Mechanisms Of Target Selection Research Articles

In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex.

A wide variety of prokaryotes possess DNA modifications consisting of sequence-specific phosphorothioates (PT) inserted by members of a five-gene cluster. Recent genome mapping studies revealed two unusual features of PT modifications: short consensus sequences and partial modification of a specific genomic site in a population of bacteria. To better understand the mechanism of target selection of PT modifications that underlies these features, we characterized the substrate recognition of the PT-modifying enzymes termed DptC, D and E in a cell extract system from Salmonella. The results revealed that double-stranded oligodeoxynucleotides underwent de novo PT modification in vitro, with the same modification pattern as in vivo, i. e., GpsAAC/GpsTTC motif. Unexpectedly, in these in vitro analyses we observed no significant effect on PT modification by sequences flanking GAAC/GTTC motif, while PT also occurred in the GAAC/GTTC motif that could not be modified in vivo. Hemi-PT DNA also served as substrate of the PT-modifying enzymes, but not single-stranded DNA. The PT-modifying enzymes were then found to function as a large protein complex, with all of three subunits in tetrameric conformations. This study provided the first demonstration of in vitro DNA PT modification by PT-modifying enzymes that function as a large protein complex.

Hybrid Brain-Computer Interface (BCI) based on the EEG and EOG signals.

Recently, the integration of different electrophysiological signals into an electroencephalogram (EEG) has become an effective approach to improve the practicality of brain-computer interface (BCI) systems, referred to as hybrid BCIs. In this paper, a hybrid BCI was designed by combining an EEG with electrocardiograph (EOG) signals and tested using a target selection experiment. Gaze direction from the EOG and the event-related (de)synchronization (ERD/ERS) induced by motor imagery from the EEG were simultaneously detected as the output of the BCI system. The target selection mechanism was based on the synthesis of the gaze direction and ERD activity. When an ERD activity was detected, the target corresponding to the gaze direction was selected; without ERD activity, no target was selected, even when a subjects gaze was directed at the target. With this mechanism, the operation of the BCI system is more flexible and voluntary. The accuracy and completion time of the target selection tasks during the online testing were 89.3% and 2.4 seconds, respectively. These results show the feasibility and practicality of this hybrid BCI system, which can potentially be used in the rehabilitation of disabled individuals.

MuB gives a new twist to target DNA selection

Transposition target immunity is a phenomenon observed in some DNA transposons that are able to distinguish the host chromosome from their own DNA sequence, thus avoiding self-destructive insertions. The first molecular insight into target selection and immunity mechanisms came from the study of phage Mu transposition, which uses the protein MuB as a barrier to self-insertion. MuB is an ATP-dependent non-specific DNA binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. However, a detailed mechanistic understanding of MuB functioning was hindered by the poor solubility of the MuB-ATP complexes. Here we comment on the recent discovery that MuB is an AAA+ ATPase that upon ATP binding assembles into helical filaments that coat the DNA. Remarkably, the helical parameters of the MuB filament do not match those of the bound DNA. This intriguing mismatch symmetry led us to propose a model on how MuB targets DNA for transposition, favoring DNA bending and recognition by the transposase at the filament edge. We also speculate on a different protective role of MuB during immunity, where filament stickiness could favor the condensation of the DNA into a compact state that occludes it from the transposase.

Abstract 1991: PDCD4 de-repression of Bcl-xl IRES-mediated translation leads to enhanced chemo-resistance of glioblastoma multiforme tumors.

