Peripheral thickening of the sarcomeres and pointed end elongation of the thin filaments are both promoted by SALS and its formin interaction partners.

During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-of-function studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.

Children's perception of visual and auditory ambiguity and its link to executive functions and creativity.

The phenomenon of perceptual bistability provides insights into aspects of perceptual processing not normally accessible to everyday experience. However, most experiments have been conducted in adults, and it is not clear to what extent key aspects of perceptual switching change through development. The current research examined the ability of 6-, 8-, and 10-year-old children (N = 66) to switch between competing percepts of ambiguous visual and auditory stimuli and links between switching rate, executive functions, and creativity. The numbers of switches participants reported in two visual tasks (ambiguous figure and ambiguous structure from motion) and two auditory tasks (verbal transformation and auditory streaming) were measured in three 60-s blocks. In addition, inhibitory control was measured with a Stroop task, set shifting was measured with a verbal fluency task, and creativity was measured with a divergent thinking task. The numbers of perceptual switches increased in all four tasks from 6 to 10 years of age but differed across tasks in that they were higher in the verbal transformation and ambigous structure-from-motion tasks than in the ambigous figure and auditory streaming tasks for all age groups. Although perceptual switching rates differed across tasks, there were predictive relationships between switching rates in some tasks. However, little evidence for the influence of central processes on perceptual switching was found. Overall, the results support the notion that perceptual switching is largely modality and task specific and that this property is already evident when perceptual switching emerges.

The effects of attention and task-relevance on the processing of syntactic violations during listening to two concurrent speech streams.

The notion of automatic syntactic analysis received support from some event-related potential (ERP) studies. However, none of these studies tested syntax processing in the presence of a concurrent speech stream. Here we present two concurrent continuous speech streams, manipulating two variables potentially affecting speech processing in a fully crossed design: attention (focused vs. divided) and task (lexical - detecting numerals vs. syntactical - detecting syntactic violations). ERPs elicited by syntactic violations and numerals as targets were compared with those for distractors (task-relevant events in the unattended speech stream) and attended and unattended task-irrelevant events. As was expected, only target numerals elicited the N2b and P3 components. The amplitudes of these components did not significantly differ between focused and divided attention. Both task-relevant and task-irrelevant syntactic violations elicited the N400 ERP component within the attended but not in the unattended speech stream. P600 was only elicited by target syntactic violations. These results provide no support for the notion of automatic syntactic analysis. Rather, it appears that task-relevance is a prerequisite of P600 elicitation, implying that in-depth syntactic analysis occurs only for attended speech under everyday listening situations.

Combining Novel Renal Injury Markers with Delta Serum Creatinine Early after Cardiac Surgery and Risk-Stratification for Serious Adverse Outcomes: An Exploratory Analysis.

OBJECTIVE: To evaluate the prognostic utility of multiple novel urinary biomarkers of renal injury when used alone, in pair-wise combination with an early delta serum creatinine (ΔSCr) term, and combined as a broad biomarker panel for the prediction of serious adverse outcomes that may reflect AKI in patients undergoing cardiac surgery. DESIGN: Post-hoc analysis of prospective observational study. SETTING: Academic medical center. PARTICIPANTS: 603 adults undergoing cardiac surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Urinary cystatin-c, kidney injury molecule-1, chemokine (C-C motif) ligand 2 and interleukin-18 were measured at baseline and <1 hour, 3 hours and 18-24 hours after separation from cardiopulmonary bypass (CPB). ΔSCr-initial was defined as the difference in SCr from baseline to first postoperative measure. The primary outcome of hospital mortality or renal replacement therapy occurred in 25 patients. Concordant elevation of any urinary biomarker measured 3 hours after CPB together with ΔSCr-initial ≥0 mg.dL-1 provided excellent early risk stratification for the primary outcome (OR ≥15.1, 95% CI 4.1-55.4). Combining four urinary biomarkers together with ΔSCr-initial and neutrophil gelatinase-associated lipocalin, previously reported from the same cohort, to provide a 6-point AKI risk score enabled early identification of patients reaching the primary outcome (ROCAUC 0.86, 95% CI 0.79-0.92) with potentially useful sensitivity and specificity at varied cut-points. CONCLUSIONS: Combining novel urinary biomarkers of renal injury with a creatinine-based metric soon after cardiac surgery provided excellent prognostic utility for serious adverse outcomes. Future studies are required to confirm these findings and determine optimal biomarker combinations for cost-effective risk stratification.

