PubMed Central (PMC3 - NLM DTD)
Archive of life sciences journal literature at the U.S. National Institutes of Health (NIH), developed and managed by NIH's National Center for Biotechnology Information (NCBI) in the National Library of Medicine (NLM).
Mostrando recursos 1 - 20 de 1,044
Rab8a and Rab8b are essential for several apical transport pathways but insufficient for ciliogenesis - Sato, Takashi; Iwano, Tomohiko; Kunii, Masataka; Matsuda, Shinji; Mizuguchi, Rumiko; Jung, Yongwook; Hagiwara, Haruo; Yoshihara, Yoshihiro; Yuzaki, Michisuke; Harada, Reiko; Harada, Akihiro
The small GTP-binding protein Rab8 is known to play an essential role in intracellular transport and cilia formation. We have previously demonstrated that Rab8a is required for localising apical markers in various organisms. Rab8a has a closely related isoform, Rab8b. To determine whether Rab8b can compensate for Rab8a, we generated Rab8b-knockout mice. Although the Rab8b-knockout mice did not display an overt phenotype, Rab8a and Rab8b double-knockout mice exhibited mislocalisation of apical markers and died earlier than Rab8a-knockout mice. The apical markers accumulated in three intracellular patterns in the double-knockout mice. However, the localisation of basolateral and/or dendritic markers of the...
Injury-triggered Akt phosphorylation of Cx43: a ZO-1-driven molecular switch that regulates gap junction size - Dunn, Clarence A.; Lampe, Paul D.
The proteins that form vertebrate gap junctions, the connexins, are highly regulated and have short (<2 hour) half-lives. Phosphorylation of connexin43 (Cx43) affects gap junction assembly, channel gating and turnover. After finding dramatic effects on gap junctions with Akt inhibitors, we created an antibody specific for Cx43 phosphorylated on S373, a potential Akt substrate. We found S373 phosphorylation in cells and skin or heart almost exclusively in larger gap-junctional structures that increased dramatically after wounding or hypoxia. We were able to mechanistically show that Akt-dependent phosphorylation of S373 increases gap junction size and communication by completely eliminating the interaction between Cx43...
Live-cell fluorescence imaging reveals high stoichiometry of Grb2 binding to the EGF receptor sustained during endocytosis - Fortian, Arola; Sorkin, Alexander
Activation of epidermal growth factor (EGF) receptor (EGFR) leads to its interaction with Grb2, a dual-function adapter mediating both signaling through Ras and receptor endocytosis. We used time-lapse three-dimensional imaging by spinning disk confocal microscopy to analyze trafficking of EGFR and Grb2 in living HeLa cells stimulated with low, physiological concentrations of EGFR ligands. Endogenous Grb2 was replaced in these cells by Grb2 fused to yellow fluorescent protein (YFP). After transient residence in the plasma membrane, Rhodamine-conjugated EGF (EGF–Rh) and Grb2–YFP were rapidly internalized and accumulated in endosomes. Quantitative image analysis revealed that on average two Grb2–YFP molecules were colocalized...
The Smad7–Skp2 complex orchestrates Myc stability, impacting on the cytostatic effect of TGF-β - Kim, Tae-Aug; Kang, Jin Muk; Hyun, Ja-Shil; Lee, Bona; Kim, Staci Jakyong; Yang, Eun-Sung; Hong, Suntaek; Lee, Ho-Jae; Fujii, Makiko; Niederhuber, John E.; Kim, Seong-Jin
In most human cancers the Myc proto-oncogene is highly activated. Dysregulation of Myc oncoprotein contributes to tumorigenesis in numerous tissues and organs. Thus, targeting Myc stability could be a crucial step for cancer therapy. Here we report Smad7 as a key molecule regulating Myc stability and activity by recruiting the F-box protein, Skp2. Ectopic expression of Smad7 downregulated the protein level of Myc without affecting the transcription level, and significantly repressed its transcriptional activity, leading to inhibition of cell proliferation and tumorigenic activity. Furthermore, Smad7 enhanced ubiquitylation of Myc through direct interaction with Myc and recruitment of Skp2. Ablation of...
