Genome-wide Maps of Nuclear Lamina Interactions in Single Human Cells

Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization.National Institutes of Health (U.S.) (Grant R01 GM114190)National Human Genome Research Institute (U.S.) (Grant R01 HG003143

Modular actin nano-architecture enables podosome protrusion and mechanosensing

Basement membrane transmigration during embryonal development, tissue homeostasis and tumor invasion relies on invadosomes, a collective term for invadopodia and podosomes. An adequate structural framework for this process is still missing. Here, we reveal the modular actin nano-architecture that enables podosome protrusion and mechanosensing. The podosome protrusive core contains a central branched actin module encased by a linear actin module, each harboring specific actin interactors and actin isoforms. From the core, two actin modules radiate: ventral filaments bound by vinculin and connected to the plasma membrane and dorsal interpodosomal filaments crosslinked by myosin IIA. On stiff substrates, the actin modules mediate long-range substrate exploration, associated with degradative behavior. On compliant substrates, the vinculin-bound ventral actin filaments shorten, resulting in short-range connectivity and a focally protrusive, non-degradative state. Our findings redefine podosome nanoscale architecture and reveal a paradigm for how actin modularity drives invadosome mechanosensing in cells that breach tissue boundaries

Hemidesmosomes modulate force generation via focal adhesions.

Hemidesmosomes are specialized cell-matrix adhesion structures that are associated with the keratin cytoskeleton. Although the adhesion function of hemidesmosomes has been extensively studied, their role in mechanosignaling and transduction remains largely unexplored. Here, we show that keratinocytes lacking hemidesmosomal integrin α6β4 exhibit increased focal adhesion formation, cell spreading, and traction-force generation. Moreover, disruption of the interaction between α6β4 and intermediate filaments or laminin-332 results in similar phenotypical changes. We further demonstrate that integrin α6β4 regulates the activity of the mechanosensitive transcriptional regulator YAP through inhibition of Rho-ROCK-MLC- and FAK-PI3K-dependent signaling pathways. Additionally, increased tension caused by impaired hemidesmosome assembly leads to a redistribution of integrin αVβ5 from clathrin lattices to focal adhesions. Our results reveal a novel role for hemidesmosomes as regulators of cellular mechanical forces and establish the existence of a mechanical coupling between adhesion complexes

Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy

<div><p>Single Molecule Localization super-resolution Microscopy (SMLM) has become a powerful tool to study cellular architecture at the nanometer scale. In SMLM, single fluorophore labels are made to repeatedly switch on and off (“blink”), and their exact locations are determined by mathematically finding the centers of individual blinks. The image quality obtainable by SMLM critically depends on efficacy of blinking (brightness, fraction of molecules in the on-state) and on preparation longevity and labeling density. Recent work has identified several combinations of bright dyes and imaging buffers that work well together. Unfortunately, different dyes blink optimally in different imaging buffers, and acquisition of good quality 2- and 3-color images has therefore remained challenging. In this study we describe a new imaging buffer, OxEA, that supports 3-color imaging of the popular Alexa dyes. We also describe incremental improvements in preparation technique that significantly decrease lateral- and axial drift, as well as increase preparation longevity. We show that these improvements allow us to collect very large series of images from the same cell, enabling image stitching, extended 3D imaging as well as multi-color recording.</p></div

pH in OxEA and Gloxy buffer.

<p>The pH in open dishes filled with 0.5 ml of OxEA (red squares) or Gloxy buffer (blue circles) is graphed at the indicated time points. Note the steep drop in pH in Gloxy buffer, which limits imaging to ~ 1 hour unless measures are taken to prevent oxygen influx.</p

Characterization of blinking in OxEA, Gloxy and MEA buffer.

<p>(A) Raw blinking frames (10 ms each, i.e. non-merged results) taken at the indicated time points. Shown are data for Alexa-488 (A488), Alexa-555 (A555) and Alexa-647 (A647) in both fresh Gloxy and OxEA buffer. Imaging was started after a 2–5 second pumping period at full laser power. Note presence of significant structured background in Gloxy buffer. (B) Mean intensity of individual blinks (merged in consecutive frames). Data are mean +/- SEM. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158884#sec010" target="_blank">Methods</a> for further details. (C) Left panel, average number of blinks per frame (calculated in blocks of 1000 frames) of a preparation labeled with Alexa-488 and imaged in Gloxy or OxEA. Similar parts of cells with similar initial labeling density were selected based on the low-intensity wide-field image. Note the much larger number of blinks in OxEA for Alexa-488. Right panel, summary of blinks per frame data for Alexa-488, Alexa-555, Alexa-647 and FITC in three different buffers. Data are mean +/- SEM; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158884#sec010" target="_blank">Methods</a> for further details. (D) Number of blinks per frame, averaged over the full duration of the acquisition movie, in experiments carried out at the indicated times after applying the buffers. Within the hour, blinking has dropped dramatically in Gloxy whereas OxEA performs well for several hours. (E) Duration of individual blinks of Alexa-488 and Alexa-647 in fresh and ageing (90 minutes) Gloxy, and in fresh and ageing (120 min) OxEA. Multi-frame blinks are very common in ageing Gloxy buffer, as witnessed from the increased average duration of blinks and the enormous increase in duration spread (Data are mean +/- standard deviation). Increased blink duration adds to the appearance of structured background.</p

Sealing cell culture dishes to prevent oxygen influx.

<p>(A) O₂ levels, detected by daily fluorescence lifetime-based recording in a WillCo Well sealed with Twinsil glue (blue) or aluminum tape (red), respectively. At 48 days, the seal was broken to test responsiveness of the O₂ sensor (black arrow). (B) A fluorescent O₂ indicator pad was covered with Twinsil (blue) or aluminum tape (red) and submerged in buffer at ambient oxygen levels. O₂ levels were recorded continuously, and at t = 8 min oxygen scavenger was added from concentrated stock. Note the rapid drop in O₂ levels below the Twinsil seal, indicating its permeability to oxygen. (C) WillCo Wells #GWSB 3512-N dish shown with 24-mm coverslip lid (right) and sealed with pieces of aluminum tape (left).</p

Optimizing drift in SR preparations.

<p>(A) Example traces of drift quantifications during 30 min in #3512 dishes (blue), #3512-N dishes (red) and #3512-N dishes sealed with coverslip and adhesive-backed aluminum tape (pink). Shown is the mean displacement away from the origin of immobilized beads during 30 min. (B) Summary of 2D (lateral; square symbols) and 3D (lateral + focus, round symbols) drift experiments. Shown are endpoint drifts at 30 min and at 60 min for the indicated imaging dishes. The WillCo Wells optimized #3512-N dishes display significantly improved stability. Data are mean +/- SEM of >3 experiments each.</p

GSDIM imaging in OxEA buffer.

<p>(A) comparison of image quality in ageing Gloxy buffer (right) to that in OxEA buffer (left). Images of Ab-labeled vimentin intermediate filaments were collected ~ 2 hours after mounting the preparation in an open dish. (B) 3-color image of keratin (green, Alexa-555), plectin (blue, Alexa-488) and β4 integrin (red, Alexa-647). Approximately 12000 frames where collected for each color channel. Full resolution images are available at <a href="https://osf.io/q684r/" target="_blank">https://osf.io/q684r/</a>.</p

Oxygen levels in OxEA and Gloxy buffers.

<p>O₂ levels were detected every two seconds using a FireSting fluorescence-lifetime based oxygen detector. Note that addition of Gloxy buffer (blue) causes a rapid drop in O₂ level to undetectable levels, whereas OxEA caused a more slow and less complete removal of O₂.</p