Molecular Characterization of Photomixotrophic Tobacco Cells Resistant to Protoporphyrinogen Oxidase-Inhibiting Herbicides

Peroxidizing herbicides inhibit protoporphyrinogen oxidase (Protox), the last enzyme of the common branch of the chlorophyll- and heme-synthesis pathways. There are two isoenzymes of Protox, one of which is located in the plastid and the other in the mitochondria. Sequence analysis of the cloned Protox cDNAs showed that the deduced amino acid sequences of plastidial and mitochondrial Protox in wild-type cells and in herbicide-resistant YZI-1S cells are the same. The level of plastidial Protox mRNA was the same in both wild-type and YZI-1S cells, whereas the level of mitochondrial Protox mRNA YZI-1S cells was up to 10 times the level of wild-type cells. Wild-type cells were observed by fluorescence microscopy to emit strong autofluorescence from chlorophyll. Only a weak fluorescence signal was observed from chlorophyll in YZI-1S cells grown in the Protox inhibitor N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide. Staining with DiOC6 showed no visible difference in the number or strength of fluorescence between wild-type and YZI-1S mitochondria. Electron micrography of YZI-1S cells showed that, in contrast to wild-type cells, the chloroplasts of YZI-1S cells grown in the presence of N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide exhibited no grana stacking. These results suggest that the herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox.     

Effect of Starvation on the Endocytic Pathway in Dictyostelium Cells

Dictyostelium discoideum amoebae have been used extensively to study the structure and dynamics of the endocytic pathway. Here, we show that while the general structure of the endocytic pathway is maintained in starved cells, its dynamics rapidly slow down. In addition, analysis of apm3 and lvsB mutants reveals that the functional organization of the endocytic pathway is profoundly modified upon starvation. Indeed, in these mutant cells, some of the defects observed in rich medium persist in starved cells, notably an abnormally slow transfer of endocytosed material between endocytic compartments. Other parameters, such as endocytosis of the fluid phase or the rate of fusion of postlysosomes to the cell surface, vary dramatically upon starvation. Studying the endocytic pathway in starved cells can provide a different perspective, allowing the primary (invariant) defects resulting from specific mutations to be distinguished from their secondary (conditional) consequences.Dictyostelium discoideum is a widely used model organism for studying the organization and function of the endocytic pathway. In Dictyostelium, the organization of the endocytic pathway is similar to that in higher eukaryotes. The pathway in Dictyostelium can be divided into four steps (see Fig. S1 in the supplemental material): uptake at the plasma membrane of particles and medium, transfer through early acidic endocytic compartments (lysosomes), passage into less acidic postlysosomes (PLs), and finally, exocytosis of undigested materials (17, 20). Thus, Dictyostelium recapitulates many of the functions of the endocytic pathway in mammalian cells, including some features observed in most cell types (lysosome biogenesis) and some observed only in specialized cells (phagocytosis, macropinocytosis, and lysosome secretion).Dictyostelium amoebae live in the soil, where they feed by ingesting and digesting other microorganisms. In addition, axenic laboratory strains can macropinocytose medium to ensure their growth. Accordingly, both in natural situations and in laboratory settings, the endocytic pathway plays a key role in the acquisition of nutrients by Dictyostelium cells. In agreement with this notion, several observations suggest that the physiology of the endocytic pathway is sensitive to nutrient availability. In particular, starvation induces secretion of lysosomal enzymes by an unknown mechanism (11). The morphology of the endocytic pathway is also sensitive to nutritional cues, as shown for example by the observation that formation of multilamellar endosomes is enhanced in cells fed with bacteria (18).Here, we analyzed the effect of starvation on the organization as well as the dynamics of the endocytic pathway. We found that, while the overall organization was not extensively modified in starved cells, the dynamics of endocytic compartments were altered. Moreover, analysis of two specific knockout mutants, the apm3 (6) and lvsB (8) strains, revealed that their phenotype was profoundly altered upon starvation, providing further insight about the role of Apm3 and LvsB in the endocytic pathway.     

