Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM cholesterol forms complexes with other PM lipids, making it inaccessible for intracellular transport. Transition of PM cholesterol between accessible and inaccessible pools maintains cellular homeostasis, but how cells monitor the accessibility of PM cholesterol remains unclear. We show that endoplasmic reticulum (ER)-anchored lipid transfer proteins, the GRAMD1s, sense and transport accessible PM cholesterol to the ER. GRAMD1s bind to one another and populate ER-PM contacts by sensing a transient expansion of the accessible pool of PM cholesterol via their GRAM domains. They then facilitate the transport of this cholesterol via their StART-like domains. Cells that lack all three GRAMD1s exhibit striking expansion of the accessible pool of PM cholesterol as a result of less efficient PM to ER transport of accessible cholesterol. Thus, GRAMD1s facilitate the movement of accessible PM cholesterol to the ER in order to counteract an acute increase of PM cholesterol, thereby activating non-vesicular cholesterol transport.

In the primary visual cortex (V1), the single-neuron response to a grating stimulus placed in the classical receptive field (CRF) is suppressed by a similar stimulus presented in the CRF surround. To assess the input mechanism underlying the surround suppression, we tested the effects of iontophoretically administered GABA A -receptor antagonist, bicuculline methiodide (BMI), for the 46 V1 neurons in anesthetized cats. First, the stimulus-size tuning curves were studied, with or without BMI administration, for each neuron by changing the size of the grating patch. During the BMI administration, the shape of the normalized size tuning curve did not change considerably. Second, the dependency of surround suppression on the orientation of the surround grating was examined. In the control, the surround suppression showed the clear orientation tuning that peaked at an orientation the same as the optimal orientation of the CRF response. The BMI administration did not change the orientation dependency of surround suppression. We also estimated the relative contribution of excitation and inhibition to the size and orientation tuning of surround suppression. It was concluded that cortical excitation and inhibition were well balanced, having similar tuning profiles for both stimulus size and orientation of the surround grating. Furthermore, surround stimuli used for V1 neurons suppressed the CRF response of neurons in the lateral geniculate nucleus. These results suggest that surround suppression is not primarily attributable to the intracortical inhibition, but because of a reduction of thalamocortical inputs, which drive the cortical excitation and inhibition, and a subsequent decrease in the cortical excitatory interactions.

Background:The enzymatic activities of the ATP10 family of mammalian P4-ATPases are unknown. Results: ATP10A catalyzes the flipping of NBD-PC and expression of ATP10A altered cell shape and inhibited cell adhesion and spreading. Conclusion: The enhanced PC flipping activity by ATP10A changes the lipid composition, which may cause a delay in cell spreading. Significance: This is the first evidence showing that PC flipping activity by P4-ATPase is associated with the plasma membrane dynamics.

