bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypothesis testing in a rule discovery problem: When a focused procedure is effective
Authors: Rossi Sandrine , Caverni Jean Paul , Girotto Vittorio
Published: July 2002

We investigated individuals’ ability to use negative evidence in hypothesis testing. We compared performance in two versions of Wason's (1960) rule discovery problem. In the original version, a triple of numbers- {2, 4, 6}- was presented as an example of a rule that the experimenter had in mind (i.e., “increasing numbers”). Participants had to discover the rule by proposing new triples. In the other version, the same triple was presented as a counterexample to the experimenter's rule (i.e., “decreasing numbers”). We predicted that, in both conditions, participants would form hypotheses based on the features of the triple, and test only instances of the hypothesized rule. However, in the counter-example condition, such focused testing would invariably produce negative evidence. As a consequence, participants would be forced to revise their hypotheses. The reported results corroborated our predictions: Participants solved the counter-example version significantly better than the original problem.
Conditionals and directionality: On the meaning of if vs. only if
Authors: Santamaría Carlos , Espino Orlando
Published: July 2002

The aim of this study was to test the predictions of the current theories of reasoning about the comprehension of conditional statements. We used two types of conditional statement that are logically equivalent: if p then q and p only if q. The model theory of reasoning considers that these conditional forms differ in their initial meaning, because the negative contingency is considered only in the p only if q form. Mental-rule theories maintain that the interpretation of p only if q depends on a rephrasing of the statement as: if not q then not p. Alternatively, a directional bias may explain the differences between if p then q and p only if q. We report three experiments that demonstrate the existence of a directional bias in the comprehension of the conditionals. The results were not predicted by either the mental-rules theories or the model theory; they could, however, be easily assimilated by the model theory.
Perceiving a strong causal relation in a weak contingency: Further investigation of the evidential evaluation model of causal judgement
Authors: White Peter A.
Published: July 2002

Contingency information is information about the occurrence or nonoccurrence of a certain effect in the presence or absence of a candidate cause. An objective measure of contingency is the δ P rule, which involves subtracting the probability of occurrence of an effect when a causal candidate is absent from the probability of occurrence of the effect when the candidate is present. Causal judgements conform closely to δ P but deviate from it under certain circumstances. Three experiments show that such deviations can be predicted by a model of causal judgement that has two components: a rule of evidence, that causal judgement is a function of the proportion of relevant instances that are judged to be confirmatory for the causal candidate, and a tendency for information about instances in which the candidate is present to have greater effect on judgement than instances in which the candidate is absent. Two experiments demonstrate how this model accounts for some recently published findings. A third experiment shows that it is possible to use the model to predict the occurrence of high causal judgements when the objective contingency is close to zero.
Verbal structure of numerals and digits handwriting: New evidence from kinematics
Authors: Lochy Aliette , Pillon Agnesa , Zesiger Pascal , Seron Xavier
Published: July 2002

Two experiments used a digitizing tablet to analyse the temporal, spatial, and kinematic characteristics of handwritten production of arabic numbers. They addressed a specific issue of the numerical domain: Does the lexical and syntactic structure of verbal numerals influence the production of arabic numerals (Experiments 1 and 2), even after enforced semantic processing in a comparison task (Experiment 2)? Subjects had to write multi-digit arabic numerals (e.g., 1200) presented in two different verbal structures: a multiplicative one (e.g., teen-hundred, douze cents (twelve hundred)) or an additive one (e.g., thousand-unit-hundred, mille deux cents (one thousand two hundred)). Results show differences in the inter-digit jumps that reflect the influence of the structure of verbal numerals, even after the semantic task. This finding is discussed with regard to different models of number transcoding (McCloskey, Caramazza, & Basili, 1985; Power & Dal Martello, 1990, 1997).
Inverting the modality effect in serial recall
Authors: Beaman C. Philip
Published: July 2002

