The Journal of cell biology.
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Figure 2. HR12-treatment induces stress-fiber formation in Rat1/ras cells. Rat1/ras cells were treated as in Fig 1 and stained with TRITC-labeled phalloidin. (a) No treatment, (b) 24 h, (c) 48 h, (d) 72 h, and (e) 48 h exposure to HR12, followed by 24 h without the inhibitor. (f) Untransformed Rat1 cells. Bar, 10 µm.
- We examined the effect of the Ras-FTI, HR12, on Rat1/ras cells (fibroblasts stably transformed with Ha-rasV12). Fig 1 Fig 2 Fig 3 show a comparison of nontransformed Rat1 cells, Rat1/ras cells, and HR12-treated Rat1/ras cells (48 h, 20 µM).
- Stress fibers, labeled by TRITC-phalloidin, were also disrupted (Fig 2).
- In Rat1 cells, F-actin was engaged as a dense web of conspicuous stress-fibers (Fig 2 f).
- Rat1/ras cells appeared elongated, with numerous F-actincontaining protrusions and ruffles, but essentially no actin bundles (Fig 2 a).
- At 48 h and later, circumferential bundles also became apparent (Fig 2c and Fig d).
- Removal of HR12 for 24 h, after a 48-h treatment, resulted in the loss of organized actin bundles and the appearance of a more diffuse pattern (Fig 2 e).
- Actin exists in a dynamic equilibrium between a Triton-soluble pool and Triton-insoluble cytoskeletal filaments. Fig 2 shows that HR12 triggers dramatic assembly of actin into stress fiber networks in Rat1/ras cells.
- In contrast to the reports cited above in support of the RhoB theory, we show here: (a) by 15 h, most of Ras population is unprocessed (Fig 6), which corresponds to the kinetics of the morphological changes (Fig 1 and Fig 2); (b) NIH3T3myr-ras cells fail to form adhesions in response to HR12, unlike NIH3T3 cells transformed by farnesylation-dependent oncogenic ras (Fig 9); and (c) HR12 treatment leads to the accumulation of high levels of oncogenic Ras in the cytoplasm, followed by potent inhibition of Mek/Erk activation (Fig 5 and Fig 6).
Journal of neurophysiology.
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Fig. 1. Line drawing of the dorsal surface of the cerebropleural and pedal ganglia indicating locations of identified neuronal somata of networks for feeding (inset: MCG, PCP, PSE, PCT, 2 I1s, and 3 I2s), escape swimming (inset: A1, As1-4, A10, A3, and A-ci1), and locomotion (G neuron cluster). Filled circles: serotonin immunoreactive somata. Whereas bilaterally symmetrical, somata are only shown unilaterally for convenience. Cell abbreviations: MCG, metacerebral giant neuron; PCP, phasic paracerebral interneuron; PSE, polysynaptic excitor of the PCP; PCT, tonic paracerebral interneuron; I1, Interneuron 1; and I2, Interneuron 2. Nerve abbreviations, cerebropleural ganglion: BWN, body wall nerve; sBWN, small body wall nerve; CBC, cerebrobuccal connective; aCPC, anterior cerebropedal connective; pCPC, posterior cerebropedal connective; CVC, cerebrovisceral connective; MN, mouth nerve; OVN, oral veil nerve; RN, rhinophore nerve; SCC, subcerebral commissure; and TN, tentacle nerve. Pedal ganglion: aLBWN, anterior lateral body wall nerve; pLBWN, posterior lateral body wall nerve; PC, pedal commissure; pPC, parapedal commissure; aPN, anterior pedal nerve; mPN, medial pedal nerve; and pPN, posterior pedal nerve.
- Figure 1 shows the relative positions of the neuron cell bodies.
- Known premotor neurons that either compose the central pattern generator for escape swimming or receive outputs from it are found on the dorsal cerebral region of the cerebropleural ganglion, in a group called the A cluster (Fig. 1).
- Feeding behavior is inhibited during swim episodes in part by spike activity in the swim neuron A1, which was previously shown to activate a strong polysynaptic inhibitory pathway to the PCp feeding command neurons (Jing and Gillette 1995) (Fig. 1).
