Myosin light chain kinase inhibitors ML-7 and ML-9 inhibit mouse lung carcinoma cell attachment to the fibronectin substratum.

We studied the effects of various protein kinase inhibitors on the attachment of mouse lung carcinoma 3LL cells to the fibronectin (FN) substratum. Calmodulin antagonists (W-7 and W-13) and myosin light chain kinase inhibitors (ML-7 and ML-9) exhibited the inhibitory effect for the attachment, while inhibitors of protein kinases A and C were ineffective. Since Arg-Gly-Asp-containing hexapeptide blocked the attachment, cell surface FN receptor appeared to be involved in this mechanism. These results support the hypothesis that the cell attachment requires the rearrangement of the cytoskeleton in association with the phosphorylation of myosin light chain which would lead to the clustering of the cell surface FN receptors.     

STUNTED PLANT 1, A Gene Required for Expansion in Rapidly Elongating but Not in Dividing Cells and Mediating Root Growth Responses to Applied Cytokinin

To understand the control of spatial patterns of expansion, we have studied root growth in wild type and in the stunted plant 1 mutant, stp1, of Arabidopsis thaliana. We measured profiles of cell length and calculated the distribution of elongation rate. Slow growth of stp1 results both from a failure of dividing cell number to increase and from low elongation rates in the zone of rapid expansion. However, elongation of dividing cells was not greatly affected, and stp1 and wild-type callus grew at identical rates. Thus, rapid cellular expansion differs in mechanism from expansion in dividing cells and is facilitated by the STP1 gene. Additionally, there was no difference between stp1 and wild-type roots for elongation in response to abscisic acid, auxin, ethylene, or gibberellic acid or for radial expansion in response to ethylene; however, stp1 responded to cytokinin much less than wild type. In contrast, both genotypes responded comparably to hormones when explants were cultured; in particular, there was no difference between genotypes in shoot regeneration in response to cytokinin. Thus, effects on root expansion mediated by cytokinin, but not effects mediated by other hormones or effects on other cytokinin-mediated responses, require the STP1 locus.     

Nuclear export and cytoplasmic processing of precursors to the 40S ribosomal subunits in mammalian cells

It is generally assumed that, in mammalian cells, preribosomal RNAs are entirely processed before nuclear exit. Here, we show that pre-40S particles exported to the cytoplasm in HeLa cells contain 18S rRNA extended at the 3' end with 20-30 nucleotides of the internal transcribed spacer 1. Maturation of this pre-18S rRNA (which we named 18S-E) involves a cytoplasmic protein, the human homolog of the yeast kinase Rio2p, and appears to be required for the translation competence of the 40S subunit. By tracking the nuclear exit of this precursor, we have identified the ribosomal protein Rps15 as a determinant of preribosomal nuclear export in human cells. Interestingly, inhibition of exportin Crm1/Xpo1 with leptomycin B strongly alters processing of the 5'-external transcribed spacer, upstream of nuclear export, and reveals a new cleavage site in this transcribed spacer. Completion of the maturation of the 18S rRNA in the cytoplasm, a feature thought to be unique to yeast, may prevent pre-40S particles from initiating translation with pre-mRNAs in eukaryotic cells. It also allows new strategies for the study of preribosomal transport in mammalian cells.     

Dl-alpha-tocopherol,a potent inhibitor of phorbol ester induced shape change of erythro- and megakaryoblastic leukemia cells

Synthetic vitamin E, dl-α-tocopherol, added to a human erythroleukemia HEL and a megakaryoblastic leukemia, Meg-01, cell culture produced potent dose-dependent inhibition of phorbol ester-induced adhesion and of the morphologic changes accompanying it. The inhibition was reversible by withdrawal of supplemental vitamin E from the medium. dl-α-Tocopherol also inhibited protein kinase C activity both at baseline and after phorbol ester stimulation. Arachidonic acid stimulated protein kinase C activity of erythroleukemia cells and promoted their adhesion, an effect that was also inhibited by dl-α-tocopherol. Introduction of a protein kinase C-neutralizing antibody or a protein kinase C-inhibitor substrate into permeabilized HEL cells inhibited phorbol ester-induced adhesion and shape change. dl-α-Tocopherol also affected the cellular distribution of protein kinase C, shifting the major portion of the enzyme to the cytosol fraction and reducing phorbol ester-induced membrane association of the enzyme. Thus, protein kinase C appears to mediate shape change and adhesion, both of which are strongly inhibited by dl-α-tocopherol. J. Cell. Physiol. 172:351–360, 1997. © 1997 Wiley-Liss, Inc.     

