Journal of cell science.
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Fig. 3. Strategies for the development of comprehensive system models. Proteomic or genomic information can be directly used to develop pathway and network models (dashed lines). Alternatively, specific cellular processes such as transcription or replication can be modeled in the form of functional modules, using quantitative cell-biological methods including combined imaging and computational approaches. Linking models of functional modules yields an integrated model.
- Two complementary strategies should eventually achieve this goal (Fig. 3).
- Genome and proteome information can be directly assembled into pathways and networks (Fig. 3).
- To obtain a more realistic, complete system-level view we must model single processes in the greatest possible detail, taking into account the networks of molecular interactions, their dynamics and spatial organization (Fig. 3).
Genome Biology
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Outlines of the pathways studied. (a) Methionine (MET); (b) nitrogen catabolite repression (NCR); (c) pseudohypal growth/mating (HYPE); (d) regulation of early meiotic genes (CCYCLE); (e) pleiotropic drug resistance (PDR). The sub-networks enlarged from Figure 2, with the identified motifs within the pathway drawn from the interaction databases, are shown on the left (colors and conventions are the same as in Figure 2). A schematic representation of the regulation mechanisms for the same pathways, based on the present experimental knowledge as discussed in the text, is shown on the right. Full lines represent transcriptional regulation, dashed lines non-transcriptional regulation, and wavy lines transformations and syntheses. Arrowheads, positive regulation; lines ending in a terminal bar, negative regulation.
- Two examples are the sub-networks methionine synthesis (MET) and nitrogen catabolite repression (NCR), shown shaded in Figure 2 and in more detail in Figure 4.
- Sub-network MET in Figures 2 and 4a shows the interaction graph for the cluster of interacting genes centered on CBF1, MET4 and MET28.
- Two loops drive the dynamics of complex availability, sketched in Figure 4a.
- This information does not unambiguously emerge from the topology of the network (Figure 4a, left), as the topology is also compatible with the three proteins acting separately.
- In conclusion, the key features of the methionine synthesis pathway do not seem to hinge on transcriptional regulation via the motifs instances shown in Figure 4a.
- The NCR system shown in Figures 2 and 4b is used by the cell to control the synthesis of proteins capable of handling poor sources of nitrogen.
- The accepted mechanisms of NCR are as follows ([26-28] and see Figure 4b).
- The sub-network HYPHE in Figure 2 and Figure 4c is formed by one motif of type III.5, involving the two genes STE12 and TEC1.
- The sub-network around IME1 in Figure 2 and Figure 4d is made of one II.1, two III.5 and one III.6 motifs and is implicated in the activation of early meiotic genes.
- The information processing is rather implemented by alternative routes and the picture of the interactions shown on the sub-network CCYCLE in Figure 2 and Figure 4d (left) appears to be insufficient and misleading.
- The corresponding sub-network (named PDR in Figure 2 and 4e) is composed of eight motifs of type III.1 (so-called feed-forward loops) and one of type II.1, showing a star-like configuration with PDR1 and PDR3 in a central position.
Endocrinology.
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FIG. 6. Regulation by E2 of genes encoding receptors and signal transduction proteins. A and B, Microarray data are shown for prostaglandin E synthase (PGES) and prostaglandin receptor EP3, which are both stimulated by E2, and for the chemokine receptor CXCR4 and the signaling protein B cell linker (BLNK), which are both down-regulated by E2. C and D, Real-time PCR data are shown for four receptors found to be either rapidly up-regulated or down-regulated by E2.
- Two pathways of note, which are regulated at several levels, include the prostaglandin E pathway (Fig. 6A) and SDF-1 (up-regulated by E2; as shown in Fig. 5) and its chemokine receptor CXCR4 (Fig. 6B) pathway.
- The enzyme prostaglandin E synthase as well as the prostaglandin E receptor (EP3) were found to be stimulated by E2 (Fig. 6A).
- In contrast, the CXCR4 receptor, which may play a role in cell motility, was down-regulated by E2, as was BLNK (Fig. 6B), a B cell scaffolding protein that is involved in regulating the activity of CXCR4 (36).
- Some other important receptors found to be regulated by E2 include the highly up-regulated calcitonin receptor (Fig. 6C) and the down-regulated erythropoietin receptor, IL-1 receptor type I, and Notch 3, which are rapidly decreased, by 4 and 8 h (Fig. 6D).