Differences in recall ability between immediate serial recall of auditorily and visually presented verbal material have traditionally been considered restricted to the end of to-be-recalled lists, the recency section of the serial position curve (e.g., Crowder & Morton, 1969). Later studies showed that—under certain circumstances–differences in recall between the two modalities can be observed across the whole of the list (Frankish, 1985). However in all these studies the advantage observed is for recall of material presented in the auditorily modality. Six separate conditions across four experiments demonstrate that a visual advantage can be obtained with serial recall if participants are required to recall the list in two distinct sections using serial recall. Judged on a list-wide basis, the visual advantage is of equivalent size to the auditory advantage of the classical modality effect. The results demonstrate that differences in representation of auditory and visual verbal material in short-term memory persist beyond lexical and phonological categorization and are problematic for current theories of the modality effect.
Responding to object files: Automatic integration of spatial information revealed by stimulus-response compatibility effects
Authors: Hommel Bernhard
Published: July 2002

Spatial information is assumed to play a central, organizing role in object perception and to be an important ingredient of object representations. Here, evidence is provided to show that automatically integrated spatial object information is also functional in guiding spatial action. In particular, retrieving nonspatial information about a previewed object facilitates responses that spatially correspond to this object. This is true whether the object is still in sight or has already disappeared. So, forming an object representation entails the integration and storage of action-related information concerning the action that the object affords.
Motor learning by observation: Evidence from a serial reaction time task
Authors: Heyes C.M. , Foster C.L.
Published: July 2002

This study sought evidence of observational motor learning, a type of learning in which observation of the skilled performance of another person not only facilitates motor skill acquisition but does so by contributing to the formation of effector-specific motor representations. Previous research has indicated that observation of skilled performance engages cognitive processes similar to those occurring during action execution or physical practice, but has not demonstrated that these include processes involved in effector-specific representation. In two experiments, observer subjects watched the experimenter performing a serial reaction time (SRT) task with a six-item unique sequence before sequence knowledge was assessed by response time and/or free generation measures. The results suggest that: (1) subjects can acquire sequence information by watching another person performing the task (Experiments 1-2); (2) observation results in as much sequence learning as task practice when learning is measured by reaction times (RTs) and more than task practice when sequence learning is measured by free generation performance (Experiment 2, Part 1); and (3) sequence knowledge acquired by model observation can be encoded motorically—that is, in an effector-specific fashion (Experiment 2, Part 2).
An Assessment of Factors Contributing to Instrumental Performance for Sexual Reward in the Rat
Authors: Woodson James C. , Balleine Bernard W.
Published: July 2002

Male and female rats were gonadectomized, implanted in adulthood with capsules containing either testosterone propionate (TP) or cholesterol, and were trained to lever press for access to an oestrous female. When lever press performance was tested in extinction, only the male rats implanted with TP displayed significantly higher levels of responding than controls, demonstrating that lever pressing for oestrous females as a reward is sexually dimorphic. Ejaculation patterns from a separate assessment of these rats’ copulatory ability were significantly correlated with their instrumental performance. In a second experiment, “masculinized” females exposed to TP postnatally and given TP implants responded in extinction at mean levels equivalent to those exhibited by adult males that were either intact or castrated with androgen replacement. These data suggest that organizational steroid exposure perinatally affects the actual reward value assigned to oestrous females inadulthood, in combination withconsummatory sexual experience.
Spatial Separation of Target and Competitor Cues Enhances Blocking of Human Causality Judgements
Authors: Glautier Steven
Published: July 2002

Three experiments were carried out. Each required subjects to make judgements about the causal status of cues following a two-stage blocking procedure. In Stage 1 a competitor cue was consistently paired with an outcome, and in Stage 2 the competitor continued to be paired with the outcome but was accompanied by a target cue. It was predicted that causal judgements for the target would be reduced by the presence of the competitor. In Experiments 1 and 2 the blocking procedure was implemented as a computer simulation of a card game during which subjects had to learn which cards produced the best payouts. The cues that subjects used to make their judgement were colours and symbols that appeared on the backs of the cards. When the target and competitor cues appeared on the same card blocking effects did not emerge, but when they appeared as part of different cards blocking effects were found. Thus, spatial separation of target and competitor cues appeared to facilitate blocking. Experiment 3 replicated the blocking result using spatially separated target and competitor cues.
Contextual Influence On The Language Production Of Children With Speech/Language Impairment
Authors: Hansson Kristina , Nettelbladt Ulrika , Nilholm Claes
Published: July 2002