Abstract Glioblastoma multiforme (GBM) is the most prevalent form of tumor of the central nervous system with an average survival time of less than one year. Given the location and characteristics of GBM only limited treatment options are available, thus understanding the mechanisms of GBM formation or progression may lead to the development of novel treatment options. Strikingly, a common feature of GBM is the loss of expression of the tumor suppressor programmed cell death 4 (PDCD4) which correlates with adverse outcomes for patients in response to chemo- or radiation therapy. Through both transcriptional and translational regulation, PDCD4 has been shown to regulate many proteins within the cell, although the precise mechanism of target selection is not known. We have hypothesised that silencing of PDCD4 in glioblastomas leads to an altered expression of apoptosis-regulating proteins resulting in the suppression of apoptotic signals and enhanced chemo-resistance. Indeed, we show that low levels of PDCD4 correlate with an increase in the anti-apoptotic Bcl-xL protein. Importantly, Bcl-xL is regulated at the level of protein synthesis through the internal ribosome entry site (IRES) in its 5’ untranslated region (UTR). We further show that PDCD4 regulates the activity of the Bcl-xL IRES, and is therefore an IRES specific translational regulator. Importantly, reintroduction of PDCD4 into glioblastoma cells or directly inhibiting Bcl-xL with the chemical inhibitor ABT-737 sensitizes these cells to doxorubicin, thus enhancing cell death. In summary, our work provides a novel role for PDCD4 as a translational regulator of Bcl-xL as well as a potential therapeutic option for the treatment of GBM. Citation Format: Urszula Liwak, Lindsay E. Jordan, Federico Roncaroli, Martin Holcik. PDCD4 de-repression of Bcl-xl IRES-mediated translation leads to enhanced chemo-resistance of glioblastoma multiforme tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1991. doi:10.1158/1538-7445.AM2013-1991

Molecular Insights into Vesicle Tethering at the Golgi by the Conserved Oligomeric Golgi (COG) Complex and the Golgin TATA Element Modulatory Factor (TMF)

Protein sorting between eukaryotic compartments requires vesicular transport, wherein tethering provides the first contact between vesicle and target membranes. Here we map and start to functionally analyze the interaction network of the conserved oligomeric Golgi (COG) complex that mediates retrograde tethering at the Golgi. The interactions of COG subunits with members of transport factor families assign the individual subunits as specific interaction hubs. Functional analysis of selected interactions suggests a mechanistic tethering model. We find that the COG complex interacts with two different Rabs in addition to each end of the golgin "TATA element modulatory factor" (TMF). This allows COG to potentially bridge the distance between the distal end of the golgin and the target membrane thereby promoting tighter docking. Concurrently we show that the central portion of TMF can bind to Golgi membranes that are liberated of their COPI cover. This latter interaction could serve to bring vesicle and target membranes into close apposition prior to fusion. A target selection mechanism, in which a hetero-oligomeric tethering factor organizes Rabs and coiled transport factors to enable protein sorting specificity, could be applicable to vesicle targeting throughout eukaryotic cells.

Attentional priorities and access to short-term memory: Parietal interactions

The intraparietal sulcus (IPS) has been implicated in selective attention as well as visual short-term memory (VSTM). To contrast mechanisms of target selection, distracter filtering, and access to VSTM, we combined behavioral testing, computational modeling and functional magnetic resonance imaging. Sixteen healthy subjects participated in a change detection task in which we manipulated both target and distracter set sizes. We directly compared the IPS response as a function of the number of targets and distracters in the display and in VSTM. When distracters were not present, the posterior and middle segments of IPS showed the predicted asymptotic activity increase with an increasing target set size. When distracters were added to a single target, activity also increased as predicted. However, the addition of distracters to multiple targets suppressed both middle and posterior IPS activities, thereby displaying a significant interaction between the two factors. The interaction between target and distracter set size in IPS could not be accounted for by a simple explanation in terms of number of items accessing VSTM. Instead, it led us to a model where items accessing VSTM receive differential weights depending on their behavioral relevance, and secondly, a suppressive effect originates during the selection phase when multiple targets and multiple distracters are simultaneously present. The reverse interaction between target and distracter set size was significant in the right temporoparietal junction (TPJ), where activity was highest for a single target compared to any other condition. Our study reconciles the role of middle IPS in attentional selection and biased competition with its role in VSTM access.

Novel applications of deep brain stimulation

The success of deep brain stimulation (DBS) surgery in treating medically refractory symptoms of some movement disorders has inspired further investigation into a wide variety of other treatment-resistant conditions. These range from disorders of gait, mood, and memory to problems as diverse as obesity, consciousness, and addiction. We review the emerging indications, rationale, and outcomes for some of the most promising new applications of DBS in the treatment of postural instability associated with Parkinson's disease, depression, obsessive–compulsive disorder, obesity, substance abuse, epilepsy, Alzheimer′s-type dementia, and traumatic brain injury. These studies reveal some of the excitement in a field at the edge of a rapidly expanding frontier. Much work still remains to be done on basic mechanism of DBS, optimal target and patient selection, and long-term durability of this technology in treating new indications.

