Breast cancer antiestrogen resistance 3-p130Cas interactions promote adhesion disassembly and invasion in breast cancer cells.

Adhesion turnover is critical for cell motility and invasion. We previously demonstrated that the adaptor molecule breast cancer antiestrogen resistance 3 (BCAR3) promotes adhesion disassembly and breast tumor cell invasion. One of two established binding partners of BCAR3 is the adaptor molecule, p130Cas. In this study, we sought to determine whether signaling through the BCAR3-Cas complex was responsible for the cellular functions of BCAR3. We show that the entire pool of BCAR3 is in complex with Cas in invasive breast tumor cells and that these proteins colocalize in dynamic cellular adhesions. Although accumulation of BCAR3 in adhesions did not require Cas binding, a direct interaction between BCAR3 and Cas was necessary for efficient dissociation of BCAR3 from adhesions. The dissociation rates of Cas and two other adhesion molecules, α-actinin and talin, were also significantly slower in the presence of a Cas-binding mutant of BCAR3, suggesting that turnover of the entire adhesion complex was delayed under these conditions. As was the case for adhesion turnover, BCAR3-Cas interactions were found to be important for BCAR3-mediated breast tumor cell chemotaxis toward serum and invasion in Matrigel. Previous work demonstrated that BCAR3 is a potent activator of Rac1, which in turn is an important regulator of adhesion dynamics and invasion. However, in contrast to wild-type BCAR3, ectopic expression of the Cas-binding mutant of BCAR3 failed to induce Rac1 activity in breast cancer cells. Together, these data show that the ability of BCAR3 to promote adhesion disassembly, tumor cell migration and invasion, and Rac1 activity is dependent on its ability to bind to Cas. The activity of BCAR3-Cas complexes as a functional unit in breast cancer is further supported by the co-expression of these molecules in multiple subtypes of human breast tumors.

Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope.

Raster image correlation spectroscopy (RICS) is a new and novel technique for measuring molecular dynamics and concentrations from fluorescence confocal images. The RICS technique extracts information about molecular dynamics and concentrations from images of living cells taken on commercial confocal systems. Here we develop guidelines for performing the RICS analysis on an analogue commercial laser scanning confocal microscope. Guidelines for typical instrument settings, image acquisition settings and analogue detector characterization are presented. Using appropriate instrument/acquisition parameters, diffusion coefficients and concentrations can be determined, even for highly dynamic dye molecules in solution. Standard curves presented herein demonstrate the ability to detect protein concentrations as low as approximately 2 nM. Additionally, cellular measurements give accurate values for the diffusion of paxillin-enhanced-green fluorescent protein (EGFP), an adhesion adaptor molecule, in the cytosol of the cell and also show slower paxillin dynamics near adhesions where paxillin interacts with immobile adhesion components. Methods are presented to account for bright immobile structures within the cell that dominate spatial correlation functions; allowing the extraction of fast protein dynamics within and near these structures. A running average algorithm is also presented to address slow cellular movement or movement of cellular features such as adhesions. Finally, methods to determine protein concentration in the presence of immobile structures within the cell are presented. A table is presented giving guidelines for instrument and imaging setting when performing RICS on the Olympus FV300 confocal and these guidelines are a starting point for performing the analysis on other commercial confocal systems.

Visualizing muscle cell migration in situ.

BACKGROUND: Cell migration has been studied extensively by manipulating and observing cells bathed in putative chemotactic or chemokinetic agents on planar substrates. This environment differs from that in vivo and, consequently, the cells can behave abnormally. Embryo slices provide an optically accessible system for studying cellular navigation pathways during development. We extended this system to observe the migration of muscle precursors from the somite into the forelimb, their cellular morphology, and the localization of green fluorescent protein (GFP)-tagged adhesion-related molecules under normal and perturbed conditions. RESULTS: Muscle precursors initiated migration synchronously and migrated in broad, rather than highly defined, regions. Bursts of directed migration were followed by periods of meandering or extension and retraction of cell protrusions. Although paxillin did not localize to discernible intracellular structures, we found that alpha-actinin localized to linear, punctate structures, and the alpha5 integrin to some focal complexes and/or vesicle-like concentrations. Alterations in the expression of adhesion molecules inhibited migration. The muscle precursors migrating in situ formed unusually large, long-lived protrusions that were polarized in the direction of migration. Unlike wild-type Rac, a constitutively active Rac localized continuously around the cell surface and promoted random protrusive activity and migration. CONCLUSIONS: The observation of cellular migration and the dynamics of molecular organization at high temporal and spatial resolution in situ is feasible. Migration from the somite to the wing bud is discontinuous and not highly stereotyped. In situ, local activation of Rac appears to produce large protrusions, which in turn, leads to directed migration. Adhesion can also regulate migration.