Blood.
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Figure 2. The endothelium as a therapeutic target. An understanding of the endothelial response to pathogens provides a foundation for therapeutic design. For purposes of illustration and discussion, the temporal sequence of events is depicted from left to right. In sepsis, the endothelium is activated by LPS-mediated engagement of the toll-like receptor (TLR4) or by the interaction of inflammatory mediators (IL-6, TNF-{alpha}, IL-1, kinins, and C5a are shown) with their respective receptors (drawn as a single representative receptor). At the same time (or later during the sepsis cascade), the endothelium may be conditioned by other environmental factors, such as hypoxia, low blood flow, changes in temperature, acid-base/electrolyte abnormalities, and/or hyperglycemia. The interaction of extracellular mediators with their receptors leads to activation of downstream signaling pathways (including MAPK and PKC), which in turn promote posttranscriptional changes in cell function or alter gene expression profiles through a number of transcription factors, including NF-{kappa}B, GATA-2, and AP-1. The up-regulation of cell adhesion molecules on the surface of the endothelium (P-selectin, E-selectin, VCAM-1, and ICAM-1 are shown) promotes increased adhesion, rolling, and transmigration of circulating leukocytes. Leukocyte-endothelial interactions further modulate the phenotype of these cells. The release of cytokines from the endothelium results in additional activation of monocytes and endothelial cells. Increased expression of procoagulants (eg, TF) and/or reduced expression of anticoagulants (eg, TM, EPCR) promote increased thrombin generation and fibrin formation. Various components of the coagulation pathway (including serine proteases, fibrin, and platelets) may signal directly in the endothelium through protease-activated receptors (PAR-1 is shown). Changes in the expression of proapoptotic and antiapoptotic genes (along with a multitude of posttranscriptional changes) may result in a shift in balance toward programmed cell death. During the process of activation, NADPH oxidase may induce the formation of reactive oxygen species (ROS), nitric oxide (NO) is released, and cell permeability is increased. In keeping with the theme of spatial and temporal dynamics, the relative activity of the various pathways will vary between different endothelial cells and from one moment to the next. Not shown are the critical interactions between the endothelium and underlying extracellular matrix and parenchymal cells. Temp indicates temperature; ICAM-1, intracellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule; EC, endothelial cell; TF, tissue factor; TM, thrombomodulin; EPCR, endothelial protein C receptor; NO, nitric oxide; PGI2, prostacyclin. Receptors are labeled in light font.
- In some cases, pathogens directly infect intact endothelial cells.81 More commonly, components of the bacterial wall (eg, lipopolysaccharide [LPS]) activate pattern recognition receptors on the surface of the endothelium.22-25 Finally, a myriad of host-derived factors activate endothelial cells, including complement, cytokines, chemokines, serine proteases, fibrin, activated platelets and leukocytes, hyperglycemia, and/or changes in oxygenation or blood flow (see Table 1 and Figure 2 for an expanded list of host-derived mediators).
- The other is to target endothelial components (eg, cell surface receptors, signaling pathways, transcriptional networks, or endothelial cell gene products) that are involved in mediating the sepsis phenotype (Figure 2; Table 1).
Blood.
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Figure 7. Genetic regulatory networks controlling early PLM and ALM development. Direct relationships, illustrated by continuous lines, are defined by 3 criteria: (1) target gene expression is affected by perturbation of activator; (2) target gene and activator are coexpressed; (3) target gene promoter/enhancer sequences contain binding sites for activators, or length of time between perturbation of activator and effect on target gene is probably insufficient to allow for synthesis of intermediates. Where criteria 1 and 2 are met but 3 is unknown, the relationship is described as indirect and depicted by a dashed line. Lmo2 does not bind DNA, but is known to be an obligate member of a multiprotein complex containing Scl (represented here by the "and" function).15,37 PLM (Erythroid): A gata1 enhancer contains binding sites for the multiprotein complex containing Scl and Lmo2.49 Our data show that initiation of gata1 expression is Scl-dependent. Initially, runx1 expression is dependent on Scl, whereas hhex and dra are Scl-independent. After 7 somites (12.5 hpf), hhex and dra become dependent on Scl, whereas runx1 expression gradually becomes Scl-independent. ALM (Myeloid): Pu.1 expression is initially dependent on Scl, whereas hhex and dra are independent. After 7 somites (12.5 hpf), hhex and dra become Scl-dependent. Unknown activators of Scl-independent genes are depicted as genes X, Y, and Z. It is possible that X and Y represent the same gene due to similarities in timing of involvement.