Molecular Investigation of the Mechanism of Non-Enzymatic Hydrolysis of Proteins and the Predictive Algorithm for Susceptibility.

A number of potential degradation routes can limit the shelf life of a biotherapeutic. While these are experimentally measurable, the tests to do so require a substantial investment in both time and material, resources rarely available early in the drug development process. To address the potential degradation route of non-enzymatic hydrolysis, we performed a molecular modeling analysis, together with an experimental study, to gain detailed insight into the reaction. On the basis of the mechanism, an algorithm for predicting the likely cleavage sites of a protein has been created. This algorithm measures four key properties during a molecular dynamics simulation, which relate to the key steps of the hydrolysis mechanism, in particular the rate-determining step (which can vary depending on the local environment). The first two properties include the secondary structure and the surface exposure of the amide bond, both of which help detect if the addition of the proton to the amide bond is possible. The second two properties relate to whether the side chain can cyclize and form a furane ring. These two properties are the orientation of the side chain relative to the amide bond and the number of hydrogen bonds between the side chain and the surrounding protein. Overall, the algorithm performs well at identifying reactive versus nonreactive bonds. The algorithm correctly classifies nearly 90% of all amide bonds following an aspartic or glutamic acid residue as reactive or nonreactive.

Assessing the validity of subjective reports in the auditory streaming paradigm.

While subjective reports provide a direct measure of perception, their validity is not self-evident. Here, the authors tested three possible biasing effects on perceptual reports in the auditory streaming paradigm: errors due to imperfect understanding of the instructions, voluntary perceptual biasing, and susceptibility to implicit expectations. (1) Analysis of the responses to catch trials separately promoting each of the possible percepts allowed the authors to exclude participants who likely have not fully understood the instructions. (2) Explicit biasing instructions led to markedly different behavior than the conventional neutral-instruction condition, suggesting that listeners did not voluntarily bias their perception in a systematic way under the neutral instructions. Comparison with a random response condition further supported this conclusion. (3) No significant relationship was found between social desirability, a scale-based measure of susceptibility to implicit social expectations, and any of the perceptual measures extracted from the subjective reports. This suggests that listeners did not significantly bias their perceptual reports due to possible implicit expectations present in the experimental context. In sum, these results suggest that valid perceptual data can be obtained from subjective reports in the auditory streaming paradigm.

Newborn infants detect cues of concurrent sound segregation.

Separating concurrent sounds is fundamental for a veridical perception of one's auditory surroundings. Sound components that are harmonically related and start at the same time are usually grouped into a common perceptual object, whereas components that are not in harmonic relation or have different onset times are more likely to be perceived in terms of separate objects. Here we tested whether neonates are able to pick up the cues supporting this sound organization principle. We presented newborn infants with a series of complex tones with their harmonics in tune (creating the percept of a unitary sound object) and with manipulated variants, which gave the impression of two concurrently active sound sources. The manipulated variant had either one mistuned partial (single-cue condition) or the onset of this mistuned partial was also delayed (double-cue condition). Tuned and manipulated sounds were presented in random order with equal probabilities. Recording the neonates' electroencephalographic responses allowed us to evaluate their processing of the sounds. Results show that, in both conditions, mistuned sounds elicited a negative displacement of the event-related potential (ERP) relative to tuned sounds from 360 to 400 ms after sound onset. The mistuning-related ERP component resembles the object-related negativity (ORN) component in adults, which is associated with concurrent sound segregation. Delayed onset additionally led to a negative displacement from 160 to 200 ms, which was probably more related to the physical parameters of the sounds than to their perceptual segregation. The elicitation of an ORN-like response in newborn infants suggests that neonates possess the basic capabilities of segregating concurrent sounds by detecting inharmonic relations between the co-occurring sounds.

Neutrophil gelatinase-associated lipocalin combined with delta serum creatinine provides early risk stratification for adverse outcomes after cardiac surgery: a prospective observational study.