Lipids in Health and Disease
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Pathway analysis. Figures 3-7 represent signaling pathways for 58 focus genes selected from Table 3 by Ingenuity Systems software (Redwood City, CA. The following signaling cascades are shown: JUN, TNF, and CDKN2A signaling cascade affecting: DNA replication; recombination and repair; immune response; and cell cycle (Fig. 3); TGFB1 signaling cascade affecting: cell morphology; cancer; and tumor morphology (Fig. 4); CTNB1 signaling cascade affecting: cell signaling; gene expression; and cell cycle (Fig. 5); INS1/hRAS signaling cascade affecting: carbohydrate metabolism; endocrine disorders; and metabolic disease (Fig. 6); and MYC signaling cascade affecting: viral function; gene expression; and cell Cycle (Fig. 7). Differentiating groups (per Table 3) are overlaid onto the signaling diagrams, and abbreviated: FU, fungal; FI, fish oil; CO, combination diet. When CO was the differentiating group, absolute differences between FU and FI are indicated. Intracellular location of focus genes (subscripts) are annotated: C, cytoplasm; E, extracellular; N, nucleus; P, plasma membrane; U, unknown. Major canonical functional/signaling categories associated with genes in the figures identified by the software, are shown in yellow boxes.
- Pathway analysis (Figs. 3, 4, 5, 6, 7) was performed on selected transcripts differentiating the groups (Table 3; described in next section).
- TGFB1 signaling (Fig. 4)
- Herein, we focus on genes implicated in pathway analysis (Figs 3, 4, 5, 6, 7) and linkable to specific functions (Tables 2, 3).
- Pathway analysis (Figs. 3, 4, 5, 6, 7)
- Network diagrams including these 58 focus genes and some connection genes were redrawn for simplicity (Figs. 3, 4, 5, 6, 7).
- The 5 networks were independent from one another, except for an indirect connection via JUN linking Figs. 3, 4.
The Journal of biological chemistry.
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Fig. 5. Evidence of the extraparasitized PfNSF A, after saponin treatment, P. falciparum cells (lane 1), an extracellular particulate fraction (lane 2), and a soluble fraction (lane 3) were isolated as described under "Experimental Procedures." After dissociation with SDS-sample buffer, each sample was analyzed by immunoblotting with the antibodies as indicated. B-G, immunohistochemical detection of PfNSF (B), PfNSF in noninfected erythrocyte (C), vacuolar H+-ATPase subunit A (V-ATPase) (D), H+-pumping pyrophosphatase (V-PPase) (E), and the serine repeat antigen protein (F) in parasitized erythrocytes. The parasitized erythrocytes were immunostained with the indicated antibodies and then observed by fluorescence microscopy. Vital staining with C5-ceramide was also performed to reveal localization of the tubovesicular membrane networks (TVM) (G). P, P. falciparum cell; EMP, erythrocyte plasma membrane. Bar, 5 µm. (Note: during preparation, H+-pumping pyrophosphatase was digested by protease, resulting in a low molecular weight fragment (A)).
- Presence of Extracellular PfNSF-- During isolation of malaria parasites from parasitized erythrocytes with saponin (see "Experimental Procedures"), we noticed that an appreciable level of anti-NSF immunoreactivity remained in the supernatant after isolation of the parasite cells (Fig. 5A).
- Western blotting experiments indicated that PfNSF was present in the extracellular particulate fraction as well as in the parasites but not in the supernatant (Fig. 5A).
- Vacuolar H+-ATPase and H+-pumping pyrophosphatase, which are known to be present in the parasite (35, 41), were detected in parasite cells but not detected in either the extracellular particulate fraction or the supernatant (Fig. 5A).
- The anti-NSF antibodies immunostained the parasite cells in parasitized erythrocytes (Fig. 5B), whereas no immunoreactivity was found in noninfected erythrocytes (Fig. 5C).
- Significantly, the PfNSF immunoreactivity was present within the vesicular structures outside the parasite cells (Fig. 5B).
- Consistent with the distribution observed on the immunoblotting shown in Fig. 5A, no such extraparasitized vesicular structures were observed in the immunoreactivities against antibodies for vacuolar H+-ATPase (Fig. 5D), H+-pumping pyrophosphatase (Fig. 5E), serine repeat antigen protein, markers for the peripheral space between the parasitophorus vacuolar membranes, and the plasma membrane of the malaria parasite (Fig. 5F).
