Cellular mechanisms of heterogeneity in NF2-mutant schwannoma.

Schwannomas are common sporadic tumors and hallmarks of familial neurofibromatosis type 2 (NF2) that develop predominantly on cranial and spinal nerves. Virtually all schwannomas result from inactivation of the NF2 tumor suppressor gene with few, if any, cooperating mutations. Despite their genetic uniformity schwannomas exhibit remarkable clinical and therapeutic heterogeneity, which has impeded successful treatment. How heterogeneity develops in NF2-mutant schwannomas is unknown. We have found that loss of the membrane:cytoskeleton-associated NF2 tumor suppressor, merlin, yields unstable intrinsic polarity and enables Nf2-/- Schwann cells to adopt distinct programs of ErbB ligand production and polarized signaling, suggesting a self-generated model of schwannoma heterogeneity. We validated the heterogeneous distribution of biomarkers of these programs in human schwannoma and exploited the synchronous development of lesions in a mouse model to establish a quantitative pipeline for studying how schwannoma heterogeneity evolves. Our studies highlight the importance of intrinsic mechanisms of heterogeneity across human cancers.

Merlin/ERM proteins regulate growth factor-induced macropinocytosis and receptor recycling by organizing the plasma membrane:cytoskeleton interface.

The architectural and biochemical features of the plasma membrane are governed by its intimate association with the underlying cortical cytoskeleton. The neurofibromatosis type 2 (NF2) tumor suppressor merlin and closely related membrane:cytoskeleton-linking protein ezrin organize the membrane:cytoskeleton interface, a critical cellular compartment that both regulates and is regulated by growth factor receptors. An example of this poorly understood interrelationship is macropinocytosis, an ancient process of nutrient uptake and membrane remodeling that can both be triggered by growth factors and manage receptor availability. We show that merlin deficiency primes the membrane:cytoskeleton interface for epidermal growth factor (EGF)-induced macropinocytosis via a mechanism involving increased cortical ezrin, altered actomyosin, and stabilized cholesterol-rich membranes. These changes profoundly alter EGF receptor (EGFR) trafficking in merlin-deficient cells, favoring increased membrane levels of its heterodimerization partner, ErbB2; clathrin-independent internalization; and recycling. Our work suggests that, unlike Ras transformed cells, merlin-deficient cells do not depend on macropinocytic protein scavenging and instead exploit macropinocytosis for receptor recycling. Finally, we provide evidence that the macropinocytic proficiency of NF2-deficient cells can be used for therapeutic uptake. This work provides new insight into fundamental mechanisms of macropinocytic uptake and processing and suggests new ways to interfere with or exploit macropinocytosis in NF2 mutant and other tumors.

EGFR-induced cytoskeletal changes drive complex cell behaviors: The tip of the iceberg.

Cytoskeletal networks are dramatically reorganized upon EGF stimulation to enable complex cell behaviors such as cell-cell communication, migration and invasion, and cell division. In this issue of Science Signaling, Roth et al. and Pike et al. use proteomic methods to identify several effectors of EGF responses. The findings show the interdependent nature of growth factor signaling and the cytoskeleton and identify potential new therapeutic targets for treating cancer and other growth factor-driven diseases.

Cell-Cell Contact and Receptor Tyrosine Kinase Signaling.

The behavior of cells within tissues is governed by the activities of adhesion receptors that provide spatial cues and transmit forces through intercellular junctions, and by growth-factor receptors, particularly receptor tyrosine kinases (RTKs), that respond to biochemical signals from the environment. Coordination of these two activities is essential for the patterning and polarized migration of cells during morphogenesis and for homeostasis in mature tissues; loss of this coordination is a hallmark of developing cancer and driver of metastatic progression. Although much is known about the individual functions of adhesion and growth factor receptors, we have a surprisingly superficial understanding of the mechanisms by which their activities are coordinated.

NF2/Merlin mediates contact-dependent inhibition of EGFR mobility and internalization via cortical actomyosin.

The proliferation of normal cells is inhibited at confluence, but the molecular basis of this phenomenon, known as contact-dependent inhibition of proliferation, is unclear. We previously identified the neurofibromatosis type 2 (NF2) tumor suppressor Merlin as a critical mediator of contact-dependent inhibition of proliferation and specifically found that Merlin inhibits the internalization of, and signaling from, the epidermal growth factor receptor (EGFR) in response to cell contact. Merlin is closely related to the membrane-cytoskeleton linking proteins Ezrin, Radixin, and Moesin, and localization of Merlin to the cortical cytoskeleton is required for contact-dependent regulation of EGFR. We show that Merlin and Ezrin are essential components of a mechanism whereby mechanical forces associated with the establishment of cell-cell junctions are transduced across the cell cortex via the cortical actomyosin cytoskeleton to control the lateral mobility and activity of EGFR, providing novel insight into how cells inhibit mitogenic signaling in response to cell contact.

p120-catenin binding masks an endocytic signal conserved in classical cadherins.