To investigate the possible contextual variability of language and interaction, data from 10 children with speech/language impairment and three different types of conversational partners (a parent, a peer and a clinician) were collected. The dialogues were analysed with respect to the characteristics of the dialogues as wholes, to the dominance and the productivity of the three types of conversational partners, and to the productivity, fluency and grammatical structure of the language production of the children with speech/language impairment. Productivity was measured as mean length of utterance (MLU) in words, number of utterances, number of different words and proportion of complete and intelligible utterances. Fluency was measured as the proportion of utterances containing a maze. Grammatical structure was analysed with respect to the use of grammatical morphemes, word order patterns, the occurrence of expansions and the complexity of verb forms. Significant differences, in particular between child‐child and adult‐child dialogues were found. The adult‐child dialogues were asymmetrical, where the adults dominated through asking many questions and talking much. The peer dialogues were more dynamic and equal. In these dialogues the children with speech/language impairment took a more active role as a conversational partner. The children produced more utterances and different words with the adult partners, but variables related to fluency and grammatical structure did not vary as a function of the conversational partner. The results imply that children with speech/language impairment practise different aspects of their communicative ability with different types of conversational partners. In particular, it is important to provide them with opportunities to interact with peers, in order for them to develop their skills as independent conversational partners.
Phonological awareness therapy and articulatory training approaches for children with phonological disorders: a comparative outcome study
Authors: Hesketh Anne , Adams Catherine , Nightingale Catherine , Hall Rebecca
Published: July 2002

Sixty‐one children, aged 3;6‐5;0, with developmental phonological disorders (PD) participated in a study comparing the effects of metaphonologically (MET) or articulation‐based (ART) therapy. Maturational effects were controlled for by the inclusion of 59 normally speaking control children of the same age range. Measures of phonological (speech) output and phonological awareness were taken before and after therapy for all subjects and at 3 months post‐therapy for PD children. Results showed that PD children improved significantly in both phonological output and awareness skills across the intervention period compared with control children, but that there was no significant difference on the awareness measure between ART and MET groups. ART and MET groups differed from each other on one measure of speech improvement only, with the ART group making more change than the MET group on individual probe scores. Follow‐up measures for both therapy groups indicated that there was little difference between the groups on phonological awareness change or speech development 3 months after intervention, though there was a trend for MET children to continue to make more long‐term change than the ART group on one output measure. Additional analysis showed that there were generally few significant implications for outcome between PD children with good initial phonological awareness skills and those who initially had poor phonological awareness skills.
NGF and Neurotrophin-3 Both Activate TrkA on Sympathetic Neurons but Differentially Regulate Survival and Neuritogenesis
Authors: Belliveau Daniel J. , Krivko Irena , Kohn Judi , Lachance Christian , Pozniak Christine , Rusakov Dmitri , Kaplan David , Miller Freda D.
Published: July 2002

In this report we examine the biological and molecular basis of the control of sympathetic neuron differentiation and survival by NGF and neurotrophin-3 (NT-3). NT-3 is as efficient as NGF in mediating neuritogenesis and expression of growth-associated genes in NGF-dependent sympathetic neurons, but it is 20–40fold less efficient in supporting their survival. Both NT-3 and NGF induce similar sustained, long-term activation of TrkA, while NGF is 10-fold more efficient than NT-3 in mediating acute, short-term TrkA activity. At similar acute levels of TrkA activation, NT-3 still mediates neuronal survival two- to threefold less well than NGF. However, a mutant NT-3 that activates TrkC, but not TrkA, is unable to support sympathetic neuron survival or neuritogenesis, indicating that NT3–mediated TrkA activation is necessary for both of these responses. On the basis of these data, we suggest that NGF and NT-3 differentially regulate the TrkA receptor both with regard to activation time course and downstream targets, leading to selective regulation of neuritogenesis and survival. Such differential responsiveness to two ligands acting through the same Trk receptor has important implications for neurotrophin function throughout the nervous system.
Proteolytic Pathways of Activation and Degradation of a Bacterial Phospholipase C during Intracellular Infection by Listeria monocytogenes
Authors: Marquis Hélène , Goldfine Howard , Portnoy Daniel A.
Published: July 2002

