Scattered Golgi Elements during Microtubule Disruption Are Initially Enriched in Trans-Golgi Proteins

We have addressed the question of whether or not Golgi fragmentation, as exemplified by that occurring during drug-induced microtubule depolymerization, is accompanied by the separation of Golgi subcompartments one from another. Scattering kinetics of Golgi subcompartments during microtubule disassembly and reassembly following reversible nocodazole exposure was inferred from multimarker analysis of protein distribution. Stably expressed α-2,6-sialyltransferase and N-acetylglucosaminyltransferase-I (NAGT-I), both C-terminally tagged with the myc epitope, provided markers for the trans-Golgi/trans-Golgi network (TGN) and medial-Golgi, respectively, in Vero cells. Using immunogold labeling, the chimeric proteins were polarized within the Golgi stack. Total cellular distributions of recombinant proteins were assessed by immunofluorescence (anti-myc monoclonal antibody) with respect to the endogenous protein, β-1,4-galactosyltransferase (GalT, trans-Golgi/TGN, polyclonal antibody). ERGIC-53 served as a marker for the intermediate compartment). In HeLa cells, distribution of endogenous GalT was compared with transfected rat α-mannosidase II (medial-Golgi, polyclonal antibody). After a 1-h nocodazole treatment, Vero α-2,6-sialyltransferase and GalT were found in scattered cytoplasmic patches that increased in number over time. Initially these structures were often negative for NAGT-I, but over a two- to threefold slower time course, NAGT-I colocalized with α-2,6-sialyltransferase and GalT. Scattered Golgi elements were located in proximity to ERGIC-53-positive structures. Similar trans-first scattering kinetics was seen with the HeLa GalT/α-mannosidase II pairing. Following nocodazole removal, all cisternal markers accumulated at the same rate in a juxtanuclear Golgi. Accumulation of cisternal proteins in scattered Golgi elements was not blocked by microinjected GTPγS at a concentration sufficient to inhibit secretory processes. Redistribution of Golgi proteins from endoplasmic reticulum to scattered structures following brefeldin A removal in the presence of nocodazole was not blocked by GTPγS. We conclude that Golgi subcompartments can separate one from the other. We discuss how direct trafficking of Golgi proteins from the TGN/trans-Golgi to endoplasmic reticulum may explain the observed trans-first scattering of Golgi transferases in response to microtubule depolymerization.

Temporary Disruption of the Plasma Membrane Is Required for c-fos Expression in Response to Mechanical Stress

Mechanically stressed cells display increased levels of fos message and protein. Although the intracellular signaling pathways responsible for FOS induction have been extensively characterized, we still do not understand the nature of the primary cell mechanotransduction event responsible for converting an externally acting mechanical stressor into an intracellular signal cascade. We now report that plasma membrane disruption (PMD) is quantitatively correlated on a cell-by-cell basis with fos protein levels expressed in mechanically injured monolayers. When the population of PMD-affected cells in injured monolayers was selectively prevented from responding to the injury, the fos response was completely ablated, demonstrating that PMD is a requisite event. This PMD-dependent expression of fos protein did not require cell exposure to cues inherent in release from cell–cell contact inhibition or presented by denuded substratum, because it also occurred in subconfluent monolayers. Fos expression also could not be explained by factors released through PMD, because cell injury conditioned medium failed to elicit fos expression. Translocation of the transcription factor NF-κB into the nucleus may also be regulated by PMD, based on a quantitative correlation similar to that found with fos. We propose that PMD, by allowing a flux of normally impermeant molecules across the plasma membrane, mediates a previously unrecognized form of cell mechanotransduction. PMD may thereby lead to cell growth or hypertrophy responses such as those that are present normally in mechanically stressed skeletal muscle and pathologically in the cardiovascular system.

