Quantitative Maximum Shear-Wave Stiffness of Breast Masses as a Predictor of Histopathologic Severity.

OBJECTIVE: The objective of our study was to compare quantitative maximum breast mass stiffness on shear-wave elastography (SWE) with histopathologic outcome. SUBJECTS AND METHODS: From September 2008 through September 2010, at 16 centers in the United States and Europe, 1647 women with a sonographically visible breast mass consented to undergo quantitative SWE in this prospective protocol; 1562 masses in 1562 women had an acceptable reference standard. The quantitative maximum stiffness (termed "Emax") on three acquisitions was recorded for each mass with the range set from 0 (very soft) to 180 kPa (very stiff). The median Emax and interquartile ranges (IQRs) were determined as a function of histopathologic diagnosis and were compared using the Mann-Whitney U test. We considered the impact of mass size on maximum stiffness by performing the same comparisons for masses 9 mm or smaller and those larger than 9 mm in diameter. RESULTS: The median patient age was 50 years (mean, 51.8 years; SD, 14.5 years; range, 21-94 years), and the median lesion diameter was 12 mm (mean, 14 mm; SD, 7.9 mm; range, 1-53 mm). The median Emax of the 1562 masses (32.1% malignant) was 71 kPa (mean, 90 kPa; SD, 65 kPa; IQR, 31-170 kPa). Of 502 malignancies, 23 (4.6%) ductal carcinoma in situ (DCIS) masses had a median Emax of 126 kPa (IQR, 71-180 kPa) and were less stiff than 468 invasive carcinomas (median Emax, 180 kPa [IQR, 138-180 kPa]; p = 0.002). Benign lesions were much softer than malignancies (median Emax, 43 kPa [IQR, 24-83 kPa] vs 180 kPa [IQR, 129-180 kPa]; p < 0.0001). Usual benign lesions were soft, including 62 cases of fibrocystic change (median Emax, 32 kPa; IQR, 24-94 kPa), 51 cases of fibrosis (median Emax, 36 kPa; IQR, 22-102 kPa), and 301 fibroadenomas (median Emax, 45 kPa; IQR, 30-79 kPa). Eight lipomas (median Emax, 14 kPa; IQR, 8-15 kPa), 154 cysts (median Emax, 29 kPa; IQR, 10-58 kPa), and seven lymph nodes (median Emax, 17 kPa; IQR, 9-40 kPa) were softer than usual benign lesions (p < 0.0001 for lipomas and cysts; p = 0.007 for lymph nodes). Risk lesions were slightly stiffer than usual benign lesions (p = 0.002) but tended to be softer than DCIS (p = 0.14). Fat necrosis and abscesses were relatively stiff. Conclusions were similar for both small and large masses. CONCLUSION: Despite overlap in Emax values, maximum stiffness measured by SWE is a highly effective predictor of the histopathologic severity of sonographically depicted breast masses.

[Epigenetics and aging: How is epigenetics linked to aging?] / Épigénétique et vieillissement - Comment l'épigénétique est associée au vieillissement.

Links between aging and epigenetics have been revealed by bio-mathematicians. Methylation of cytosine, which is a characteristic of the epigenome, varies with age on some ADN loci, increasing or decreasing. From an analysis of the methylome, algorithms giving an "epigenetic age" were obtained, strongly correlated with the age. Surprisingly, this approach could be applied consistently to different tissues or unpurified cells. It was successfully applied to tissues of 185 mammalian species. The epigenetic age of embryonic pluripotent stem cells is nearly zero and it decreases to "ground zero" during gastrulation. The average methylation curve as a function of age allows discrimination between slowly or rapidly aging individuals. At the present time, more than 10 different epigenetic clocks have been proposed for medical applications. The localization of aging-sensitive CpG pairs on the genome revealed networks of "co-methylation", involved in different functions such as regulation of morphogenesis or cell differentiation. From these studies, aging appears as a continuous process, with the epigenetic clock starting to "tick" in the embryo.