Listeria monocytogenes is a facultative intracellular bacterial pathogen that spreads cell to cell without exposure to the extracellular environment. Bacterial cell-to-cell spread is mediated in part by two secreted bacterial phospholipases C (PLC), a broad spectrum PLC (PC-PLC) and a phosphatidylinositolspecific PLC (PI-PLC). PI-PLC is secreted in an active state, whereas PC-PLC is secreted as an inactive proenzyme (proPC-PLC) whose activation is mediated in vitro by an L. monocytogenes metalloprotease (Mpl). Analysis of PI-PLC, PC-PLC, and Mpl single and double mutants revealed that Mpl also plays a role in the spread of an infection, but suggested that proPC-PLC has an Mpl-independent activation pathway. Using biochemical and microscopic approaches, we describe three intracellular proteolytic pathways regulating PCPLC activity. Initially, proPC-PLC secreted in the cytosol of infected cells was rapidly degraded in a proteasome-dependent manner. Later during infection, PCPLC colocalized with bacteria in lysosome-associated membrane protein 1–positive vacuoles. Activation of proPC-PLC in vacuoles was mediated by Mpl and an Mpl-independent pathway, the latter being sensitive to inhibitors of cysteine proteases. Lastly, proPC-PLC activation by either pathway was sensitive to bafilomycin A1, a specific inhibitor of vacuolar ATPase, suggesting that activation was dependent on acidification of the vacuolar compartment. These results are consistent with a model in which proPC-PLC activation is compartment specific and controlled by a combination of bacterial and host factors.
Actinin-associated LIM Protein: Identification of a Domain Interaction between PDZ and Spectrin-like Repeat Motifs
Authors: Xia Houhui , Winokur Sara T. , Kuo Wen-Lin , Altherr Michael R. , Bredt David S.
Published: July 2002

PDZ motifs are protein–protein interaction domains that often bind to COOH-terminal peptide sequences. The two PDZ proteins characterized in skeletal muscle, syntrophin and neuronal nitric oxide synthase, occur in the dystrophin complex, suggesting a role for PDZ proteins in muscular dystrophy. Here, we identify actinin-associated LIM protein (ALP), a novel protein in skeletal muscle that contains an NH2-terminal PDZ domain and a COOH-terminal LIM motif. ALP is expressed at high levels only in differentiated skeletal muscle, while an alternatively spliced form occurs at low levels in the heart. ALP is not a component of the dystrophin complex, but occurs in association with α-actinin-2 at the Z lines of myofibers. Biochemical and yeast two-hybrid analyses demonstrate that the PDZ domain of ALP binds to the spectrin-like motifs of α-actinin-2, defining a new mode for PDZ domain interactions. Fine genetic mapping studies demonstrate that ALP occurs on chromosome 4q35, near the heterochromatic locus that is mutated in fascioscapulohumeral muscular dystrophy.
A Yeast Mutant Showing Diagnostic Markers of Early and Late Apoptosis
Authors: Madeo Frank , Fröhlich Eleonore , Fröhlich Kai-Uwe
Published: July 2002

A Saccharomyces cerevisiae mutant in cell division cycle gene CDC48 shows typical markers of apoptosis: membrane staining with annexin V, indicating an exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane; intense staining, using the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling method, indicating DNA fragmentation; and chromatin condensation and fragmentation. The coordinate occurrence of these events at different locations in the cell, which have no obvious connection except their relation to apoptosis, implies the presence of the molecular machinery performing the basic steps of apoptosis already in yeast. Saccharomyces cerevisiae may prove a suitable model to trace the roots of apoptosis.
The Mammalian Protein (rbet1) Homologous to Yeast Bet1p Is Primarily Associated with the Pre-Golgi Intermediate Compartment and Is Involved in Vesicular Transport from the Endoplasmic Reticulum to the Golgi Apparatus
Authors: Zhang Tao , Wong Siew Heng , Tang Bor Luen , Xu Yue , Peter Frank , Subramaniam V. Nathan , Hong Wanjin
Published: July 2002