Phosphorylation of the E3 ubiquitin ligase RNF41 by the kinase Par-1b is required for epithelial cell polarity - Lewandowski, Katherine T.; Piwnica-Worms, Helen
The establishment and maintenance of cell polarity is an essential property governing organismal homeostasis, and loss of polarity is a common feature of cancer cells. The ability of epithelial cells to establish apical-basal polarity depends on intracellular signals generated from polarity proteins, such as the Par-1 family of proteins, as well as extracellular signals generated through cell contacts with the extracellular matrix (ECM). The Par-1 family has a well-established role in regulating cell–cell contacts in the form of tight junctions by phosphorylating Par-3. In addition, Par-1 has been shown to impact on cell–ECM interactions by regulating laminin receptor localization and...
Photobleaching imprinting microscopy: seeing clearer and deeper - Gao, Liang; Garcia-Uribe, Alejandro; Liu, Yan; Li, Chiye; Wang, Lihong V.
We present a generic sub-diffraction-limited imaging method – photobleaching imprinting microscopy (PIM) – for biological fluorescence imaging. A lateral resolution of 110 nm was measured, more than a twofold improvement over the optical diffraction limit. Unlike other super-resolution imaging techniques, PIM does not require complicated illumination modules or specific fluorescent dyes. PIM is expected to facilitate the conversion of super-resolution imaging into a routine lab tool, making it accessible to a much broader biological research community. Moreover, we show that PIM can increase the image contrast of biological tissue, effectively extending the fundamental depth limit of multi-photon fluorescence microscopy.
Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation - Bhave, Madhura; Papanikou, Effrosyni; Iyer, Prasanna; Pandya, Koushal; Jain, Bhawik Kumar; Ganguly, Abira; Sharma, Chandrakala; Pawar, Ketakee; Austin, Jotham; Day, Kasey J.; Rossanese, Olivia W.; Glick, Benjamin S.; Bhattacharyya, Dibyendu
Regulation of the size and abundance of membrane compartments is a fundamental cellular activity. In Saccharomyces cerevisiae, disruption of the ADP-ribosylation factor 1 (ARF1) gene yields larger and fewer Golgi cisternae by partially depleting the Arf GTPase. We observed a similar phenotype with a thermosensitive mutation in Nmt1, which myristoylates and activates Arf. Therefore, partial depletion of Arf is a convenient tool for dissecting mechanisms that regulate Golgi structure. We found that in arf1Δ cells, late Golgi structure is particularly abnormal, with the number of late Golgi cisternae being severely reduced. This effect can be explained by selective changes in...
Actin network disassembly powers dissemination of Listeria monocytogenes - Talman, Arthur M.; Chong, Ryan; Chia, Jonathan; Svitkina, Tatyana; Agaisse, Hervé
Several bacterial pathogens hijack the actin assembly machinery and display intracellular motility in the cytosol of infected cells. At the cell cortex, intracellular motility leads to bacterial dissemination through formation of plasma membrane protrusions that resolve into vacuoles in adjacent cells. Here, we uncover a crucial role for actin network disassembly in dissemination of Listeria monocytogenes. We found that defects in the disassembly machinery decreased the rate of actin tail turnover but did not affect the velocity of the bacteria in the cytosol. By contrast, defects in the disassembly machinery had a dramatic impact on bacterial dissemination. Our results suggest...
Phosphorylation regulates VCIP135 function in Golgi membrane fusion during the cell cycle - Zhang, Xiaoyan; Zhang, Honghao; Wang, Yanzhuang
The Golgi apparatus in mammalian cells consists of stacks that are often laterally linked into a ribbon-like structure. During cell division, the Golgi disassembles into tubulovesicular structures in the early stages of mitosis and reforms in the two daughter cells by the end of mitosis. Valosin-containing protein p97–p47 complex-interacting protein, p135 (VCIP135), an essential factor involved in p97-mediated membrane fusion pathways, is required for postmitotic Golgi cisternae regrowth and Golgi structure maintenance in interphase. However, how VCIP135 function is regulated in the cell cycle remains unclear. Here, we report that VCIP135 depletion by RNA interference results in Golgi fragmentation. VCIP135...
14-3-3θ facilitates plasma membrane delivery and function of mechanosensitive connexin 43 hemichannels - Batra, Nidhi; Riquelme, Manuel A.; Burra, Sirisha; Jiang, Jean X.
