor. As we showed that the two N-terminal immunoglobulin modules of NCAM, which are known to bind to heparin, were responsible for this inhibition, we presume that this receptor is a heparan sulfate proteoglycan. A model for the inhibitory effect of NCAM is proposed, which involves competition between NCAM and extracellular components for the binding to membrane-associated heparan sulfate proteoglycan.
Increase of cAMP upon release from prophase arrest in surf clam oocytes
Authors: Yi Jae-Hyuk , Lefièvre Linda , Gagnon Claude , Anctil Michel , Dubé François
Published: April 2021

Surf clam (Spisula solidissima) oocytes are spawned at the prophase I stage of meiosis, and they remain arrested at this stage until fertilization. Full oocyte meiosis reinitiation, first evidenced by germinal vesicle breakdown (GVBD), may be induced by artificial activators mimicking sperm, such as high K+ or serotonin. Previous reports indicated that treatments thought to increase the level of oocyte cAMP inhibited sperm- or serotonin-induced, but not KCl-induced, GVBD in clam oocytes. These observations extend the well known requirement for a drop in occyte cAMP levels in mammalian, amphibian or starfish oocytes and support the view that such a drop is universally important throughout the animal kingdom. We have re-examined the cAMP dependency of GVBD in clam oocytes and found that various treatments that raise oocyte cAMP levels did not, surprisingly, affect either KCl- or serotonin-induced GVBD. Such treatments, however, inhibited GVBD upon insemination of the oocytes, but this was due to the failure of sperm to fuse/penetrate the oocytes; thus, it was not an inhibition of oocyte activation as such. Direct measurements of oocyte cAMP levels after activation by serotonin, KCl or sperm showed that, contrary to expectations, there is a rise in cAMP levels before GVBD. Using SQ22536, an adenylyl cyclase inhibitor, the increase in oocyte cAMP level was partly prevented and GVBD proceeded, but with a significant retardation, indicating that the normal cAMP rise facilitates GVBD. Our work sheds light on the diversity of upstream pathways leading to activation of MPF and provides a unique model whereby the onset of meiosis reinitiation is associated with an increase, not a decrease, in oocyte cAMP levels.