Neural mechanisms of saccade target selection: gated accumulator model of the visual–motor cascade

We review a new computational model developed to understand how evidence about stimulus salience in visual search is translated into a saccade command. The model uses the activity of visually responsive neurons in the frontal eye field as evidence for stimulus salience that is accumulated in a network of stochastic accumulators to produce accurate and timely saccades. We discovered that only when the input to the accumulation process was gated could the model account for the variability in search performance and predict the dynamics of movement neuron discharge rates. This union of cognitive modeling and neurophysiology indicates how the visual-motor transformation can occur, and provides a concrete mapping between neuron function and specific cognitive processes.

Abstract 2113: Programmed cell death 4 (PDCD4) regulates apoptotic resistance of human gliomas

Abstract Glioblastoma multiforme (GBM) is the most prevalent form of tumors of the central nervous system with an average survival rate of less than one year. Given the location and characteristics of GBM only limited treatment options are available, thus understanding the mechanisms of GBM formation or progression may lead to the development of novel treatment options. Strikingly, a common feature of GBM is the loss of expression of the tumor suppressor programmed cell death gene 4 (PDCD4), which correlates with a poor prognosis. Through both transcriptional and translational regulation, PDCD4 has been shown to regulate many proteins within the cell, although the precise mechanism of target selection is not known. We have hypothesised that silencing of PDCD4 in glioblastomas leads to an altered expression of apoptosis-regulating proteins resulting in the suppression of apoptotic signals and enhanced chemo-resistance of glioblastomas. Indeed, we show that low levels of PDCD4 correlate with an increase in the anti-apoptotic XIAP and Bcl-xL proteins, and decrease in pro-apoptotic Apaf-1. Importantly, these proteins are regulated at the level of protein synthesis through the internal ribosome entry sites (IRESs) in the 5’ untranslated region (UTR) of their respective mRNAs. IRES elements allow for translation to occur through a cap-independent mechanism during times of cellular stress when global translation is attenuated. We further show that PDCD4 regulates the activity of Bcl-xL, XIAP and Apaf-1 IRES and is therefore an IRES specific translational regulator. We further dissect the signalling pathways that regulate PDCD4 levels and show that activation of S6K2 results in the proteolytic degradation of PDCD4 and concomitant upregulation of XIAP and Bcl-xL, and downregulation of Apaf-1 levels. Importantly, reintroduction of PDCD4 into glioblastoma cell lines results in a reduction in both XIAP and Bcl-xL protein levels and enhanced cell death. In summary, our work provides a novel role for PDCD4 as a translational regulator of XIAP, Bcl-xL and Apaf-1, as well as a mechanistic understanding into the regulation of PDCD4 expression. Our research offers insight into the biology of glioblastomas in the hopes that we can identify new pathways as novel targets for anticancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2113. doi:10.1158/1538-7445.AM2011-2113

Visualizing Axons in the Drosophila Central Nervous System Using Immunohistochemistry and Immunofluorescence: Figure 1.

How axons in the developing nervous system navigate to their correct targets is a fundamental question in neuroscience. Studies of axon guidance in the embryonic central nervous system (CNS) of Drosophila melanogaster have proved instrumental to the identification of molecules and mechanisms that regulate wiring. The relative simplicity of the Drosophila embryonic CNS, the advantages of genetic approaches, and the ability to analyze the pathfinding decisions of well-defined axon groups or even individual axons have all contributed to our understanding of the fundamental mechanisms of axon guidance and target selection. In this protocol, we describe methods for using fluorescent antibody staining as well as horseradish peroxidase (HRP) immunohistochemistry to reveal axon-projection patterns and identify specific guidance molecules. In the presence of 3,3'-diaminobenzidine tetrahydrochloride (DAB), HRP forms a brown, stable precipitate that allows visualization of axon populations under white light. This can be quite advantageous when teaching basic embryonic anatomy and dissection techniques, because unlike fluorescent antibody staining, HRP can be visualized in a lighted room. HRP detection does not allow precise colocalization of signals, nor can multiple colors be visualized simultaneously. These objectives are easily achieved with fluorescent antibody staining.