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.
Cytoskeletal Protein ABP-280 Directs the Intracellular Trafficking of Furin and Modulates Proprotein Processing in the Endocytic Pathway
Authors: Liu Gseping , Thomas Laurel , Warren Robin A. , Enns Caroline A. , Cunningham C. Casey , Hartwig John H. , Thomas Gary
Published: July 2002

Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system. Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes). Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein. The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein. Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface. Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor. However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280. By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280. In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic. Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways. The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed.
Identification of p130Cas as a Mediator of Focal Adhesion Kinase–promoted Cell Migration
Authors: Cary Leslie A. , Han Dong Cho , Polte Thomas R. , Hanks Steven K. , Guan Jun-Lin
Published: July 2002

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130Cas, two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130Cas association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130Cas and its subsequent binding to several SH2 domains, which depended on both the p130Cas binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130Cas further increased cell migration on FN and coexpression of the p130Cas SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130Cas, but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130Cas complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.
β-Filagenin, a Newly Identified Protein Coassembling with Myosin and Paramyosin in Caenorhabditis elegans
Authors: Liu Feizhou , Bauer Christopher C. , Ortiz Irving , Cook Richard G. , Schmid Michael F. , Epstein Henry F.
Published: July 2002

Muscle thick filaments are stable assemblies of myosin and associated proteins whose dimensions are precisely regulated. The mechanisms underlying the stability and regulation of the assembly are not understood. As an approach to these problems, we have studied the core proteins that, together with paramyosin, form the core structure of the thick filament backbone in the nematode Caenorhabditis elegans. We obtained partial peptide sequences from one of the core proteins, β-filagenin, and then identified a gene that encodes a novel protein of 201–amino acid residues from databases using these sequences. β-Filagenin has a calculated isoelectric point at 10.61 and a high percentage of aromatic amino acids. Secondary structure algorithms predict that it consists of four β-strands but no α-helices. Western blotting using an affinity-purified antibody showed that β-filagenin was associated with the cores. β-Filagenin was localized by immunofluorescence microscopy to the A bands of body–wall muscles, but not the pharynx. β-filagenin assembled with the myosin homologue paramyosin into the tubular cores of wild-type nematodes at a periodicity matching the 72-nm repeats of paramyosin, as revealed by immunoelectron microscopy. In CB1214 mutants where paramyosin is absent, β-filagenin assembled with myosin to form abnormal tubular filaments with a periodicity identical to wild type. These results verify that β-filagenin is a core protein that coassembles with either myosin or paramyosin in C. elegans to form tubular filaments.
Mammalian Granulocyte–Macrophage Colony-stimulating Factor Receptor Expressed in Primary Avian Hematopoietic Progenitors: Lineage-specific Regulation of Proliferation and Differentiation
Authors: Wessely Oliver , Deiner Eva-Maria , Lim Kim Chew , Mellitzer Georg , Steinlein Peter , Beug Hartmut
Published: July 2002