- This period of time is unlikely to be long enough for the synthesis of intermediates; therefore, our data suggest that Scl activates gata1 directly in embryonic precursors to erythroid cells (Figure 7).
- Thus, pu.1 looks like a direct target for Scl, acting with Lmo2 and Gata2, in myeloid precursors (Figure 7).
- By 7 somites, however, hhex, dra, and all other hematopoietic gene expression measured in this study had become dependent on Scl, either directly or indirectly (Figure 7).
- Expression of runx1 in the PLM follows that of scl by less than 1 hour, making it another possible direct target (Figure 7).
- Over time, an Scl-independent pathway is able to compensate for the absence of Scl, and runx1 expression levels in both the ALM and PLM increase (Figure 7).
The Journal of biological chemistry.
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FIG. 1. Global trace residues identify a canonical signal transduction pathway with three functional subdomains: a ligand trigger region, an allosteric linking core and a G protein-coupling region. A shows the top 20% of class A determinants (C{{alpha}} atoms) mapped onto the rhodopsin structure (1HZX [PDB] ) with retinal depicted as a yellow stick model. B shows exclusively the subset that affects ligand binding (cyan spheres) on mutation, forming the trigger region. C shows in blue the residues that cause constitutive activity or folding/expression effects on mutation. They cluster to form an intermediate linking core involved in conformational activation linking the trigger region to a coupling region shown in D (magenta spheres) consisting of residues that affect G protein coupling/activation. Ile-75 and Leu-79, which had not previously been assigned any function, are depicted as yellow spheres.
- Subtle changes were not taken into account, and given multiple functional effects a residue was classified by the one most frequently observed (Fig. 1 and Table II).
- The 39 residues ranked in the top 20th percentile (Fig. 1 and Tables I and II) are predicted to be generically important.
- To correlate the structural location of these trace residues with their function, we further sub-classified them according to their most frequently observed mutational effect and mapped this information onto the rhodopsin structure (see Fig. 1 and Supplementary Material).
- Fourteen residues, colored cyan in Fig. 1B, predominantly affect ligand binding (Table II) and segregate in the extracellular half of the cluster.
- Sixteen residues, colored blue in Fig. 1C, cause constitutive activity, folding, or expression defects, and these segregate roughly in the middle of the cluster (Table II).
- Seven residues, colored magenta in Fig. 1D, primarily affect G protein coupling and signaling, and those fill the cytoplasmic base of the transmembrane domain.
- We traced 129 rhodopsin sequences and subtracted from the resulting trace residues the global ET residues (Fig. 1) at the same percentile rank (Fig. 2).
- B shows the trace of class A sequences from Fig. 1A.
- In addition, correlation of functional effects of mutations with location revealed that the switch involves three functionally distinct but structurally connected sub-clusters: a trigger region (Fig. 1B), near the retinal binding pocket of rhodopsin, a coupling region (Fig. 1D) near the G protein-coupling site, and a linking core (Fig. 1C) between the first two.
- The seven coupling region residues fill the cytoplasmic base of the transmembrane domain, presumably forming a platform for G protein interactions, and six residues border the key G protein coupling loops 2 and 3 (Fig. 1D).
- The generic trigger region extends into the transmembrane area nearly up to the retinal binding pocket in rhodopsin (Fig. 1B), suggesting an intimate role in ligand sensing, which is supported by the mutagenesis data (Table II).
