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1339 Janelia Publications

Showing 81-90 of 1339 results
11/10/17 | Semisynthetic fluorescent pH sensors for imaging exocytosis and endocytosis.
Martineau M, Somasundaram A, Grimm JB, Gruber TD, Choquet D, Taraska JW, Lavis LD, Perrais D
Nature Communications. 2017 Nov 10;8(1):1412. doi: 10.1038/s41467-017-01752-5

The GFP-based superecliptic pHluorin (SEP) enables detection of exocytosis and endocytosis, but its performance has not been duplicated in red fluorescent protein scaffolds. Here we describe "semisynthetic" pH-sensitive protein conjugates with organic fluorophores, carbofluorescein, and Virginia Orange that match the properties of SEP. Conjugation to genetically encoded self-labeling tags or antibodies allows visualization of both exocytosis and endocytosis, constituting new bright sensors for these key steps of synaptic transmission.

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11/08/17 | Fully integrated silicon probes for high-density recording of neural activity.
Jun JJ, Steinmetz NA, Siegle JH, Denman DJ, Bauza M, Barbarits B, Lee AK, Anastassiou CA, Andrei A, Aydın Ç, Barbic M, Blanche TJ, Bonin V, Couto J, Dutta B, Gratiy SL, Gutnisky DA, Häusser M, Karsh B, Ledochowitsch P, Lopez CM, Mitelut C, Musa S, Okun M, Pachitariu M, Putzeys J, Rich PD, Rossant C, Sun W, Svoboda K, Carandini M, Harris KD, Koch C, O'Keefe J, Harris TD
Nature. 2017 Nov 08;551(7679):232-236. doi: 10.1038/nature24636

Sensory, motor and cognitive operations involve the coordinated action of large neuronal populations across multiple brain regions in both superficial and deep structures. Existing extracellular probes record neural activity with excellent spatial and temporal (sub-millisecond) resolution, but from only a few dozen neurons per shank. Optical Ca(2+) imaging offers more coverage but lacks the temporal resolution needed to distinguish individual spikes reliably and does not measure local field potentials. Until now, no technology compatible with use in unrestrained animals has combined high spatiotemporal resolution with large volume coverage. Here we design, fabricate and test a new silicon probe known as Neuropixels to meet this need. Each probe has 384 recording channels that can programmably address 960 complementary metal-oxide-semiconductor (CMOS) processing-compatible low-impedance TiN sites that tile a single 10-mm long, 70 × 20-μm cross-section shank. The 6 × 9-mm probe base is fabricated with the shank on a single chip. Voltage signals are filtered, amplified, multiplexed and digitized on the base, allowing the direct transmission of noise-free digital data from the probe. The combination of dense recording sites and high channel count yielded well-isolated spiking activity from hundreds of neurons per probe implanted in mice and rats. Using two probes, more than 700 well-isolated single neurons were recorded simultaneously from five brain structures in an awake mouse. The fully integrated functionality and small size of Neuropixels probes allowed large populations of neurons from several brain structures to be recorded in freely moving animals. This combination of high-performance electrode technology and scalable chip fabrication methods opens a path towards recording of brain-wide neural activity during behaviour.

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11/08/17 | Ultra-selective looming detection from radial motion opponency.
Klapoetke NC, Nern A, Peek MY, Rogers EM, Breads P, Rubin GM, Reiser MB, Card GM
Nature. 2017 Nov 08;551(7679):237-241. doi: 10.1038/nature24626

Nervous systems combine lower-level sensory signals to detect higher-order stimulus features critical to survival, such as the visual looming motion created by an imminent collision or approaching predator. Looming-sensitive neurons have been identified in diverse animal species. Different large-scale visual features such as looming often share local cues, which means loom-detecting neurons face the challenge of rejecting confounding stimuli. Here we report the discovery of an ultra-selective looming detecting neuron, lobula plate/lobula columnar, type II (LPLC2) in Drosophila, and show how its selectivity is established by radial motion opponency. In the fly visual system, directionally selective small-field neurons called T4 and T5 form a spatial map in the lobula plate, where they each terminate in one of four retinotopic layers, such that each layer responds to motion in a different cardinal direction. Single-cell anatomical analysis reveals that each arm of the LPLC2 cross-shaped primary dendrites ramifies in one of these layers and extends along that layer's preferred motion direction. In vivo calcium imaging demonstrates that, as their shape predicts, individual LPLC2 neurons respond strongly to outward motion emanating from the centre of the neuron's receptive field. Each dendritic arm also receives local inhibitory inputs directionally selective for inward motion opposing the excitation. This radial motion opponency generates a balance of excitation and inhibition that makes LPLC2 non-responsive to related patterns of motion such as contraction, wide-field rotation or luminance change. As a population, LPLC2 neurons densely cover visual space and terminate onto the giant fibre descending neurons, which drive the jump muscle motor neuron to trigger an escape take off. Our findings provide a mechanistic description of the selective feature detection that flies use to discern and escape looming threats.

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11/07/17 | Immature HIV-1 lattice assembly dynamics are regulated by scaffolding from nucleic acid and the plasma membrane.
Pak AJ, Grime JM, Sengupta P, Chen AK, Durumeric AE, Srivastava A, Yeager M, Briggs JA, Lippincott-Schwartz J, Voth GA
Proceedings of the National Academy of Sciences of the United States of America. 2017 Nov 07:. doi: 10.1073/pnas.1706600114

The packaging and budding of Gag polyprotein and viral RNA is a critical step in the HIV-1 life cycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from subnanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA-SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while overexpression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding.