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.     

EVIDENCE For THE CYTOPLASMIC SYNTHESIS OF NUCLEAR HISTONE DURING SPERMIOGENESIS IN THE GRASSHOPPER CHORTOPHAGA VIRIDIFASCIATA (DE GEER)

Histone synthesis during spermiogenesis in the grasshopper Chortophaga viridifasciata was studied using autoradiographic and cytochemical methods. It was found that meiosis is followed by a cessation of RNA synthesis, an elimination of RNA from the nucleus, and, during the cytoplasmic sloughing accompanying the initial cytoplasmic elongation, a loss of most of the RNA from the cell. The initial phase of cell elongation results in a long spermatid headed by a spherical RNA-less nucleus bounded by a thin RNA-containing layer of cytoplasm. Subsequent nuclear elongation is accompanied by a replacement of the typical histones by others rich in arginine. This replacement is the result of synthesis of new protein. Incorporation of arginine is first seen to occur in the thin cytoplasmic layer surrounding the nucleus. This layer was shown by staining and electron microscopy to contain aggregations of ribosome-like particles. These observations support the conclusion that the histone is synthesized in association with the RNA granules in the cytoplasm, then migrates into the nucleus where it combines with the DNA.     

Messenger ribonucleoproteins (informosomes) and RNA-binding proteins

Messenger ribonucleoproteins, first discovered in 1964 in our laboratory as free mRNA-containing particles of fish embryo cytoplasm and designated as informosomes, proved to have a universal occurrence in eukaryotic cells. Messenger ribonucleoproteins of different intracellular localization such as free cytoplasmic non-translatable informosomes, translatable messenger ribonucleoproteins in polyribosomes and nuclear pre-mRNA-containing particles are characterized by a number of features common for all of them. However, the transport from the nucleus into the cytoplasm as well as the transition from the free non-translatable state into the polyribosome-bound translatable state are accompanied by essential changes in the protein moiety of the particles. The existance of free RNA-binding proteins in eukaryotic cells has also been shown. These proteins seem to represent a pool for the formation of messenger ribonucleoproteins (informosomes).It has recently been demonstrated that the eukaryotic translation factors and, in particular, both the elongation factors and some initiation factors are among the cytoplasmic RNA-binding proteis. It is suggested that the mRNA in eukaryotic cells at different stages of its life time carries on itself the proteins which are required for its own biogenesis, processing and transport (nuclear informosomes), for its existence in a temporarily inactive state (free cytoplasmic informosomes) and for its functioning as a template (polyribosomal informosomes):omnia mea mecum porto.     

Active ras triggers death in glioblastoma cells through hyperstimulation of macropinocytosis

Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death.     

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4)

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cell's undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.     

Bidirectional morphological changes induced by dexamethasone in Morris hepatoma cell lines McA-RH7777 and McA-RH8994: independence of fibronectin and its receptor.

Two Morris hepatoma-derived cell lines, McA-RH7777 (7777) and McA-RH8994 (8994), exhibit different alterations in morphology upon exposure to glucocorticoid. After treatment with synthetic glucocorticoid dexamethasone (DEX), 7777 cells show increased adhesiveness and more flattened shape, while DEX-treated 8994 cells show decreased adhesiveness to substratum and exhibit a marked increase of round and detached cells. Since fibronectin has been thought to play an important role in cell adhesiveness to substratum in hepatoma cell culture, we have also compared the effects of DEX on the biosynthesis of fibronectin (FN) and the functional level of FN receptor in 7777 and 8994 cells. Northern blot analysis and immunofluorescent studies showed that 7777 cells have a high basal expression level of FN synthesis and that DEX treatment induces FN expression two- to threefold with establishment of an extensive fibrillar FN network around the cells. On the other hand, 8994 cells were shown to express little FN and no apparent FN was localized on nonstimulated 8994 cells. However, DEX-treatment drastically increased FN expression in 8994 cells to the level of more than that of DEX-treated 7777 cells and induced a detectable level of cell-associated FN around DEX-treated 8994 cells, which appears to be contradictory to the decreased adhesiveness to the substratum in DEX-treated 8994 cells. Cell attachment assays using FN-coated plates demonstrated that DEX does not exhibit significant effects on the attachment of either 7777 or 8994 cells to FN-coated dishes. Our results suggest that decrease of adhesiveness to the substratum and increase of round detached cells in DEX-treated 8994 cells are independent of changes in the FN expression and the function of FN receptor.     