Investigating Grid-Based Navigation

Hands-free speech-based technology can be a useful alternative for individuals that find traditional input devices, such as keyboard and mouse, difficult to use. Various speech-based navigation techniques have been examined, and several are available in commercial software applications. Among these alternatives, grid-based navigation has demonstrated both potential and limitations. In this article, we discuss an empirical study that assessed the efficacy of two enhancements to grid-based navigation: magnification and fine-tuning. The magnification capability enlarges the selected region when it becomes sufficiently small, making it easier to see the target and cursor. The fine-tuning capability allows users to move the cursor short distances to position the cursor over the target. The study involved one group of participants with physical disabilities, an age-matched group of participants without disabilities, and a third group that included young adults without disabilities. The results confirm that both magnification and fine-tuning significantly improved the participants’ performance when selecting targets, especially small targets. Providing either, or both, of the proposed enhancements substantially reduced the gaps in performance due to disability and age. The results will inform the design of speech-based target selection mechanism, allowing users to select targets faster while making fewer errors.

Searching for Targets within the Spatial Layout of Visual Short-Term Memory

Recent studies have revealed that the internal representations that we construct from the environment and maintain in visual short-term memory (VSTM) to guide behavior are highly flexible and can be selectively modulated according to our task goals and expectations. In the current study, we conducted two experiments to compare and contrast neural mechanisms of selective attention related to searching for target items within perceptual versus VSTM representations. We used event-related potentials to investigate whether searching for relevant target items from within VSTM representations involves spatially specific biasing of neural activity in a manner analogous to that which occurs during visual search for target items in perceptual arrays. The results, replicated across the two experiments, revealed that selection of a target object within a search array maintained in VSTM proceeds through a similar mechanism as that in the perceptual domain. In line with previous results, N2pc potentials were obtained when targets were identified within a perceptual visual-search array. Interestingly, equivalent N2pcs, with similar time courses and scalp distributions, were also elicited when target items were identified within a VSTM representation. The findings reinforce the notion of highly flexible VSTM representations that can be modulated according to task goals and suggest a large degree of overlap in the spatially specific neural mechanisms of target selection across the perceptual and VSTM domains.

At the next stop sign turn right: the metalloprotease Tolloid-related 1 controls defasciculation of motor axons inDrosophila

Navigation of motoneuronal growth cones toward the somatic musculature in Drosophila serves as a model system to unravel the molecular mechanisms of axon guidance and target selection. In a large-scale mutagenesis screen, we identified piranha, a motor axon guidance mutant that shows strong defects in the neuromuscular connectivity pattern. In piranha mutant embryos, permanent defasciculation errors occur at specific choice points in all motor pathways. Positional cloning of piranha revealed point mutations in tolloid-related 1 (tlr1), an evolutionarily conserved gene encoding a secreted metalloprotease. Ectopic expression of Tlr1 in several tissues of piranha mutants, including hemocytes, completely restores the wild-type innervation pattern, indicating that Tlr1 functions cell non-autonomously. We further show that loss-of-function mutants of related metalloproteases do not have motor axon guidance defects and that the respective proteins cannot functionally replace Tlr1. tlr1, however, interacts with sidestep, a muscle-derived attractant. Double mutant larvae of tlr1 and sidestep show an additive phenotype and lack almost all neuromuscular junctions on ventral muscles, suggesting that Tlr1 functions together with Sidestep in the defasciculation process.

Distractor interference during smooth pursuit eye movements.