OBJECTIVES: Novel biomarkers of renal injury appear inconsistent in identifying a creatinine-based diagnosis of acute kidney injury. To be clinically useful, novel acute kidney injury biomarkers should identify patients at increased risk for adverse outcomes that are a consequence of acute kidney injury earlier and with greater utility than conventional creatinine-based metrics. We sought to determine the prognostic utility of both urinary neutrophil gelatinase-associated lipocalin and varying creatinine-based metrics of renal injury at multiple time points associated with cardiac surgery. DESIGN: Prospective observational study. SETTING: Academic medical center. PATIENTS: Six hundred three adults undergoing cardiac surgery. INTERVENTIONS: Nil. MEASUREMENTS AND MAIN RESULTS: Urinary neutrophil gelatinase-associated lipocalin was measured at baseline and again less than 1 hour, 3 hours, and 18-24 hours after separation from cardiopulmonary bypass. Creatinine-based metrics included a Kidney Disease: Improving Global Outcomes definition of acute kidney injury through 7 days postoperatively as well as ΔSCr-initial, defined as the incremental change in SCr from baseline to first postoperative measure. Multivariable regression determined the prognostic utility of neutrophil gelatinase-associated lipocalin and creatinine, alone and in combination, for the primary composite outcome of hospital mortality or renal replacement therapy. The primary outcome occurred in 25 patients. Adjusted for covariates ΔSCr-initial greater than or equal to 0.0 mg/dL provided early prognostic utility for the primary outcome (odds ratio, 8.9; 95% CI, 3.0-26.6), the odds ratio comparable to a creatinine-based Kidney Disease: Improving Global Outcomes definition of acute kidney injury applied over 7 days postoperatively. The upper quartile of urinary neutrophil gelatinase-associated lipocalin best predicted the primary outcome when measured 18-24 hours post-cardiopulmonary bypass (odds ratio, 18.6; 95% CI, 5.1-68.4; p = 0.001) with earlier post-cardiopulmonary bypass measures of uncertain utility. Combining both ΔSCr-initial and neutrophil gelatinase-associated lipocalin measured 3 hours after cardiopulmonary bypass provided excellent early risk stratification for the primary outcome (odds ratio, 18.3; 95% CI, 4.5-75.0). CONCLUSIONS: Combining urinary neutrophil gelatinase-associated lipocalin with a novel creatinine-based metric, both available soon after completion of surgery, may provide previously unavailable early and effective risk stratification for serious adverse outcomes after cardiac surgery.

Decline in serum hemoglobin in the 7 days after cardiac catheterization.

OBJECTIVES: Bleeding is an established complication following cardiac catheterization and lower preoperative hemoglobin concentration is a potentially modifiable risk factor for adverse outcomes after cardiac surgery. However, typical changes in serum hemoglobin concentration after cardiac catheterization are poorly defined. The authors sought to identify the pattern of change in serum hemoglobin concentration within 7 days after cardiovascular catheterization, factors associated with this change and any association with adverse outcomes. DESIGN: Retrospective observational study over a 1-year period. SETTING: U.S. academic medical institution. PARTICIPANTS: Participants were 284 adult patients with baseline hemoglobin concentration≥12 g/dL undergoing nonemergent cardiac surgery after cardiovascular catheterization via the femoral arterial route. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Lowest daily hemoglobin concentration was recorded where available for up to 7 days after catheterization and before surgery. Generalized estimating equations identified the pattern of change in serum hemoglobin while regression models identified factors associated with hemoglobin decline. Following cardiovascular catheterization average serum hemoglobin declined over time, reaching a nadir 1.4 g/dL (95% CI 1.0-1.8) below baseline 6 days after catheterization. A higher baseline hemoglobin concentration and lower body mass index were associated with greater maximal decline in hemoglobin concentration after catheterization. Acute preoperative hemoglobin decline was not associated with acute kidney injury (AKI) or a composite adverse outcome that may reflect organ ischemia. CONCLUSIONS: In a cohort of patients before cardiac surgery serum hemoglobin declines during the week after cardiac catheterization, with maximal average decline observed 5 to 7 days after catheterization.

Incorporating oliguria into the diagnostic criteria for acute kidney injury after on-pump cardiac surgery: impact on incidence and outcomes.