Title: Épigénétique et vieillissement - Comment l'épigénétique est associée au vieillissement. Abstract: L'épigénétique étudie les modifications chimiques qui régulent l'expression des gènes sans changement de séquence de l'ADN. L'existence d'un lien entre méthylation des paires CpG de l'ADN, une caractéristique de l'épigénome, et vieillissement a été montrée : un nombre limité de ces paires voit en effet sa méthylation augmenter ou diminuer, avec l'âge. La variation de méthylation sur l'ensemble de l'ADN permet ainsi de définir un « âge épigénétique ¼, qui est corrélé avec l'âge chronologique. Chez des individus jeunes, la courbe moyenne de méthylation selon l'âge permet de repérer la qualité de leur vieillissement. Ces résultats sont surprenants car ils sont obtenus sur des tissus dont la durée de vie est pourtant variable. Certaines paires CpG sont organisées en réseaux dans lesquels on observe des co-méthylations, réseaux qui impliquent des gènes participant à la régulation de la morphogenèse ou de la différenciation cellulaire. Ces réseaux seraient les effecteurs de cette horloge épigénétique.

Shear-wave elastography improves the specificity of breast US: the BE1 multinational study of 939 masses.

PURPOSE: To determine whether adding shear-wave (SW) elastographic features could improve accuracy of ultrasonographic (US) assessment of breast masses. MATERIALS AND METHODS: From September 2008 to September 2010, 958 women consented to repeat standard breast US supplemented by quantitative SW elastographic examination in this prospective multicenter institutional review board-approved, HIPAA-compliant protocol. B-mode Breast Imaging Reporting and Data System (BI-RADS) features and assessments were recorded. SW elastographic evaluation (mean, maximum, and minimum elasticity of stiffest portion of mass and surrounding tissue; lesion-to-fat elasticity ratio; ratio of SW elastographic-to-B-mode lesion diameter or area; SW elastographic lesion shape and homogeneity) was performed. Qualitative color SW elastographic stiffness was assessed independently. Nine hundred thirty-nine masses were analyzable; 102 BI-RADS category 2 masses were assumed to be benign; reference standard was available for 837 category 3 or higher lesions. Considering BI-RADS category 4a or higher as test positive for malignancy, effect of SW elastographic features on area under the receiver operating characteristic curve (AUC), sensitivity, and specificity after reclassifying category 3 and 4a masses was determined. RESULTS: Median participant age was 50 years; 289 of 939 (30.8%) masses were malignant (median mass size, 12 mm). B-mode BI-RADS AUC was 0.950; eight of 303 (2.6%) BI-RADS category 3 masses, 18 of 193 (9.3%) category 4a lesions, 41 of 97 (42%) category 4b lesions, 42 of 57 (74%) category 4c lesions, and 180 of 187 (96.3%) category 5 lesions were malignant. By using visual color stiffness to selectively upgrade category 3 and lack of stiffness to downgrade category 4a masses, specificity improved from 61.1% (397 of 650) to 78.5% (510 of 650) (P<.001); AUC increased to 0.962 (P=.005). Oval shape on SW elastographic images and quantitative maximum elasticity of 80 kPa (5.2 m/sec) or less improved specificity (69.4% [451 of 650] and 77.4% [503 of 650], P<.001 for both), without significant improvement in sensitivity or AUC. CONCLUSION: Adding SW elastographic features to BI-RADS feature analysis improved specificity of breast US mass assessment without loss of sensitivity.

Shear wave elastography for breast masses is highly reproducible.

OBJECTIVES: To evaluate intra- and interobserver reproducibility of shear wave elastography (SWE) for breast masses. METHODS: For intraobserver reproducibility, each observer obtained three consecutive SWE images of 758 masses that were visible on ultrasound. 144 (19%) were malignant. Weighted kappa was used to assess the agreement of qualitative elastographic features; the reliability of quantitative measurements was assessed by intraclass correlation coefficients (ICC). For the interobserver reproducibility, a blinded observer reviewed images and agreement on features was determined. RESULTS: Mean age was 50 years; mean mass size was 13 mm. Qualitatively, SWE images were at least reasonably similar for 666/758 (87.9%). Intraclass correlation for SWE diameter, area and perimeter was almost perfect (ICC ≥ 0.94). Intraobserver reliability for maximum and mean elasticity was almost perfect (ICC = 0.84 and 0.87) and was substantial for the ratio of mass-to-fat elasticity (ICC = 0.77). Interobserver agreement was moderate for SWE homogeneity (κ = 0.57), substantial for qualitative colour assessment of maximum elasticity (κ = 0.66), fair for SWE shape (κ = 0.40), fair for B-mode mass margins (κ = 0.38), and moderate for B-mode mass shape (κ = 0.58), orientation (κ = 0.53) and BI-RADS assessment (κ = 0.59). CONCLUSIONS: SWE is highly reproducible for assessing elastographic features of breast masses within and across observers. SWE interpretation is at least as consistent as that of BI-RADS ultrasound B-mode features. KEY POINTS: • Shear wave ultrasound elastography can measure the stiffness of breast tissue • It provides a qualitatively and quantitatively interpretable colour-coded map of tissue stiffness • Intraobserver reproducibility of SWE is almost perfect while intraobserver reproducibility of SWE proved to be moderate to substantial • The most reproducible SWE features between observers were SWE image homogeneity and maximum elasticity.