Inhibition of Rho Is Required for cAMP-induced Melanoma Cell Differentiation

Up-regulation of the cAMP pathway by forskolin or α-melanocyte stimulating hormone induces melanocyte and melanoma cell differentiation characterized by stimulation of melanin synthesis and dendrite development. Here we show that forskolin-induced dendricity is associated to a disassembly of actin stress fibers. Since Rho controls actin organization, we studied the role of this guanosine triphosphate (GTP)-binding protein in cAMP-induced dendrite formation. Clostridium botulinum C3 exotransferase, which inhibits Rho, mimicked the effect of forskolin in promoting dendricity and stress fiber disruption, while the Escherichia coli toxin cytotoxic necrotizing factor-1 (CNF-1), which activates Rho and the expression of a constitutively active Rho mutant, blocked forskolin-induced dendrite outgrowth. In addition, overexpression of a constitutively active form of the Rho target p160 Rho-kinase (P160ROCK) prevented the dendritogenic effects of cAMP. Our results suggest that inhibition of Rho and of its target p160ROCK are required events for cAMP-induced dendrite outgrowth in B16 cells. Furthermore, we present evidence that Rho is involved in the regulation of melanogenesis. Indeed, Rho inactivation enhanced the cAMP stimulation of tyrosinase gene transcription and protein expression, while Rho constitutive activation impaired these cAMP-induced effects. This reveals that, in addition to controlling dendricity, Rho also participates in the regulation of melanin synthesis by cAMP.

Mutational Effect of Fission Yeast Polα on Cell Cycle Events

Polα is the principal DNA polymerase for initiation of DNA replication and also functions in postinitiation DNA synthesis. In this study, we investigated the cell cycle responses induced by mutations in polα+. Germinating spores carrying either a deletion of polα+ (polαΔ) or a structurally intact but catalytically dead polα mutation proceed to inappropriate mitosis with no DNA synthesis. This suggests that the catalytic function, and not the physical presence of Polα, is required to generate the signal that prevents the cells from entering mitosis prematurely. Cells with a polαts allele arrest the cell cycle near the hydroxyurea arrest point, but, surprisingly, polαts in cdc20 (polε mutant) background arrested with a cdc phenoytpe, not a polαts-like phenotype. At 25°C, replication perturbation caused by polαts alleles induces Cds1 kinase activity and requires the checkpoint Rads, Cds1, and Rqh1, but not Chk1, to maintain cell viability. At 36°C, replication disruption caused by polαts alleles induces the phosphorylation of Chk1; however, mutant cells arrest with heterogeneous cell sizes with a population of the cells entering aberrant mitosis. Together, our results indicate that the initiation DNA structure synthesized by Polα is required to bring about the S phase to mitosis checkpoint, whereas replication defects of different severity caused by polαts mutations induce differential downstream kinase responses.

Radiation-induced Release of Transforming Growth Factor α Activates the Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase Pathway in Carcinoma Cells, Leading to Increased Proliferation and Protection from Radiation-induced Cell Death

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0–10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90–240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor α receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor α (TGFα) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFα cleavage 120–180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFα. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFα. Neutralization of TGFα function by an anti-TGFα antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFα–EGFR–MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.

Fission Yeast Pob1p, Which Is Homologous to Budding Yeast Boi Proteins and Exhibits Subcellular Localization Close to Actin Patches, Is Essential for Cell Elongation and Separation

The fission yeast pob1 gene encodes a protein of 871 amino acids carrying an SH3 domain, a SAM domain, and a PH domain. Gene disruption and construction of a temperature-sensitive pob1 mutant indicated that pob1 is essential for cell growth. Loss of its function leads to quick cessation of cellular elongation. Pob1p is homologous to two functionally redundant Saccharomyces cerevisiae proteins, Boi1p and Boi2p, which are necessary for cell growth and relevant to bud formation. Overexpression of pob1 inhibits cell growth, causing the host cells to become round and swollen. In growing cells, Pob1p locates at cell tips during interphase and translocates near the division plane at cytokinesis. Thus, this protein exhibits intracellular dynamics similar to F-actin patches. However, Pob1p constitutes a layer, rather than patches, at growing cell tips. It generates two split discs flanking the septum at cytokinesis. The pob1-defective cells no longer elongate but swell gradually at the middle, eventually assuming a lemon-like morphology. Analysis using the pob1-ts allele revealed that Pob1p is also essential for cell separation. We speculate that Pob1p is located on growing plasma membrane, possibly through the function of actin patches, and may recruit proteins required for the synthesis of cell wall.