OBJECTIVES: Consensus definitions represent an important step toward defining the epidemiology of acute kidney injury (AKI). However, the oliguric component of these definitions remains of uncertain impact and utility after cardiac surgery. The authors sought to define the specific impact of oliguric criteria, both alone and in combination with serum creatinine criteria, on the observed incidence of AKI and associated adverse outcomes following adult cardiac surgery. DESIGN: Retrospective observational study over a 1-year period. SETTING: Academic medical institution. PARTICIPANTS: A total of 311 adult patients undergoing elective valve and/or coronary artery bypass graft surgery with cardiopulmonary bypass. INTERVENTIONS: No interventions were performed as part of the study. MEASUREMENTS AND MAIN RESULTS: Hourly urine output and daily serum creatinine were recorded in the 2 days following surgery. AKI was defined by Acute Kidney Injury Network oliguric and serum creatinine criteria. Defined by serum creatinine criteria alone, the incidence of AKI was 17.7% and was associated strongly with in-hospital mortality (odds ratio 6.6, 95% confidence interval 1.4-30.5, p = 0.02) and renal replacement therapy (odds ratio 12.7, 95% confidence interval 2.4-67.3, p = 0.003) as well as time to discharge from the intensive care unit and hospital. Defined by oliguric criteria alone through 48 hours following surgery, the incidence of AKI dramatically increased to 55.6% but was not associated with mortality, renal replacement therapy, or time to discharge. CONCLUSIONS: Acute Kidney Injury Network oliguric criteria applied over 48 hours after surgery dramatically increased the measured incidence of AKI after cardiac surgery, but was not associated with adverse outcomes.

Acute kidney injury after cardiac surgery: does the time interval from contrast administration to surgery matter?

OBJECTIVE: The authors sought to evaluate the association between the time interval from contrast administration to cardiac surgery and postoperative acute kidney injury (AKI). DESIGN: A retrospective observational study over a 1-year period. SETTING: A US academic medical institution. PARTICIPANTS: Six hundred forty-four adult patients undergoing nonemergent cardiac surgery. INTERVENTIONS: No interventions were performed as part of the study. MEASUREMENTS AND MAIN RESULTS: AKI was defined as an increase in serum creatinine by ≥0.3 mg/dL or ≥50% above baseline within the first 2 postoperative days or the commencement of renal replacement therapy within the same period. Using a contrast-to-surgery time interval >7 days as the baseline, multivariable logistic regression analysis determined the association between a contrast-to-surgery time interval ≤1 day or 2 to 7 days and postoperative AKI adjusting for potential confounding variables. The incidence of AKI within the study cohort was 21.9%. After adjusting for other covariates, there was no association between the contrast-to-surgery time and AKI (odds ratio [OR] ≤1 day = 0.93; 95% confidence interval [CI], 0.52-1.66; p = 0.81; OR = 2-7 days = 1.28; 95% CI, 0.78-2.11; p = 0.34). CONCLUSIONS: In an appropriately selected population, cardiac surgery can be performed within 1 day of cardiovascular catheterization and contrast administration without an increase in the incidence of postoperative AKI. Recommendations to delay cardiac surgery for a specified period after contrast administration to reduce the risk of postoperative AKI are premature. Additional evidence is required before making recommendations on optimal surgical timing after contrast exposure.

Do we parse the background into separate streams in the cocktail party?

In the cocktail party situation, people with normal hearing usually follow a single speaker among multiple concurrent ones. However, there is no agreement in the literature as to whether the background is segregated into multiple streams/speakers. The current study varied the number of concurrent speech streams and investigated target detection and memory for the contents of a target stream as well as the processing of distractors. A male-voiced target stream was either presented alone (single-speech), together with one male-voiced distractor (one-distractor), or a male- and a female-voiced distractor (two-distractor). Behavioral measures of target detection and content tracking performance as well as target- and distractor detection related event-related brain potentials (ERPs) were assessed. We found that the N2 amplitude decreased whereas the P3 amplitude increased from the single-speech to the concurrent speech streams conditions. Importantly, the behavioral effect of distractors differed between the conditions with one vs. two distractor speech streams and the non-zero voltages in the N2 time window for distractor numerals and in the P3 time window for syntactic violations appearing in the non-target speech stream significantly differed between the one- and two-distractor conditions for the same (male) speaker. These results support the notion that the two background speech streams are segregated, as they show that distractors and syntactic violations appearing in the non-target streams are processed even when two speech non-target speech streams are delivered together with the target stream.

Differentiation of patients with mild cognitive impairment and healthy controls based on computer assisted hand movement analysis: a proof-of-concept study.