- Most trace residues, however, lie in the intermediate linking core (Fig. 1C) drawn heavily from residues forming key hydrogen bond networks such as TM1-TM2-TM7 (critical role for Asn-55), TM2-TM3-TM4 (Asn-78), and TM6-TM7 (Met-257, Asn-302, and Pro-303) (5052).
Journal of neurophysiology.
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Fig. 7. Carbachol (CCH)-induced {theta} GABAergic depolarization of hippocampal CA1 pyramidal cells enables GABAergic inputs to entrain CA1 pyramidal cells. A: {theta} rhythm stimulation evoked IPSPs before CCH application (Control). During CCH application (50 µM, 30 min; middle panel), the same pattern of stimulation entrained activity of the pyramidal cell. Action potentials were evoked at the 2nd, 3rd, and 5th pulses, with action potentials truncated. The evoked postsynaptic responses were abolished by BIC (1 µM, 30 min). B: schematic diagram of network and discharge relationship between CA1 pyramidal cells and GABAergic interneurons. Top: cholinergic inputs (synaptic or diffuse transmission) act on pyramidal cells, inducing HCO3{-} accumulation and enhanced HCO3{-} conductance through the GABAA receptor channels. The {theta} rhythmic activity of GABAergic interneurons can then directly be transmitted to the pyramidal cells, entraining their activity and altering signal processing. Bottom: peak discharge relationship of pyramidal cells (black rectangle) and interneurons (shadow oval) in {theta} rhythm in behaving and rapid eye movement (REM) sleep (based on O'Keefe and Recce 1993; Shen et al. 1997; Csicsvari et al. 1999). The arrow indicates the discharge shift of a place cell, starting from shadow rectangle, in relation to the {theta} activity as the animal travels into the place field of the place cell. GABA, GABAergic interneurons; Pyr, CA1 pyramidal cells; SCH, Schaffer collateral pathway. C: top left, top right, and bottom left are examples of traces without truncation, showing that the brief pulse of stimulation at 5.5 Hz elicited action potentials even though the 1st brief pulse of stimulation was insufficient to evoke action potential. Bottom right illustrates responses to co-stimulation of Sch at below-threshold intensity and GABAergic inputs at pre-CCH (Control) and post-CCH (CCH) periods. Inset shows the initial segment at ×3 amplification with action potential truncated. Arrowheads indicate the time when the brief pulse of stimulation was delivered.
- On the other hand, in the presence of CCH, stimulation of GABAergic inputs elicited instantly phase-locked firing of pyramidal cells (Fig. 7, A and C; n = 14).
- The postsynaptic GABAergic response to the first stimulation pulse usually did not reach action potential threshold (Fig. 7, A and C).
- The postsynaptic GABAergic responses were sensitive to BIC, indicating the involvement of the same receptor channels (Fig. 7A).
- Before the CCH administration, co-stimulation of Sch at the set intensity (50% below the threshold) and GABAergic inputs (50 µA, 50 µs) largely abolished the Sch stimulation-induced excitatory potential (by 89.5 ħ 4.3%, n = 8, P < 0.05; Fig. 7C).
- Action potentials, however, were evoked by co-stimulation of Sch at below-threshold intensity together with reversed GABAergic inputs in all cases (n = 8, P < 0.05; Fig. 7C, bottom right).
- 1999) and reconfigures the operations of hippocampal networks into patterns of activity associated with GABAergic inputs (Fig. 7B).
- Place cells thus fire in phase with progressively stronger GABAergic inputs from interneurons and at earlier phases of the cycles as the rat moves toward the center of their place field (Fig. 7B) (Csicsvari et al.
- The reversed response, although often not strong enough to reach threshold by itself, can entrain the pyramidal cells when stimulated at a frequency (Fig. 7).
- Furthermore, the reversed response can effectively enhance weak excitatory inputs to reach threshold (Fig. 7C) (Sun et al.
The Journal of cell biology.
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Figure 3. Attenuation of PI3K activity results in cross-modulation of other signaling pathways and intermediates. Cell lysates from S-1, T4-2, and T4-2+LY grown in 3D lrBM or on 2D plastic substrata for 10 d were analyzed for expression of (A) EGFR, ß1 integrin, phosphorylated Akt (serine 473)/total, phosphorylated GSK-3ß (serine 9)/total, and (B) PTEN (n = 3); E-cadherin was used as the loading control. It was shown previously that the total level of E-cadherin does not change under these conditions (Weaver et al., 1997).