Caldesmon Inhibits Nonmuscle Cell Contractility and Interferes with the Formation of Focal Adhesions

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca2+–calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca2+–calmodulin or Ca2+–S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

The Rho-GEF Rom2p Localizes to Sites of Polarized Cell Growth and Participates in Cytoskeletal Functions in Saccharomyces cerevisiae

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1Δ, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11°C and 37°C. rom2Δ cells exhibit morphological defects. At permissive temperatures, rom2Δ cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1Δ and kar3Δ strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore, rom2Δ strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.

Glutathione synthesis is essential for mouse development but not for cell growth in culture

Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of γ-glutamylcysteine synthetase (γGCS-HStm1), an essential enzyme in GSH synthesis. Embryos homozygous for γGCS-HStm1 fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.

Poly(ADP-ribose) polymerase gene disruption conferred mice resistant to streptozotocin-induced diabetes

Streptozotocin (STZ), a glucose analogue known to induce diabetes in experimental animals, causes DNA strand breaks and subsequent activation of poly(ADPribose) polymerase (Parp). Because Parp uses NAD as a substrate, extensive DNA damage will result in reduction of cellular NAD level. In fact, STZ induces NAD depletion and cell death in isolated pancreatic islets in vitro. Activation of Parp therefore is thought to play an important role in STZ-induced diabetes. In the present study, we established Parp-deficient (Parp−/−) mice by disrupting Parp exon 1 by using the homologous recombination technique. These mice were used to examine the possible involvement of Parp in STZ-induced β-cell damage in vivo. The wild-type (Parp+/+) mice showed significant increases in blood glucose concentration from 129 mg/dl to 218, 370, 477, and 452 mg/dl on experimental days 1, 7, 21, and 60, respectively, after a single injection of 180 mg STZ/kg body weight. In contrast, the concentration of blood glucose in Parp−/− mice remained normal up to day 7, slightly increased on day 21, but returned to normal levels on day 60. STZ injection caused extensive necrosis in the islets of Parp+/+ mice on day 1, with subsequent progressive islet atrophy and loss of functional β cells from day 7. In contrast, the extent of islet β-cell death and dysfunction was markedly less in Parp−/− mice. Our findings clearly implicate Parp activation in islet β-cell damage and glucose intolerance induced by STZ in vivo.

Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling by the transforming growth factor-β and Wnt pathways

The transforming growth factor-β (TGFβ) and Wnt/wingless pathways play pivotal roles in tissue specification during development. Activation of Smads, the effectors of TGFβ superfamily signals, results in Smad translocation from the cytoplasm into the nucleus where they act as transcriptional comodulators to regulate target gene expression. Wnt/wingless signals are mediated by the DNA-binding HMG box transcription factors lymphoid enhancer binding factor 1/T cell-specific factor (LEF1/TCF) and their coactivator β-catenin. Herein, we show that Smad3 physically interacts with the HMG box domain of LEF1 and that TGFβ and Wnt pathways synergize to activate transcription of the Xenopus homeobox gene twin (Xtwn). Disruption of specific Smad and LEF1/TCF DNA-binding sites in the promoter abrogates synergistic activation of the promoter. Consistent with this observation, introduction of Smad sites into a TGFβ-insensitive LEF1/TCF target gene confers cooperative TGFβ and Wnt responsiveness to the promoter. Furthermore, we demonstrate that TGFβ-dependent activation of LEF1/TCF target genes can occur in the absence of β-catenin binding to LEF1/TCF and requires both Smad and LEF1/TCF DNA-binding sites in the Xtwn promoter. Thus, our results show that TGFβ and Wnt signaling pathways can independently or cooperatively regulate LEF1/TCF target genes and suggest a model for how these pathways can synergistically activate target genes.

Genetic disruption of PPARδ decreases the tumorigenicity of human colon cancer cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that have been implicated in a variety of biologic processes. The PPARδ isotype was recently proposed as a downstream target of the adenomatous polyposis coli (APC)/β-catenin pathway in colorectal carcinogenesis. To evaluate its role in tumorigenesis, a PPARδ null cell line was created by targeted homologous recombination. When inoculated as xenografts in nude mice, PPARδ −/− cells exhibited a decreased ability to form tumors compared with PPARδ +/− and wild-type controls. These data suggest that suppression of PPARδ expression contributes to the growth-inhibitory effects of the APC tumor suppressor.