Mild cognitive impairment (MCI) is the prodromal phase of dementia, and it is highly underdiagnosed in the community. We aimed to develop an automated, rapid (< 5 min), electronic screening tool for the recognition of MCI based on hand movement analysis. Sixty-eight individuals participated in our study, 46 healthy controls and 22 patients with clinically defined MCI. All participants underwent a detailed medical assessment including neuropsychology and brain MRI. Significant differences were found between controls and MCI groups in mouse movement characteristics. Patients showed higher level of entropy for both the left (F = 5.24; p = 0.001) and the right hand (F = 8.46; p < 0.001). Longer time was required in MCI to perform the fine motor task (p < 0.005). Furthermore, we also found significant correlations between mouse movement parameters and neuropsychological test scores. Correlation was the strongest between motor parameters and Clinical Dementia Rating scale (CDR) score (average r: - 0.36, all p's < 0.001). Importantly, motor parameters were not influenced by age, gender, or anxiety effect (all p's > 0.05). Our study draws attention to the utility of hand movement analysis, especially to the estimation of entropy in the early recognition of MCI. It also suggests that our system might provide a promising tool for the cognitive screening of large populations.

Who said what? The effects of speech tempo on target detection and information extraction in a multi-talker situation: An ERP and functional connectivity study.

People with normal hearing can usually follow one of the several concurrent speakers. Speech tempo affects both the separation of concurrent speech streams and information extraction from them. The current study varied the tempo of two concurrent speech streams to investigate these processes in a multi-talker situation. Listeners performed a target-detection and a content-tracking task, while target-related ERPs and functional brain networks sensitive to speech tempo were extracted from the EEG signal. At slower than normal speech tempo, building the two streams required longer processing times, and possibly the utilization of higher-order, for example, syntactic and semantic cues. The observed longer reaction times and higher connectivity strength in a theta band network associated with frontal control over auditory/speech processing are compatible with this notion. With increasing tempo, target detection performance decreased and the N2b and the P3b amplitudes increased. These data suggest an increased need for strictly allocating target-detection-related resources at higher tempo. This was also reflected by the observed increase in the strength of gamma-band networks within and between frontal, temporal, and cingular areas. At the fastest tested speech tempo, there was a sharp drop in recognition memory performance, while target detection performance increased compared to the normal speech tempo. This was accompanied by a significant increase in the strength of a low alpha network associated with the suppression of task-irrelevant speech. These results suggest that participants prioritized the immediate target detection task over the continuous content tracking, likely due to some capacity limit reached the fastest speech tempo.

Linguistic predictability influences auditory stimulus classification within two concurrent speech streams.

Acoustic predictability has been shown to affect auditory stream segregation, while linguistic predictability is known to be an important factor in speech comprehension. We tested the effects of linguistic predictability on auditory stream segregation and target detection by assessing the event-related potentials elicited by targets and distractors in participants presented with two concurrent speech streams. The linguistic cues of predictability varied over four levels. In the three real speech conditions, natural speech was presented with intact phonotactics and sentence prosody: normal speech, word-salad (randomized word order within each sentence), and pseudo-words (randomized syllable order within each sentence). The fourth (control) condition delivered a spectrally rotated version of the normal speech condition. Participants were instructed to attend one stream and respond to the natural cough sounds embedded in it. Coughs were present in both streams, serving as targets in the attended and as distractors in the unattended stream. We expected improved target detection with increasing linguistic predictability. The target-related N2b component's amplitude monotonically increased from the pseudo-word to the word-salad and normal speech condition, while no predictability effects were observed for the P3b amplitude or for behavioral responses. The dissociation between the N2b and P3b/behavioral effect suggests that while linguistic predictability enhanced the process of classifying stimuli as potential targets, this did not affect their detection. Furthermore, the observed nonmonotonic distractor N2 (probably MMN) amplitude increase with increasing linguistic predictability is compatible with the notion that linguistic predictability can modulate auditory stream segregation and/or stream selection.

The effects of speech processing units on auditory stream segregation and selective attention in a multi-talker (cocktail party) situation.