p120-catenin (p120) binds to the cytoplasmic tails of classical cadherins and inhibits cadherin endocytosis. Although p120 regulation of cadherin internalization is thought to be important for adhesive junction dynamics, the mechanism by which p120 modulates cadherin endocytosis is unknown. In this paper, we identify a dual-function motif in classical cadherins consisting of three highly conserved acidic residues that alternately serve as a p120-binding interface and an endocytic signal. Mutation of this motif resulted in a cadherin variant that was both p120 uncoupled and resistant to endocytosis. In endothelial cells, in which dynamic changes in adhesion are important components of angiogenesis and inflammation, a vascular endothelial cadherin (VE-cadherin) mutant defective in endocytosis assembled normally into cell-cell junctions but potently suppressed cell migration in response to vascular endothelial growth factor. These results reveal the mechanistic basis by which p120 stabilizes cadherins and demonstrate that VE-cadherin endocytosis is crucial for endothelial cell migration in response to an angiogenic growth factor.

Evaluation of implanted gold seeds for breast radiotherapy planning and on treatment verification: a feasibility study on behalf of the IMPORT trialists.

BACKGROUND AND PURPOSE: We describe a feasibility study testing the use of gold seeds for the identification of post-operative tumour bed after breast conservation surgery (BCS). MATERIALS AND METHODS: Fifty-three patients undergoing BCS for invasive cancer were recruited. Successful use was defined as all six seeds correctly positioned around the tumour bed during BCS, unique identification of all implanted seeds on CT planning scan and ≥ 3 seeds uniquely identified at verification to give couch displacement co-ordinates in 10/15 fractions. Planning target volume (PTV) margin size for four correction strategies were calculated from these data. Variability in tumour bed contouring was investigated with five radiation oncologists outlining five CT datasets. RESULTS: Success in inserting gold seeds, identifying them at CT planning and using them for on-treatment verification was recorded in 45/51 (88%), 37/38 (97%) and 42/43 (98%) of patients, respectively. The clinicians unfamiliar with CT breast planning consistently contoured larger volumes than those already trained. Margin size ranged from 10.1 to 1.4mm depending on correction strategy. CONCLUSION: It is feasible to implant tumour bed gold seeds during BCS. Whilst taking longer to insert than surgical clips, they have the advantage of visibility for outlining and verification regardless of the ionising radiation beam quality. Appropriate correction strategies enable margins of the order of 5mm as required by the IMPORT trials however, tackling clinician variability in contouring is important.

Auditory contact cues improve performance when grasping augmented and virtual objects with a tool.

This experiment investigated the roles of haptic and auditory contact information for the control and execution of reaching to grasp augmented (physical plus graphic) and virtual (graphic only) objects of different sizes using a tool. Haptic contact information was present when grasping augmented objects only. In half of the trials, auditory cues were provided when contact was made between the tip of the tool and the target object. Transport and grasp kinematics, as well as on spatial errors at the end of the movement were examined. Providing auditory cues at contact led to faster movements; moreover, when haptic information was not available (when grasping virtual objects), auditory cues improved spatial accuracy. Movement times increased as the object size decreased for the augmented objects, when haptic information was available, and also for the virtual objects when auditory contact cues were provided, thus following Fitts' law. However, movement times were similar for all object sizes when neither haptic nor auditory contact information was provided. These results emphasize the importance of contact information when grasping with a tool, and the benefits of auditory contact cues for improving performance.

Grasping at 'thin air': multimodal contact cues for reaching and grasping.

Two experiments investigated the effects of haptic, auditory and graphic contact cues on reaching to grasp augmented objects (physical and graphic) and virtual objects (graphic only) of various sizes. In Experiment 1, auditory contact cues were presented either to enhance or to replace natural haptic contact cues in grasping. In Experiment 2, graphic contact cues were presented alone or in combination with auditory cues, and were provided either to enhance or to replace haptic contact information. Visual information of the hand was not available. Experiment 1 showed that enhancing haptic contact information with redundant auditory cues (augmented object) led to faster movement times than haptic cues alone. When haptic information was not available (virtual object), it could be replaced to some extent by auditory contact cues. In Experiment 2 movement times were fastest when both auditory and graphic cues were provided, and slowest when no contact cues were provided. Further, movement times were scaled to target width when reaching to grasp augmented objects, thus following Fitts' law. In contrast, movement times showed a less pronounced decrease with increasing object size for virtual objects. However, even in the absence of haptic information, movement times showed a more pronounced scaling to object size when auditory contact cues were provided. These results emphasize the importance of contact information, especially haptic and auditory information, for planning and control of reaching and grasping.

A reexamination of the size-weight illusion induced by visual size cues.