The cytokine Granulocyte–Macrophage Colony-Stimulating Factor (GM-CSF) regulates proliferation, differentiation, and apoptosis during myelopoiesis and erythropoiesis. Structure–function relationships of GM-CSF interactions with its receptor (GM-R), the biochemistry of GM-R signal transduction, and GM-CSF action in vivo are relatively well understood. Much less is known, however, about GM-R function in primary hematopoietic cells. In this paper we show that expression of the human GM-R in a heterologous cell system (primary avian erythroid and myeloid cells) confirms respective results in murine or human cell lines, but also provides new insights how the GM-R regulates progenitor proliferation and differentiation. As expected, the hGM-CSF stimulated myeloid progenitor proliferation and differentiation and enhanced erythroid progenitor proliferation during terminal differentiation. In the latter cells, however, the hGM-R only partially substituted for the activities of the erythropoietin receptor (EpoR). It failed to replace the EpoR in its cooperation with c-Kit to induce long-term proliferation of erythroid progenitors. Furthermore, the hGM-R α chain specifically interfered with EpoR signaling, an activity neither seen for the βc subunit of the receptor complex alone, nor for the α chain of the closely related Interleukin-3 receptor. These results point to a novel role of the GM-R α chain in defining cell type–specific functions of the GM-R.
Death-effector Filaments: Novel Cytoplasmic Structures that Recruit Caspases and Trigger Apoptosis
Authors: Siegel Richard M. , Martin David A. , Zheng Lixin , Ng Samuel Y. , Bertin John , Cohen Jeffrey , Lenardo Michael J.
Published: July 2002

The death-effector domain (DED) is a critical protein interaction domain that recruits caspases into complexes with members of the TNF-receptor superfamily. Apoptosis can also be induced by expressing certain DED-containing proteins without surface receptor cross-linking. Using Green Fluorescent Protein to examine DED-containing proteins in living cells, we show that these proteins cause apoptosis by forming novel cytoplasmic filaments that recruit and activate pro-caspase zymogens. Formation of these filaments, which we term death-effector filaments, was blocked by coexpression of viral antiapoptotic DED-containing proteins, but not by bcl-2 family proteins. Thus, formation of death-effector filaments allows a regulated intracellular assembly of apoptosis-signaling complexes that can initiate or amplify apoptotic stimuli independently of receptors at the plasma membrane.
Motile Properties of Vimentin Intermediate Filament Networks in Living Cells
Authors: Yoon Miri , Moir Robert D. , Prahlad Veena , Goldman Robert D.
Published: July 2002

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.
Localization of the Kinesin-like Protein Xklp2 to Spindle Poles Requires a Leucine Zipper, a Microtubule-associated Protein, and Dynein
Authors: Wittmann Torsten , Boleti Haralabia , Antony Claude , Karsenti Eric , Vernos Isabelle
Published: July 2002

Xklp2 is a plus end–directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49–59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein–dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.
Transportin-SR, a Nuclear Import Receptor for SR Proteins
Authors: Kataoka Naoyuki , Bachorik Jennifer L. , Dreyfuss Gideon
Published: July 2002

The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. We found that RS domain–bearing proteins do not utilize any of the known nuclear import receptors and identified a novel nuclear import receptor specific for SR proteins. The SR protein import receptor, termed transportin-SR (TRN-SR), binds specifically and directly to the RS domains of ASF/SF2 and SC35 as well as several other SR proteins. The nuclear transport regulator RanGTP abolishes this interaction. Recombinant TRN-SR mediates nuclear import of RS domain– bearing proteins in vitro. TRN-SR has amino acid sequence similarity to several members of the importin β/transportin family. These findings strongly suggest that TRN-SR is a nuclear import receptor for the SR protein family.
Recycling of E-Cadherin
Authors: Le Tam Luan , Yap Alpha S. , Stow Jennifer L.
Published: July 2002

E-Cadherin plays critical roles in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. The fate of E-cadherin once it is delivered to the basolateral cell surface, and the mechanisms which govern its participation in adherens junctions, are not well understood. Using surface biotinylation and recycling assays, we observed that some of the cell surface E-cadherin is actively internalized and is then recycled back to the plasma membrane. The pool of E-cadherin undergoing endocytosis and recycling was markedly increased in cells without stable cell-cell contacts, i.e., in preconfluent cells and after cell contacts were disrupted by depletion of extracellular Ca2+, suggesting that endocytic trafficking of E-cadherin is regulated by cell-cell contact. The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment. The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18°C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway. We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.
Gemin4
Authors: Charroux Bernard , Pellizzoni Livio , Perkinson Robert A. , Yong Jeongsik , Shevchenko Andrej , Mann Matthias , Dreyfuss Gideon
Published: July 2002