Generation of Transgenic Rats through Induced Pluripotent Stem Cells

The rat is an important animal model for human disease research. Using inhibitors of glycogen synthase kinase 3 and MAPK signaling pathways, rat embryonic stem cells and rat induced pluripotent stem cells (riPSCs) have been derived. However, unlike rat embryonic stem cells, germ line competent riPSCs have only been derived from Wistar rats at low efficiency. Here, we found that an optimized induction medium containing knock-out serum replacement and vitamin C improved the rate and efficiency of riPSCs generation from Dark Agouti rat fibroblasts and Sertoli cells. riPSCs maintained an undifferentiated status for >30 passages and could differentiate into various cells types including germ cells when injected into rat blastocysts. Moreover, transgenic riPSCs could be generated through the PiggyBac transposon, which could be used to generate transgenic rats through germ line transmission. riPSCs can be used as a novel tool in genetic and genomic studies of the rat.     

Mass Spectrometric Identification of In Vivo Phosphorylation Sites of Differentially Expressed Proteins in Elongating Cotton Fiber Cells

Two-dimensional gel electrophoresis (2-DE)-based proteomics approach was applied to extensively explore the molecular basis of plant development and environmental adaptation. These proteomics analyses revealed thousands of differentially expressed proteins (DEPs) closely related to different biological processes. However, little attention has been paid to how peptide mass fingerprinting (PMF) data generated by the approach can be directly utilized for the determination of protein phosphorylation. Here, we used the software tool FindMod to predict the peptides that might carry the phosphorylation modification by examining their PMF data for mass differences between the empirical and theoretical peptides and then identified phosphorylation sites using MALDI TOF/TOF according to predicted peptide data from these DEP spots in the 2-D gels. As a result, a total of 48 phosphorylation sites of 40 DEPs were successfully identified among 235 known DEPs previously revealed in the 2-D gels of elongating cotton fiber cells. The 40 phosphorylated DEPs, including important enzymes such as enolase, transketolase and UDP-L-rhamnose synthase, are presumed to participate in the functional regulation of numerous metabolic pathways, suggesting the reverse phosphorylation of these proteins might play important roles in elongating cotton fibers. The results also indicated that some different isoforms of the identical DEP revealed in our 2-DE-based proteomics analysis could be annotated by phosphorylation events. Taken together, as the first report of large-scale identification of phosphorylation sites in elongating cotton fiber cells, our study provides not only an excellent example of directly identifying phosphorylation sites from known DEPs on 2-D gels but also provides a valuable resource for future functional studies of phosphorylated proteins in this field.     

The Superoxide Synthases of Rose Cells : Comparison of Assays

In an effort to identify the enzymatic mechanism responsible for the synthesis of reactive oxygen species produced during the hypersensitive response, preparations of rose (Rosa damascena) cell plasma membranes, partially solubilized plasma membrane protein, and cytosol were assayed for the NADH- and NADPH-dependent synthesis of superoxide using assays for the reduction of cytochrome c (Cyt c), assays for the reduction of nitroblue tetrazolium, and assays for the chemiluminescence of N,N′-dimethyl-9,9′-biacridium dinitrate (lucigenin). Each assay ascribed the highest activity to a different preparation: the Cyt c assay to cytosol, the nitroblue tetrazolium assay to plasma membrane, and the lucigenin assay to the partially solubilized plasma membrane protein (with NADH). This suggests that no two assays measure the same set of enzymes and that none of the assays is suitable for comparisons of superoxide synthesis among different cell fractions. With the plasma membrane preparation, the presence of large amounts of superoxide-dismutase-insensitive Cyt c reductase confounded attempts to use Cyt c to measure superoxide synthesis. With the partially solubilized membrane protein, direct reduction of lucigenin probably contributed to the chemiluminescence. Superoxide synthesis detected with lucigenin should be confirmed by superoxide-dismutase-sensitive Cyt c reduction.     