Intracellular signaling in osteocytes activated by mechanical loading is important for bone formation and remodeling. These signaling events are mediated by small modulators released from Cx43 hemichannels (HC). We have recently shown that integrin α5 senses the mechanical stimulation and induces the opening of Cx43 HC; however, the underlying mechanism is unknown. Here, we show that both Cx43 and integrin α5 interact with 14-3-3θ, and this interaction is required for the opening of Cx43 HC upon mechanical stress. The absence of 14-3-3θ prevented the interaction between Cx43 and integrin α5, and blocked HC opening. Furthermore, it decreased the transport of...
S-adenosylmethionine limitation induces p38 mitogen-activated protein kinase and triggers cell cycle arrest in G1 - Lin, Da-Wei; Chung, Benjamin P.; Kaiser, Peter
The primary methyl group donor S-adenosylmethionine (SAM) is important for a plethora of cellular pathways including methylation of nucleic acids, proteins, and the 5′ cap structure of mRNAs, as well as biosynthesis of phospholipids and polyamines. In addition, because it is the cofactor for chromatin methylation, SAM is an important metabolite for the establishment and maintenance of epigenetic marks. Here, we demonstrate that cells halt proliferation when SAM levels become low. Cell cycle arrest occurs primarily in the G1 phase of the cell cycle and is accompanied by activation of the mitogen-activated protein kinase p38 (MAPK14) and subsequent phosphorylation of...
Protein kinase Darkener of apricot and its substrate EF1γ regulate organelle transport along microtubules - Serpinskaya, Anna S.; Tuphile, Karine; Rabinow, Leonard; Gelfand, Vladimir I.
Regulation of organelle transport along microtubules is important for proper distribution of membrane organelles and protein complexes in the cytoplasm. RNAi-mediated knockdown in cultured Drosophila S2 cells demonstrates that two microtubule-binding proteins, a unique isoform of Darkener of apricot (DOA) protein kinase, and its substrate, translational elongation factor EF1γ, negatively regulate transport of several classes of membrane organelles along microtubules. Inhibition of transport by EF1γ requires its phosphorylation by DOA on serine 294. Together, our results indicate a new role for two proteins that have not previously been implicated in regulation of the cytoskeleton. These results further suggest that the...
Paracrine-rescued lobulogenesis in chimeric outgrowths comprising progesterone-receptor-null mammary epithelium and redirected wild-type testicular cells - Bruno, Robert D.; Boulanger, Corinne A.; Rosenfield, Sonia M.; Anderson, Lisa H.; Lydon, John P.; Smith, Gilbert H.
We have previously shown that non-mammary and tumorigenic cells can respond to the signals of the mammary niche and alter their cell fate to that of mammary epithelial progenitor cells. Here we tested the hypothesis that paracrine signals from mammary epithelial cells expressing progesterone receptor (PR) are dispensable for redirection of testicular cells, and that re-directed wild-type testicular-derived mammary cells can rescue lobulogenesis of PR-null mammary epithelium by paracrine signaling during pregnancy. We injected PR-null epithelial cells mixed with testicular cells from wild-type adult male mice into cleared fat-pads of recipient mice. The testicular cells were redirected in vivo to...
Interaction of 4.1G and cGMP-gated channels in rod photoreceptor outer segments - Cheng, Christiana L.; Molday, Robert S.
In photoreceptors, the assembly of signaling molecules into macromolecular complexes is important for phototransduction and maintaining the structural integrity of rod outer segments (ROSs). However, the molecular composition and formation of these complexes are poorly understood. Using immunoprecipitation and mass spectrometry, 4.1G was identified as a new interacting partner for the cyclic-nucleotide gated (CNG) channels in ROSs. 4.1G is a widely expressed multifunctional protein that plays a role in the assembly and stability of membrane protein complexes. Multiple splice variants of 4.1G were cloned from bovine retina. A smaller splice variant of 4.1G selectively interacted with CNG channels not associated...
Regulation of endoplasmic reticulum Ca2+ oscillations in mammalian eggs - Wakai, Takuya; Zhang, Nan; Vangheluwe, Peter; Fissore, Rafael A.