- T4-2 cells treated with LY294002 show reduced levels of EGFR and ß1 integrin (Fig. 3 A).
- This effect depended upon 3D lrBM as it is not observed in cells cultured on two-dimensional (2D) plastic substrata (it should be noted that inhibition of PI3K activity, as measured by activation of downstream mediators Akt and GSK-3ß, was equally effective in cells on 2D or in 3D; Fig. 3 A).
- In addition, our results revealed that PTEN, the antagonist of PI3K that acts to dephosphorylate PIP3 and which becomes down-regulated in many carcinomas (Simpson and Parsons, 2001; Yamada and Araki, 2001), is also a component of the cross-modulated signaling network, as treatment of T4-2 cells with LY294002 resulted in an increase of PTEN to the level of the nonmalignant cells; this modulation, too, was seen only in cells cultured on 3D lrBM (Fig. 3 B).
- The evidence that inhibition of PI3K can affect crossmodulation of a number of distinct signaling pathways is a demonstration that pathways downstream of PI3K are integrated into transduction networks when cells are grown in the physiological 3D lrBM; consistent with this model, we found that reversion of the tumor cells to a normal phenotype was associated with increased expression of PTEN, the PI3K antagonist (Fig. 3 B).
- Normalization of signaling pathways in T4-2 cells in response to inhibition of PI3K is dependent upon culture in 3D lrBM, as T4-2 cells grown on 2D tissue culture plastic do not show the dramatic downmodulation of ß1 integrin and EGFR (Fig. 3 A), up-regulation of PTEN (Fig. 3 B), or the alterations in cellular morphology in response to treatment with inhibitors of PI3K (Figs.
- 2 and 3).
- We now show that components of the PI3K signaling pathway are involved in this cross-modulation process, as phenotypic reversion by inhibition of PI3K is associated with, and presumably, supported by, up-regulation of the PI3K antagonist, PTEN (Fig. 3).
- This also requires the establishment of organized structures in 3D lrBM, as treatment of T4-2 cells with PI3K inhibitors does not result in up-regulation of PTEN when cells are grown on 2D plastic substrata (Fig. 3 B).
The functional genomic response of developing embryonic submandibular glands to NF-kappaB inhibition
BMC Developmental Biology
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Connections Map. This signaling map reflects the pathways investigated in SMGs. Known and putative connections are based on references [6], [11], [23], [36], [76]-[108].
- These SMG cellular and extracellular components may be visualized as a Connections Map which details the functional relationships within and between pathways (Fig. 1).
- Complex networks of biological signaling pathways (Fig. 1) emerge from the interconnections of simple pathways under local control [15-17].
- With the present experiments, we sought a glimpse of the extraordinarily complex behaviors of a focused signaling network (Fig. 1).
- Of these, we focused our attention on those signal transduction, cell cycle, and apoptosis transcripts related to the Connections Map (Fig. 1).
- Cyclin D2, Cdc25a, and PCNA promote cell division; p57 inhibits cell division (Fig. 1).
- BMPs inhibit cell proliferation via downstream Smad1/5/8 proteins whereas Smad7 inhibits TGF-β and activin signaling (Fig. 1).
- Further, we utilized PNN analysis to determine the iterated composite relative importance among Connections Map (Fig. 1) transcripts which have altered expression as a consequence of inhibition of NF-κB translocation into the nucleus (Fig. 10).
- The declining PCNA and GR reflect the sharp decline in cell proliferation and branching; the increasing BMP1 and BMP3b similarly reflects inhibition of cell proliferation (Fig. 1).
- As shown in Table 2, we find 18 proteins which have both a 1.5-fold or greater change with NF-κB inhibition and are specifically related to the Connections Map (Fig. 1).
- Raf plays a key role in the Ras signaling pathway (Fig. 1).