Nonselective Suppression of Voltage-gated Currents by Odorants in the Newt Olfactory Receptor Cells

Effects of odorants on voltage-gated ionic channels were investigated in isolated newt olfactory receptor cells by using the whole cell version of the patch–clamp technique. Under voltage clamp, membrane depolarization to voltages between −90 mV and +40 mV from a holding potential (V_h) of −100 mV generated time- and voltage-dependent current responses; a rapidly (< 15 ms) decaying initial inward current and a late outward current. When odorants (1 mM amyl acetate, 1 mM acetophenone, and 1 mM limonene) were applied to the recorded cell, the voltage-gated currents were significantly reduced. The dose-suppression relations of amyl acetate for individual current components (Na⁺ current: I_Na, T-type Ca²⁺ current: I_Ca,T, L-type Ca²⁺ current: I_Ca,L, delayed rectifier K⁺ current: I_Kv and Ca²⁺-activated K⁺ current: I_K(Ca)) could be fitted by the Hill equation. Half-blocking concentrations for each current were 0.11 mM (I_Na), 0.15 mM (I_Ca,T), 0.14 mM (I_Ca,L), 1.7 mM (I_Kv), and 0.17 mM (I_K(Ca)), and Hill coefficient was 1.4 (I_Na), 1.0 (I_Ca,T), 1.1 (I_Ca,L), 1.0 (I_Kv), and 1.1 (I_K(Ca)), suggesting that the inward current is affected more strongly than the outward current. The activation curve of I_Na was not changed significantly by amyl acetate, while the inactivation curve was shifted to negative voltages; half-activation voltages were −53 mV at control, −66 mV at 0.01 mM, and −84 mV at 0.1 mM. These phenomena are similar to the suppressive effects of local anesthetics (lidocaine and benzocaine) on I_Na in various preparations, suggesting that both types of suppression are caused by the same mechanism. The nonselective blockage of ionic channels observed here is consistent with the previous notion that the suppression of the transduction current by odorants is due to the direst blockage of transduction channels.     

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34⁺) leukemic versus normal specimens. Our data indicate that CD34⁺ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34⁺ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34⁺ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34⁺ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34⁺ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.     

Non-targeted Identification of Prions and Amyloid-forming Proteins from Yeast and Mammalian Cells

The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin.     

Plasma Membrane-Associated Actin in Bright Yellow 2 Tobacco Cells : Evidence for Interaction with Microtubules

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K⁺ and Ca²⁺ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.     

Using in Vivo Biotinylated Ubiquitin to Describe a Mitotic Exit Ubiquitome from Human Cells