Lipid transport is an essential process with manifest importance to human health and disease. Phospholipid flippases (P4-ATPases) transport lipids across the membrane bilayer and are involved in signal transduction, cell division, and vesicular transport. Mutations in flippase genes cause or contribute to a host of diseases, such as cholestasis, neurological deficits, immunological dysfunction, and metabolic disorders. Genomewide association studies have shown that ATP10A and ATP10D variants are associated with an increased risk of diabetes, obesity, myocardial infarction, and atherosclerosis. Moreover, ATP10D SNPs are associated with elevated levels of glucosylceramide (GlcCer) in plasma from diverse European populations. Although sphingolipids strongly contribute to metabolic disease, little is known about how GlcCer is transported across cell membranes. Here, we identify a conserved clade of P4-ATPases from Saccharomyces cerevisiae (Dnf1, Dnf2), Schizosaccharomyces pombe (Dnf2), and Homo sapiens (ATP10A, ATP10D) that transport GlcCer bearing an sn2 acyl-linked fluorescent tag. Further, we establish structural determinants necessary for recognition of this sphingolipid substrate. Using enzyme chimeras and site-directed mutagenesis, we observed that residues in transmembrane (TM) segments 1, 4, and 6 contribute to GlcCer selection, with a conserved glutamine in the center of TM4 playing an essential role. Our molecular observations help refine models for substrate translocation by P4-ATPases, clarify the relationship between these flippases and human disease, and have fundamental implications for membrane organization and sphingolipid homeostasis. Phospholipid flippases in the P4-ATPase family establish and repair the phospholipid asymmetry of cellular membranes and facilitate import of phospholipid from extracellular sources (1). These enzymes are integral membrane proteins that harness energy from ATP catalysis to translocate lipids from the exofacial leaflet to the cytofacial leaflet of cellular membranes. There are 14 distinct P4-ATPases in humans with important differences in tissue-specific expression, subcellular localization, and substrate specificity (2). These differences in P4-ATPase localization and enzymology are important for coordinating physiological processes, and their impairment results in diverse pathologies (3). For example, the P4-ATPase ATP8B1 transports phosphatidylcholine at the apical membrane of canalicular hepatocytes (4), and mutations in the ATP8B1 gene lead to membrane damage and hepatic cholestasis (3, 5). Conversely, ATP8A2 translocates phosphatidylserine within the central nervous system, and mutations in ATP8A2 cause cerebellar ataxia mental retardation and disequilibrium syndrome in humans and motor neuron degeneration in mice (6-8). Thus, defining the substrate specificity of the P4-ATPases is crucial to understanding their role in disease. Genome-wide association (GWA) 3 studies have found that SNPs in the human P4-ATPase genes ATP10A and ATP10D are strongly linked with meta...

To study the molecular mechanism how cortical areas are specialized in adult primates, we searched for area-specific genes in macaque monkeys and found striking enrichment of serotonin (5-hydroxytryptamine, 5-HT) 1B receptor mRNA, and to a lesser extent, of 5-HT2A receptor mRNA, in the primary visual area (V1). In situ hybridization analyses revealed that both mRNA species were highly concentrated in the geniculorecipient layers IVA and IVC, where they were coexpressed in the same neurons. Monocular inactivation by tetrodotoxin injection resulted in a strong and rapid (<3 h) downregulation of these mRNAs, suggesting the retinal activity dependency of their expression. Consistent with the high expression level in V1, clear modulatory effects of 5-HT1B and 5-HT2A receptor agonists on the responses of V1 neurons were observed in in vivo electrophysiological experiments. The modulatory effect of the 5-HT1B agonist was dependent on the firing rate of the recorded neurons: The effect tended to be facilitative for neurons with a high firing rate, and suppressive for those with a low firing rate. The 5-HT2A agonist showed opposite effects. These results suggest that this serotonergic system controls the visual response in V1 for optimization of information processing toward the incoming visual inputs.

Pedestrian detection is one of the key technologies for autonomous driving systems and driving assistance systems. To predict the possibility of a future collision, these systems have to accurately recognize pedestrians as far away as possible. Moreover, the function to detect not only people walking but also people who are standing near the road is also required. This paper proposes a method for recognizing pedestrians by using a high-definition LIDAR (light detection and ranging). Two novel features are introduced to improve the classification performance. One is the slice feature, which represents the profile of a human body by widths at the different height levels. The other is the distribution of the reflection intensities of points measured on the target. This feature can contribute to the pedestrian identification because each substance has its own unique reflection characteristics in the near-infrared region of the laser beam. Our approach applies a support vector machine (SVM) to train a classifier from these features. The classifier identifies the clusters of the laser range data that are the pedestrian candidates, generated by pre-processing. A quantitative evaluation in a road environment confirms the effectiveness of the proposed method.

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The ARF1+ARF4 and ARF1+ARF3 pairs are both required for integrity of recycling endosomes but are involved in distinct transport pathways: the former pair regulates retrograde transport from endosomes to the TGN, whereas the latter is required for the transferrin recycling pathway from endosomes to the plasma membrane.