Particles that bud off from the cell surface, including viruses and microvesicles, typically have a unique membrane protein composition distinct from that of the originating plasma membrane. This selective protein composition enables viruses to evade the immune response and infect other cells. But how membrane proteins sort into budding viruses such as human immunodeficiency virus (HIV) remains unclear. Proteins could passively distribute into HIV-assembly-site membranes producing compositions resembling pre-existing plasma-membrane domains. Here, we demonstrate that proteins instead sort actively into HIV-assembly-site membranes, generating compositions enriched in cholesterol and sphingolipids that undergo continuous remodeling. Proteins are recruited into and removed from the HIV assembly site through lipid-based partitioning, initiated by oligomerization of the HIV structural protein Gag. Changes in membrane curvature at the assembly site further amplify this sorting process. Thus, a lipid-based sorting mechanism, aided by increasing membrane curvature, generates the unique membrane composition of the HIV surface.

Developmental signals such as Wnts are often presented to cells in an oriented manner. To examine the consequences of local Wnt signaling, we immobilized Wnt proteins on beads and introduced them to embryonic stem cells in culture. At the single-cell level, the Wnt-bead induced asymmetric distribution of Wnt-β-catenin signaling components, oriented the plane of mitotic division, and directed asymmetric inheritance of centrosomes. Before cytokinesis was completed, the Wnt-proximal daughter cell expressed high levels of nuclear β-catenin and pluripotency genes, whereas the distal daughter cell acquired hallmarks of differentiation. We suggest that a spatially restricted Wnt signal induces an oriented cell division that generates distinct cell fates at predictable positions relative to the Wnt source.

Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

Smart camera networks have recently emerged as a new class of sensor network infrastructure that is capable of supporting high-power in-network signal processing and enabling a wide range of applications. In this article, we provide an exposition of our efforts to build a low-bandwidth wireless camera network platform, called CITRIC, and its applications in smart camera networks. The platform integrates a camera, a microphone, a frequency-scalable (up to 624 MHz) CPU, 16 MB FLASH, and 64 MB RAM onto a single device. The device then connects with a standard sensor network mote to form a wireless camera mote. With reasonably low power consumption and extensive algorithmic libraries running on a decent operating system that is easy to program, CITRIC is ideal for research and applications in distributed image and video processing. Its capabilities of in-network image processing also reduce communication requirements, which has been high in other existing camera networks with centralized processing. Furthermore, the mote easily integrates with other low-bandwidth sensor networks via the IEEE 802.15.4 protocol. To justify the utility of CITRIC, we present several representative applications. In particular, concrete research results will be demonstrated in two areas, namely, distributed coverage hole identification and distributed object recognition.

Rapid evolution of genitalia shape, a widespread phenomenon in animals with internal fertilization, offers the opportunity to dissect the genetic architecture of morphological evolution linked to sexual selection and speciation. Most quantitative trait loci (QTL) mapping studies of genitalia divergence have focused on Drosophila melanogaster and its three most closely related species, D. simulans, D. mauritiana, and D. sechellia, and have suggested that the genetic basis of genitalia evolution involves many loci. We report the first genetic study of male genitalia evolution between D. yakuba and D. santomea, two species of the D. melanogaster species subgroup. We focus on male ventral branches, which harm females during interspecific copulation. Using landmark-based geometric morphometrics, we characterized shape variation in parental species, F1 hybrids, and backcross progeny and show that the main axis of shape variation within the backcross population matches the interspecific variation between parental species. For genotyping, we developed a new molecular method to perform multiplexed shotgun genotyping (MSG), which allowed us to prepare genomic DNA libraries from 365 backcross individuals in a few days using little DNA. We detected only three QTL, one of which spans 2.7 Mb and exhibits a highly significant effect on shape variation that can be linked to the harmfulness of the ventral branches. We conclude that the genetic architecture of genitalia morphology divergence may not always be as complex as suggested by previous studies.