[Neurodegenerative diseases and brain aging]. / Les maladies neurodégénératives et le vieillissement.

Neurodegenerative diseases such as Alzheimer's, Parkinson's or Amyotrophic Lateral Sclerosis are generally of sporadic origin, but the risk of being affected increases dramatically with age. Progress in understanding brain aging contributes to define precisely the relationships between aging associated physiological and pathological processes. Aging is induced by various stresses, which turn somatic cells to a senescent state. Senescent cells cannot divide but acquire a typical phenotype and release chemicals that trigger inflammation. In the brain, glial cells such as astrocytes or microglia as well as neurons are affected. Dendritic branching declines, resulting in a decrease of synaptic plasticity and cognitive performances, associated with brain aging. Aging alters also glymphatic washing, the process by which brain catabolites are eliminated. The neurodegenerative diseases are proteinopathies resulting from pathological conformations of physiological proteins. Normally washed by the glymphatic flux, these proteins accumulate and aggregate in its absence and then trigger neuron death.

Title: Les maladies neurodégénératives et le vieillissement. Abstract: Les maladies neurodégénératives, maladies d'Alzheimer, de Parkinson et de Charcot, qui affectent principalement la population âgée, sont le plus souvent d'origine sporadique, c'est-à-dire sans causes identifiées. L'avancée des connaissances sur le vieillissement permet de préciser les rapports entre vieillissement physiologique et pathologique. Le vieillissement est un phénomène cellulaire, marqué par l'apparition de cellules à l'état de sénescence et d'une inflammation chronique. Les cellules gliales, astrocytes et microglie, mais aussi les neurones sont affectés, entraînant une diminution de la plasticité synaptique, à l'origine de la diminution des performances cognitives. Ce vieillissement affecte aussi le lavage glymphatique par lequel sont éliminés les déchets métaboliques. Les maladies neurodégénératives sont des protéinopathies dues à ces déchets et la dégradation du lavage glymphatique participe au développement des pathologies.

[Is understanding the mechanism of perception possible?] / Peut-on comprendre les mécanismes de la perception ?

Perception is the understanding by the brain of the different sensory information. In humans, information is mostly visual and is conveyed to the occipital cortex. MRI techniques suggest that it is deciphered in different areas of the temporal cortex, depending upon the observed scene. The neural codes used by neurons of these areas will be discussed in two instances: Recognition of written words and of faces. In the first case, an hypothesis using basic properties of neurons and hierarchy of local combination detectors, accounts for the invariance of their visual form. That it is relevant is emphasized by its successful application to reading mechanism. In the second more recent instance, recognition of human faces by macaques is shown to reside in neuron patches in the inferior temporal cortex, forming a system identifying faces with view invariance. The code allowing facial identification using the electrical activity of 200 neurons was cracked.

TITLE: Peut-on comprendre les mécanismes de la perception ? ABSTRACT: La perception est la compréhension des informations que les organes sensoriels envoient au cerveau. Visuelles, elles sont transmises au cortex visuel dans le lobe occipital. L'imagerie par résonance magnétique montre que cette information est traitée dans le cortex temporal. Deux exemples seront discutés dans cette revue : la reconnaissance des mots écrits, et celle des visages. Dans le premier cas, une hypothèse, la hiérarchie de la combinaison de détecteurs locaux, explique les propriétés d'invariance de la forme des lettres. Dans le second, le code utilisé dans le cerveau du singe macaque pour reconnaître les visages a été déchiffré : à partir de l'activité électrique de 200 neurones du cortex temporal inférieur, il est en effet possible à un singe de connaître le visage qui lui a été présenté.

[Does neuroscience understand dreaming?] / Comment les neurosciences recherchent la clé des songes.