Use of context by young and aged adults with normal hearing.

Word recognition in sentences with and without context was measured in young and aged subjects with normal but not identical audiograms. Benefit derived from context by older adults has been obscured, in part, by the confounding effect of even mildly elevated thresholds, especially as listening conditions vary in difficulty. This problem was addressed here by precisely controlling signal-to-noise ratio across conditions and by accounting for individual differences in signal-to-noise ratio. Pure-tone thresholds and word recognition were measured in quiet and threshold-shaped maskers that shifted quiet thresholds by 20 and 40 dB. Word recognition was measured at several speech levels in each condition. Threshold was defined as the speech level (or signal-to-noise ratio) corresponding to the 50 rau point on the psychometric function. As expected, thresholds and slopes of psychometric functions were different for sentences with context compared to those for sentences without context. These differences were equivalent for young and aged subjects. Individual differences in word recognition among all subjects, young and aged, were accounted for by individual differences in signal-to-noise ratio. With signal-to-noise ratio held constant, word recognition for all subjects remained constant or decreased only slightly as speech and noise levels increased. These results suggest that, given equivalent speech audibility, older and younger listeners derive equivalent benefit from context.

Feasibility of using fMRI to study mothers responding to infant cries.

While parenting is a universal human behavior, its neuroanatomic basis is currently unknown. Animal data suggest that the cingulate may play an important function in mammalian parenting behavior. For example, in rodents cingulate lesions impair maternal behavior. Here, in an attempt to understand the brain basis of human maternal behavior, we had mothers listen to recorded infant cries and white noise control sounds while they underwent functional MRI (fMRI) of the brain. We hypothesized that mothers would show significantly greater cingulate activity during the cries compared to the control sounds. Of 7 subjects scanned, 4 had fMRI data suitable for analysis. When fMRI data were averaged for these 4 subjects, the anterior cingulate and right medial prefrontal cortex were the only brain regions showing statistically increased activity with the cries compared to white noise control sounds (cluster analysis with one-tailed z-map threshold of P < 0.001 and spatial extent threshold of P < 0.05). These results demonstrate the feasibility of using fMRI to study brain activity in mothers listening to infant cries and that the anterior cingulate may be involved in mothers listening to crying babies. We are currently replicating this study in a larger group of mothers. Future work in this area may help (1) unravel the functional neuroanatomy of the parent-infant bond and (2) examine whether markers of this bond, such as maternal brain response to infant crying, can predict maternal style (i.e., child neglect), offspring temperament, or offspring depression or anxiety.

Psychometric functions for gap detection in noise measured from young and aged subjects.

Psychometric functions for gap detection of temporal gaps in wideband noise were measured in a "yes/no" paradigm from normal-hearing young and aged subjects with closely matched audiograms. The effects of noise-burst duration, gap location, and uncertainty of gap location were tested. A typical psychometric function obtained in this study featured a steep slope, which was independent of most experimental conditions as well as age. However, gap thresholds were generally improved with increasing duration of the noise burst for both young and aged subjects. Gap location and uncertainty had no significant effects on the thresholds for the young subjects. For the aged subjects, whenever the gap was sufficiently away from the onset or offset of the noise burst, detectability was robust despite uncertainty about the gap location. Significant differences between young and aged subjects could be observed only when the gap was very close to the signal onset and offset.

The time course of hearing aid benefit.

OBJECTIVE: The present study sought to determine whether benefit derived from hearing aid use increases with hearing aid experience. This question is of considerable interest to both researchers and clinicians. Several previous investigations recently published have obtained mixed results. DESIGN: Control conditions were provided to address the potential influences of practice effects and changes in preferred hearing aid gain. The experimental group consisted of 13 hearing-impaired listeners about to be fit with their first hearing-aid. The control group consisted of 13 hearing aid users with at least 1 yr's experience with their hearing aids. The control group permitted examination of practice effects that may have confounded previous results showing increases in benefit with experience. Hearing aid benefit was defined as aided speech recognition ability minus unaided speech recognition ability and was assessed repeatedly over 18 wk. Two measures of hearing aid benefit were employed: an objective syllable recognition task and a subjective questionnaire. For the objective measure, hearing aid benefit was assessed for the condition of fixed hearing aid gain and also for the condition of subject-adjusted hearing aid gain to examine effects of changes in audibility that may have influenced benefit and confounded previous results. RESULTS: The objective measure of group mean hearing aid benefit increased significantly over time for both gain conditions for the new hearing aid users, but did not increase for the long-standing control group. Subjective benefit increased over time, but without statistical significance for the new hearing aid users, and was essentially unchanged for the long-standing control group. CONCLUSIONS: Results suggest that the observed improvements in speech recognition are not due to increases in audibility nor to simple practice effects. The overall improvements in benefit over time were of statistical significance and also practical importance for studies of group differences. However, the improvements are too small to be observed consistently for individual hearing aid users.