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/ trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.     

Growth cone advance mediated by fibronectin-associated filopodia is inhibited by a phorbol ester tumor promoter

In serum-supplemented medium, exposure to the tumor promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) increases the proportion of SH-SY5Y neuroblastoma cells with neurites and increases the average neurite length. In the present study, under serum-free conditions, PMA treatment had the opposite effects, i.e., retarded neurite sprouting and partially inhibited neurite elongation. This inhibition in neurite outgrowth was partially antagonized by the addition of serum fibronectin (FN) to the medium or substratum. In the absence of PMA, SH-SY5Y cells grown under serum-free conditions showed extensive neurite outgrowth as well as the capacity to secrete FN into their microenvironment and form FN-containing substratum-attachment sites. Immunogold labeling and whole mount transmission electron microscopy (WMTEM) demonstrated FN-containing contact pads at sites where filopodia attached to the substratum and focal plaques on the underside of growth cone margins. The appearance and abundance of FN-containing contact pads and focal plaques were increased by the addition of exogenous FN to defined medium. Focal plaques appeared in close association with microfilament bundles, and nearly always with bundles that projected into filopodia attached to the substratum by contact pads. A method for immunolabeling FN in the filopodial contact pads of living cultures provided more direct evidence that filopodia and contact pads have a major role in FN-mediated attachment and are central in determining growth cone shape and the rate and direction of advance. In support of this view, we show that PMA treatment retards neurite sprouting, alters growth cone morphology and motility, and eliminates the appearance of microfilament bundles, filopodia, and FN-containing substratum-attachment plaques.     

Study of the interaction of soluble and particle-bound nicotinamide adenine dinucleotide pyrophosphorylase with cytoplasmic ribosomes

The cytoplasm of cells infected with EMC virus contains new structures which possess activity of the nuclear enzyme NAD pyrophosphorylase [14]. An attempt was made to understand the mode of formation of these structures in the infected cell. It was found that soluble NAD pyrophosphorylase manifests a strong affinity for cytoplasmic ribosomes, sedimenting at 90S. When cytoplasmic ribosomes were dissociated to the 60S and 40S subunits, the enzyme was found to be adsorbed only to the 60S unit. In extracts of rat liver nuclei, NAD pyrophosphorylase is associated with 35S particles, composed mainly of protein and DNA. The bond between enzyme and particle is of a loose nature. When ribosomes are mixed with 35S nuclear particles, most of the enzyme activity is transferred from the nuclear particles to the ribosomes, thus forming particles with an average sedimentation coefficient of 90S. Similar structures are obtained when either soluble NAD pyrophosphorylase or 35S nuclear particles are mixed with preparations of cytoplasm isolated from non-infected cells. The results of these experiments suggest that the 90S cytoplasmic structures found in virus-infected cells could result from an association between either free or particle-bound NAD pyrophosphorylase with cytoplasmic ribosomes.     

Nucleotide and deduced amino acid sequence of stp: the bacteriophage T4 anticodon nuclease gene

Pre-existing host tRNAs are reprocessed during bacteriophage T4 infection of certain Escherichia coli strains. In this pathway, tRNALys is cleaved 5' to the wobble base by anticodon nuclease and is later restored in polynucleotide kinase and RNA ligase reactions. Anticodon nuclease depends on prr, a locus found only in host strains that restrict T4 mutants lacking polynucleotide kinase and RNA ligase; and on stp, the T4 suppressor of prr restriction. stp was cloned and the nucleotide sequences of its wild-type and mutant alleles determined. Their comparison defined an stp open reading frame of 29 codons at 162.8 to 9 kb of T4 DNA (1 kb = 10(3) base-pairs). We suggest that stp encodes a subunit of anticodon nuclease, perhaps one that harbors the catalytic site; while additional subunits, such as a putative prr gene product, impart protein folding environment and tRNA substrate recognition.     