When 2 targets for pursuit eye movements move in different directions, the eye velocity follows the vector average (S. G. Lisberger & V. P. Ferrera, 1997). The present study investigates the mechanisms of target selection when observers are instructed to follow a predefined horizontal target and to ignore a moving distractor stimulus. Results show that at 140 ms after distractor onset, horizontal eye velocity is decreased by about 25%. Vertical eye velocity increases or decreases by 1 degrees /s in the direction opposite from the distractor. This deviation varies in size with distractor direction, velocity, and contrast. The effect was present during the initiation and steady-state tracking phase of pursuit but only when the observer had prior information about target motion. Neither vector averaging nor winner-take-all models could predict the response to a moving to-be-ignored distractor during steady-state tracking of a predefined target. The contributions of perceptual mislocalization and spatial attention to the vertical deviation in pursuit are discussed.

Structure of a Putative Lipoate Protein Ligase from Thermoplasma acidophilum and the Mechanism of Target Selection for Post-translational Modification

Lipoyl-lysine swinging arms are crucial to the reactions catalysed by the 2-oxo acid dehydrogenase multienzyme complexes. A gene encoding a putative lipoate protein ligase (LplA) of Thermoplasma acidophilum was cloned and expressed in Escherichia coli. The recombinant protein, a monomer of molecular mass 29 kDa, was catalytically inactive. Crystal structures in the absence and presence of bound lipoic acid were solved at 2.1 A resolution. The protein was found to fall into the alpha/beta class and to be structurally homologous to the catalytic domains of class II aminoacyl-tRNA synthases and biotin protein ligase, BirA. Lipoic acid in LplA was bound in the same position as biotin in BirA. The structure of the T.acidophilum LplA and limited proteolysis of E.coli LplA together highlighted some key features of the post-translational modification. A loop comprising residues 71-79 in the T.acidophilum ligase is proposed as interacting with the dithiolane ring of lipoic acid and discriminating against the entry of biotin. A second loop comprising residues 179-193 was disordered in the T.acidophilum structure; tryptic cleavage of the corresponding loop in the E.coli LplA under non-denaturing conditions rendered the enzyme catalytically inactive, emphasizing its importance. The putative LplA of T.acidophilum lacks a C-terminal domain found in its counterparts in E.coli (Gram-negative) or Streptococcus pneumoniae (Gram-positive). A gene encoding a protein that appears to have structural homology to the additional domain in the E.coli and S.pneumoniae enzymes was detected alongside the structural gene encoding the putative LplA in the T.acidophilum genome. It is likely that this protein is required to confer activity on the LplA as currently purified, one protein perhaps catalysing the formation of the obligatory lipoyl-AMP intermediate, and the other transferring the lipoyl group from it to the specific lysine residue in the target protein.

Targeted transposition by the V(D)J recombinase.

Cleavage by the V(D)J recombinase at a pair of recombination signal sequences creates two coding ends and two signal ends. The RAG proteins can integrate these signal ends, without sequence specificity, into an unrelated target DNA molecule. Here we demonstrate that such transposition events are greatly stimulated by--and specifically targeted to--hairpins and other distorted DNA structures. The mechanism of target selection by the RAG proteins thus appears to involve recognition of distorted DNA. These data also suggest a novel mechanism for the formation of alternative recombination products termed hybrid joints, in which a signal end is joined to a hairpin coding end. We suggest that hybrid joints may arise by transposition in vivo and propose a new model to account for some recurrent chromosome translocations found in human lymphomas. According to this model, transposition can join antigen receptor loci to partner sites that lack recombination signal sequence elements but bear particular structural features. The RAG proteins are capable of mediating all necessary breakage and joining events on both partner chromosomes; thus, the V(D)J recombinase may be far more culpable for oncogenic translocations than has been suspected.

Search target selection in monkey prefrontal cortex.

To explore a visual scene, the brain must detect an object of interest and direct the eyes to it. To investigate the brain's mechanism of saccade target selection, we trained monkeys to perform a visual search task with a response delay and recorded neuronal activity in the prefrontal (PF) cortex. Even though the monkey was not allowed to express its choice until after a delay, the response field of a class of PF neurons was able to differentiate between target and distractors from the very beginning of their response (135 ms). Strong responses were obtained only when the target was presented at the field. Neurons responded much less during a nonsearch task in which saccade target was presented alone in this response field. These results suggest that the PF cortex may be involved in the decision-making process and the focal attention for saccade target selection.

Target selection for pursuit and saccadic eye movements in humans.