Changes in the intracellular concentration of free calcium ([Ca2+]i) regulate diverse cellular processes including fertilization. In mammalian eggs, the [Ca2+]i changes induced by the sperm unfold in a pattern of periodical rises, also known as [Ca2+]i oscillations. The source of Ca2+ during oscillations is the endoplasmic reticulum ([Ca2+]ER), but it is presently unknown how [Ca2+]ER is regulated. Here, we show using mouse eggs that [Ca2+]i oscillations induced by a variety of agonists, including PLCζ, SrCl2 and thimerosal, provoke simultaneous but opposite changes in [Ca2+]ER and cause differential effects on the refilling and overall load of [Ca2+]ER. We also found that...
TAK1 regulates SOX9 expression in chondrocytes and is essential for postnatal development of the growth plate and articular cartilages - Gao, Lin; Sheu, Tzong-jen; Dong, Yufeng; Hoak, Donna M.; Zuscik, Michael J.; Schwarz, Edward M.; Hilton, Matthew J.; O’Keefe, Regis J.; Jonason, Jennifer H.
TAK1 is a MAP3K that mediates non-canonical TGF-β and BMP signaling. During the embryonic period, TAK1 is essential for cartilage and joint development as deletion of Tak1 in chondro-osteo progenitor cells leads to severe chondrodysplasia with defects in both chondrocyte proliferation and maturation. We have investigated the role of TAK1 in committed chondrocytes during early postnatal development. Using the Col2a1-CreERT2; Tak1f/f mouse model, we induced deletion of Tak1 at postnatal day 7 and characterized the skeletal phenotypes of these mice at 1 and 3 months of age. Mice with chondrocyte-specific Tak1 deletion exhibited severe growth retardation and reduced proteoglycan and...
Oncogene PKCε controls INrf2–Nrf2 interaction in normal and cancer cells through phosphorylation of INrf2 - Niture, Suryakant K.; Gnatt, Averell; Jaiswal, Anil K.
The INrf2 (Keap1)–Nrf2 cell sensor complex has a crucial role in protection against chemical- and radiation-induced oxidative stress and cellular transformation. INrf2, in association with Cul3–Rbx1, ubiquitylates and degrades Nrf2. Exposure to stressors leads to stabilization of Nrf2 and the coordinated activation of cytoprotective proteins and cellular protection. However, the molecular signal(s) that regulate control of Nrf2 by INrf2 remain elusive. In this report, we demonstrate that phosphorylation of INrf2 at Ser599 and Ser602 by the oncoprotein PKCε is essential for INrf2–Nrf2 interaction, and the subsequent ubiquitylation and degradation of Nrf2. Inhibition of PKCε, knockdown of PKCε and the INrf2S602A...
Insulin receptor isoform switching in intestinal stem cells, progenitors, differentiated lineages and tumors: evidence that IR-B limits proliferation - Andres, Sarah F.; Simmons, James G.; Mah, Amanda T.; Santoro, M. Agostina; Van Landeghem, Laurianne; Lund, P. Kay
Despite evidence for the impact of insulin on intestinal epithelial physiology and pathophysiology, the expression patterns, roles, and regulation of insulin receptor (IR) and IR isoforms in the intestinal epithelium are not well characterized. IR-A is thought to mediate the proliferative effects of insulin or insulin growth factors (IGFs) in fetal or cancer cells. IR-B is considered to be the metabolic receptor for insulin in specialized tissues. This study used a novel Sox9-EGFP reporter mouse that permits isolation of intestinal epithelial stem cells (IESCs), progenitors, enteroendocrine cells and differentiated lineages, the ApcMin/+ mouse model of precancerous adenoma and normal human...
Keratin 8 modulates β-cell stress responses and normoglycaemia - Alam, Catharina M.; Silvander, Jonas S. G.; Daniel, Ebot N.; Tao, Guo-Zhong; Kvarnström, Sofie M.; Alam, Parvez; Omary, M. Bishr; Hänninen, Arno; Toivola, Diana M.
Keratin intermediate filament (IF) proteins are epithelial cell cytoskeletal components that provide structural stability and protection from cell stress, among other cellular and tissue-specific functions. Numerous human diseases are associated with IF gene mutations, but the function of keratins in the endocrine pancreas and their potential significance for glycaemic control are unknown. The impact of keratins on β-cell organisation and systemic glucose control was assessed using keratin 8 (K8) wild-type (K8+/+) and K8 knockout (K8−/−) mice. Islet β-cell keratins were characterised under basal conditions, in streptozotocin (STZ)-induced diabetes and in non-obese diabetic (NOD) mice. STZ-induced diabetes incidence and islet damage...