Sleep is a succession of two stages: slow-wave and rapid eye-movement sleep. The later has mixed characteristics between sleep and wakefulness. Therefore, dreams have been proposed to occur during this stage. This hypothesis is now considered as oversimplified. Dreaming may occur during the two stages though with different characteristics. Deciphering brain structures associated with dreaming is difficult. However, during the two stages, a decrease in low-frequency and an increase in high-frequency electrical activity in posterior cortical regions has been reported that might be the neural correlate of dreaming. The origin of cortex stimulation is under debate, but the mechanisms involved are similar to those acting during wakefulness. Dream function is not known and it might be an epiphenomenon originating from synaptic transmission noise. Depriving subjects of rapid-eye movement sleep for two weeks has no apparent effect on their behavior.

TITLE: Comment les neurosciences recherchent la clé des songes. ABSTRACT: Le sommeil est une succession de deux phases : le sommeil profond d'une part et le sommeil paradoxal d'autre part, qui possède des caractères propres à la veille et au sommeil, ce qui a conduit à proposer que cette phase hébergerait les rêves. Cette hypothèse est maintenant considérée comme simplificatrice, le sommeil profond abritant aussi des rêves aux caractéristiques différentes. Dans ces conditions, déterminer les structures cérébrales associées au rêve est difficile. Le rêve et la veille impliquent les mêmes mécanismes. L'origine des stimulations du cortex, en l'absence de stimulation externe, reste débattue et la fonction du rêve incomprise. Pour certains, le rêve pourrait être un épiphénomène provoqué par le bruit de fond de la transmission synaptique.

Rab27A and its effector MyRIP link secretory granules to F-actin and control their motion towards release sites.

The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex.

[Genetics and origin of Homo sapiens]. / Génétique et origine d'Homo sapiens.

Usually, paleoanthropology studies remains and artefacts. However, more recently, genetics offer new avenues. Information on humanisation mechanisms has been obtained from comparison with primate or archaic Homo DNA sequences. Likewise, the 1 000 Genomes Project has characterized the geographic spectrum of human genetic variation offering a basis for a genomic study of Homo sapiens phylogeny. From these studies, a model, Out of Africa, was derived. His origin is Africa, where he lived 200 000 years ago. A small fraction of the population left Africa between 50 and 100 000 years ago that have populated the rest of the world, to Europe, coastal Asia to Australia and mainland Asia to Behring Land Bridge and America. The model is supported by the decrease of genetic diversity with the distance to Eastern Africa (serial founder effect). In Europe and Asia, Homo sapiens met archaic Homo neanderthalis and H denisova. The presence of 1-3% neanderthalis sequences in modern Homo ADN indicates admixtures between these groups. Some archaic sequences are on positive selection pressure, thus suggesting that the extinct hominins might have facilitated the adaptation of H sapiens to new environments.

A 20-nm step toward the cell membrane preceding exocytosis may correspond to docking of tethered granules.

In endocrine cells, plasma membrane (PM)-bound secretory granules must undergo a number of maturation stages (i.e., priming) to become fusion-competent. Despite identification of several molecules involved in binding granules to the PM and priming them, the exact nature of events occurring at the PM still largely remains a mystery. In stimulated BON cells, we used evanescent wave microscopy to study trajectories of granules shortly before their exocytoses, which provided a physical description of vesicle-PM interactions at an unprecedented level of detail, and directly lead to an original mechanistic model. In these cells, tethered (T), nonfusogenic, vesicles are prevented from converting to fusogenic, docked (D) ones in resting conditions. Upon elevation of calcium, T-vesicles perform a 21-nm step toward the PM to become D, and fuse approximately 3 s thereafter. Our ability to directly visualize different modes of PM-attachment paves the way for clarifying the exact role of various molecules implicated in attachment and priming of granules in future studies.

Myosin va mediates docking of secretory granules at the plasma membrane.

Myosin Va (MyoVa) is a prime candidate for controlling actin-based organelle motion in neurons and neuroendocrine cells. Its function in secretory granule (SG) trafficking was investigated in enterochromaffin cells by wide-field and total internal reflection fluorescence microscopy. The distribution of endogenous MyoVa partially overlapped with SGs and microtubules. Impairing MyoVa function by means of a truncated construct (MyoVa tail) or RNA interference prevented the formation of SG-rich regions at the cell periphery and reduced SG density in the subplasmalemmal region. Individual SG trajectories were tracked to analyze SG mobility. A wide distribution of their diffusion coefficient, D(xy), was observed. Almost immobile SGs (D(xy) < 5 x 10(-4) microm2 x s(-1)) were considered as docked at the plasma membrane based on two properties: (1) SGs that undergo exocytosis have a D(xy) below this threshold value for at least 2 s before fusion; (2) a negative autocorrelation of the vertical motion was found in subtrajectories with a D(xy) below the threshold. Using this criterion of docking, we found that the main effect of MyoVa inhibition was to reduce the number of docked granules, leading to reduced secretory responses. Surprisingly, this reduction was not attributable to a decreased transport of SGs toward release sites. In contrast, MyoVa silencing reduced the occurrence of long-lasting, but not short-lasting, docking periods. We thus propose that, despite its known motor activity, MyoVa directly mediates stable attachment of SGs at the plasma membrane.