Selective adhesion of osteoblastic cells to different integrin ligands induces osteopontin gene expression.

Skeletal homeostasis is partly regulated by the mechanical environment and specific signals generated by a cell's adhesion to the matrix. Previous studies demonstrated that osteopontin (OPN) expression is stimulated in response to both cellular adhesion and mechanical stimulation. The present studies examine if specific integrin ligands mediate osteoblast selective adhesion and whether opn mRNA expression is induced in response to these same ligands. Embryonic chicken calvaria osteoblastic cells were plated on bacteriological dishes coated with fibronectin (FN), collagen type I (Col1), denatured collagen/gelatin (G), OPN, vitronectin (VN), laminin (LN) or albumin (BSA). Osteoblastic cells were shown to selectively adhere to FN, Col1, G and LN, yet not to VN, OPN or BSA. Opn mRNA expression was induced by adhesion to Col1, FN, LN and G, but neither OPN nor VN induced this expression. Examination of the activation of the protein kinases A and C second signaling systems showed that only adhesion to FN induced protein kinase A and protein kinase C (PKC) activity while adherence to Col1 induced PKC. Evaluation of the intracellular distribution of focal adhesion kinase (FAK) and p-tyrosine within cells after adherence to FN, VN or BSA demonstrated that adherence to FN stimulated FAK translocation from the nucleus to the cytoplasm and high levels of p-tyrosine localization at the cell surface. However, cell adherence to VN or BSA did not show these morphological changes. These data illustrate that osteoblast selective adhesion is mediated by specific integrin ligands, and induction of intracellular second signal kinase activity is related to the nature of the ligand.     

Control of intracellular pH and growth by fibronectin in capillary endothelial cells

The aim of this work was to analyze the mechanism by which fibronectin (FN) regulates capillary endothelial cell proliferation. Endothelial cell growth can be controlled in chemically-defined medium by varying the density of FN coated on the substratum (Ingber, D. E., and J. Folkman. J. Cell Biol. 1989. 109:317-330). In this system, DNA synthetic rates are stimulated by FN in direct proportion to its effect on cell extension (projected cell areas) both in the presence and absence of saturating amounts of basic FGF. To investigate direct growth signaling by FN, we carried out microfluorometric measurements of intracellular pH (pHi), a cytoplasmic signal that is commonly influenced by soluble mitogens. pHi increased 0.18 pH units as FN coating densities were raised and cells progressed from round to spread. Intracellular alkalinization induced by attachment to FN was rapid and followed the time course of cell spreading. When measured in the presence and absence of FGF, the effects of FN and FGF on pHi were found to be independent and additive. Furthermore, DNA synthesis correlated with pHi for all combinations of FGF and FN. Ethylisopropylamiloride, a specific inhibitor of the plasma membrane Na+/H+ antiporter, completely suppressed the effects of FN on both pHi and DNA synthesis. However, cytoplasmic pH per se did not appear to be a critical determinant of growth since DNA synthesis was not significantly inhibited when pHi was lowered over the physiological range by varying the pH of the medium. We conclude that FN and FGF exert their growth-modulating effects in part through activation of the Na+/H+ exchanger, although they appear to trigger this system via separate pathways.     

Identification and cloning of a CD43-associated serine/threonine kinase

CD43 is expressed on most hematopoetic cells and has been shown to regulate the activation and adhesion of T cells. We have cloned a serine/threonine kinase that can interact with the cytoplasmic domain of CD43. This protein is expressed in multiple tissues, including lymphoid cells. Analysis of the subcellular localization reveals it to be present in both the nucleus and the cytoplasm of the cell. The identification of this protein suggests that CD43 may mediate its biologic effects through activation of a kinase cascade, resulting in the regulation of cell growth.