- Further, both the SHP-2/Ras and JAK/STAT3 pathways are activated by IL-6R/gp130 signaling (Fig. 1).
- Considering the outcome of this study relative to the Connections Map (Fig. 1), it is apparent that NF-κB nuclear translocation is functionally integral to a genetic network with broadly related, rather than independent, components.
- Specifically, we assigned those genes related to the Connections Map (Fig. 1) that have a 1.5 or greater fold-change to functional groups (i.e., cell cycle, apoptosis, signal transduction, etc.) which have biological significance.
- We used PNN analyses to determine which Connection Map (Fig. 1) transcripts or proteins with altered expression best discriminate CONT from SN50-treated explants with 100% sensitivity and specificity [69].
The Journal of cell biology.
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Figure 1. Basic functions for plus and minus end-directed myosins in cells. Myosin drives oriented transportation of cargo (A) and sliding of actin filaments (B). Myosin head (black balls) and tail (black stick) domains, actin filaments (red, blue lines), vesicle (circle), plasma membrane (curved and straight lines). Both indicated types of actin organization (A and B) exist in nonmuscle motile cells. A, Myosin pulls cargo on a uniform filament polarity actin network. Plus end- (upward arrow) and minus end- (downward arrows) directed myosins transport cargo in opposite directions. Myosins I and V are known plus end-directed transport motors. Myosin VI, the minus end-directed actin motor, also has transport motor activity in cells, but oriented movement of cargo remains to be demonstrated (see text). B, Myosin slides actin filaments in an opposite filament polarity (antiparallel) actin network. A plus end-directed myosin (top) pulls actin filaments together (thick arrows move together), generating pulling or contraction force in the cell. A minus end-directed myosin (bottom) pushes filaments apart (thick arrows move apart), generating pushing or expansion force in the cell. Contraction force generated by myosin II has been documented in many systems. However, expansion force resulting from minus end-directed myosins is, at this point, hypothetical.
- Two distinct types of actin organization that exist in cells, uniform and opposite filament polarity actin networks (see below for descriptions), allow two extreme types of myosin function, respectively (Fig 1): oriented transportation of cargo (Fig 1 A) and sliding of actin filaments (Fig 1 B).
- To drive cargo transport, myosin (Fig 1 A, single black ball and stick) moves (Fig 1 A, arrows) attached cargo (Fig 1 A, circles, curved line) over the surface of a uniform filament polarity actin network (Fig 1 A, red lines).
- In this actin organization, all actin filament plus ends face towards the cell surface (Fig 1 A, as indicated).
- This allows oriented transportation of cargo into or out of the cell (e.g., Fig 1 A, compare upward and downward arrows).
- Thus, a plus end-directed myosin will transport attached cargo outwards towards the cell surface, such as a vesicle on the exocytic pathway (Fig 1 A, upward arrow).
- Conversely, a minus end-directed myosin will transport attached cargo inwards, away from the cell surface, such as a vesicle on the endocytic pathway or a region of the plasma membrane that is being retracted or tethered by the myosin (Fig 1 A, downward arrows).
- To drive actin filament sliding, myosin oligomers (Fig 1 B, two black balls and stick) sit between repeating units of actin filament subbundles (e.g., two subbundles are represented by red and blue lines in Fig 1 B) in an opposite filament polarity actin network, and move subbundles relative to one another (e.g., Fig 1 B top, compare thick arrows).
- In this actin organization, actin filament plus ends from adjacent subbundles face opposite directions (Fig 1 B, compare red and blue lines) and myosin sits between overlapping/interdigitating filament minus ends from adjacent subbundles (Fig 1 B, as indicated).
- Also, net filament plus ends are anchored to the plasma membrane (e.g., Fig 1 B, junction of vertical and horizontal lines) via adhesion complexes or other connections.
- Thus, filament sliding driven by a plus end-directed myosin in an opposite filament polarity actin network exerts net pulling force or cell contraction force (Fig 1 B, upper panel, arrows move together).
- Conversely a minus end-directed myosin in the same actin network exerts net pushing force or cell expansion force (Fig 1 B, bottom, arrows move apart).