The survival of motor neurons (SMN) protein, the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, is localized both in the cytoplasm and in discrete nuclear bodies called gems. In both compartments SMN is part of a large complex that contains several proteins including Gemin2 (formerly SIP1) and the DEAD box protein Gemin3. In the cytoplasm, the SMN complex is associated with snRNP Sm core proteins and plays a critical role in spliceosomal snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing by serving in the regeneration of spliceosomes. These functions are likely impaired in cells of SMA patients because they have reduced levels of functional SMN. Here, we report the identification by nanoelectrospray mass spectrometry of a novel component of the SMN complex that we name Gemin4. Gemin4 is associated in vivo with the SMN complex through a direct interaction with Gemin3. The tight interaction of Gemin4 with Gemin3 suggests that it could serve as a cofactor of this DEAD box protein. Gemin4 also interacts directly with several of the Sm core proteins. Monoclonal antibodies against Gemin4 efficiently immunoprecipitate the spliceosomal U snRNAs U1 and U5 from Xenopus oocytes cytoplasm. Immunolocalization experiments show that Gemin4 is colocalized with SMN in the cytoplasm and in gems. Interestingly, Gemin4 is also detected in the nucleoli, suggesting that the SMN complex may also function in preribosomal RNA processing or ribosome assembly.
Nuclear Import of the Ran Exchange Factor, Rcc1, Is Mediated by at Least Two Distinct Mechanisms
Authors: Nemergut Michael E. , Macara Ian G.
Published: July 2002

RCC1, the only known guanine-nucleotide exchange factor for the Ran GTPase, is an ∼45-kD nuclear protein that can bind chromatin. An important question concerns how RCC1 traverses the nuclear envelope. We now show that nuclear RCC1 is not exported readily in interphase cells and that the import of RCC1 into the nucleoplasm is extremely rapid. Import can proceed by at least two distinct mechanisms. The first is a classic import pathway mediated by basic residues within the NH2-terminal domain (NTD) of RCC1. This pathway is dependent upon both a preexisting Ran gradient and energy, and preferentially uses the importin-α3 isoform of importin-α. The second pathway is not mediated by the NTD of RCC1. This novel pathway does not require importin-α or importin-β or the addition of any other soluble factor in vitro; however, this pathway is saturable and sensitive only to a subset of inhibitors of classical import pathways. Furthermore, the nuclear import of RCC1 does not require a preexisting Ran gradient or energy. We speculate that this second import pathway evolved to ensure that RCC1 never accumulates in the cytoplasm.
The Centrosomal Protein C-Nap1 Is Required for Cell Cycle–Regulated Centrosome Cohesion
Authors: Mayor Thibault , Stierhof York-Dieter , Tanaka Kayoko , Fry Andrew M. , Nigg Erich A.
Published: July 2002

Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle–regulated manner. To test this model, we have performed a detailed structure–function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle–regulated structure that mediates centriole–centriole cohesion.
A Caveolin Dominant Negative Mutant Associates with Lipid Bodies and Induces Intracellular Cholesterol Imbalance
Authors: Pol Albert , Luetterforst Robert , Lindsay Margaret , Heino Sanna , Ikonen Elina , Parton Robert G.
Published: July 2002

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3DGV, specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1–3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cavDGV to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.
Nup2p Dynamically Associates with the Distal Regions of the Yeast Nuclear Pore Complex
Authors: Dilworth David J. , Suprapto Adisetyantari , Padovan Julio C. , Chait Brian T. , Wozniak Richard W. , Rout Michael P. , Aitchison John D.
Published: July 2002

Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC). Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them. Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and export-bound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner. When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils. On the nucleoplasmic face, where the Ran–GTP levels are predicted to be high, Nup2p binds to Nup60p. Deletion of NUP60 renders Nup2p nucleoplasmic and compromises Nup2p-mediated recycling of Kap60p/Srp1p. Depletion of Ran–GTP by metabolic poisoning, disruption of the Ran cycle, or in vitro by cell lysis, results in a shift of Nup2p from the nucleoplasm to the cytoplasmic face of the NPC. This mobility of Nup2p was also detected using heterokaryons where, unlike nucleoporins, Nup2p was observed to move from one nucleus to the other. Together, our data support a model in which Nup2p movement facilitates the transition between the import and export phases of nucleocytoplasmic transport.
A t-SNARE of the endocytic pathway must be activated for fusion
Authors: Paumet Fabienne , Brügger Britta , Parlati Francesco , McNew James A. , Söllner Thomas H. , Rothman James E.
Published: July 2002