Speech unfolds at different time scales. Therefore, neuronal mechanisms involved in speech processing should likewise operate at different (corresponding) time scales. The present study aimed to identify speech units relevant for selecting speech streams in a multi-talker situation. Functional connectivity was extracted from the continuous EEG while young adults detected targets within one stream in the presence of a different, task-irrelevant stream. In two separate groups, either the attended or the ignored stream was manipulated so that it contained intact, word-wise scrambled, syllable-wise scrambled, or spectrally scrambled speech. We found functional brain networks that were sensitive to the difference between the situations when speech was meaningful at sentence vs. at word level, but not between when speech was meaningful at word vs. only valid at syllable level, irrespective of whether the speech units were manipulated in the attended or the ignored stream. These functional brain networks operated in the delta and theta bands corresponding to integrating information from longer time windows. Further, the networks, which could be linked with suppressing information from the to-be-ignored stream included brain areas associated with high-level processing of speech. These results are compatible with late filtering models of auditory attention, as they suggest that the length of intact speech units in the to-be-ignored stream affects processes of attentional selection. However, we found no evidence for speech-to-brain coupling differences as a function of the intact unit of speech in either stream. Thus, although the current results do not rule out that early processes of speech processing affect stream selection in a cocktail party situation, neither do they provide supporting for it.

Nanoscopy reveals the layered organization of the sarcomeric H-zone and I-band complexes.

Sarcomeres are extremely highly ordered macromolecular assemblies where structural organization is intimately linked to their functionality as contractile units. Although the structural basis of actin and Myosin interaction is revealed at a quasiatomic resolution, much less is known about the molecular organization of the I-band and H-zone. We report the development of a powerful nanoscopic approach, combined with a structure-averaging algorithm, that allowed us to determine the position of 27 sarcomeric proteins in Drosophila melanogaster flight muscles with a quasimolecular, ∼5- to 10-nm localization precision. With this protein localization atlas and template-based protein structure modeling, we have assembled refined I-band and H-zone models with unparalleled scope and resolution. In addition, we found that actin regulatory proteins of the H-zone are organized into two distinct layers, suggesting that the major place of thin filament assembly is an M-line-centered narrow domain where short actin oligomers can form and subsequently anneal to the pointed end.

The Activities of the Gelsolin Homology Domains of Flightless-I in Actin Dynamics.

Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the biological functioning of Flightless-I we studied the actin activities of Drosophila Flightless-I by in vitro bulk fluorescence spectroscopy and single filament fluorescence microscopy, as well as in vivo genetic approaches. Flightless-I was found to interact with actin and affects actin dynamics in a calcium-independent fashion in vitro. Our work identifies the first three gelsolin homology domains (1-3) of Flightless-I as the main actin-binding site; neither the other three gelsolin homology domains (4-6) nor the leucine-rich repeats bind actin. Flightless-I inhibits polymerization by high-affinity (∼nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (∼µM) monomer binding, and does not sever actin filaments. Our work reveals that in the presence of profilin Flightless-I is only able to cap actin filament barbed ends but fails to promote actin assembly. In line with the in vitro data, while gelsolin homology domains 4-6 have no effect on in vivo actin polymerization, overexpression of gelsolin homology domains 1-3 prevents the formation of various types of actin cables in the developing Drosophila egg chambers. We also show that the gelsolin homology domains 4-6 of Flightless-I interact with the C-terminus of Drosophila Disheveled-associated activator of morphogenesis formin and negatively regulates its actin assembly activity.

The spindle positioning protein Kar9p interacts with the sumoylation machinery in Saccharomyces cerevisiae.

Accurate positioning of the mitotic spindle is important for the genetic material to be distributed evenly in dividing cells, but little is known about the mechanisms that regulate this process. Here we report that two microtubule-associated proteins important for spindle positioning interact with several proteins in the sumoylation pathway. By two-hybrid analysis, Kar9p and Bim1p interact with the yeast SUMO Smt3p, the E2 enzyme Ubc9p, an E3 Nfi1p, as well as Wss1p, a weak suppressor of a temperature-sensitive smt3 allele. The physical interaction between Kar9p and Ubc9p was confirmed by in vitro binding assays. A single-amino-acid substitution in Kar9p, L304P disrupted its two-hybrid interaction with proteins in the sumoylation pathway, but retained its interactions with the spindle positioning proteins Bim1p, Stu2p, Bik1p, and Myo2p. The kar9-L304P mutant showed defects in positioning the mitotic spindle, with the spindle located more distally than normal. Whereas wild-type Kar9p-3GFP normally localizes to only the bud-directed spindle pole body (SPB), Kar9p-L304P-3GFP was mislocalized to both SPBs. Using a reconstitution assay, Kar9p was sumoylated in vitro. We propose a model in which sumoylation regulates spindle positioning by restricting Kar9p to one SPB. These findings raise the possibility that sumoylation could regulate other microtubule-dependent processes.