- Vital staining with C5-ceramide revealed tubovesicular membrane networks (6) (Fig. 5G).
- Consistent with the immunohistochemistry described in Fig. 5A, immunogold particles for PfNSF were selectively and intensely labeled in infected P. falciparum cells, whereas few immunogold particles were observed in erythrocyte cytoplasm (Fig. 8A).
- The antibodies immunostained the whole body of malaria parasites infecting erythrocytes (Fig. 5).
American journal of physiology. Heart and circulatory physiology.
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Fig. 1. Schematic of experimental site. A transverse arteriole gives rise to a branch arteriole. Branch arterioles, from which capillary networks arise, give rise to daughter arterioles termed module inflow arterioles, which in turn feed groups of capillaries (modules). Number of modules in a capillary network is usually 5-8 (see Ref. 3). For clarity, only two modules are shown in this schematic. A, transmission pathway for module inflow arteriole. B, transmission pathway for branch arteriole.
- The relevant anatomy for these protocols is schematized in Fig. 1.
- Capillary modules for study were also selected according to the orientation of the muscle fibers relative to the vasculature (generally perpendicular to the module inflow arteriole, see Fig. 1) and the ability to clearly visualize all relevant parts of the vasculature.
- In all experiments, diameters were measured in module inflow and branch arterioles (i.e., in the two arteriolar generations upstream of the selected capillary module) at rest and in response to muscle contraction underlying the capillary module (Fig. 1).
- Coral L. Murrant for critical reading of the manuscript, helpful discussions, and for generation of Fig. 1.
Journal of cell science.
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Fig. 5. sec21-3 mutant maintained for 5 minutes (a) and 20 minutes (b) at 37°C, blocked for 20 minutes at 37°C and returned for 5 (c,d) or 10 minutes (e,f,g) to permissive temperature (24°C). (a) After 5 minutes at 37°C, tubular fragments with nodular dilations (arrowhead) are interspersed within the cytoplasm. An ovoid mass made up of interconnected short ribbon-like ER elements (*) is continuous with the nuclear envelope. V, small vacuole; N, nucleus; SG, free secretion granules. Magnification x40,800. (b) After 20 minutes at 37°C, the secretion granules and tubular fragments with or without dilations are no longer seen. A mass of interconnected ribbon-like ER elements (*) forms a bridge between the nuclear envelope and the subplasmalemmal ER at the top-right. Two vacuole-like structures (V) are present next to the nucleus (N). Magnification x35,400. (c) As early as 5 minutes after shifting the temperature down to 24°C, fenestrations (white arrow) and tubular networks (arrow) with dilations (arrowheads) are seen at the periphery of an unperforated sheet of ER, which is itself continuous with the subplasmalemmal ER (ER). Magnification x42,000. (d) After 5 minutes at 24°C, a tubular network (arrow) with more intensely stained dilations (arrowhead) is observed. SG, a free secretion granule; CW, cell wall. Magnification x48,000. (e) At 10 minutes after shifting the cells back to 24°C, numerous secretion granules (SG) are interspersed within the cytoplasm and accumulate in the bud on the right. At the center left, an ER sheet seen in face view shows peripheral tubules (arrow) and dilations (arrowheads) with size and staining properties similar to those of secretion granules. Magnification x59,100. (f) After 10 minutes at 24°C, a broken tubular network, presumably a Golgi element (G), with dilations of various sizes and staining intensities is observed at the top-left. SG, free secretion granules; ER, an ER sheet with a fenestrated periphery. Magnification x35,000. (g) Small poorly contrasted vacuole-like structures (*) accumulate inside or in close proximity to a large intensely reactive vacuole (V). Magnification x23,300.
- When the sec21-3 mutant was incubated for 5 minutes at the restrictive temperature (37°C), the non-perforated ER elements extending from the nuclear envelope remained unaffected (Fig. 5a).
- Within the cytoplasm, tubular fragments in which large, strongly stained nodules were interconnected by less reactive areas were frequently observed (Fig. 5a).
- Ovoid masses of tightly anastomosed membranes were frequently encountered in close contact with the nuclear envelope (Fig. 5a).