Characterization of sequential exocytosis in a human neuroendocrine cell line using evanescent wave microscopy and "virtual trajectory" analysis.

Secretion of hormones and other bioactive substances is a fundamental process for virtually all multicellular organisms. Using total internal reflection fluorescence microscopy (TIRFM), we have studied the calcium-triggered exocytosis of single, fluorescently labeled large, dense core vesicles in the human neuroendocrine BON cell line. Three types of exocytotic events were observed: (1) simple fusions (disappearance of a fluorescent spot by rapid diffusion of the dye released to the extracellular space), (2) "orphan" fusions for which only rapid dye diffusion, but not the parent vesicle, could be detected, and (3) events with incomplete or multi-step disappearance of a fluorescent spot. Although all three types were reported previously, only the first case is clearly understood. Here, thanks to a combination of two-color imaging, variable angle TIRFM, and novel statistical analyses, we show that the latter two types of events are generated by the same basic mechanism, namely shape retention of fused vesicle ghosts which become targets for sequential fusions with deeper lying vesicles. Overall, approximately 25% of all exocytotic events occur via sequential fusion. Secondary vesicles, located 200-300 nm away from the cell membrane are as fusion ready as primary vesicles located very near the cell membrane. These findings call for a fundamental shift in current models of regulated secretion in endocrine cells. Previously, sequential fusion had been studied mainly using two-photon imaging. To the best of our knowledge, this work constitutes the first quantitative report on sequential fusion using TIRFM, despite its long running and widespread use in studies of secretory mechanisms.

Serotonin secretion by human carcinoid BON cells.

BON cells are human carcinoid cells that secrete serotonin (5-HT) and various peptides. Secretion of [(3)H]5-HT by cell cultures was investigated. Acetylcholine (Ach) stimulated secretion through a somatostatin-sensitive muscarinic pathway, whereas isoproterenol was inefficient. [(3)H]5-HT secretion also was induced by Ca(2+) in the presence of the ionophore A-23187 or after digitonin permeabilization. These two processes were insensitive to stomatostatin. Ba(2+) induced an efficient somatostatin-sensitive [(3)H]5-HT secretory response. Secretion also was analyzed at the single-cell level, using carbon fiber amperometry and evanescent-field fluorescence microscopy, after labeling the secretory vesicles by transfection of the cells with a NPY-GFP construct. Both techniques revealed slow kinetics of secretory responses, suggesting that ready-to-fuse vesicles do not accumulate in these cells. Single secretory vesicles were imaged either in resting conditions or after addition of Ca(2+) ions to digitonin-permeabilized cells. The three-dimensional movements of the vesicles before exocytosis were analyzed. The mean velocity of vesicles that released their content was lower than that of silent ones. Even in the case of mobile vesicles, exocytosis often was preceded by a period of arrest lasting at least 15 seconds, consistent with a docking/priming step.

Are proteins translocated through pores? An historical overview.

Most proteinaceous pores are characterized as ionic channels. However, some are also involved in protein translocation through phospholipidic membranes. This concept has evolved slowly in cell biology and in biophysics, requiring the development of adapted electrical and biochemical methods. Protein translocation in mitochondria biogenesis, secretion by endoplasmic reticulum or bacteria, and bacterial toxins internalization are the main fields where proteinconducting pores have been described. The concept is now well established and progress at the molecular and atomic levels have shown how different this paradigm is from ionic channels involved in neurobiology. Protein-conducting pores are often parts of large complexes and electrical analysis gives on-line information at the single-molecule level. They have a large conductance that, in certain membranes, should be highly regulated to prevent ionic leaking through the membrane. Finally, they are involved not only in protein translocation, but also in membrane protein insertion (α-helix and ß-barrel types).

Analysis of transient behavior in complex trajectories: application to secretory vesicle dynamics.