The size-weight illusion induced by visually perceived sizes was reexamined to investigate whether this illusion is a sensory based or cognitive-based phenomenon. A computer-augmented environment was utilized to manipulate visual size information of target objects independently of their haptic information. Two physical cubes of equal mass (30.0 g) and size (3.0 x 3.0 x 3.0 cm) were suspended in parallel by wires attached to small graspable rings, in order to keep haptically obtained information constant between lifts. Instead of directly seeing each physical cube, subjects viewed 3D graphics of a cube with a wire and a ring that were precisely superimposed onto each physical cube. Seventeen subjects vertically lifted these augmented cubes, one after the other, by grasping the attached rings, and then reported their perception of cube heaviness. The graphical size of a comparison cube pseudo randomly varied for every comparison from 1.0 x 1.0 x 1.0 to 9.0 x 9.0 x 9.0 cm, while that of a standard cube remained constant (5.0 x 5.0 x 5.0 cm). Results indicated that the size-weight illusion frequently and systematically occurred for all the subjects such that when the comparison cube was relatively smaller than the standard cube, it was perceived to be heavier, and vice versa. As the size difference increased between the standard cube and the comparison cube, more subjects experienced the illusion, and vice versa. Follow-up tests showed occurrence of the size-weight illusion was significantly correlated with subject's sensitivity to discriminate weight, but not with sensitivity to discriminate visual size. Results suggest that the size-weight illusion induced by only visual size cues in an augmented environment is sensory based, and depends on an individual's integrated perception based on multimodal sensory information.

Effects of assembling virtual fixtures on learning a navigation task.

An approach to enhance navigation task performance is to integrate sensory guidance (virtual fixtures) into a virtual training system. To evaluate the effects of adding virtual fixtures to skill acquisition, 32 subjects were required to use a PHANToM input device, to transport a virtual object through a computer generated 3-D graphic maze. Subjects practiced navigation under 4 conditions: the maze was augmented with either a graphic fixture (G), attractive force field (F), both graphic and force field (GF), or no (N) virtual fixture. Fifteen practice trials were given before subjects were transferred to a situation with no virtual fixtures. Results showed that the implementation of the force field assisted task performance during practice; however, it failed to show positive transfer effects. In contrast, adding a graphic fixture to the virtual maze helped subjects to define the optimal pathway throughout navigation, which facilitated skill acquisition.

Grip forces when passing an object to a partner.

The goal of the present study was to investigate how grip forces are applied when transferring stable control of an object from one person to another. We asked how grip forces would be modified by the passer to (1) control for inertial forces as the object was transported toward the receiver and (2) control for the impending perturbation when the receiver made contact with the object. Twelve volunteers worked in pairs during this experiment. One partner, playing the role of passer, transported an object with embedded load cells forward or held the object at an interception location. The second partner, playing the role of receiver, waited at an interception location or reached toward the passed object. Kinematic results indicated that while passers performed a stereotypical movement, receivers were sensitive to the motion of the object as they reached to make contact. Grip force results indicated that passers' grip forces and grip/load force ratios were variable on a trial-to-trial basis, suggesting that a refined internal model of the passing task was not achieved within the timeframe of the experiment. Furthermore, a decoupling of the temporal and magnitude characteristics of the grip and inertial forces was noted in conditions where passers transported the object toward the receiver. During object transfer, it was noted that passers used visual feedback-based anticipatory control to precisely time initial grip force release, while somatosensory control was used by both the passer and receiver to precisely coordinate transfer rate.

On defining metrics for assessing laparoscopic surgical skills in a virtual training environment.

One of the key components of any training environment for surgical education is a method that can be used for assessing surgical skills. Traditionally, defining such a method has been difficult and based mainly on observations. However, through advances in modeling techniques and computer hardware and software, such methods can now be developed using combined visual and haptic rendering of a training scene. This paper presents some ideas on how metrics may be defined and used in the assessment of surgical skills in a virtual laparoscopic training environment.

Grasping an augmented object to analyse manipulative force control.

Augmented reality allows changes to be made to the visual perception of object size even while the tangible components remain completely unaltered. It was, therefore, utilized in a study whose results are being reported here to provide the proper environment required to thoroughly observe the exact effect that visual change to object size had on programming fingertip forces when objects were lifted with a precision grip. Twenty-one participants performed repeated lifts of an identical grip apparatus to a height of 20 mm, maintained each lift for 8 seconds, and then replaced the grip apparatus on the table. While all other factors of the grip apparatus remained unchanged, visual appearance was altered graphically in a 3-D augmented environment. The grip apparatus measured grip and load forces independently. Grip and load forces demonstrated significant rates of increase as well as peak forces as the size of graphical images increased; an aspect that occurred in spite of the fact that extraneous haptic information remained constant throughout the trials. By indicating a human tendency to rely - even unconsciously - on visual input to program the forces in the initial lifting phase, this finding provides further confirmation of previous research findings obtained in the physical environment; including the possibility of extraneous haptic effects (Gordon et al. 1991a, Mon-Williams and Murray 2000, Kawai et al. 2000). The present results also suggest that existing knowledge concerning human manipulation tasks in the physical world may be applied to an augmented environment where the physical objects are enhanced by computer generated visual components.