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11/03/17 | Topological and modality-specific representation of somatosensory information in the fly brain.
Tsubouchi A, Yano T, Yokoyama TK, Murtin C, Otsuna H, Ito K
Science (New York, N.Y.). 2017 11 03;358(6363):615-623. doi: 10.1126/science.aan4428

Insects and mammals share similarities of neural organization underlying the perception of odors, taste, vision, sound, and gravity. We observed that insect somatosensation also corresponds to that of mammals. In Drosophila, the projections of all the somatosensory neuron types to the insect's equivalent of the spinal cord segregated into modality-specific layers comparable to those in mammals. Some sensory neurons innervate the ventral brain directly to form modality-specific and topological somatosensory maps. Ascending interneurons with dendrites in matching layers of the nerve cord send axons that converge to respective brain regions. Pathways arising from leg somatosensory neurons encode distinct qualities of leg movement information and play different roles in ground detection. Establishment of the ground pattern and genetic tools for neuronal manipulation should provide the basis for elucidating the mechanisms underlying somatosensation.

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11/02/17 | A moving source of matrix components is essential for de novo basement membrane formation.
Matsubayashi Y, Louani A, Dragu A, Sánchez-Sánchez BJ, Serna-Morales E, Yolland L, Gyoergy A, Vizcay G, Fleck RA, Heddleston JM, Chew T, Siekhaus DE, Stramer BM
Current Biology : CB. 2017 Nov 02:. doi: 10.1016/j.cub.2017.10.001

The basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3-5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6-9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components.

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11/02/17 | Network-size independent covering number bounds for deep networks.
Kabra M, Branson KM
arXiv. 2017 Nov 02:arXiv:1711.00753

We give a covering number bound for deep learning networks that is independent of the size of the network. The key for the simple analysis is that for linear classifiers, rotating the data doesn't affect the covering number. Thus, we can ignore the rotation part of each layer's linear transformation, and get the covering number bound by concentrating on the scaling part.

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11/02/17 | Nuclear microenvironments modulate transcription from low-affinity enhancers.
Tsai A, Muthusamy AK, Alves MR, Lavis LD, Singer RH, Stern DL, Crocker J
eLife. 2017 Nov 02;6:. doi: 10.7554/eLife.28975

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogastershavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.

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10/31/17 | Membrane dynamics and organelle biogenesis-lipid pipelines and vesicular carriers.
Stefan CJ, Trimble WS, Grinstein S, Drin G, Reinisch K, De Camilli P, Cohen S, Valm AM, Lippincott-Schwartz J, Levine TP, Iaea DB, Maxfield FR, Futter CE, Eden ER, Judith D, van Vliet AR, Agostinis P, Tooze SA, Sugiura A, McBride HM
BMC Biology. 2017 Oct 31;15(1):102. doi: 10.1186/s12915-017-0432-0

Discoveries spanning several decades have pointed to vital membrane lipid trafficking pathways involving both vesicular and non-vesicular carriers. But the relative contributions for distinct membrane delivery pathways in cell growth and organelle biogenesis continue to be a puzzle. This is because lipids flow from many sources and across many paths via transport vesicles, non-vesicular transfer proteins, and dynamic interactions between organelles at membrane contact sites. This forum presents our latest understanding, appreciation, and queries regarding the lipid transport mechanisms necessary to drive membrane expansion during organelle biogenesis and cell growth.

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10/24/17 | Intercellular mRNA trafficking via membrane nanotube-like extensions in mammalian cells.
Haimovich G, Ecker CM, Dunagin MC, Eggan E, Raj A, Gerst JE, Singer RH
Proceedings of the National Academy of Sciences of the United States of America. 2017 Oct 24:. doi: 10.1073/pnas.1706365114

RNAs have been shown to undergo transfer between mammalian cells, although the mechanism behind this phenomenon and its overall importance to cell physiology is not well understood. Numerous publications have suggested that RNAs (microRNAs and incomplete mRNAs) undergo transfer via extracellular vesicles (e.g., exosomes). However, in contrast to a diffusion-based transfer mechanism, we find that full-length mRNAs undergo direct cell-cell transfer via cytoplasmic extensions characteristic of membrane nanotubes (mNTs), which connect donor and acceptor cells. By employing a simple coculture experimental model and using single-molecule imaging, we provide quantitative data showing that mRNAs are transferred between cells in contact. Examples of mRNAs that undergo transfer include those encoding GFP, mouse β-actin, and human Cyclin D1, BRCA1, MT2A, and HER2. We show that intercellular mRNA transfer occurs in all coculture models tested (e.g., between primary cells, immortalized cells, and in cocultures of immortalized human and murine cells). Rapid mRNA transfer is dependent upon actin but is independent of de novo protein synthesis and is modulated by stress conditions and gene-expression levels. Hence, this work supports the hypothesis that full-length mRNAs undergo transfer between cells through a refined structural connection. Importantly, unlike the transfer of miRNA or RNA fragments, this process of communication transfers genetic information that could potentially alter the acceptor cell proteome. This phenomenon may prove important for the proper development and functioning of tissues as well as for host-parasite or symbiotic interactions.

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