The t-SNARE in a late Golgi compartment (Tlg2p) syntaxin is required for endocytosis and localization of cycling proteins to the late Golgi compartment in yeast. We show here that Tlg2p assembles with two light chains, Tlg1p and Vti1p, to form a functional t-SNARE that mediates fusion, specifically with the v-SNAREs Snc1p and Snc2p. In vitro, this t-SNARE is inert, locked in a nonfunctional state, unless it is activated for fusion. Activation can be mediated by a peptide derived from the v-SNARE, which likely bypasses additional regulatory proteins in the cell. Locking t-SNAREs creates the potential for spatial and temporal regulation of fusion by signaling processes that unleash their fusion capacity.
Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform
Authors: Mallard Frédéric , Tang Bor Luen , Galli Thierry , Tenza Danièle , Saint-Pol Agnès , Yue Xu , Antony Claude , Hong Wanjin , Goud Bruno , Johannes Ludger
Published: July 2002

The molecular mechanisms underlying early/recycling endosomes-to-TGN transport are still not understood. We identified interactions between the TGN-localized putative t-SNAREs syntaxin 6, syntaxin 16, and Vti1a, and two early/recycling endosomal v-SNAREs, VAMP3/cellubrevin, and VAMP4. Using a novel permeabilized cell system, these proteins were functionally implicated in the post-Golgi retrograde transport step. The function of Rab6a' was also required, whereas its closely related isoform, Rab6a, has previously been implicated in Golgi-to-endoplasmic reticulum transport. Thus, our study shows that membrane exchange between the early endocytic and the biosynthetic/secretory pathways involves specific components of the Rab and SNARE machinery, and suggests that retrograde transport between early/recycling endosomes and the endoplasmic reticulum is critically dependent on the sequential action of two members of the Rab6 subfamily.
Suppression of Leukotriene B4 Biosynthesis by Endogenous Adenosine in Ligand-activated Human Neutrophils
Authors: Krump Eric , Picard Serge , Mancini Joseph , Borgeat Pierre
Published: July 2002

Adenosine (Ado) has been shown to suppress several functional responses of human polymorphonuclear leukocytes (PMNs). The current study investigated whether endogenous Ado regulates the biosynthesis of leukotriene (LT)B4 in ligand-stimulated PMNs. Measurements of Ado in PMN resuspended in Hanks' buffered salt solution (HBSS) or plasma showed a cell concentration– and time–dependent accumulation of the nucleoside. The removal of endogenous Ado with either Ado deaminase or the blockade of its action by the Ado A2a receptor antagonist, 8-(3-chlorostyryl) caffeine, markedly increased LTB4 biosynthesis upon ligand stimulation in HBSS. Similarly, LTB4 synthesis by ligand-stimulated PMNs in plasma (containing recombinant LTA4 hydrolase to allow the conversion of protein-bound LTA4) was strongly enhanced by addition of Ado deaminase. Addition of red blood cells to suspensions of PMNs in plasma mimicked the effect of adding Ado deaminase and LTA4 hydrolase in enhancing LTB4 biosynthesis upon ligand stimulation. This effect of red blood cells on LTB4 biosynthesis was blocked by dipyridamole, an inhibitor of Ado transport, or captopril, an inhibitor of LTA4 hydrolase. These results demonstrate that endogenous Ado efficiently downregulates ligand-stimulated LTB4 biosynthesis in PMN suspensions, pointing out a potentially important regulatory function of Ado in inflammatory exudates. These results also unveil a dual role for red blood cells in upregulating LTB4 biosynthesis, namely, the removal of endogenous Ado and the conversion of LTA4 released by activated PMNs.