The Catastrophe-promoting Activity of Ectopic Op18/Stathmin Is Required for Disruption of Mitotic Spindles But Not Interphase Microtubules

Oncoprotein18/stathmin (Op18) is a microtubule (MT) destabilizing protein that is inactivated during mitosis by phosphorylation at four Ser-residues. Op18 has at least two functions; the N-terminal region is required for catastrophe-promotion (i.e., transition from elongation to shortening), while the C-terminal region is required to inhibit MT-polymerization rate in vitro. We show here that a “pseudophosphorylation” derivative of Op18 (i.e., four Ser- to Glu-substitutions at phosphorylation sites) exhibits a selective loss of catastrophe-promoting activity. This is contrasted to authentic phosphorylation, which efficiently attenuates all activities except tubulin binding. In intact cells, overexpression of pseudophosphorylated Op18, which is not phosphorylated by endogenous kinases, is shown to destabilize interphase MTs but to leave spindle formation untouched. To test if the mitotic spindle is sensitive only to the catastrophe-promoting activity of Op18 and resistant to C-terminally associated activities, N- and C-terminal truncations with defined activity-profiles were employed. The cell-cycle phenotypes of nonphosphorylatable mutants (i.e., four Ser- to Ala-substitutions) of these truncation derivatives demonstrated that catastrophe promotion is required for interference with the mitotic spindle, while the C-terminally associated activities are sufficient to destabilize interphase MTs. These results demonstrate that specific Op18 derivatives with defined activity-profiles can be used as probes to distinguish interphase and mitotic MTs.

Disruption of the FG nucleoporin NUP98 causes selective changes in nuclear pore complex stoichiometry and function

The NUP98 gene encodes precursor proteins that generate two nucleoplasmically oriented nucleoporins, NUP98 and NUP96. By using gene targeting, we have selectively disrupted the murine NUP98 protein, leaving intact the expression and localization of NUP96. We show that NUP98 is essential for mouse gastrulation, a developmental stage that is associated with rapid cell proliferation, but dispensable for basal cell growth. NUP98−/− cells had an intact nuclear envelope with a normal number of embedded nuclear pore complexes. Typically, NUP98-deficient cells contained on average approximately 5-fold more cytoplasmic annulate lamellae than control cells. We found that a set of cytoplasmically oriented nucleoporins, including NUP358, NUP214, NUP88, and p62, assembled inefficiently into nuclear pores of NUP98−/− cells. Instead, these nucleoporins were prominently associated with the annulate lamellae. By contrast, a group of nucleoplasmically oriented nucleoporins, including NUP153, NUP50, NUP96, and NUP93, had no affinity for annulate lamellae and assembled normally into nuclear pores. Mutant pores were significantly impaired in transport receptor-mediated docking of proteins with a nuclear localization signal or M9 import signal and showed weak nuclear import of such substrates. In contrast, the ability of mutant pores to import ribosomal protein L23a and spliceosome protein U1A appeared intact. These observations show that NUP98 disruption selectively impairs discrete protein import pathways and support the idea that transport of distinct import complexes through the nuclear pore complex is mediated by specific subsets of nucleoporins.

Oncogenic ras activates the ARF-p53 pathway to suppress epithelial cell transformation

Chemically induced skin carcinomas in mice are a paradigm for epithelial neoplasia, where oncogenic ras mutations precede p53 and INK4a/ARF mutations during the progression toward malignancy. To explore the biological basis for these genetic interactions, we studied cellular responses to oncogenic ras in primary murine keratinocytes. In wild-type keratinocytes, ras induced a cell-cycle arrest that displayed some features of terminal differentiation and was accompanied by increased expression of the p19ARF, p16INK4a, and p53 tumor suppressors. In ARF-null keratinocytes, ras was unable to promote cell-cycle arrest, induce differentiation markers, or properly activate p53. Although oncogenic ras produced a substantial increase in both nucleolar and nucleoplasmic p19ARF, Mdm2 did not relocalize to the nucleolus or to nuclear bodies but remained distributed throughout the nucleoplasm. This result suggests that p19ARF can activate p53 without overtly affecting Mdm2 subcellular localization. Nevertheless, like p53-null keratinocytes, ARF-null keratinocytes were transformed by oncogenic ras and rapidly formed carcinomas in vivo. Thus, oncogenic ras can activate the ARF-p53 program to suppress epithelial cell transformation. Disruption of this program may be important during skin carcinogenesis and the development of other carcinomas.