Chemical and genetic engineering of selective ion channel-ligand interactions

Ionic flux mediates essential physiological and behavioral functions in defined cell populations. Cell type-specific activators of diverse ionic conductances are needed for probing these effects. We combined chemistry and protein engineering to enable the systematic creation of a toolbox of ligand-gated ion channels (LGICs) with orthogonal pharmacologic selectivity and divergent functional properties. The LGICs and their small-molecule effectors were able to activate a range of ionic conductances in genetically specified cell types. LGICs constructed for neuronal perturbation could be used to selectively manipulate neuron activity in mammalian brains in vivo. The diversity of ion channel tools accessible from this approach will be useful for examining the relationship between neuronal activity and animal behavior, as well as for cell biological and physiological applications requiring chemical control of ion conductance.     

BAR domains as sensors of membrane curvature: the amphiphysin BAR structure

The BAR (Bin/amphiphysin/Rvs) domain is the most conserved feature in amphiphysins from yeast to human and is also found in endophilins and nadrins. We solved the structure of the Drosophila amphiphysin BAR domain. It is a crescent-shaped dimer that binds preferentially to highly curved negatively charged membranes. With its N-terminal amphipathic helix and BAR domain (N-BAR), amphiphysin can drive membrane curvature in vitro and in vivo. The structure is similar to that of arfaptin2, which we find also binds and tubulates membranes. From this, we predict that BAR domains are in many protein families, including sorting nexins, centaurins, and oligophrenins. The universal and minimal BAR domain is a dimerization, membrane-binding, and curvature-sensing module.     

Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy

Thymocytes are selected to mature according to their ability to interact with self major histocompatibility complex (MHC)-peptide complexes displayed on the thymic stroma. Using two-photon microscopy, we performed real-time analysis of the cellular contacts made by developing thymocytes undergoing positive selection in a three-dimensional thymic organ culture. A large fraction of thymocytes within these cultures were highly motile. MHC recognition was found to increase the duration of thymocyte-stromal cell interactions and occurred as both long-lived cellular associations displaying stable cell-cell contacts and as shorter, highly dynamic contacts. Our results identify the diversity and dynamics of thymocyte interactions during positive selection.     

Myosin I can act as a molecular force sensor

The ability to sense molecular tension is crucial for a wide array of cellular processes, including the detection of auditory stimuli, control of cell shape, and internalization and transport of membranes. We show that myosin I, a motor protein that has been implicated in powering key steps in these processes, dramatically alters its motile properties in response to tension. We measured the displacement generated by single myosin I molecules, and we determined the actin-attachment kinetics with varying tensions using an optical trap. The rate of myosin I detachment from actin decreases >75-fold under tension of 2 piconewtons or less, resulting in myosin I transitioning from a low (<0.2) to a high (>0.9) duty-ratio motor. This impressive tension sensitivity supports a role for myosin I as a molecular force sensor.     

A common allele on chromosome 9 associated with coronary heart disease

Coronary heart disease (CHD) is a major cause of death in Western countries. We used genome-wide association scanning to identify a 58-kilobase interval on chromosome 9p21 that was consistently associated with CHD in six independent samples (more than 23,000 participants) from four Caucasian populations. This interval, which is located near the CDKN2A and CDKN2B genes, contains no annotated genes and is not associated with established CHD risk factors such as plasma lipoproteins, hypertension, or diabetes. Homozygotes for the risk allele make up 20 to 25% of Caucasians and have a approximately 30 to 40% increased risk of CHD.     

Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak

A central issue in the regulation of apoptosis by the Bcl-2 family is whether its BH3-only members initiate apoptosis by directly binding to the essential cell-death mediators Bax and Bak, or whether they can act indirectly, by engaging their pro-survival Bcl-2-like relatives. Contrary to the direct-activation model, we show that Bax and Bak can mediate apoptosis without discernable association with the putative BH3-only activators (Bim, Bid, and Puma), even in cells with no Bim or Bid and reduced Puma. Our results indicate that BH3-only proteins induce apoptosis at least primarily by engaging the multiple pro-survival relatives guarding Bax and Bak.     

Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy

Cytotoxic chemotherapy targets elements common to all nucleated human cells, such as DNA and microtubules, yet it selectively kills tumor cells. Here we show that clinical response to these drugs correlates with, and may be partially governed by, the pretreatment proximity of tumor cell mitochondria to the apoptotic threshold, a property called mitochondrial priming. We used BH3 profiling to measure priming in tumor cells from patients with multiple myeloma, acute myelogenous and lymphoblastic leukemia, and ovarian cancer. This assay measures mitochondrial response to peptides derived from proapoptotic BH3 domains of proteins critical for death signaling to mitochondria. Patients with highly primed cancers exhibited superior clinical response to chemotherapy. In contrast, chemoresistant cancers and normal tissues were poorly primed. Manipulation of mitochondrial priming might enhance the efficacy of cytotoxic agents.