Mitotic division requires highly regulated morphological and biochemical changes to the cell. Upon commitment to exit mitosis, cells begin to remove mitotic regulators in a temporally and spatially controlled manner to bring about the changes that reestablish interphase. Ubiquitin-dependent pathways target these regulators to generate polyubiquitin-tagged substrates for degradation by the 26S proteasome. However, the lack of cell-based assays to investigate in vivo ubiquitination limits our knowledge of the identity of substrates of ubiquitin-mediated regulation in mitosis. Here we report an in vivo ubiquitin tagging system used in human cells that allows efficient purification of ubiquitin conjugates from synchronized cell populations. Coupling purification with mass spectrometry, we have identified a series of mitotic regulators targeted for polyubiquitination in mitotic exit. We show that some are new substrates of the anaphase-promoting complex/cyclosome and validate KIFC1 and RacGAP1/Cyk4 as two such targets involved respectively in timely mitotic spindle disassembly and cell spreading. We conclude that in vivo biotin tagging of ubiquitin can provide valuable information about the role of ubiquitin-mediated regulation in processes required for rebuilding interphase cells.Ubiquitination has emerged as a major post-translational modification determining the fate of cellular proteins. One of these fates is proteolysis, whereby the assembly of polyubiquitin chains creates signatures on target proteins that specify delivery to the 26S proteasome for proteolytic destruction. Targeted proteolysis is critical to the control of cell division. For example, the universally conserved mechanism of mitotic exit depends upon rapid proteolysis of mitotic cyclins and securins to drive the transition from mitosis to interphase. This transition is under surveillance by the spindle assembly checkpoint (SAC),¹ which controls the activity of a multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) (1, 2).Much of the known specificity in the ubiquitin-proteasome system (UPS) is mediated at the level of substrate targeting by ubiquitin ligase (E3) enzymes, of which there are more than 600 in human cells. Given these facts, it is perhaps surprising that the APC/C is almost the only known engineer of the protein landscape after anaphase onset, targeting mitotic regulators for destruction with high temporal specificity (2–4). Some roles for nondegradative ubiquitination in regulating the localization of mitotic kinases Aurora B and Plk1 have been described (5–9), and a growing list of reported ubiquitin interactors can modulate ubiquitin-dependent events during mitosis (10). However, the majority of ubiquitination events that have so far been described as occurring at the transition from mitosis to interphase are APC/C-dependent.Two co-activator subunits, Cdc20 and Cdh1, play vital roles in APC/C-dependent substrate recognition (11) by recognizing two widely characterized degrons, the D-box and the KEN motif (12, 13). Computational approaches that have been used to calculate the total number of APC/C substrates from the prevalence of degrons in the human proteome estimate that there are between 100 and 200 substrates (14), and experiments using in vitro ubiquitination of protein arrays have given rise to estimates in the same range (15). Most of the mitotic regulators targeted by the APC/C during mitotic exit in human cells have been identified via in vitro degradation assays or ubiquitination assays on in vitro–expressed pools of substrates (15–18). These approaches have identified several important substrates, but in the absence of in vivo parameters they may not identify substrates whose targeting depends on post-translational modifications or substrates that are only recognized in vivo as components of higher-order complexes. Not all substrates identified in this way have been validated as polyubiquitinated proteins in vivo. Multiple recent proteomic studies have identified large numbers of in vivo ubiquitin-modified sites from yeast (19–21) and human cells (22–29). None of these studies have used synchronized cell populations to provide information on the timing or regulation of substrate ubiquitination.We reasoned that a better view of ubiquitin-mediated processes that regulate mitotic exit would come from identifying proteins that are ubiquitinated in vivo during mitotic exit. With this goal in mind we adopted a system for in vivo tagging of ubiquitin chains with biotin, previously used to identify ubiquitin-conjugated proteins from the Drosophila neural system (30), and applied it to a human cell line (U2OS) that can be tightly synchronized at mitosis. In contrast to several recent studies that employed antibodies specific to the diGly-Lys remnant that marks ubiquitination sites following trypsin digestion (19, 25), an in vivo ubiquitin tagging strategy allows direct validation of candidate ubiquitinated proteins (whether mono- or polyubiquitinated) through immunoblotting of samples. Moreover, in contrast to other methods for affinity tagging of ubiquitin, or affinity purification via ubiquitin-binding domains, the use of the biotin tag enables purification under highly denaturing conditions for stringent isolation of ubiquitin-conjugated material from higher eukaryotes. His₆-tagged ubiquitin is also available for use under denaturing conditions, but it is not generally useful in higher eukaryotic cells, where a high frequency of proteins containing multiple histidine residues confounds the specificity of nickel-affinity pulldowns (as discussed in detail in Ref. 30). Therefore, in this paper we describe the reproducible identification and validation of mitoticphase-specific polyubiquitinated proteins via the in vivo biotinylation of ubiquitin. A large number of polyubiquitinated proteins that we identified are specific to mitotic exit, when the APC/C is active, and we expect that many of them are substrates for the APC/C. We formally identified KIFC1/HSET and Cyk4/RACGAP1 as targets of APC/C-dependent ubiquitin-mediated proteolysis after anaphase onset and investigated the role of their ubiquitination in the regulation of mitotic exit. Cell cycle phase-specific information on protein ubiquitination and the generation of ubiquitinated protein networks provides a framework for further investigation of ubiquitin-controlled processes occurring during the rebuilding of interphase cells.     