Eye movements were recorded from three subjects as they initiated tracking of a small circle ("target") moving leftward or rightward, above or below the horizontal meridian, either alone or in the presence of a small square ("distractor") moving leftward or rightward on the other side of the horizontal meridian. At the start of each trial, subjects were provided with either a "form" cue (always centrally positioned and having the circular shape and color of the upcoming moving target) or a "location" cue (a small white square positioned where the upcoming target would appear). The latency of pursuit increased in the presence of an oppositely moving distractor when subjects were provided the form cues but not when they were provided the location cues. The latency of saccades showed similar, but smaller, increases when subjects were given the form cues. On many trials with the form cues, pursuit started in the direction of the distractor and then reversed to follow the target. On these trials, the initial saccade often, but not always, also followed the distractor. These results indicate that the mechanisms of target selection for pursuit and saccades are tightly coordinated but not strictly yoked. The shared effects of the distractor on the latencies of pursuit and saccades probably reflect the common role of visual attention in filtering the inputs that guide these two types of eye movements. The differences in the details of the effects on pursuit and saccades suggest that the neural mechanisms that trigger these two movements can be independently regulated.

Mechanism of regulatory target selection by the SOX high-mobility-group domain proteins as revealed by comparison of SOX1/2/3 and SOX9.

SOX proteins bind similar DNA motifs through their high-mobility-group (HMG) domains, but their action is highly specific with respect to target genes and cell type. We investigated the mechanism of target selection by comparing SOX1/2/3, which activate delta-crystallin minimal enhancer DC5, with SOX9, which activates Col2a1 minimal enhancer COL2C2. These enhancers depend on both the SOX binding site and the binding site of a putative partner factor. The DC5 site was equally bound and bent by the HMG domains of SOX1/2 and SOX9. The activation domains of these SOX proteins mapped at the distal portions of the C-terminal domains were not cell specific and were independent of the partner factor. Chimeric proteins produced between SOX1 and SOX9 showed that to activate the DC5 enhancer, the C-terminal domain must be that of SOX1, although the HMG domains were replaceable. The SOX2-VP16 fusion protein, in which the activation domain of SOX2 was replaced by that of VP16, activated the DC5 enhancer still in a partner factor-dependent manner. The results argue that the proximal portion of the C-terminal domain of SOX1/2 specifically interacts with the partner factor, and this interaction determines the specificity of the SOX1/2 action. Essentially the same results were obtained in the converse experiments in which COL2C2 activation by SOX9 was analyzed, except that specificity of SOX9-partner factor interaction also involved the SOX9 HMG domain. The highly selective SOX-partner factor interactions presumably stabilize the DNA binding of the SOX proteins and provide the mechanism for regulatory target selection.

Shared target selection for combined version-vergence eye movements.

Primates frequently make rapid binocular eye movements to reorient gaze in both direction and depth. To explain the unequal movements made by the two eyes, it often is assumed that they result from the combined action of a conjugate saccadic system and a vergence contribution. Clearly such a scheme can only yield coordinated binocular movements if both systems are guided by a shared or coupled target selection mechanism. To investigate the degree of cooperation at this level, we studied binocular refixations to target-nontarget double-stimuli in three-dimensional (3-D) space. Binocular eye movements were recorded in seven subjects using the scleral coil technique. In the experiments, 20% of trials were composed of a green target and a red nontarget, presented at the same time, but at different locations in 3-D space. These were alternated randomly with single-target trials (80%) in which the green stimulus was presented randomly at one of eight possible positions in 3-D space. Instructions to the subject emphasized either the speed or accuracy of response. Our findings show that typical features of the saccadic response to double-stimuli (bistability, averaging, and a speed-accuracy trade-off), as found in earlier two-dimensional studies, are also prevalent for initial binocular refixations to double stimuli in 3-D space. When the first saccadic response is directed to one of the two stimuli, the vergence system almost invariably makes the same choice. Likewise, when the saccadic system makes a short-latency averaging response, the vergence system shows a similar compromise. Statistical analysis shows a high correlation between saccadic and vergence target selection, strongly suggesting that the amplitude computation process of both subsystems is due to a common target selection stage that has access to information about stimulus location in 3-D space.