Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically-Encoded Probes for DNA-PAINT

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super-resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of circa 3 nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy

Antitrust: Will it Change the Lives of Telecommunications Executives?

Good afternoon. This is the last panel of the afternoon. I would like to introduce myself. I\u27m Deborah Ellenberg, one of the hearing examiners at the State Corporation Commission, and I might add, who has a heightened appreciation for the Virginia Commission\u27s wise decision to handle those arbitrations. I am sure on behalf of Howard, Glenn and myself, we thank you for that decision

Using species attributes to characterize late-glacial and early-Holocene environments at Kråkenes, western Norway

Aim: We aim to use species attributes such as distributions and indicator values to reconstruct past biomes, environment, and temperatures from detailed plant‐macrofossil data covering the late glacial to the early Holocene (ca. 14–9 ka). Location: Kråkenes, western Norway. Methods: We applied attributes for present‐day geographical distribution, optimal July and January temperatures, and Ellenberg indicator values for plants in the macrofossil data‐set. We used assemblage weighted means (AWM) to reconstruct past biomes, changes in light (L), nitrogen (N), moisture (F), and soil reaction (R), and temperatures. We compared the temperature reconstructions with previous chironomid‐inferred temperatures. Results: After the start of the Holocene around 11.5 ka, the Arctic‐montane biome, which was stable during the late‐glacial period, shifted successively into the Boreo‐arctic montane, Wide‐boreal, Boreo‐montane, Boreo‐temperate, and Wide‐temperate biomes by ca. 9.0 ka. Circumpolar and Eurasian floristic elements characteristic of the late‐glacial decreased and the Eurosiberian element became prominent. Light demand (L), soil moisture (F), nitrogen (N), and soil reaction (R) show different, but complementary responses. Light‐demanding plants decreased with time. Soil moisture was relatively stable until it increased during organic soil development during the early Holocene. Soil nitrogen increased during the early Holocene. Soil reaction (pH) decreased during the Allerød, but increased during the Younger Dryas. It decreased markedly after the start of the Holocene, reaching low but stable levels in the early Holocene. Mean July and January temperatures show similar patterns to the chironomid‐inferred mean July temperature trends at Kråkenes, but chironomids show larger fluctuations and interesting differences in timing. Conclusion: Assigning attributes to macrofossil species is a useful new approach in palaeoecology. It can demonstrate changes in biomes, ecological conditions, and temperatures. The late‐glacial to early‐Holocene transition may form an analogue for changes observed in the modern arctic and in mountains, with melting glaciers, permafrost thaw, and shrub encroachment into tundra.publishedVersio

Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes

Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to ∼40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to ∼100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina

Communication

Visualizing the functional interactions of biomolecules such as proteins and nucleic acids is key to understanding cellular life on the molecular scale. Spatial proximity is often used as a proxy for the direct interaction of biomolecules. However, current techniques to visualize spatial proximity are either limited by spatial resolution, dynamic range, or lack of single-molecule sensitivity. Here, we introduce Proximity-PAINT (pPAINT), a variation of the super-resolution microscopy technique DNA-PAINT. pPAINT uses a split-docking-site configuration to detect spatial proximity with high sensitivity, low false-positive rates, and tunable detection distances. We benchmark and optimize pPAINT using designer DNA nanostructures and demonstrate its cellular applicability by visualizing the spatial proximity of alpha- and beta-tubulin in microtubules using super-resolution detection. © 2020 Wiley-VCH GmbH

Testing Hardy nonlocality proof with genuine energy-time entanglement

We show two experimental realizations of Hardy ladder test of quantum nonlocality using energy-time correlated photons, following the scheme proposed by A. Cabello \emph{et al.} [Phys. Rev. Lett. \textbf{102}, 040401 (2009)]. Unlike, previous energy-time Bell experiments, these tests require precise tailored nonmaximally entangled states. One of them is equivalent to the two-setting two-outcome Bell test requiring a minimum detection efficiency. The reported experiments are still affected by the locality and detection loopholes, but are free of the post-selection loophole of previous energy-time and time-bin Bell tests.Comment: 5 pages, revtex4, 6 figure

Averages of Fourier coefficients of Siegel modular forms and representation of binary quadratic forms by quadratic forms in four variables

Let $-d$ be a a negative discriminant and let $T$ vary over a set of representatives of the integral equivalence classes of integral binary quadratic forms of discriminant $-d$. We prove an asymptotic formula for $d \to \infty$ for the average over $T$ of the number of representations of $T$ by an integral positive definite quaternary quadratic form and obtain results on averages of Fourier coefficients of linear combinations of Siegel theta series. We also find an asymptotic bound from below on the number of binary forms of fixed discriminant $-d$ which are represented by a given quaternary form. In particular, we can show that for growing $d$ a positive proportion of the binary quadratic forms of discriminant $-d$ is represented by the given quaternary quadratic form.Comment: v5: Some typos correcte

Protecting Clinical Trial Participants and Protecting Data Integrity: Are We Meeting the Challenges?

Susan Ellenberg discusses alternative approaches towards evaluating data as it accumulates in clinical trials, and to protecting the integrity and preventing undue risks to participants, as the trial continues