Comprehensive Analysis of MicroRNA (miRNA) Targets in Breast Cancer Cells

MicroRNAs (miRNAs) regulate mRNA stability and translation through the action of the RNAi-induced silencing complex. In this study, we systematically identified endogenous miRNA target genes by using AGO2 immunoprecipitation (AGO2-IP) and microarray analyses in two breast cancer cell lines, MCF7 and MDA-MB-231, representing luminal and basal-like breast cancer, respectively. The expression levels of ∼70% of the AGO2-IP mRNAs were increased by DROSHA or DICER1 knockdown. In addition, integrated analysis of miRNA expression profiles, mRNA-AGO2 interaction, and the 3′-UTR of mRNAs revealed that >60% of the AGO2-IP mRNAs were putative targets of the 50 most abundantly expressed miRNAs. Together, these results suggested that the majority of the AGO2-associated mRNAs were bona fide miRNA targets. Functional enrichment analysis uncovered that the AGO2-IP mRNAs were involved in regulation of cell cycle, apoptosis, adhesion/migration/invasion, stress responses (e.g. DNA damage and endoplasmic reticulum stress and hypoxia), and cell-cell communication (e.g. Notch and Ephrin signaling pathways). A role of miRNAs in regulating cell migration/invasion and stress response was further defined by examining the impact of DROSHA knockdown on cell behaviors. We demonstrated that DROSHA knockdown enhanced cell migration and invasion, whereas it sensitized cells to cell death induced by suspension culture, glucose depletion, and unfolding protein stress. Data from an orthotopic xenograft model showed that DROSHA knockdown resulted in reduced growth of primary tumors but enhanced lung metastasis. Taken together, these results suggest that miRNAs collectively function to promote survival of tumor cells under stress but suppress cell migration/invasion in breast cancer cells.     

Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells

Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation.     

Extending the Microtubule/Microfibril Paradigm : Cellulose Synthesis Is Required for Normal Cortical Microtubule Alignment in Elongating Cells

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25–2.5 μm) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 μg mL⁻¹ fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information.     

Structural and Quantitative Evidence for Dynamic Glycome Shift on Production of Induced Pluripotent Stem Cells

We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N- and O-linked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from α-2–3 to α-2–6, and the expression of α-1–2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a “glycome shift” upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs.Induced pluripotent stem cells (iPSCs)¹ are genetically manufactured pluripotent cells obtained by the transfection of reprogramming factors. Such iPSCs were first reported in 2006 for the mouse (1) and in 2007 for humans (2, 3). Although iPSCs have already been used in the fields of drug development and disease models (4–7), basic aspects of iPSCs largely remain to be elucidated to provide us with a fuller understanding of their properties and for therapeutic applications to be developed in the field of regenerative medicine. These aspects include the need for a definitive system to be established to evaluate their properties; e.g. pluripotency, differentiation propensity, risk of possible contamination of xenoantigens, and even the potential for tumorigenesis. Cell surface glycans are often referred to as the “cell signature,” which changes dramatically depending on the cell properties and conditions (8) as a result of changes in gene expression, including epigenetic modifications of glycan-related molecules. Glycans, because of their outermost cell-surface locations and structural complexity, are considered to be most advantageous communication molecules, playing roles in various biological phenomena. Indeed, SSEA3/4 and Tra-1–60/81, which have been used to discriminate pluripotency, are cell surface glycan epitopes that respond to some specific antibodies (9–12).Glycan-mediated cell-to-cell interactions have been shown to play important roles in various biological phenomena including embryogenesis and carcinogenesis (13–16). This might also be the case for the acquisition and maintenance of iPSC and ESC pluripotency, although there remains much to clarify concerning the roles of cell surface glycans in these events. Thus, the development of novel cell surface markers to evaluate the properties of iPSCs and ESCs is keenly required. Toward this goal, a glycomic approach has been made by several groups (17–20). In our previous study using an advanced lectin microarray technique (21), thirty-eight lectins capable of discriminating between iPSCs and SCs were statistically selected, and the characteristic features of the pluripotent state were obtained. The glycan profiles of the parent SCs, derived from four different tissues, were totally different from one another and from those of the iPSCs. Despite this observation, the technique used lacks the ability to determine detailed glycan structures or allow their quantification. For this purpose, a conventional approach based on high performance liquid chromatography (HPLC) combined with matrix-assisted laser desorption-ionization (MALDI) - time of flight (TOF) mass spectrometry (MS) was undertaken for both the definitive identification of glycan structures and their quantitative comparison, which remained unclear in the previous analysis (21).We report here structural data on N-linked and O-linked glycans derived from the human iPSC 201B7 cell line (2) and human dermal fibroblasts (SC) representing undifferentiated and differentiated cells, respectively. For quantitative comparison, the glycans were liberated by gas-phase hydrazinolysis from similar numbers of cells (22–25) fluorescently tagged with 2-aminopyridine (2-AP) at their reducing terminus (26, 27), following which the derived pyridylaminated (PA-) glycans were purified by multiple-mode (i.e. anion-exchange, size-fractionation and reverse-phase) HPLC. Their structures were determined and quantified by HPLC mapping assisted with MALDI-TOF-MS and exoglycosidase digestion analyses. This report thus provides the first structural evidence showing the occurrence of a dynamic “glycome shift” upon induction of pluripotency.     