Developmental genetic analysis has shown that embryos of the parasitoid wasp Nasonia vitripennis depend more on zygotic gene products to direct axial patterning than do Drosophila embryos. In Drosophila, anterior axial patterning is largely established by bicoid, a rapidly evolving maternal-effect gene, working with hunchback, which is expressed both maternally and zygotically. Here, we focus on a comparative analysis of Nasonia hunchback function and expression. We find that a lesion in Nasonia hunchback is responsible for the severe zygotic headless mutant phenotype, in which most head structures and the thorax are deleted, as are the three most posterior abdominal segments. This defines a major role for zygotic Nasonia hunchback in anterior patterning, more extensive than the functions described for hunchback in Drosophila or Tribolium. Despite the major zygotic role of Nasonia hunchback, we find that it is strongly expressed maternally, as well as zygotically. Nasonia Hunchback embryonic expression appears to be generally conserved; however, the mRNA expression differs from that of Drosophila hunchback in the early blastoderm. We also find that the maternal hunchback message decays at an earlier developmental stage in Nasonia than in Drosophila, which could reduce the relative influence of maternal products in Nasonia embryos. Finally, we extend the comparisons of Nasonia and Drosophila hunchback mutant phenotypes, and propose that the more severe Nasonia hunchback mutant phenotype may be a consequence of differences in functionally overlapping regulatory circuitry.

There is a continuing need for driver strains to enable cell type-specific manipulation in the nervous system. Each cell type expresses a unique set of genes, and recapitulating expression of marker genes by BAC transgenesis or knock-in has generated useful transgenic mouse lines. However since genes are often expressed in many cell types, many of these lines have relatively broad expression patterns. We report an alternative transgenic approach capturing distal enhancers for more focused expression. We identified an enhancer trap probe often producing restricted reporter expression and developed efficient enhancer trap screening with the PiggyBac transposon. We established more than 200 lines and found many lines that label small subsets of neurons in brain substructures, including known and novel cell types. Images and other information about each line are available online (enhancertrap.bio.brandeis.edu).

Ribonuclease mitochondrial RNA processing, a site-specific endoribonuclease involved in primer RNA metabolism in mammalian mitochondria, requires an RNA component for its activity. On the basis of copurification and selective inactivation with complementary oligonucleotides, a 135-nucleotide RNA species, not encoded in the mitochondrial genome, is identified as the RNA moiety of the endoribonuclease. This finding implies transport of a nucleus-encoded RNA, essential for organelle DNA replication, to the mitochondrial matrix.

Activity in the mouse anterior lateral motor cortex (ALM) instructs directional movements, often seconds before movement initiation. It is unknown whether this preparatory activity is localized to ALM or widely distributed within motor cortex. Here we imaged activity across motor cortex while mice performed a whisker-based object localization task with a delayed, directional licking response. During tactile sensation and the delay epoch, object location was represented in motor cortex areas that are medial and posterior relative to ALM, including vibrissal motor cortex. Preparatory activity appeared first in deep layers of ALM, seconds before the behavioral response, and remained localized to ALM until the behavioral response. Later, widely distributed neurons represented the outcome of the trial. Cortical area was more predictive of neuronal selectivity than laminar location or axonal projection target. Motor cortex therefore represents sensory, motor, and outcome information in a spatially organized manner.

Early stages of sensory systems face the challenge of compressing information from numerous receptors onto a much smaller number of projection neurons, a so called communication bottleneck. To make more efficient use of limited bandwidth, compression may be achieved using predictive coding, whereby predictable, or redundant, components of the stimulus are removed. In the case of the retina, Srinivasan et al. (1982) suggested that feedforward inhibitory connections subtracting a linear prediction generated from nearby receptors implement such compression, resulting in biphasic center-surround receptive fields. However, feedback inhibitory circuits are common in early sensory circuits and furthermore their dynamics may be nonlinear. Can such circuits implement predictive coding as well? Here, solving the transient dynamics of nonlinear reciprocal feedback circuits through analogy to a signal-processing algorithm called linearized Bregman iteration we show that nonlinear predictive coding can be implemented in an inhibitory feedback circuit. In response to a step stimulus, interneuron activity in time constructs progressively less sparse but more accurate representations of the stimulus, a temporally evolving prediction. This analysis provides a powerful theoretical framework to interpret and understand the dynamics of early sensory processing in a variety of physiological experiments and yields novel predictions regarding the relation between activity and stimulus statistics.