- After 20 minutes at 37°C, the tubular fragments were no longer visible, whereas extensive masses of anastomosed ribbon-like elements formed bridges between the nuclear envelope and the poorly perforated sheets underlying the plasma membrane (Fig. 5b).
- As early as 5 minutes after returning the mutant to permissive temperature, oval fenestrae (Fig. 5c) and polygonal arrays of membranous tubules (Fig. 5c,d) were seen at the periphery of nonperforated ER sheets.
- Dilations reappeared at the intersections of the anastomosed tubules (Fig. 5c,d).
- After 10 minutes, numerous, more or less fragmented tubular networks with dilations of various size and density were interspersed throughout the cytoplasm (Fig. 5f).
- Occasionally, dilations similar to mature secretion granules were present at the edges of ER fenestrae (Fig. 5e).
- Secretion granules were numerous at this time; they were dispersed throughout the cytoplasm (Fig. 5e,f) but sometimes, as in wild-type cells, accumulated in the bud (Fig. 5e).
- Vacuole-like structures appeared instead to accumulate in large, intensely stained vacuoles or in their immediate vicinity (Fig. 5g).
Endocrinology.
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FIG. 5. Regulation of growth factor gene expression by E2. Microarray data over the 48-h E2 treatment time reveals distinct time courses for the four up-regulated (SDF-1, amphiregulin, stanniocalcin 2, and VEGF) and the three down-regulated (TGFß3, BMP4, and inhibin ßB) genes encoding growth factors.
- Amphiregulin, stromal cell-derived factor 1 (SDF-1; also known as chemokine ligand 12), stanniocalcin 2, and vascular endothelial growth factor (VEGF), all previously identified as E2-stimulated genes, showed early increases after E2 exposure, and as shown in Fig. 5, each of these genes had a unique time course of regulation, with amphiregulin and SDF-1 showing sustained high fold stimulated levels over the 48 h, and VEGF and stanniocalcin being elevated primarily at early times only.
- Several novel genes associated with the suppression of breast cancer cell proliferation were found to be down-regulated by E2, including TGFß3, bone morphogenic protein 4 (BMP4) and the inhibin ßB-subunit, which is common to both activin and inhibin (Table 3 and Fig. 5).
- Two pathways of note, which are regulated at several levels, include the prostaglandin E pathway (Fig. 6A) and SDF-1 (up-regulated by E2; as shown in Fig. 5) and its chemokine receptor CXCR4 (Fig. 6B) pathway.
Journal of cell science.
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Fig. 1. GFP-Rab22a localizes to early and late endosomes. CHO cells were transfected with pEGFP-Rab22aWT and 24 hours later the distribution of the GFP fluorescence was recorded. Early endosomes were labeled by a 5 minute uptake of Rh-HRP (A-C) or Rh-Tf (D-F). Late endosomes were labeled by a 5 minute uptake of Rh-HRP followed by 15 minute chase (G-I) or by immunostaining with an anti CI-M6PR antibody (J-L). Notice the large size of some Rab22a-positive vacuoles (arrow heads) and the altered morphology of early and late compartments in cells overexpressing Rab22a (t) when compared with untransfected cells (u). The inset in B shows tubular projections attached to a large vesicle. The bars represent 7 µm.
- Twenty-four hours after transfection of CHO cells, the fluorescent protein associated with small and large round-shaped structures, and with tubular networks (Fig. 1A,D,G,J).
- Rab22a clearly labeled the perimeter of several large structures, indicating that they correspond to large vesicles and not to aggregates of small compartments (arrowheads, Fig. 1D,G).
- Tubular projections were frequently observed (inset, Fig. 1B).
- The GFP-tagged protein partially colocalized with early endosomes loaded by fluid phase (Rh-HRP, 5 minute uptake, Fig. 1C) and receptor-mediated (Rh-tf, 5 minute uptake, Fig. 1F) uptake.
- Rab22a was also found associated with late endosomes (Rh-HRP, 5 minute uptake, 15 minute chase, Fig. 1I).
- Localization with late endosomes was confirmed by immunocytochemistry using an antibody that recognizes the CI-M6PR (Fig. 1L).
- It was interesting to observe that cells overexpressing Rab22a presented enlarged structures loaded with early and late endosomal markers (compare the size of rhodamine-labeled vesicles in transfected and untransfected cells in Fig. 1C,I,L).