Determination of in Vivo Dissociation Constant, KD, of Cdc42-Effector Complexes in Live Mammalian Cells Using Single Wavelength Fluorescence Cross-correlation Spectroscopy

The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downstream of membrane-bound receptors through distinct effector pathways. Cdc42-effector protein interactions represent important elements of cell signaling pathways that regulate cell biology in systems as diverse as yeast and humans. To derive mechanistic insights into cell signaling pathways, it is vital that we generate quantitative data from in vivo systems. We need to be able to measure parameters such as protein concentrations, rates of diffusion, and dissociation constants (K_D) of protein-protein interactions in vivo. Here we show how single wavelength fluorescence cross-correlation spectroscopy in combination with Förster resonance energy transfer analysis can be used to determine K_D of Cdc42-effector interactions in live mammalian cells. Constructs encoding green fluorescent protein or monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain (CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP) and insulin receptor substrate protein (IRSp53), were expressed as pairs in Chinese hamster ovary cells, and concentrations of free protein as well as complexed protein were determined. The measured K_D for Cdc42V12-N-WASP, Cdc42V12-CRIB, and Cdc42V12-IRSp53 was 27, 250, and 391 nm, respectively. The determination of K_D for Cdc42-effector interactions opens the way to describe cell signaling pathways quantitatively in vivo in mammalian cells.Over the last 2 decades, we have been successful in describing a myriad of cell signaling pathways that regulate the biology of cells. These pathways are made of elements incorporating protein-protein, protein-lipid and protein-ligand interactions. With the advent of GFP² (1, 2) and its variants (3), it is now possible to genetically encode fluorescent probes into any protein of interest. GFP fusion proteins can be used in live cells giving spatial and temporal resolution to cell signaling pathways (4). To gain mechanistic insights into cellular processes, it is crucial that we measure quantitative parameters to describe cell signaling. In this study, we present an approach based on fluorescence cross-correlation spectroscopy (FCCS) (5, 6) and Förster resonance energy transfer (FRET) to determine quantitative parameters of cell signaling pathways, including the determination of the K_D for Cdc42-effector interactions in live CHO-K-1 (hereafter referred to as CHO) mammalian cells.The RhoGTPase Cdc42 (7, 8) regulates pathways that coordinate cell cycle, morphogenesis, and polarity. Cdc42 is a molecular switch that cycles between an inactive (GDP-bound) and active (GTP-bound) state. The V12 Cdc42 point mutation freezes the protein in an activated GTP-bound form, which binds effectors strongly. In contrast, Cdc42N17 is a dominant negative protein that is GDP-bound and interacts with effectors weakly if at all (9). A major Cdc42 binding site/domain in effector proteins is known as Cdc42- and Rac-interacting binding region (CRIB)³ and was originally found in activated Cdc42 kinase, p21 activated kinase (PAK), and neural Wiskott-Aldrich syndrome protein (N-WASP) (10). The inverse Bin-amphiphysins-Rvs domain adaptor protein IRSp53 is also an effector but binds Cdc42 through a partial CRIB domain (11, 12). Cdc42 interaction with its effectors has two main consequences, which are not mutually exclusive: (i) unfolding of effector to expose the active site and (ii) relocalization of effector to membrane compartments. Thus Cdc42-effector interactions serve as a good model for cell signaling as a whole.Fluorescence correlation spectroscopy and FCCS measure fluctuations in fluorescence of a small number of molecules as they pass through a defined confocal volume, respectively (13, 14, 15). Since the number of molecules in the confocal volume and the confocal volume itself can be determined, concentrations of protein can be measured by fluorescence correlation spectroscopy. Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS) is an FCCS variant in which excitation of two or more probes is achieved by single wavelength one-photon excitation. To date SW-FCCS has been used successfully to follow receptors and receptor-ligand interactions in vitro and in vivo (6, 16, 17).In the present analysis, we take a two-step approach to determining the K_D of Cdc42 binding to CRIB (domain of PAK), N-WASP, and IRSp53. First, we show that the proteins under investigation are indeed interacting with each other directly in vivo by FRET analysis. Here we use acceptor photobleaching (AP)-FRET as well as changes in lifetime (through fluorescence lifetime imaging microscopy (FLIM)) as indicators of FRET. Second, we use SW-FCCS to determine the K_D of Cdc42 interacting with its effectors by measuring the concentration of free protein versus complexed protein. Thus, the combined use of FRET and FCCS allows quantitative analysis of cell signaling pathways in vivo.     

