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1287 Janelia Publications

Showing 41-50 of 1287 results
Wu Lab
10/01/17 | Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres.
Xiao H, Wang F, Wisniewski J, Shaytan AK, Ghirlando R, Fitzgerald PC, Huang Y, Wei D, Li S, Landsman D, Panchenko AR, Wu C
Genes & Development. 2017 Oct 01;31(19):1958-1972. doi: 10.1101/gad.304782.117

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.

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09/26/17 | Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes.
Fritz-Laylin LK, Riel-Mehan M, Chen B, Lord SJ, Goddard TD, Ferrin TE, Nicholson-Dykstra SM, Higgs H, Johnson GT, Betzig E, Mullins RD
eLife. 2017 Sep 26;6:. doi: 10.7554/eLife.26990

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

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09/25/17 | Cell volume change through water efflux impacts cell stiffness and stem cell fate.
Guo M, Pegoraro AF, Mao A, Zhou EH, Arany PR, Han Y, Burnette DT, Jensen MH, Kasza KE, Moore JR, Mackintosh FC, Fredberg JJ, Mooney DJ, Lippincott-Schwartz J, Weitz DA
Proceedings of the National Academy of Sciences of the United States of America. 2017 Sep 25;114(41):E8618-27. doi: 10.1073/pnas.1705179114

Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its volume decreases, while its stiffness concomitantly increases. We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations, including varying substrate stiffness, cell spread area, and external osmotic pressure. The reduction of cell volume is a result of water efflux, which leads to a corresponding increase in intracellular molecular crowding. Furthermore, we find that changes in cell volume, and hence stiffness, alter stem-cell differentiation, regardless of the method by which these are induced. These observations reveal a surprising, previously unidentified relationship between cell stiffness and cell volume that strongly influences cell biology.

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09/21/17 | Genomic probes.
Singer RH, Deng W, Lionnet T
USPTO. 2017 Sep 21;A1:

Labeled probes, and methods of use thereof, comprise a Cas polypeptide conjugated to gRNA that is specific for target nucleic acid sequences, including genomic DNA sequences. The probes and methods can be used to label nucleic acid sequences without global DNA denaturation. The presently-disclosed subject matter meets some or all of the above identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

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09/19/17 | Cohesin can remain associated with chromosomes during DNA replication.
Rhodes JD, Haarhuis JH, Grimm JB, Rowland BD, Lavis LD, Nasmyth KA
Cell Reports. 2017 Sep 19;20(12):2749-55. doi: 10.1016/j.celrep.2017.08.092

To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We developed a non-fluorescent HaloTag ligand to discriminate the fluorescence recovery signal from labeling of newly synthesized Halo-tagged Scc1 protein (pulse-chase or pcFRAP). In cells where cohesin turnover is inactivated by deletion of WAPL, Scc1 can remain associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to un-replicated DNA.

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09/19/17 | Synthesis of Janelia Fluor HaloTag and SNAP-Tag Ligands and Their Use in Cellular Imaging Experiments.
Grimm JB, Brown TA, English BP, Lionnet T, Lavis LD
Methods in Molecular Biology (Clifton, N.J.). 2017;1663:179-188. doi: 10.1007/978-1-4939-7265-4_15

The development of genetically encoded self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in microscopy. Intracellular labeling using these systems requires small, cell-permeable dyes with high brightness and photostability. We recently discovered a general method to improve the properties of classic fluorophores by replacing N,N-dimethylamino groups with four-membered azetidine rings to create the "Janelia Fluor" dyes. Here, we describe the synthesis of the HaloTag and SNAP-tag ligands of Janelia Fluor 549 and Janelia Fluor 646 as well as standard labeling protocols for use in ensemble and single-molecule cellular imaging.

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09/14/17 | Q&A: The brain under a mesoscope: the forest and the trees.
Sofroniew NJ
BMC Biology. 2017 Sep 14;15(1):82. doi: 10.1186/s12915-017-0426-y

Neurons relevant to a particular behavior are often widely dispersed across the brain. To record activity in groups of individual neurons that might be distributed across large distances, neuroscientists and optical engineers have been developing a new type of microscope called a mesoscope. Mesoscopes have high spatial resolution and a large field of view. This Q&A will discuss this exciting new technology, highlighting a particular instrument, the two-photon random access mesoscope (2pRAM).

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09/09/17 | A deep structured learning approach towards automating connectome reconstruction from 3D electron micrographs.
Funke J, Tschopp FD, Grisaitis W, Singh C, Saalfeld S, Turaga SC
arXiv. 2017 Sep 09:

We present a deep learning method for neuron segmentation from 3D electron microscopy (EM), which improves significantly upon state of the art in terms of accuracy and scalability. Our method consists of a fully 3D extension of the U-NET architecture, trained to predict affinity graphs on voxels, followed by a simple and efficient iterative region agglomeration. We train the U-NET using a structured loss function based on MALIS that encourages topological correctness. The resulting affinity predictions are accurate enough that we obtain state-of-the-art results by a simple new learning-free percentile-based iterative agglomeration algorithm. We demonstrate the accuracy of our method on three different and diverse EM datasets where we significantly improve over the current state of the art. We also show for the first time that a common 3D segmentation strategy can be applied to both well-aligned nearly isotropic block-face EM data, and poorly aligned anisotropic serial sectioned EM data. The runtime of our method scales with O(n) in the size of the volume and is thus ready to be applied to very large datasets.

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Simpson Lab
09/09/17 | Simultaneous activation of parallel sensory pathways promotes a grooming sequence in Drosophila.
Hampel S, McKellar CE, Simpson JH, Seeds AM
eLife. 2017 Sep 09;6:. doi: 10.7554/eLife.28804

A central model that describes how behavioral sequences are produced features a neural architecture that readies different movements simultaneously, and a mechanism where prioritized suppression between the movements determines their sequential performance. We previously described a model whereby suppression drives a Drosophila grooming sequence that is induced by simultaneous activation of different sensory pathways that each elicit a distinct movement (Seeds et al. 2014). Here, we confirm this model using transgenic expression to identify and optogenetically activate sensory neurons that elicit specific grooming movements. Simultaneous activation of different sensory pathways elicits a grooming sequence that resembles the naturally induced sequence. Moreover, the sequence proceeds after the sensory excitation is terminated, indicating that a persistent trace of this excitation induces the next grooming movement once the previous one is performed. This reveals a mechanism whereby parallel sensory inputs can be integrated and stored to elicit a delayed and sequential grooming response.

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09/08/17 | Behavioral time scale synaptic plasticity underlies CA1 place fields.
Bittner KC, Milstein AD, Grienberger C, Romani S, Magee JC
Science (New York, N.Y.). 2017 Sep 08;357(6355):1033-1036. doi: 10.1126/science.aan3846

Learning is primarily mediated by activity-dependent modifications of synaptic strength within neuronal circuits. We discovered that place fields in hippocampal area CA1 are produced by a synaptic potentiation notably different from Hebbian plasticity. Place fields could be produced in vivo in a single trial by potentiation of input that arrived seconds before and after complex spiking. The potentiated synaptic input was not initially coincident with action potentials or depolarization. This rule, named behavioral time scale synaptic plasticity, abruptly modifies inputs that were neither causal nor close in time to postsynaptic activation. In slices, five pairings of subthreshold presynaptic activity and calcium (Ca(2+)) plateau potentials produced a large potentiation with an asymmetric seconds-long time course. This plasticity efficiently stores entire behavioral sequences within synaptic weights to produce predictive place cell activity.

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