Forward- and backward-masked intensity discrimination measured using forced-choice and adjustment procedures.

A "midlevel" hump in the intensity jnd has been reported for pure tones preceded [e.g., Zeng et al., Hear. Res. 55, 223-230 (1991)] or followed [Plack and Viemeister, J. Acoust. Soc. Am. 92, 3097-3101 (1992)] by an intense masker where the signal-masker interval was 100 ms. These previous studies used forced-choice procedures, in which subjects were required to indicate the more intense tone. Plack and Viemeister [J. Acoust. Soc. Am. 92, 3097-3101 (1992)] have proposed that the task of judging the intensity of the short probe tone, when it is presented along with an intense masker, may lead to cognitive or central factors influencing the results. The present experiments attempted to reduce these possible effects by measuring intensity jnd's using two additional paradigms. First, a "multiple-look" forced-choice method, in which subjects listened to the stimulus pairs several times before responding, was used to obtain only forward-masked intensity jnd's. Second, the method of adjustment was used to obtain both forward- and backward-masked intensity jnd's. Both the standard forced-choice method and the multiple-look forced-choice method yielded jnd data with a midlevel hump, when compared to jnd's measured without a masker. In contrast, jnd's obtained with the method of adjustment yielded jnd data with no midlevel hump. The present results suggest that the traditional method of adjustment for intensity discrimination, where subjects adjust the signal level to a point of subjective equality, may measure a fundamentally different quantity than that measured by forced-choice procedures.

Frequency discrimination in forward and backward masking.

Frequency difference limens for pure tones preceded by a forward masker or followed by a backward masker were obtained across a wide range of signal levels. Relkin and Doucet [Hear. Res. 55, 215-222 (1991)] have shown that at a masker-signal delay of 100 ms, the thresholds of high-SR (spontaneous rate) auditory-nerve fibers are recovered, while the low-SR fiber thresholds are not. Therefore, forward-masked frequency discrimination potentially offers a method to investigate the role of low-SR fibers in the coding of frequency. It has been shown that when an intense forward masker is presented 100 ms before a pure-tone signal, intensity difference limens are elevated for mid-level signals [Zeng et al., Hear. Res. 55, 223-230 (1991)]. However, Plack and Viemeister [J. Acoust. Soc. Am. 92, 3097-3101 (1992)] have shown that a similar elevation in the intensity difference limen is obtained under conditions of backward masking, where selective adaptation of the auditory neurons would not be expected to occur. A condition of backward-masked frequency discrimination was therefore included to investigate the role of interference resulting from adding additional stimuli to a discrimination task. For signals at 1000 and 6000 Hz, there was no effect of a forward masker upon frequency difference limens. For the backward-masked conditions, an elevation of the frequency difference limen was observed at all signal levels, demonstrating that the effects of forward and backward maskers upon frequency discrimination are dissimilar and suggesting that cognitive effects are present in backward-masked discrimination tasks.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection and recognition of stop consonants by normal-hearing and hearing-impaired listeners.

This study examined the possibility that hearing-impaired listeners, in addition to displaying poorer-than-normal recognition of speech presented in background noise, require a larger signal-to-noise ratio for the detection of the speech sounds. Psychometric functions for the detection and recognition of stop consonants were obtained from both normal-hearing and hearing-impaired listeners. Expressing the speech levels in terms of their short-term spectra, the detection of consonants for both subject groups occurred at the same signal-to-noise ratio. In contrast, the hearing-impaired listeners displayed poorer recognition performance than the normal-hearing listeners. These results imply that the higher signal-to-noise ratios required for a given level of recognition by some subjects with hearing loss are not due in part to a deficit in detection of the signals in the masking noise, but rather are due exclusively to a deficit in recognition.

Effects of different frequency response strategies upon recognition and preference for audible speech stimuli.

The purpose of this study was to examine whether the amount of low- versus high-frequency amplification should change as a function of input level, as is done in some recently developed hearing aids. Adults with high-frequency sensorineural hearing loss served as subjects. Both identification performance and preference judgments for audible CV syllables were assessed as a function of input level for three different signal processing conditions both in quiet and in noise. The first signal processing condition was a conventional high-pass frequency response that did not change its transfer function as the input level increased; the second condition was similar to a typical adaptive frequency response (AFR) hearing aid: a high-pass frequency response that became increasingly high-pass as the input level increased; the third condition was similar to the K-Amp hearing aid recommended by Killion (1988): a high-pass frequency response that became more broadband as the input level increased. Results indicated no significant differences among the three different processing conditions for syllable recognition and a strong listener preference for the syllables presented via the conventional amplification scheme.