Down-regulation of Mitochondrial Acyl Carrier Protein in Mammalian Cells Compromises Protein Lipoylation and Respiratory Complex I and Results in Cell Death

The objective of this study was to evaluate the physiological importance of the mitochondrial fatty acid synthesis pathway in mammalian cells using the RNA interference strategy. Transfection of HEK293T cells with small interfering RNAs targeting the acyl carrier protein (ACP) component reduced ACP mRNA and protein levels by >85% within 24 h. The earliest phenotypic changes observed were a marked decrease in the proportion of post-translationally lipoylated mitochondrial proteins recognized by anti-lipoate antibodies and a reduction in their catalytic activity, and a slowing of the cell growth rate. Later effects observed included a reduction in the specific activity of respiratory complex I, lowered mitochondrial membrane potential, the development of cytoplasmic membrane blebs containing high levels of reactive oxygen species and ultimately, cell death. Supplementation of the culture medium with lipoic acid offered some protection against oxidative damage but did not reverse the protein lipoylation defect. These observations are consistent with a dual role for ACP in mammalian mitochondrial function. First, as a key component of the mitochondrial fatty acid biosynthetic pathway, ACP plays an essential role in providing the octanoyl-ACP precursor required for the protein lipoylation pathway. Second, as one of the subunits of complex I, ACP is required for the efficient functioning of the electron transport chain and maintenance of normal mitochondrial membrane potential.Eukaryotes employ two distinct systems for the synthesis of fatty acids de novo. The bulk of fatty acids destined for membrane biogenesis and energy storage are synthesized in the cytosolic compartment by megasynthases in which the component enzymes are covalently linked in very large polypeptides; this system is referred to as the type I fatty acid synthase (FAS)² (1, 2). A second system localized in mitochondria is composed of a suite of discrete, freestanding enzymes that closely resemble their counterparts in prokaryotes (3–10), which are characterized as type II FASs (11). Most of the constituent enzymes of the mitochondrial fatty acid biosynthetic system have been identified and characterized in fungi and animals; all are nuclear-encoded proteins that are transported to the matrix compartment of mitochondria. Fungi with deleted mitochondrial FAS genes fail to grow on non-fermentable carbon sources, have low levels of lipoic acid and elevated levels of mitochondrial lysophospholipids (12, 13). These observations indicate that the mitochondrial FAS may serve to provide the octanoyl precursor required for the biosynthesis of lipoyl moieties de novo, as well as providing fatty acids that are utilized in remodeling of mitochondrial membrane phospholipids (14). The mitochondrial FAS system in animals is less well characterized. However, kinetic analysis of the β-ketoacyl synthase enzyme responsible for catalysis of the chain extension reaction in human mitochondria suggested that this system is uniquely engineered to produce mainly octanoyl moieties and has limited ability to form long-chain products (9). Indeed, studies with a reconstituted system from bovine heart mitochondrial matrix extracts confirmed that octanoyl moieties are the main product and are utilized for the synthesis of lipoyl moieties (15). One of the key components of the prokaryotic and mitochondrial FAS systems is a small molecular mass, freestanding protein, the ACP, that shuttles substrates and pathway intermediates to each of the component enzymes. The mitochondrial ACP is localized primarily in the matrix compartment (16), but a small fraction is integrated into complex I of the electron transport chain (17–23). As is the case with many of the other 45 subunits of complex I, the role of the ACP subunit is unclear (24). To clarify the physiological importance of the mitochondrial FAS, and the mitochondrial ACP in particular, in mammalian mitochondrial function we have utilized an RNA interference strategy to knockdown the mitochondrial ACP in cultured HEK293T cells.