Analysis of trajectories of dynamical biological objects, such as breeding ants or cell organelles, is essential to reveal the interactions they develop with their environments. Many previous works used a global characterization based on parameters calculated for entire trajectories. In cases where transient behavior was detected, this usually concerned only a particular type, such as confinement or directed motion. However, these approaches are not appropriate in situations in which the tracked objects may display many different types of transient motion. We have developed a method to exhaustively analyze different kinds of transient behavior that the tracked objects may exhibit. The method discriminates stalled periods, constrained and directed motions from random dynamics by evaluating the diffusion coefficient, the mean-square displacement curvature, and the trajectory asymmetry along individual trajectories. To detect transient motions of various durations, these parameters are calculated along trajectories using a rolling analysis window whose width is variable. The method was applied to the study of secretory vesicle dynamics in the subplasmalemmal region of human carcinoid BON cells. Analysis of transitions between transient motion periods, combined with plausible assumptions about the origin of each motion type, leads to a model of dynamical subplasmalemmal organization.

Transmitter uptake and release in PC12 cells overexpressing plasma membrane monoamine transporters.

Transmitter uptake and exocytosis of secretory vesicles are two essential aspects of neurotransmission. Here we show that transient overexpression of plasma membrane monoamine transporters in rat pheochromocytoma PC12 cells induced an approximate 20-fold enhancement of cellular uptake of monoamines. Intravesicular amine concentration was greatly increased, as demonstrated directly by carbon fibre amperometry. However, the amount of stored monoamines diminished over a 5-h period, unless monoamine oxidase was inhibited, indicating that monoamines leak out from secretory vesicles. This efflux of monoamines accounts for the reported dependence of vesicular monoamine content (the quantal size) on the kinetics of vesicular monoamine uptake. Measuring radiolabelled monoamines release from the cell population provided accurate determination of the secretory activity of the subpopulation (10-20%) of cells transfected with monoamine transporters, since they contained about 95% of the radiolabel. Accordingly, significant modification of the secretory responses was observed, at the cell population level, upon transient expression of the serotonin transporter and of proteins known to interfere with exocytosis, such as botulinum neurotoxin C1, GTPase-deficient Rab3 proteins, truncated Rabphilin constructs or Rim. The co-transfection assay described here, based on transient expression of monoamine transporters, should prove useful in functional studies of the secretory machinery.

Novel organization and properties of annexin 2-membrane complexes.

Annexin 2 belongs to the annexin family of proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Here we show that, under mild acidic conditions, annexin 2 binds to and aggregates membranes containing anionic phospholipids, a fact that questions the mechanism of its interaction with membranes via Ca(2+) bridges only. The H(+) sensitivity of annexin 2-mediated aggregation is modulated by lipid composition (i.e. cholesterol content). Cryo-electron microscopy of aggregated liposomes revealed that both the monomeric and the tetrameric forms of the protein form bridges between the liposomes at acidic pH. Monomeric annexin 2 induced two different organizations of the membrane junctions. The first resembled that obtained at pH 7 in the presence of Ca(2+). For the tetramer, the arrangement was different. These bridges seemed more flexible than the Ca(2+)-mediated junctions allowing the invagination of membranes. Time-resolved fluorescence analysis at mild acidic pH and the measurement of Stokes radius revealed that the protein undergoes conformational changes similar to those induced by Ca(2+). Labeling with the lipophilic probe 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine indicated that the protein has access to the hydrophobic part of the membrane at both acidic pH in the absence of Ca(2+) and at neutral pH in the presence of Ca(2+). Models for the membrane interactions of annexin 2 at neutral pH in the presence of Ca(2+) and at acidic pH are discussed.

MyRIP, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes.

Defects of the myosin VIIa motor protein cause deafness and retinal anomalies in humans and mice. We report on the identification of a novel myosin-VIIa-interacting protein that we have named MyRIP (myosin-VIIa- and Rab-interacting protein), since it also binds to Rab27A in a GTP-dependent manner. In the retinal pigment epithelium cells, MyRIP, myosin VIIa and Rab27A are associated with melanosomes. In transfected PC12 cells, overexpression of MyRIP was shown to interfere with the myosin VIIa tail localization. We propose that a molecular complex composed of Rab27A, MyRIP and myosin VIIa bridges retinal melanosomes to the actin cytoskeleton and thereby mediates the local trafficking of these organelles. The defect of this molecular complex is likely to account for the perinuclear mislocalization of the melanosomes observed in the retinal pigment epithelium cells of myosinVIIa-defective mice.